studies related to identification of toxic metabolites of

Studies Related to Identification of Toxic Metabolites of Benzene and

Their Chemical Reactivity

SUMMARY SUBMITTED TO THE

DEPARTMENT OF BIOCHEMISTRY (Faculty of Life Sciences)

ALIGARH MUSLIM UNIVERSITY, ALIGARH FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY

By

PETROLEUM TOXICITY DIVISION

Industrial Toxicology Research Centre Lucknow (India)

m

Summary

Small amounts of a large variety of substances normally regarded as foreign to the

body are always existent in the environment. These are often toxic and occur frequently.

In the modern world, man is increasingly dependent upon the use of synthetic chemicals

and other domestic products in larger number.

Benzene is frequently used as an industrial solvent of the modem time. Its excellent

solvent properties and its use as a starting material for wide ranging chemicals and

domestic products of commercial importance has made it indispensable. Its value in our

modern society is great, but due to its toxic nature, the use of t)enzene would undoubtedly

have an impact on our way of life. The use of benzene has been on the rise and

obviously the risk of exposure is also increasing. Though the exposure levels to benzene

can be brought down to acceptable levels but can not be eliminated.

Benzene is a well known genotoxic and carcinogenic agent. But the precise

mechanism of its genotoxicity and carcinogenicity are yet to be known. Therefore, in this

study an attempt has been made to explain the probable metabolite(s) of benzene to

induce haematotoxicity and role of transition metal ions (iron and copper) to explain the

Summary 2

possible biochemical mechanisms of genoloxicity and carcinogenicity expressed during

benzene exposure.

Release of iron from ferritin by 1,2,4-benzenetriol

Iron is a vital metallic cofactor of several important biomolecules. It is normally

localized in ferritin (main iron storage protein) or transferrin (iron transport protein). But

this iron, when it gets decompartmentalized manifests lethally by triggering off free radical

reactions by means of Fenton and Haber-Weiss type of reactions:

Fe(lll) + Reductant > Fe(ll) + Oxidized Reductant

2Fe(ll) + O2+ 2H* > 2Fe(lll) + H A

Fe(ll) + HjOj -— > Fe(lll) +-OH + OH"

Large human prospective studies suggest that excess body free iron is associated

v/ith carcinogenesis, such as primary hepatocellular carcinoma, colon cancer, lung cancer,

bladder cancer, acute lymphocytic leukemia and neuroblastoma. Bleomycin detectable

iron has been shown to accumulate in significant concentrations in the bone marrow of

benzene exposed rats. Polyphenolic metabolite of benzene, particularly 1,2,4-

benzenetriol (a Irihydroxy benzene) was found to release significant amount of iron from

horse spleen ferritin under aerobic and anaerobic conditions. The release of iron from

ferritin during autooxidation of 1,2,4-benzenetriol in wvo may result in increased

intracellular concentrations of free iron. Although, the physiological mechanism of iron

mobilization from ferritin is pooriy understood, the observed iron release from ferritin by

1,2,4-benzenetriol could occur through the direct reduction of iron by the hydroxy

hydroquinone form or via its autooxidation intermediates i.e., superoxide radical and

semiquinone It was observed that the iron released from ferritin in the presence of

1,2,4-benzenelriol was capable of inducing lipid peroxidation and catalyzing

bleomycin-dependenl calf thymus DNA degradation. The mechanism by which iron is

released from bone marrow cells and whether it acts as a prooxidant during benzene

toxicity is not known. However, the present observation offer a new mechanism that the

iron released from ferntin by 1,2,4-benzenetriol could contribute to the better

Summary 3

Polyphenol-iron complex: A probable toxic metabolite of benzene

An attempt was made to examine in vitro formation of polyphenoi:iron complex.

Complex formation was fractionated on Sephadex G-10 column chromatography. Results

clearly show the formation of polyphenoUron complex under in vitro condition. Polyphenol

iron complex formation was also observed during the release of iron from ferritin in the

presence of 1,2,4-benzenetriol. Although, benzene exposure leads to accumulation of

iron as well as polyphenolic metabolites of benzene in the bone marrow against the

concentration gradient, the formation of such complex in vivo is yet to be identified.

We evaluated the haematotoxicity of hydroquinone:iron complex as a possible

haematotoxic intermediate during benzene exposure. Results indicate that hydroquinone

iron chelate showed marginal increase in haematological parameters in experimental

animals But Ihe precise mechanism of marginal, yet significant increase in haema

tological indices is not clear, Atleast, some of the effects observed in hydroquinone:iron

treated group showed similarities to some extent with what has been shown to occur

following exposure of benzene to mice.

Glutathionyl hydroquinone: A possible toxic metabolite of benzene

Glutathionyl hydroquinone has been isolated as one of the urinary metabolites of

hydroquinone, indicating that glutathionyl hydroquinone formation occurs in vivo and

autooxidized at several fold higher than the parent hydroquinone to glutathionyl

benzoquinone We noticed that glutathionyl hydroquinone is a potent prooxidant to

damage both calf thymus as well as plasmid pUC 18 DNA and this observation may be

of toxicological importance in relation to benzene induced genotoxicity.

Haematotoxicity of glutathionyl hydroquinone was studied in male mice at dose 100

mg/kg body weight, intraperitoneally for one month. Increased pattern of relative liver and

spleen weight and WBC were noticed, but these results were not conclusive to explain

GHQ as potent haematotoxic metabolite of benzene The pharmacokinetics and

distribution of administered GHQ Is not known. Whether GHQ administered I.p. reaches

Summary 4

understanding of the toxicity of benzene. Further, elucidation of the mechanisms of

iron-mediated oxidative damage may be important for the prevention of carcinogenesis.

Prooxidant and antioxidant properties of iron: polyphenol complex

Polyphenolic metabolites of benzene viz, hydroquinone and 1,2,4- benzenetriol have

been shown to be toxic and the suggested mechanism includes free radical fonnation via

superoxide during their autooxidation and the covalent binding of semiquinone to DNA,

RNA and other cellular macromolecules. Benzene exposure also leads to an

accumulation of bleomycin sensitive iron in bone marrow. 1,2.4-Benzenetriol, a trihydroxy

metabolite of benzene was capable of releasing iron from horse spleen ferritin, which may

lead to accumulation of iron in bone marrow during benzene exposure, as the

polyphenolic metabolites are accumulated against the concentration gradient in the bone

marrow. The ferrous iron may be chelated to the polyhydroxy metabolites of benzene viz.

hydroquinone and 1,2,4-benzenetriol.

Studies carried out with hydroquinone/1,2,4-benzenetriol and iron showed several

fold increase in iron-catalyzed bleomycin- dependent degradation of calf thymus DNA as

compared to iron alone. Complex formation some how increase the availability of iron to

degrade DNA. It is also possible that the hydroquinone and 1,2,4-benzenetriol possess

combined iron-chelating and iron ion reducing properties to stimulate DNA degradation

in the presence of bleomycin. Further, iron:polyphenol complex appears to be stable and

intact as the recovery from bone marrow lysate was complete. On the other hand,

iron polyphenol complexes have shown an antioxidant activity by inhibiting of

iron-dependent lipid peroxidation in rat brain homogenate and glutamate degradation.

This is similar to the antioxidant properties exhibited by flavonoids due to their ability to

chelate iron. Thus the polyphenolic chelates limiting the availability of iron for lipid

peroxidation and at the same time facilitate bleomycin- dependent degradation of DNA.

Summmy

the target organ i.e. bone marrow is yet to be evaluated. Other reason may be due to

difference in sensitivity of species towards different benzene metabolites.

Role of transition metal ions (Fe.Cu) during benzene toxicity

Earlier studies have provided enough evidence that transition metals including iron

and copper at e capable of directly mediating the activation or metabolism of several

xenobiotics via a metal redox mechanism, which leads to the formation of more reactive

oxygen and other organic free radicals. We observed that exposure to benzene leads to

increase in total iron content in liver and isolated rat liver nuclei. Benzene metabolites

induced the DNA damage particularly in the presence of iron and copper. This study

would help in better understanding of the chromosomal aberrations/abnormalities

associated with benzene exposure. Copper which is associated with DNA, may play a

potential role in nuclear DNA damage in presence of hydroquinone. However, the

molecular mechanism of its damage is not well understood. Significant increase in iron

content in liver and in isolated liver nuclei during benzene exposure and the copper which

is associated with DNA may have toxicological implications for cytotoxicity of liver cells

and liver nuclear DNA damage in wVo.

In the light of our investigations following conclusions can be made;

• 1,2,4-Benzenetriol is a potent reductant of ferric iron of ferritin and releases iron from

ferritin core. The release of iron from bone marrow lysate by 1,2,4-benzenetriol may

be of toxicological significance during benzene exposure as this could lead to

disruption of intracellular iron homeostasis in bone marrow cells.

• The iron:polyphenol chelate, on one hand, is a more potent DNA cleaving agent in

the presence of bleomycin, and on the other hand, it is a less effective free radical

generator as compared to iron alone. However, the formation of such a complex in

VIVO IS not known. Further studies are needed to characterize such complexes in

vivo and evaluate their toxicological significance during benzene exposure.

B Polyphenol forms complex with iron under in vitro condition and may be a possible

h««»rri8t!iit©i«iQ interma^iista during benzene exposure.

Siunmary

The present in vitro studies revealed that glutathionyl hydroquinone is a potent toxic

metabolite of benzene and acts as a prooxidant due to its faster autooxidation to

glutathionyl benzoquinone and oxyradicals which may induce DNA damage.

Exposure to benzene resulted in significant increase in total iron content in liver

tissue and in isolated liver nuclei.

Benzene metabolites (particularly hydroquinone) induced genotoxic effect in

presence of copper which is associated with the nuclear DNA. However, further

studies are needed to explain the precise mechanism of DNA damage and

chromosomal aberration in wVo during benzene exposure and effect of transition

metal ions in benzene induced genotoxicity and human leukemia.

Studies Related to Identification of Toxic Metabolites of Benzene and

Their Chemical Reactivity

THESIS SUBMITTED TO THE

DEPARTMENT OF BIOCHEMISTRY (Faculty of Life Sciences)

ALIGARH MUSLIM UNIVERSITY, ALIGARH FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY

By

PETROLEUM TOXICITY DIVISION

Industrial Toxicology Research Centre Lucknow (India)

1996

, r , . ^ ^ . M , , g ^ ^

'^^^.

' • / Ace No. ^

« * / , . , ••'•^ UNIVtR^^"^^

T4873

DEPARTMENT OF BIOCHEMISTRY F A C U L T Y O F L I F E S C I E N C E S

ALIGARH MUSLIM UNIVERSITY ALIGARH—202002 (INDIA)

TELEPHONE : 4 00 74 1 TELEX: 564-230 AMU IN

Ref. No- Dated-

Dr. Jawaid Iqbal Lecturer Department of Biochemistry

CERTIFICATE

This is to certify that the work embodied in this thesis entitled "Studies related to identification of toxic metabolites of benzene and their chemical reactivity" has been carried out by Mr. Sarfaraz Ahmad under my supervision.

He has fulfilled all the requirements of the Aligarh Muslim University, Aligarh regarding the nature and period prescribed for investigational work for the degree of Doctor of Philosophy in Biochemistry.

The work included in this thesis is original unless stated othenwise and has not been submitted for any other degree.

Dated: T^ov. c^fh , /'-f7^ (Jawaid Iqbal)

Industrial Toxicology Research Centre

F M NO. - 0522-278227 Telex -0535-2456 ^JJLM M A H A T M A GANDHI MARC

12 2 0 1 0 7 y S ^ ^ POST BOX No. 80

2 i 7 5 8 6 3 ^ ^ a » L U C K N O W-226001 T e l e g r a n i - I N T O X r ^ — ^ U.P.INDIA

Dr. G.S. Rao Assistant Director Petroleum Toxicity Division

CERTIFICATE

This is to certify that the work embodied in this thesis entitled "Studies related to identification of toxic metabolites of benzene and their chemical reactivity" has been carried out by Mr. Sarfaraz Ahmad under my supervision

He has fulfilled all the requirements of the Aligarh Muslim University, Aligarh regarding the nature and period prescribed for investigational work for the degree of Doctor of Philosophy in Biochemistry

The work included in this thesis is onginal unless stated otherwise and has not been submitted for any other degree. .

Dated: | ^ "^ ^ ' (Dr. G.S. Rao)

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CONTENTS

Page 1

i i i

Abbreviations

Preface

Chapter -1 Benzene an Overview 1-25

Chapter - U Materials and Methods 26-40

Chapter - HI Release of Iron from Ferritin by 1,2,4-benzenetriol 41-54

Chapter - IV Prooxidant and Antioxidant Properties of Polyphenol-iron 55-64 Complex: an Implication for Benzene Toxicity

Chapter - V Polyphenol - iron Complex: a Probable Toxic Metabolite 65-78 of Benzene.

Chapter - VI Glutathionyl Hydroquinone: a Possible Toxic Metabolite 79-97 of Benzene.

Chapter - VII Role of Transition Metal Ions (Fe, Cu) During Benzene Toxicity 98-114

Summary 115-120

Bibliography 121-149

List of Publications 150

IIII till till It

8-ALA

AML

BQ

BSA

BT

b.w.

CML

dl

DLC

DNA

DTNB

DTPA

EPO

GHQ

gm

GSH

HO

H,0,

HQ

hr

I.p.

Kg

LMI

M

mg

min

ml

mM

mmole

MPO

N

ABBREVIATIONS

5 -Aminolevulinic acid

Acute myelogenous leukemia

1,4-Ben2oquinone

Bovine serum albumin

1,2,4-Benzenetriol

Body weight

Chronic myelogenous leukemia

Decilitre

Differential leukocyte count

Deoxyribonucleic acid

5,5'-Dithiobis-(2-nitrobenzoic acid)

Diethylenetriaminepentaacetic acid

Eosinophil peroxidase

Glutathionyl hydroquinone

Gram(s)

Glutathione (reduced)

Hydroxyl radical

Hydrogen peroxide

Hydroquinone

Hour(s)

Intraperitoneal

Kilogram

Low molecular weight iron

Molar

Milligram

Minute(s)

Millilitre

Millimolar

Millimole

Myeloperoxidase

Normal

ii

NADH

nmole

8-OHdG

ppm

RNA

rpm

SD

SE

TBA

TBAR

TCA

WBC

w/v

A°

nm

Mg

Ml

JM

%

x p

6

A

Nicotinamide adenine dinucleotide

Nanomole

8-Hydroxydeoxyguanosine

Part per million

Ribonucleic acid

Rotations per minute

Standard deviation

Standard error

Thiobarbituric acid

Thiobarbituric acid reactive products

Trichloroacetic acid

White blood corpuscle

Weight by volume

Angstrom

Nanometre

Microgram

Microlitre

Micromolar

Percent

Degree centigrade

Beta

Delta

Lembda

Phi

i i i

After the advent of industrial revolution there has tjeen a massive

proliferation of industries to cater to the needs of the modern society But,

unfortunately man has been exposed knowlingly and unknowlingly to many

hazardous chemicals. Benzene is a v /eil known xenobiotic environmental pollutant

and is a natural constituent of crude oil.

Benzene is extensively used as an industrial solvent and a common additive

in gasoline and other solvents. Emissions from burning coal and oil, benzene

waste and storage operations motor vehicle exhaust, evaporation from gasoline

service stations and use of industrial solvents increase benzene level in the air.

Since tobacco contains high level of benzene, tobacco smoke is another source

of benzene in air. People living around hazardous waste sites, petroleum refining

operations, petrochemical manu-facturing sites, or gas station may be exposed to

higher levels of benzene in air.

i v

Benzene causes disorder in the blood and is well known human leukemogen.

People who breath benzene for long periods may experience hannful effects in the

tissues that form blood cells. Understanding and preventing the threat of benzene

to human health is one of the most important environmental issues facing national

and international regulatory authorities. Uncertainties about health effects must

be balanced against the potential for substantial economic and societal costs in

regulating benzene.

Despite the extensive research in the field of benzene induced genotoxicity

and carcinogenesis, the precise mechanism of its toxicity is largely unknown.

Therefore, we studied in this respect to explain the possible mechanisms of

benzene toxicity and role of metal ions (particulariy iron and copper) in benzene

induced genotoxicity and carcinogenesis.

Chapter- I

Benzene an Overvieiv

Introduction

Exposure

Route of Exposure

Biomarkers

Health Hazards

Human Exposure

Metabolism

Toxicity

Mechanism of Toxicity

Current Trend and Future Research

Chapter -1

INTRODUCTION

Benzene (CgHg) is the smallest and most stable aromatic hydrocarbon. It is a

colourless liquid with a sweet odor. Due to high vapour pressure, benzene evaporates

into air very quickly and is highly flammable. Today benzene is commercially recovered

from both coal and petroleum sources. In the past benzene was widely used as a solvent

but this use is now decreasing due to its toxic nature. The major uses of benzene are in

the production of ethylbenzene, cumene, cyclohexane, styrene, caprolactam, linear alkyl

benzene, phenol, maleic anhydride, DDT, BHC and various chemical synthesis. Other

industrial use of the benzene is in the production of rubber plastics, paints, adhesives,

nylon fibers and artificial leather (Eveleth, 1990). Benzene is also a component of

gasoline since it occurs naturally in crude oil and since it is a by product of oil refining

processes. Benzene is especially important for unleaded gasoline because of its

anti-knock characteristics. For this reason, the concentration of aromatics such as

benzene in unleaded fuels has increased (Brief et al., 1980).

Chapter - / 2

EXPOSURE Benzene is a ubiquitous agent. As a component of petroleum it is widely distributed.

Virtually all (99.9%) of the benzene released into the environment is emitted into the air.

Benzene exposure occurs in the workplace, in the general environment, and through the

use of consumer products. Occupational exposures present the highest risks, but most

individuals are exposed through the use of petrochemicals, including gasoline evaporation

and automobile exhaust, and through direct or indirect exposure to cigarette smoke. The

general population may also be exposed to t)enzene in a number of ways.

(a) Cigarette smoking

Cigarette smoking is the most significant source of t>enzene exposure for the general

population from tx)th active and passive smoke. Smoking accounts for about half of the

total population burden of exposure to benzene (Wallace, 1989). Blood benzene levels

have been shown to be higher in smokers than in nonsmokers (Brugnone et al., 1989).

One pack a day contributes about 600 ug benzene to the smoker (WHO, 1987).

(b) Home use of solvents or gasoline

Gasoline is frequently used in daily domestic purpose, particularly those who work

on gasoline-fueled machinery. Gasoline contain variable amount of benzene and can be

a significant source of exposure to the individual, involving dermal exposure as well as

inhalation. Other source of benzene exposure include consumer products such as

paints, adhesives rubber products, tapes and household cleaning agents.

(c) Leaky underground storage tanks

Leakage of underground storage tanks of gasoline is a major source of water

contamination with benzene. Contamination of ground water has occurred, leading to

human exposure through the three routes of ingestion, inhalation and skin absorption.

General population may be exposed with benzene through the supply of contaminated

drinking water supply.

Chapter -1 4

some exposure to benzene, 60-70% indicate a dangerous level of exposure, and less

than 60% indicate an extremely hazardous exposure. Urinary sulphate levels are

however, quite variable, and are not being used to identify exposure levels of benzene

associated with minimal toxic effect. Additionally, the test for urinary sulphates is not

specific for benzene.

Urinary phenol measurements have routinely been used for monitoring occupational

exposure to benzene, and there is some evidence that urinary levels can be correlated

with exposure level (Inoue et al., 1988a; Karacic et at., 1987). However, correlating

urinary phenol with benzene exposure is complicated by potentially high and variable

background levels that result from ingestion of vegetables, exposure to other aromatic

compounds, ingestion of ethanol, and inhalation of cigarette smoke (Nakajima et al.,

1987). In addition, coexposure to toluene, a common solvent, has been shown to inhibit

transformation of benzene to phenol (Inoue et al., 1988a). Analysis of data collected for

49 chemical woricers in the coke oven industry led the examiners to conclude that urinary

phenol could not be used as an indicator of benzene uptake for exposures of about 1

ppm or less (Drummond et al., 1988). The American Conference of Governmental

Industrial Hygienists has established 50 mg phenol/liter urine as the biological exposure

indices for benzene exposure in the workplace (Lowry, 1986),

Other urinary metabolites of benzene have been measured, but it is not clear if any

of these additional metabolites can be used to monitor exposure to benzene. In addition

to phenol, other phenolic metabolites have been investigated as biomarkers of benzene

exposure (Inoue et al., 1988b). Results from data collected on 152 chemical workers

indicated that, for benzene concentrations greater than 10 ppm, there was a linear

relationship between the concentration of benzene in breathing zone air and urinary

concentrations of catechol and quinol. Urinary trans, trans-muconic acid (an open-ring

benzene metabolite) has also been investigated as an indicator of benzene exposure

(Inoue et al., 1989a). In a study of 152 shoe and paint manufacturers exposed to

benzene, it was shown that urinary trans, trans-muconic acid correlated linearty with the

time weighted averages of benzene. Measurements of trans, trans-muconic acid were

able to distinguish the groups of workers exposed to benzene at concentrations of 6-7

Chapter -1 3

(d) Automobile source

Automobile emissions remain a substantial source of community exposure to

benzene. However, release of benzene and ottier gasoline vapors during refueling

remains a significant source of community t>enzene exposure as well as exposure to the

individual doing the refueling. The general population is exposed to t^enzene mainly

through inhalation of contaminated air particularly in areas of heavy motor traffic and

around gas stations. All outdoor source, including automobile exhaust and stationary

source emissions, account for only at>out 20% of the total population exposure to

benzene (Wallace, 1989).

ROUTE OF EXPOSURE

Benzene exposure occurs through inhalation, ingestion and dermal routes.

Inhalation is the dominant pathv*/ay of human exposure, accounting for more than 99%

of the total daily intake of benzene (Hattemer-Frey et al., 1990). Benzene is readily

absorbed in the lung, directly entering the blood stream and taeing distributed to the

tissues. Benzene within the blood is in direct equilibrium with the tsenzene in expired air.

Thus, measurement of end alveolar breath l>enzene concentration is a good indicator

of body benzene concentration. Approximately 50% of benzene taken up into the body

by any route is eventually exhaled, the extent being dependent on benzene dose and the

rate of metabolism and respiratory mechanics. Ingested benzene is also assumed to be

fully absort>ed but the dermal absorption of t>enzene is likely to be minimal (P<1.0%)

(Loden, 1986; Susten et al., 1985).

BIOMARKERS

Biomarkers of exposure are available for tsenzene. One selective test is the urinary

sulphate ratio test which is based on the premise that with increasing exposure there will

be an increase in benzene metabolites conjugated with sulphate moieties (Hammond

and Herman, 1960). Estimates of benzene exposure can be made by comparing the

ratio of inorganic to organic sulphates in the urine. Inorganic sulphate levels amounting

to 80-95% of total urinary sulphates are considered nomnal background, 70-80% indicate

Chapter • I ^

ppm from the group of nonexposed workers. The high sensitivity of trans, trans-muconic

acid is due to the very low urinary background levels of trans, trans-muconic acid among

the general population in contrast to the high background levels found for phenol,

catechol, and quinol. Inoue ef a/., (1989a) determined that, while trans, trans-muconic

acid measurements were useful for determining group exposures to benzene, they could

not t>e used to monitor individual exposure because of the large variations in individuals

urinary trans, trans-muconic acid values. In addition, urinary trans, trans-muconic acid

excretion was suppressed by coexposure \o toluene. However, recent studies (Bartczak

et al., 1994; Weaver et al., 1996) supported that trans, trans-muconic acid may be useful

in the evaluation of environmental benzene exposure. Weaver et al., (1996) assessed

benzene exposure by monitoring urinary trans, trans-muconic add in urban children with

elevated blood lead level. Inoue et al., (1989b) reported that the urinary concentration

of 1,2,4-t)enzenetriol related lineariy to the intensity of exposure to benzene both in men

and women workers exposed to benzene and however was suppressed by toluene

coexposure among male workers exposed to a mixture of benzene and toluene.

Another b>enzene-specific urinary metabolite is S-phenyl-N-acetylcysteine (Jonge-

neelen et al., 1987). No background excretion of S-phenyl-N-acetylcysteine was found

in rats or in humans. However, the authors concluded that biological monitoring of

industrial exposure to benzene by the determination of S-phenyl-N-acetylcysteine in the

urine is not better than the determination of phenol in urine. An improvement in the

biological monitoring of benzene exposure at levels even <1 ppm has been indicated by

determining S-phenylmercapturic acid in the urine (Stommel et al., 1989),

Red and white blood cell counts have been used as an indicator of high occupational

exposures. Monitoring of benzene exposed wori<ers has included monthly blood counts,

with workers being removed from areas of potential exposure when white blood cell

counts fell below 4,000/mm^ or erythrocyte counts fell below 4,000,000/mm' (OSHA,

1987). Additionally, it has been suggested that chromosomal aberrations in the peripheral

lymphocytes and sister chromatid exchanges could be used as monitoring end points for

benzene effects (Van Sitter! and de Jong, 1985). Benzene metabolites have also been

found to form adducts with DNA (Lutz and Schlatter, 1977, Norpoth et al., 1988; Snyder

Chapter • I 6

et ai, 1987). Furthermore, quantitation of benzene derived adducts on haemoglobin

shows potential as a marker for assessing the possible health effects of occupational

exposure to inhaled benzene (Sun et al., 1990). Measurement of protein adducts in

blood also offers the potential for monitoring the bioavailability of reactive, and

presumably toxic metabolites of benzene in workers and other persons. Recently

Yeowell-O'Connell et al., (1996) reported a method for measuring cysteinyl adducts of

the benzene metabolite benzene oxide with haemoglobin in blood from humans or

rodents exposed to benzene. This new procedure is currently being applied to the

analysis of albumin adducts of benzene oxide in rats.

Exposure to benzene causes toxic effects in the bone marrow, either from benzene

or its metabolites (Eastmond et al., 1987). Therefore, it is possible that haematological

tests could be used as markers of haernatotoxicity. To date, surveillance and eariy

diagnosis of benzene haematotoxicity depend primarily on the complete blood count,

including haemoglobin, hematocrit, erythrocyte indices, erythrocyte count, white blood cell

count, and differential and platelet count. In effect, complete blood counts and marrow

examinations should be good for eariy detection of preleukemic lesions. Additionally,

cytogenetic tests of marrow cell are being used more often, but are not yet of diagnostic

significance. Workers exposed to benzene in the air have shown elevated levels of

6-aminolevulinic acid in erythrocytes and elevated levels of coproporphyrin in the urine

(Khan and Muzyka, 1973). These may be biomari<ers for disruption of porphyrin synthesis

and may be eariy indicators of adverse haematological effects.

HEALTH HAZARDS

(I) Acute exposure effects

High concentration of benzene inhaled affect the central nervous system. At high

levels benzene has an anesthetic effect (Walker, 1976). Fatalities due to benzene are

the result of its concentration in the lipid of brain cells, effecting circulating problems,

some instances of convulsions and/or paralysis, the loss of consciousness, coma and

finally death. Damage to brain has been found to be a primary cause of death. At times

death wasprecededby periods of violent convulsion (Feil, 1933). The narcotic threshold

Chapter -1 7

concentration for laboratory animals is approximately 4,000 ppm and concentrations

above 10,000 ppm are usually fatal. Acute LDM value for new bom rats is as low as 1.0

ml/kg, while for adult rats it is in the range of 5.6-6.8 ml/kg (Kimura e a/., 1971). The

experimental results obtained indicate that the putative mechanism of acute benzene

toxicity is the rapid release of the adrenal hormones,epinephrine and nor-epinephrine,

into the blood stream, sensitizing the myocardium to its action and the most prominent

effect is central nervous system stimulation followed by depression and respiratory

failure. Direct exposure to high benzene concentration may lead to tissue destruction

due to its lipid solubility.

(II) Chronic exposure effects

(a) Haematotoxjcity

Benzene is a myelotoxin, chronic exposure of humans and experimental animals

results in a wide vanety of blood dyscrasias including; aplastic anemia, leukopenia,

pancytopenia, thrombocytopenia etc. (Snyder etai, 1977; Aksoy, 1989).

Le Noir (1897) and Selling (1910) were the first to report on haematopoietic disorders

associated with benzene in humans. These disorders were usually manifested by a

decrease in the number of one or more of the three major circulating blood cell types.

Irons et al., (1979) and Snyder et al., (1982) have shown that lymphocytes to be the most

susceptible of all the blood elements and granulocytes to be the most resistant of the

circulating cells to benzene toxicity. Granulocyte levels got depressed only on doses, 880

mg/day for 10 days (Irons et al., 1979). On the other hand exposures to concentration

sufficient to produce lymphocytopenia and anemia, even for life time, failed to effect the

granulocyte levels (Snyder, 1982).

Evidence that benzene acts primarily on committed progenitor cells was indicated

by a decrease in the number of differentiating erythroid and myeloid progenitors in the

bone man-ow (Irons e/a/., 1979). Studies by Bolcsack and Nerland (1983) have shown

that phenol, catechol and hydroquinone reduce the ^'Fe incorporation into developing

erythrocytes. The macrophages, which are a major source of polypeptide growth factors

required for the proliferation, development and survival of progenitor cells may be the

possible target of benzene toxicity (Moore, 1978). Post era/., (1986) demonstrated that

Chapter -1 8

phenol is metabolized in adherent mouse marrow maaophages by a peroxidase activity

to one or more covalently binding species and that micromolar concentrations of

hydroquinone, p-benzoquinone inhibit macrophage RNA synthesis. Later on Lewis et al.,

(1988 a) showed that benzene metabolites, catechol, hydroquinone, p-t)enzoquinone and

1,2,4-benzenetriol have potent and varied toxic effects on macrophage function and

activation. Sabourin et al., (1990) showed that inhalation exposure of mice resulted in

benzene metabolite DNA and haemoglobin adducts formation.

(b) Genotoxicity

Evidence for the genotoxicity of benzene in humans comes from studies of

occupationally exposed populations (Sasiadek et al., 1989; Yardley-Jones et al., 1990).

The convenient parameters that have been monitored include chromosomal abnor

malities, sister chromatid exchanges and micronuclei induction. Benzene induced

cytogenetic effects, including chromosome and chromatid at>errations, sister chromatid

exchanges, and micronuclei, have been consistently found in in vivo animal studies

(Erexson eta!.. 1986, Toftetal., 1982).

Forni et al., (1971) observed chromosomal aberrations in workers occupationally

exposed to benzene. Siou et al., (1981) demonstrated that nicks and breaks were the

most common in the chromosomal aben-ations studied in mice. Tice et al., (1980) have

presented evidence that the sister chromatid exchanges were due to both persistent and

new chromosomal lesions. Morimoto et al., (1983) observed that benzene biotrans

formation is a prerequisite for inducing sister chromatid exchange in human whole blood

cultures. Tunek and coworkers (1982) have related benzene ability to induce micronuclei

in bone marrow cells with benzene biotransformation In vivo. Zhang et al., (1993)

investigated that induction of both kinetochore-positive and kinetochore-negative

micronuclei by 1,2,4-benzenetriol was mediated via active oxygen species formation.

These data indicate the involvement of active oxygen species in 1,2,4-benzenetriol-

induced DNA damage and implicate the potential importance of 1,2,4-benzenetriol-

mediated genetic damage in benzene-induced human leukemia. Toft et al., (1982)

exposed male mice to benzene vapours (1 to 200 ppm) for varying periods of time and

reported that continuous exposure to 14 ppm benzene after one week resulted in a

Chapter -1

significant elevation in micronuclei. Gad-EI-Karim et al., (1984) while studying the

clastogenic effects of benzene, observed two fold increase in miaonuclei in bone man-ow

of CD-1 mice. Luke et al., (1988) evaluated the genotoxic effects of benzene (3,000

ppm) on DBA/2 mice by evaluation of micronuclei frequencies in peripheral blood,

polychromatic erythrocytes and non-chromatic erythrocytes. Delayed cell cycle in mouse

bone marrow and DMA adducts with benzene metabolites in mouse and rat haemoglobin

adducts or rat liver cell DNA adducts were also found after benzene inhalation (Tice et

al.. 1982; Sabourin et al., 1990). Recently Pathak et al., (1995) have noticed the DNA

adduct formation in the bone marrow of Isenzene administered mice. Futher Levay et al.,

(1996) hypothesized that the DNA adduct formation might depend on both the dose and

duration of benzene exposure. DNA adduct formation was also observed in HL-60 cell

culture treated with hydroquinone (Hedii et al., 1996; Pongracz and Bodell, 1996).

Further HedIi e al., (1996) demonstrated that hydroquinone and 1,2,4-t)enzenetriol, each

inhibits retinoic acid-induced granulocytic differentation in HL-60 treated cells but, DNA

adducts formation was only observed in case of hydroquinone. These findings suggest

that adduct formation might play a significant role in hydroquinone but not in 1,2,4-

benzenetriol toxicity, and that different mechanisms might be responsible for the

contributions of each metabolite to benzene toxicity.

(c) Carcinogenicity

Benzene is a well known leukemogenic and carcinogenic agent. Goldstein (1977)

and Aksoy (1985) have demonstrated that acute myelogenous leukemia (AML) and its

variants are commonly associated with t>enzene exposed workers. Paradoxically, the

occun^ence of myeloid leukemia in rats and mice is exceedingly low. But for a few stray

reports of myeloid leukemia in rats, most of them, instead, exhibited tumors. The first

example of malignant tumors experimentally produced by tjenzene was reported by

Maltoni and Scarnato (1979). The predominant tumor was not of haematopoietic origin

but rather a carcinoma of the rat zymbal gland. Further studies by Maltoni et al., (1985),

demonstrated the formation of zymbal gland carcinoma by administering benzene

through different routes. Studies by Cronkite (1987) confirmed that benzene is multipotent

carcinogen in experimental animals. The first experimental demonstration of lympho-

Chapter -1 10

reticular tumors produced by benzene was reported by Snyder et al., (1980). Life time

exposures of C57B1 mice to 300 ppm benzene (6hr/for 5 days/week) produced 6 out of

40 cases of thymic lymphoma. Cronkite et al., (1984) later showed that a shorter

exposure period (16 weeks) to 300 ppm was sufficient to produce thymic lymphoma in

these animals, although the tumor induction time was lengthened

In rodents, benzene induces a variety of tumors in organs including the zymbal

gland, harderian gland, mammary gland, lung, liver, nose and oral cavity (Maltoni e al.,

1983). Despite extensive research the mechanisms of benzene induced myelotoxicity

and leukemogenicity are unclear. Bone marrow cells contain high levels of peroxidase

activity, most of which is attributed to myeloperoxidase (MPO) and eosinophilperoxidase

(EPO) (Kariya et al., 1987). It is interesting that several target organs of benzene induced

carcinogenesis contain appreciable levels of peroxidase activity. Subrahmanyam e al.,

(1991a) have shown that phenolic metabolites of benzene are substrates for human

MPO, and that their interactions result in enhanced production of 1,4-benzoquinone. But

the toxicological significance of such reactions is not clear. They suggested that inter

action of phenolic compounds, presumably by hydrogen-bonding with the activity limiting

distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important

contributing factor in the enhanced myeloperoxidase-dependent metabolism of

hydroquinone in the presence of other phenolic compounds. In sum, these provide ample

evidence to demonstrate that benzene is a carcinogen for rats and mice.

(d) Immunotoxicity

Animal studies show that benzene affects humoral and cellular immunity. Benzene

decreases the fonnation of the lymphocytes that produce the seaim immunoglobulins or

antibodies. Exposure to benzene at 10 ppm and above for 6 days decreased the ability

of bone man-ow cells to produce mature B-lymphocytes of C57BL/6 mice (Rozen et al.,

1984). The spleen was also inhibited from forming mature T-lymphocytes at exposure

levels of 31 ppm and above. Mitogen-induced blastogenesis B-and T-lymphocytes was

depressed at 10 ppm and above. Peripheral lymphocyte counts were depressed at all

levels, whereas erythrocyte counts were depressed only at 100-300 ppm (Rozen and

Chapter -I "

Snyder, 1985). A continuation of this line of study for 6 and 23 weeks at 300 ppm

showed continuous decreases in numt)ers of mature B- and T- lymphocytes produced in

the bone marrow, spleen, and thymus. Another series of experiments revealed that

exposures as low as 25 ppm for 2 weeks caused decrease in the numbers of circulating

lymphocytes (Cronkite e a/., 1989). Intemiediate-duration exposure to benzene also

deaeased leukocyte counts in rats and mice exposed to 300 ppm for 2-13 weeks (Ward

ef a/., 1985). Chronic exposure to benzene caused bone marrow hypoplasia,

lymphocytopenia, and anemia in mice exposed to 100 ppm for a lifetime (Snyder e a/.,

1980).

Studies by Irons et al., (1981) and Pfeifer and Irons (1983) have shown that phenol,

hydroquinone and catechol to suppress lymphocyte growth and function in vitro, which

correlates with their capacity to undergo autooxidation and with their concentration in the

bone man ow or lymphoid organs. They also showed that hydroquinone and its oxidation

product, p-benzoquinone inhibit proliferation and differentiation in lectin stimulated

lymphocytes in culture. Further, they showed that these compounds interfere with

microtubule assembly at concentrations that are not cytotoxic, while, phenol and catechol

suppress lymphocyte activation only at cytotoxic concentrations. They postulated that the

suppression of lymphocyte blastogenesis by hydroquinone to be mediated by the

interaction of p-benzoquinone with sulphahydryl groups on tubulin. This binding interferes

with microtubular integrity, which is essential in cell division via spindle formation and in

the regulation of surface receptor movement and signal transduction across the plasma

membrane.

Wierda and Irons (1982) showed that hydroquinone and catechol also to be

immunotoxic in vivo. Benzene, if metabolized in the lymphocyte to a reactive inter

mediate such as p-benzoquinone, could inhibit the production of lymphokines. Post et

al.. (1985) demonstrated that hydroquinone and p-benzoquinone affected the dose-

dependent inhibition of RNA synthesis in mouse spleen lymphocytes in vitro at micromolar

concentrations that were not cytotoxic. They also showed that exposure to p-

benzoquinone completely inhibited the proliferation and production of the T-cell

lymphokine, interieukin-2 by concanavalin A stimulated T-lymphocytes. It has been

Chapter -1 12

observed that benzene can modify both host resistance to a bacterial infectious agent

and T-cell mediated tumor resistance (Rosenthal and Snyder, 1986). Pandya et al.,

(1986) observed that the immunosuppressive effects of benzene have been modulated

by the prior administration of fungal product 6-MFA from Aspergillus ochraceous and

polyinosinic-polycytidilic acid (Pandya et al., 1989) that have interferon inducing

properties.

HUMAN EXPOSURE Inhalation is the dominant pathway of human exposure. Occupational exposure to

benzene has been associated with aplastic anemia, leukemia, and other related blood

disorders (Aksoy, 1988). For certain cancers, increased mortality was noted among

benzene exposed males in comparison with that among unexposed male. Lung cancer,

stomach cancer and primary hepatocarcinoma were reported by numerous investigators

during the benzene exposure, whereas, for female only leukemia occun'ed in excess

among the exposed (Yin et al., 1989). Risk of leukemia rose as duration of benzene

exposure increases. In acute myeloid leukemia (AML) there is diminished production of

normal erythrocytes, granulocytes, and platelets, which leads to death by anemia,

infection, or hemorrhage. Whereas in chronic myeloid leukemia (CML) the leukemic cells

retain the ability to differentiate and perform function; later there is a loss of ability to

differentiate. Case reports and epidemiological studies of workers have established as

a causal relationship between benzene exposure and AML. Study by Yin et al., (1996)

of benzene-exposed workers in China provides further support for the association of

tjenzene exposure with an increased risk for myelogenous leukemia. While some studies

have implicated other types of leukemia or even lymphomas, only the incidence of AML

and its variants has consistently been increased In groups of workers with excess

benzene exposure (Goldstein, 1988). The evidence linking benzene exposure with

leukemia was considered sufficient proof of human carcinogenicity (Dosemeci et al.,

1994). Haematotoxicity among Chinese workers were also observed who were exposed

to high concentration of benzene (Rothman et al., 1996) But precise mechanism for

benzene toxicity is not known.

Chapter -1 13

Benzene and its metabolites do, however, produce chromosomal damage in a

variety of systems. The incidence of chromatid deletions and gaps v\/ere slightly increased

in workers exposed to benzene levels of 10-100 ppm compared with control groups

(Yardley-Jones et ai, 1990). Many investigators (Glatt et at., 1989; Witz et at., 1990;

Yager et ai, 1990) have demonstrated that benzene exposure induce chromosomal

aben'ations, micronuclei and sister chromatid exchanges in human lymphocytes in vitro.

Benzene and some of its metabolites have been tested for genotoxicity in Salmonella

typhimurium and V79 Chinese hamster cells Dihydrodiol, hydroquinone, 1,2,4- benzene-

triol and catechol, the potent benzene metabolites showed genotoxic and mutagenic

effects in V79 cells as well as sister chromatid exchanges (Glatt et al., 1989). It was

further suggested that metabolites of b>enzene can induce heritable functional changes

(gene mutation) in bacterial and mammalian cells. Cytogenetic studies of benzene

exposed wori<ers have reported a similar profile of genotoxicity. Increased frequencies

of structural chromosomal aberration in the lymphocytes of l)enzene-exposed workers

have been reported (Sorsa and Yager, 1987). Fenech and Morley (1988) investigated

the ability of the principal phenolic and quinoid metabolites of benzene to induce staictural

and numerical chromosomal aben-ation by utilizing a cytokinesis-block micronuclei assay

in treated human peripheral lymphocytes. The higher efficacy of hydroquinone in inducing

both total micronuclei and kinetochore-positive miaonucleated cell when compared with

catechol, phenol and 1,4-benzoquinone suggests that hydroquinone is a major contributor

to the clastogenicity and aneuploidy observed in the lymphocytes of benzene-exposed

workers. However, other metabolites of benzene may also contribute to the genetic

effect caused by exposure to benzene.

Since consistent chromosomal aben-ations are often observed in human leukemias,

the ability of phenolic metabolites of benzene to induce chromosomal damage in human

cell also implicates them in benzene induced leukemia (Yager e/a/., 1990). Many studies

have also shown that benzene produces both structural and numerical chromosomal

aberration in the peripheral blood lymphocytes of occupationally exposed individual (Ding

et a/., 1983). Linear regression analysis demonstrated significant decrease in white and

red blood cell counts, as well as haemoglobin content (Kipen et al., 1989).

Chapter • I 14

Because consistent chromosomal aberrations have Ijeen observed in human

leukemias and lymphomas, the induction of chromosomal damage by benzene or its

metabolites may be critical in the development of benzene induced leukemia. However,

the nature of mutations produced at specific gene site in the bone marrow, the target

organ for benzene toxicity, has not been studied to date in humans (Rothman et al.,

1995). Recently, Chen and Eastmond (1995) demonstrated that inhibition of enzymes

involved in DNA replication and repair, such as topoisomerase enzymes, by the

metabolites of benzene represent a potential mechanism for the formation of chro

mosomal al)en-ations. Further in vitro and in vivo studies will be required to demonstrate

the involvement of topoisomerase inhibition in the myelotoxic and carcinogenic effects

of benzene. Earlier, Levay et a!., (1993) demonstrated the DNA adduct fomnation by the

benzene metabolites, hydroquinone and 1,4-benzoquinone in human bone marrow, and

suggested that in human bone marrow also, as in other model system, peroxidase

enzymes play an important role in the activation of hydroquinone to form DNA adducts.

These results support the general hypothesis that benzene metabolites are activated to

form DNA adducts that may contribute to the myelotoxicity and leukemia observed in

workers occupationally exposed to benzene (Cronkite et a!., 1989; Goldstein, 1977).

Besides the genotoxic and carcinogenic effect, adverse immunological effects have

been reported in human with occupational exposure to tjenzene. There are two types of

acquired immunity, humoral and cellular and benzene damages both. First, benzene has

been shown to alter humoral immunity, i.e. to produce changes in blood levels of

antibodies. Painters who were exposed to benzene (3-7 ppm), toluene, and xylene in the

workplace for 1-21 years showed increased serum immunoglobulin values for IgM and

decreased values for IgG and IgA (Lange et a!., 1973a). Other adverse reaction,

characterized by a reaction between leukocyte agglutinins and autoleukocytes, occurred

in 10 out of 35 of these workers (Lange et al., 1973b). One of the problems with these

studies is that the workers were exposed io multiple solvents, so that benzene alone may

not be responsible for the noted effects.

The second type of immunity, cellular immunity, is affected by changes in circulating

leukocytes (White blood cells) and a subcategory of leukocytes, called lymphocytes.

Chapter -I 15

Loss of leukocytes was found in a series of studies of workers exposed to benzene at

levels as low as 30 ppm in various manufacturing processes (Aksoy et ai, 1987). Other

studies in chronically exposed workers also showed loss of lynnphocytes and other blood

elements (Kipen et ai, 1989; Yin et al.. 1987).

METABOLISM

Benzene itself is not the actual toxicant and is relatively inert to confer chemical

reactivity to the aromatic ring. Benzene requires metabolism to induce its toxic effects

and follows similar pathways in humans and animals. The metabolism of benzene

occurs in the liver and, to a lesser extent in the bone marrow by cytochrome P-450

dependent mixed-function oxidase enzymes. Although, the metabolism of benzene is

very complex, but is well understood.

Schrenk et al.. (1941) studied the absorption, distribution and elimination of benzene

after exposure of benzene to the dog. The purpose of the study was to gain better

understanding of the physicochemical phenomena underiying the physiological action of

benzene. They observed that benzene concentration in fat and fatty tissue was many

times than that in blood and vital organs. The fat, acting as a reservoir, apparently is

saturated slowly and loses its benzene slowly. In a way this makes the fat, the master

tissue as regards absorption, distribution, and elimination of benzene by the body tissues

and fluids. This phenomenon may be a partial explanation of the fundamental action of

benzene on the body, namely, its deleterious effect on the haematopoietic system.

Moreover, the high benzene concentration in the urine indicates that benzene is

concentrated by the kidneys. Porteous and Williams (1949) found that phenol, catechol,

quinol and hydroxyquinol are excreted as ethereal sulphates in the unne of rabbits orally

exposed with benzene. However, the first metabolite of benzene is phenol which may

then be oxidized to catechol and quinol. One or both of these dihydric phenols may then

give rise to hydroxyquinol. It is clear that the longer benzene remains in the body, the

greater will be the extent of its oxidation to the toxic polyhydnc phenols, and continuous

feeding to benzene will give rise to a continuous and increased production of catechol

and quinol

Chapter -1 16

According to Fabre (1947a; 1947b) rat liver tissue oxidizes tDenzene to phenol in vitro

more rapidly than any other rat tissue. Under same conditions toluene is not oxidized to

phenolic substances. On the basis of this difference in toxicity between benzene and

toluene and in their metabolic fates, support to the view that benzene is toxic because

of its, phenolic metabolites. Later on urinary benzene metabolites were quantified in a

series of studies by Parke and Williams (1953). Rabbits treated orally with '*C-labelled

benzene, eliminated about 43% of the dose as unchanged benzene in the expired air and

excreted about 35% of the radioactivity in the urine in the form of metabolites. Phenol

accounted for the largest percentage of radioactivity (68%) followed by hydroquinone

(14%), catechol (6%), trans, trans-muconic acid (1%). The similar general profile of

urinary metabolites was subsequently observed in rats (Cornish and Ryan, 1965), mice

(Longacreefa/., 1981), cats and dog (Oehme, 1969), and human (Teisingere/a/., 1952).

Capel et al., (1972) have studied the variation in the metabolic fate of '"C-labelled phenol

in eight different species namely the rat, mouse, jerboes, gerbil, hamster, lemming,

guinea pig and man. In most of the species examined, phenylglucuronide and phenyl

sulphate are indeed the main metabolites except in the case of cat and guinea-pig due

to a defective glucuronic acid and sulphate conjugation mechanism respectively (Dutton,

1966: Capel e/a/., 1972).

Initially, benzene is metabolized to benzene oxide by P-450 IIE1 monooxygenase

enzymes in the liver. Benzene oxide gives rise to phenolic metabolites, such as phenol,

catechol, hydroquinone, 1,2,4-ben2enetriol and open-ring products, such as trans,

trans-muconaldehyde and trans, trans-muconic acid (Subrahmanyam e/a/., 1991b). The

majority of these phenolic metabolites are excreted in unne as glucuronide and sulphate

conjugates . In experiments with rabbits, accumulation of several phenolic metabolites

of benzene have been observed in bone marrow against the concentration gradient

(Greenlee ef a/., 1981a).

The major pathv^ays of benzene metabolism are shown in Figure-1.1. The formation

of benzene oxide is the initial step and it is further metabolized by one of the four

pathways. The first metabolite is formed by rearrangement of the benzene oxide to

phenol spontaneously. Phenol is either conjugated to glucuronide and sulphate directly

-> i

CO CD

TO

D I E o

•D 0) Q. O

• D ra 0) c 0) N c XI »•—

o

03

5 TO Q.

O

m

V

Chapter -1 17

or is oxidized to hydroquinone and followed by conjugation to hydroquinone glucuronide

or sulphate (Huff et al., 1989). The second pathway for benzene oxide metabolism

consisted of reaction with glutathione and subsequent modification of the mercapturic acid

(Henderson et al., 1989). The third pathway for t>enzene metabolism involved in

conversion of benzene oxide into tjenzene dihydrodiol by epoxide hydrase (Tunek et al.,

1981). Benzene dihydrodiol is converted into catechol by oxidation of benzene

dihydrodiol by a soluble NADP*- dependent enzyme, tjenzene dihydrodiol dehydrogenase

(Ayengar et al., 1959) and finally conjugation to glucuronide and sulphate. Benzene

dihydrodiol is also converted into 1,2,4-b)enzenetriol by some unknown mechanism, which

is one of the potent toxic metabolites of t>enzene. However, Inoue et al., (1989b)

observed that phenol and hydroquinone, but not catechol, are precursors of

1,2,4-benzenetriol in urine after the intraperitoneal injection of the three phenolic

compounds to rats followed by urine analysis for 1,2,4-benzenetriol. A similar result was

also observed in case of rabbits (Inoue ef a/., 1989c). The fourth pathway of benzene

oxide metabolism consists of the ring opening reaction resulting in the fonmation of trans,

trans-muconic acid. However, the metabolic pathway of trans, trans-muconic acid in vivo

is yet to be identified (Latriano et al., 1986). The corresponding dialdehyde, trans,

trans-muconaldehyde has been shown to be a precursor of muconic acid in vivo (Witz

etal., 1990).

Although, the pathway of benzene ring opening is not known, but a number of

possible mechanisms for oxidation of benzene ring-opening have been postulated

(Snyder and Chatterjee, 1991). One such postulated pathway involves the formation of

trans, trans-muconaldehyde from benzene dihydrodiol in a Fenton system, presumably

via hydroxyl radical-mediated ring-opening (Latriano et al., 1985; Zhang et al., 1995a).

Zhang et al., (1995b) further demonstrated the role of iron in the mechanism of ring-

opening of benzene in a mouse liver microsomal system.

TOXICITY

Benzene is a well known xenobiotic environmental pollutant. Exposure to benzene

leads to the development of bone marrow depression characterized by progressive

Chapter -1 18

leucopenia, anemia and pancytopenia (Snyder, 1987). The responses to chronic

benzene exposure have been classified as either bone marrow depression and aplastic

anemia or the generation of acute myeloblastic leukemia (AML) and related forms of acute

lymphocytic/non-lymphocytic leukemia. Collectively, these abnonnalities have been

termed as preleukemic syndrome or myelodysplastic syndrome (Tricot, 1991). Irons

(1988) and Tricot (1991) suggested that the spectrum of bone marrow toxicities produced

by exposure to benzene represented a form of myelodysplastic syndrome resulted in

bone marrow depression and that might lead to fatal aplastic anemia. Anon (1982)

reported that benzene is a human leukemogen. Chronic tjenzene poisoning exhibited a

variety of changes in the number of basophils, eosinophils, lymphocytes and other bone

marrow function in workers (Aksoy ef a/., 1971).

Goldstein et al., (1982) reported on the production of a limited number of

myelogenous leukemias in rats and mice exposed to benzene by inhalation. The reports

by Maltoni et al., (1989) and Huff ef al., (1989) of Ijenzene induced carcinogenicity in rats

and mice given benzene orally focussed on solid tumors and multiple carcinogenic foci.

The zymbal gland was a primary carcinogenic site in both sexes of both species. Later

studies using the inhalation route (Maltoni ef al., 1989) also demonstrated the

multipotential carcinogenic activity of benzene. Excluding the studies by Yin ef al., (1987)

there is no strong evidence for benzene-induced solid tumors of these types in human.

Snyder ef al., (1980) also reported the production of thymic lymphoma after inhalation of

benzene in C57B1/6J mice but not in AKR mice. Fanis ef al., (1993) observed the

malignant lymphoma along with preputial gland carcinomas and lung adenomas in

CBA/Ca mice exposed to benzene through inhalation. Neither the Cronkite ef al. (1989)

nor the Fams etal., (1993) studies demonstrated acute granulocytic leukemia in tjenzene

inhaled mice.

Snyder and Chatterjee (1991) and Yardley-Jones ef al., (1991) have studied the

pathway of benzene metabolism and the role of benzene metabolites in the production

of benzene toxicity. Lee ef al., (1981) reported that early erythroblasts are sensitive to

benzene and its metabolites. Benzene has long l5een known to produce chromosomal

abnormalities (Eastmond, 1993) and suggested that benzene may have caused

Chapter • I 19

chromosome breakages and rearrangements in a stem cell that proliferated, leading to

erythroleukemia. Eastmond (1993) also observed that the quinone metabolites of

benzene can cause aneuploidy to chromosome nondisjunction and nonrandom chromo

somal alteration in individuals with advanced forms of Isenzene-induced myelotoxicity.

The association between chromosomal abnormalities and leukemia in tienzene exposed

rats has been reviewed by Le Beau and Larson (1991). Tice et al.. (1980) exposed mice

to benzene by inhalation and demonstrated an increased frequency of sister chromatid

exchanges in bone marrow cells and suggested that benzene metabolites were

responsible for these effects. Muconaldehyde, a hypothesized ring open benzene

metabolite also produced sister chromatid exchanges in B6C3F1 mice (Witz et al.,

1990).

The highly electrophilic metabolites such as 1,4-benzoquinone and others are

generated during Ijenzene exposure in experimental animals and form covalently bound

adducts with both nuclear and mitochondrial DNA (Eastmond, 1993). Kolachana et al.,

(1993) have demonstrated that benzene and its phenolic metabolites; phenol, hydro-

quinone and 1,2,4-benzenetriol induce oxidative damage in the genome in the form of 8-

hydroxydeoxyguanosine in HL-60 promyelocytic leukemic cells and in the bone marrow

of mice. Cheng et al., (1992) demonstrated that benzene induced active oxygen radical

production resulted in 8-hydroxydeoxyguanosine formation in vivo which may play a role

in genotoxicity and leukemogenesis. Formation of 8-hydroxydeoxyguanosine adduct is

known to cause G-T and A-C base substitutions.

MECHANISM OF TOXICITY

Any hypothesis of benzene toxicity must account for the requirement of hepatic

metabolism and selective toxicity of benzene in the bone marrow. As mentioned above,

benzene is metabolized in the liver mainly to one or more phenolic metabolites and

accumulated against the concentration gradient in bone man-ow and exerts its toxic

effects (Smith et al., 1989). It is believed that the effect of benzene is likely to be exerted

through the action of multiple metabolites on multiple targets through multiple biological

pathways (Goldstein, 1989).

Chapter -1 20

Rao and Pandya (1989) observed that polyphenolic metabolites as the toxic inter

mediates due to their capability to undergo autooxidation. One mechanism by which

benzene induces the toxic effects may be mediated by the generation of one more active

oxygen species such as superoxide anion radical (Oj), hydrogen peroxide {Hfii),

hydroxyl radical (HO) and singlet oxygen C^fii) (Subrahmanyam et al., 1991b).

Myeloperoxidase and eosinophil peroxidase are the two principal enzymes in bone

man-ow which are responsible for the formation of quinone and semiquinone free radicals

and activation of oxygen to superoxide radicals. These free radical metabolites of

benzene with dioxygen and cellular targets such as DNA, GSH, protein and lipid are

responsible for the benzene-induced myelotoxicity and leukemia (Subrahmanyam et al.,

1991b). Legathe et al., (1994) have demonstrated pharmacokinetic interaction between

benzene metabolites, phenol and hydroquinone, in B6C3F1 mice. The consequence of

which may be a higher quantity of 1,4-benzoquinone formation from hydroquinone.

Hydroquinone and catechol have been shown to be much more toxic to bone marrow

cell cultures than benzene (Parmentier and Dustin, 1953). Phenol and hydroquinone

have been shown to cause significant decrease in bone man-ow cellularity when

coadminis-tered to mice (Eastmond et al., 1987). Since the bone man'ow is rich in

peroxidative enzymes including myeloperoxidase and potential oxidants such as

hydrogen peroxide derived from leukocytes, it has been suggested (Eastmond et al.,

1987; Smith et al., 1989) that a bone man-ow-localized phenol-dependent stimulation of

hydroquinone metabolism results in the fomnation of 1,4-benzoquinone, the ultimate toxic

metabolite of benzene. 1,4-Benzoquinone has been shown to induce single strand breaks

in DNA of cultured cells (Pellak-Walker and Blumer 1986), genotoxic in V79 cells (Glatt

et al., 1989), inhibit DNA and RNA synthesis (kalf, 1987) and mitogen stimulation of

lymphocyte growth (Wierda and Irons, 1982). Levay et al., (1993) observed the DNA

adduct formation in all cell types tested in presence of 1,4-benzoquinone. Recently Sze

et al., (1996) noticed that 1,4- benzoquinone is more potent to induce DNA strand breaks

in Chinese Hamster Ovary cell. However, hydroquinone also exhibited a significant

effect as well. Although, only less than 1-5% of the benzene inhaled is metabolized to

1,4-benzoquinone in the body (Huff et al., 1989), the amount of 1,4-benzoquinone

Chapter • I 21

produced is perhaps sufficient to cause significant biological effects. On the other hand,

hydroquinone has been known to undergo autooxidation or myeloperoxidase-catalyzed

oxidation to form 1,4-benzoquinone (Subrahmanyam et al., 1991b). As such, the

cytotoxicity of hydroquinone could be due, in part, to the production of 1,4-t)enzoquinone

in the Chinese Hamster Overay cells (Sze et al., 1996).

Quinone and semiquinone metabolites of polyphenols, through their capacity to react

with nucleophilic groups of amino acids are reported to inactivate critical enzymes like

DMA polymerase (Graham et al., 1978), RNA polymerase II (Nagaraja and Shaw, 1982)

and reverse transcriptase (Wick and Fitzgerald, 1981). Quinone or semiquinone

metabolites of hydroquinone and 1,2,4-benzerietriol have been shown to inhibit mRNA

synthesis (Kalf et al., 1982), microtubule polymerization (Irons and Neptun, 1980),

mitogen stimulation of lymphocyte growth (Wierda and Irons, 1982), effect macrophage

function and activation (Lewis et al., 1988a) and form adducts with DlslA (Jowa et al.,

1986). Autooxidation of polyphenolic compounds in presence of low concentrations of

transition metal ion copper resulted in an increased production of active oxygen species

which can release aldehydic products from glutamate or DNA (Rao and Pandya, 1989).

Experimental studies have suggested that involvement of iron or copper in benzene

metabolite induced DNA damage through a mechanism involving the generation of

organic free radicals (Kawanishi et al., 1989; Rao and Pandya, 1989). In vitro studies

by Rahman et al., (1989) and Ahmad et al., (1992) have shown that naturally occurring

flavonoid, quercetin, in the presence of Cu(ll) and molecular oxygen caused breakage

of calf thymus DNA, supercoiled pBR 322 plasmid DNA and single stranded Ml 3 phage

DNA. Copper is distributed throughout the body with the liver and bone marrow being the

two major copper storage organs (Linder, 1991). Copper has been identified as the

essential redox-active centre in a variety of metalloproteins such as ceruloplasmin,

Cu-Zn superoxide dismutase, cytochrome oxidase, dopamine p-hydroxylase, tyrosinase,

lysyloxidase and ascorbate oxidase (Linder, 1991). Since copper also exists in the

nucleus and is closely associated with chromosomes and DNA (particulariy Guanine)

(Prutz et al., 1990) the chemical-metal redox system might be responsible for strand

breaks in vivo during benzene exposure. In vitro, among hydroquinone, 1,2,4-

Chapter - / 22

benzenetriol, catechol and phenol; hydroquinone/Cu^* and 1.2,4-benzenetrioiyCu^* were

the two efficient DNA cleaving systems (Li and Trush, 1993a). Recently Zhang et al.,

(1996) described that the oxidation of 1,2,4-benzenetriol and catalysis by Cu^* may play

an important role in 1,2,4-ben2enetriol-induced genotoxicity.

It was also observed that Cu(ll) strongly induces the oxidation of hydroquinone as

such may be factor involved in the oxidative action and toxic to primary bone marrow

stromal cells of DBA/2 J mice (Li and Trush, 1993b). They further observed that Cu(ll)

is capable of directly reacting with hydroquinone, causing the one electron oxidation of

hydroquinone to semiquinone radical (SQ) and reactive oxygen species generated in

chemical metal redox system were responsible for in vitro plasmid DNA damage (Li et

al., 1995).

Many investigators have shown that t>enzene exposure interferes with iron

metabolism and heme biosynthesis in experimental animals. These studies include

decreased incorporation of ^ Fe into circulating erythrocytes (Lee et al., 1974), inhibition

of heme biosynthesis enzyme at the level of 6-aminolevulinic acid (ALA) dehydratase

resulting in ALA accumulation and its loss in urine (Rao and Pandya, 1980), and

depletion of the regulatory heme pool (Siddiqui et al., 1988). Abraham et al., (1986)

demonstrated that benzene exposure leads to increase in heme oxygenase activity, a

rate limiting enzyme for heme degradation. Existence of an intracellular pool of low

molecular weight iron (LMI) compounds maintaining a dynamic equilibrium between iron

uptake, iron storage and iron incorporation into its final biochemical form have been

described (Jacobs, 1977). Benzene administration led to an accumulation of iron in bone

marrow cells which can catalyze the formation of aldehydic products (base propenal)

from DNA in presence of bleomycin (Rao et al., 1990). In all probability benzene alters

heme metabolism of erythroid cells resulting in the increased internalization of iron

transferrin-receptor complex and uptake of internalized iron is almost entirely restricted

to erythroblasts. In an/n v/fro study, Rao (1991) observed that hemin catalyzed the

autooxidation of hydroquinone or 1,2,4- benzenetriol in the presence of reducing agent

and resulted in the formation of aldehydic products from glutamate, deoxyuridine or DNA

through the formation of reactive oxygen species. Hemin is essential for the erythroid

Chapter -1 23

differentiation process and accumulates in the immature red blood cells in nanomolar

quantities as these cells differentiate (Lo et aJ., 1981). In immature red blood cells in bone

marrow, it has been noted that heme accumulates in the nucleus as these cells

differentiate and nicking of nuclear DMA has been reported as it binds covalently to both

protein and DNA (Mager and Bernstein, 1979; Lo et al., 1981). it has been postulated

that this action of hemin might account for its influence in the differentiation of these and

other cell types (Chen and London, 1981). n view of the accumulation of polyphenolic

metabolites after exposure to benzene and the presence of excessive amounts of heme

in bone marrow cells which may catalyze autooxidation of polyphenols and offer an

alternative mechanism for the bone marrow depressant effect of benzene (Rao, 1991).

CURRENT TREND AND FUTURE RESEARCH

Benzene metabolism in experimental animals are well understood, but the precise

mechanism of benzene toxicity remain largely unknown. The problem of attempting to

extrapolate to the humans is complicated by the lack of metabolism or toxicity data in

humans. In attempt to bridge the data gap, Sabourin et al. (1992) examined benzene

metabolism in cynomolgus monkey and chimpanzee. They concluded that the mouse

forms the highest levels of hydroquinone conjugate and the chimpanzee forms the

lowest. It will be important to determine the sensitivity of the monkeys and the

chimpanzees to benzene. Although, the relative sensitivity of humans to benzene toxicity

is unknown, a study of benzene metabolism in humans exposed in an industrial setting

to evaluate the urinary excretion of phenyl conjugates vs polyhydroxylate and ring open

metabolites might help to estimate how humans relate to test species in this respect

(Sabounnef al., 1992).

Since no single ring hydroxylated metabolite of benzene can produce the whole

spectrum of benzene toxicity, more and more attention has focused on the interactions

of benzene metabolites (Kolachana et al., 1993), reactive ring-opened metabolite i.e.,

trans, trans-muconaldehyde (Goon et al., 1992). Further studies are needed to

demonstrate that trans, trans-muconaldehyde is a precursor of trans, trans-muconic acid

in vivo. Recently Zhang ef al., (1995b) demonstrated the involvement of iron in the

Chapter -1 24

mechanism of ring opening of benzene in mouse liver microsomal system. Although it

is generally believed that there is no "free" iron in vh/o, there is large body of evidence that

supports the existence of a so-called "lov»/-moiecular weight-iron pool" in cells, including

hepatocytes. Earlier Rao et al., (1990) demonstrated the accumulation of bleomycin

detectable low molecular weight iron components in bone marrow in experimental rats

during benzene exposure. The liver is a tissue rich in iron, which is stored in ferritin and

present in many heme containing proteins. Under normal condition, the mobilization of

iron is strictly regulated. However, recently Ahmad et al.. (1995) observed the reductive

release of iron from horse spleen femtin by 1,2,4-benzenetriol. Oxidative stress produces

conditions that interfere with this regulation, and which may result in the release of iron

from femtin. Since there is an animal model for iron accumulation (Nielsen et al., 1993)

and since methods for assessing benzene haematotoxicity and for measuring

metabolism are available, it would be of interest to evaluate the role of iron in benzene

haematotoxicity in vivo.

The molecular mechanism of benzene toxicity is also not very well understood.

Many studies suggest that DNA damage may be playing a role in the myelotoxicity during

benzene exposure. Further studies measuring the formation of both DNA adducts and

oxidative base damage will be required to evaluate the contribution of each of these

forms of DNA damage to the myelotoxic effects of benzene. Accordingly, the interaction

oi phenolic compound with DNA associated copper may result in a spectrum of lesion

in DNA, including site specific oxidative DNA base modifications, DNA strand breaks, and

DNA adducts of phenolic intermediates (Li et al., 1995; Zhang et al., 1996). These

lesions in DNA might contribute to mutation in DNA and to the initiation of carcinogenesis

induced by phenolic compounds. However, benzene induced DNA strand-breaks in vivo

is yet to be identified.

Further studies are needed of large populations of exposed workers to produce

benzene-induced leukemia and to examine the ability of benzene metabolites to induce

mitotic recombination in human cells. Recently Chen and Eastmond (1995) have shown

that a number of polyphenolic metabolites or postulated metabolites (such as 2,2'- and

4,4'-biphenol) of benzene inhibit human topo isomerase II enzyme in vitro when activated

Chapter -1 25

by a peroxidase metabolizing system and hypothesized that the quinone and phenolic

metabolites of benzene might exert their clastogenic effects through inhibition of topo-

isomerase enzymes. Topoisomerases are major chromosomal proteins which modulate

the topological state of DNA by breaking and releasing DNA strands (Pommier and

Bertrand, 1993). These enzymes participate in a range of cellular processes including

DNA replication and transcription (Anderson and Rot)erge, 1992) and DNA repair

(Stevnsner and Bohr, 1993; Thielmann et a/., 1993). Topoisomerase II also play a role

in maintaining genomic stability (Wang et al., 1990). The inhibition or interference with

topoisomerase II activity at critical stages of the cell cycle could lead to chromosome

breakage, aneuploidy or cell death. Several inhibitors of topoisomerase II are quinone

or quinone-forming compounds which exhibit some structural similarity to metabolites of

benzene possessing hydroxyl or keto groups on the aromatic ring (Li et al., 1993;

Stoyanovsky ef a/., 1993). Furthermore, quinone and phenolic compounds have been

identified at relatively high concentrations in the peripheral blood and bone marrow of

rodents following benzene exposure (Rickert et al., 1981). Additional In vitro and In vivo

studies will be required to demonstrate the involvement of topoisomerase inhibition in the

myelotoxic and carcinogenic effects of benzene.

Finally, the possible mechanisms of benzene-induced leukemogenesis are

suggested and areas of further research are indicated that may provide new information

as well as permit the application of existing knowledge to the understanding of the

leukemogenic process.

Chapter - / / Materials & Methods]

L

Chemicals

Instnuncnts

Animals

Experimental Procedures

Statistical Analysis

Chapter - II 26

CHEMICALS

Benzene of high purity was obtained from Glaxo Laboratories India (ANLAR).

Ferritin (horse spleen), ferrozine (3-(2-pyhdyl)-5,6-bis(4-phenyl sulfonic acid)-1,2,4-

tnazine), bathocuproine, dopamine, 6-hydroxydopamine, catalase (bovine liver;

2,000-5,000 units/mg protein), superoxide dismutase (bovine erythrocytes; 2,500-5,000

units/mg protein), bovine serum albumin, calf thymus DNA, 5,5'-dithiobis-(2-nitrobenzoic

acid), Sephadex G-10 beads, agarose gel and L-glutamate monosodium were obtained

from Sigma Chemical Company, St, Louis, MO, USA. Hydroquinone, 1,2,4-benzenethol

and 1,4-benzoquinone were obtained from Aldrich, Milwaukee, USA, Bleomycin

hydrochloride was obtained from Nippon Kayaku Co, Ltd., Japan, 2-Thiobarbituric acid

was obtained from BDH Chemical Limited, England, Supercoiled plasmid pUC 18 DNA

was obtained from Bangalore GENE! Pvt, Ltd., India, whereas tris-hydroxymethyl amino

methane, glutathione reduced, Folin-Ciocalteu reagent and tnchloroacetic acid (TCA)

were obtained from Sisco Laboratohes, India, All other chemicals were either obtained

from CSIR Centre for Biochemicals, New Delhi, India or were of analytical grade.

Chapter - / / 27

INSTRUMENTS Spectronic-2001 spectrophotometer, Bausch and Lomb, Ultra Turrax T-25

homogenizer, UV-transilluminator Poloroid MP4*, gel electrophoretic apparatus, electro

phoresis power supply SPS-3000, mettler balance, digital pH meter, and microcentrifuge

(REMI) were the major instruments used in this study.

ANIMALS Male mice of 25-30 gms bred at the animal house unit of Industrial Toxicology

Research Centre were divided into four groups (10 mice in each group). One group was

kept as control which received only 0.1 ml normal saline. Second and third groups

received hydroquinone or iron (25 mg each/kg body weight) for 20 days (single dose i.p).

The last group received hydroquinone : iron complex (25 mg hydroquinone ; 25 mg

iron/kg body weight) 1:1 ratio intraperitoneally for 20 days (single dose). After the

termination of the treatment blood was collected into clean heparinized tuljes from jugular

vein and kept at 4°C for haematological studies. Liver homogenate was made in 0.15

M KCI for the estimation of sulphahydryl content and lipid peroxidation.

Female albino rats were treated with FeS04.7H20 (100 mg/kg body weight, i.p.) for

three days consecutively. Rats were sacrificed on 4th day and ferritin was isolated from

liver to study the mobilization of iron from ferritin by 1,2,4-benzenetriol. Bone marrow

cells were flushed out by 0.15 M NaCI from four femurs of control female rats to evaluate

the mobilizationof iron by 1,2,4-benzenetriol. The cells of bone marrow pellet were lysed

by resuspension in approximately 1.5 ml distilled water. This suspension was homo

genized in Ultra Turrax T-25 for 30 sec. and the volume was made upto 2.0 ml with

acetate buffer to a final buffer concentration of 0.033 M, Brain homogenate (2%) of

control rat was made in 0.15 M KCI for 1,2,4-t>enzenetriol concentration dependent iron

release and subsequent lipid peroxidation in the presence of ferritin.

Femur bone marrow cells were also isolated from control albino rats and cell lysate

was made to demonstrate the reco\jevj of iron.polyphenol complex from bone marrow

lysate.

Chapter - II 28

Albino rats of either sex (Swiss wistar strain, 100-110 gm) were treated with

benzene (0.5 ml/kg body weight) intraperitoneally daily for 10 days. Control and

experimental rats were sacrificed by cervical dislocation and liver were immediately

excised and liver homogenate (4 gm liver/30 ml of 50 mM Tris -10 mM EDTA buffer, pH

8.0) was prepared to isolate nuclei.

In another set of experiment male mice (25-30 gm) were exposed with glutathionyl

hydroquinone (GHQ) (100 mg/kg body weight) in 0.1 ml normal saline intraperitoneally

daily for one month (single dose) and control group received only 0.1 ml normal saline.

After the termination of the treatment mice were sacrificed and haematological as well

as other parameters were studied.

EXPERIMENTAL PROCEDURES

Release of thiobarbituric acid reactive products (TBAR) from DNA

Iron-catalyzed bleomycin-dependent DNA degradation was performed according to

the method of Gutteridge (1987). Principally, bleomycin in the presence of low molecular

weight iron degrades DNA with the release of base propenals which react with

2-thiobarbituric acid (TBA) to give a chromogen similar to that given by malonaldehyde

on reaction with TBA. Briefly, the assay system in a total volume of 3.0 ml consisted of

0.033 M acetate buffer of pH 5.3, 500 pg calf thymus DNA, 50 pg bleomycin hydro

chloride and the reaction was initiated by the addition of 20 pM iron or a complex of iron

with polyphenol or of endogenous chelators, such as citrate, glucose, fructose, glycine,

glutamic acid, cysteine, aspartic acid, adenosine monophosphate, adenosine diphos

phate, adenosine triphosphate and dithiothreitol(20 pM:40 pM) which were made

immediately before use. The contents were incubated at 37°C for 30 min and the

reaction was terminated by the addition of 1.0 ml of 10% (w/v) ice-cold trichloroacetic acid

(TCA). The contents were centrifuged at 3000 rpm for 15 min. To the clear supematants,

an equal volume of 0.67% (w/v) TBA was added and heated for 15 min in a boiling

watertDath. The resultant TBA chromogen was read at 532 nm using a Spectronic-2001

spectrophotometer. The values are expressed as nmoles of malonaldehyde equivalents

generated by using £532=1.56 x 10^ cm' MV

Chapter - II 29

Formation of thiobarbituric acid reactive products (TBAR) from glutamate

Release of thiobarbituric acid reactive products from glutamate was followed

according to the method described (Gutteridge, 1981). Briefly, the reaction mixture (total

volume 1.5 ml) contained 0.033 M sodium phosphate buffer of pH 7.4 in 0.15 M NaCI,

4 mM glutamate and the reaction was started by the addition of 20 IJM iron or a complex

of iron:polyphenol (20 \iM : 40 \iM). The contents were incubated for 15 min at 37°C and

the reaction was terminated by the addition of 1.0 ml of 10% (w/v) ice-cold TCA. The

contents were centrifuged at 3000 rpm for 15 min and to the clear supematants, an equal

volume of 0,67% (w/v) TBA was added and heated for 15 min in a tx)iling waterbath. The

resultant TBA chromogen formed from glutamate was measured at 532 nm using a

Spectronic-2001 spectrophotometer. The values are expressed as nmoles of

malonaldehyde equivalents generated by using E532 = 1.56 x 10* cm ' MV

Release of Iron from ferritin

Iron release from horse spleen ferritin was investigated by using the assay procedure

previously descrifc)ed by Kadir et al., (1992). Briefly, the assay system (total volume 1.5

ml) contained 100 pg ferritin, 500 pM fen-ozine and the desired concentration of

hydroquinone/1,2,4-benzenetriol/1,2,3-benzenetriol(pyrogallol)/1,3,5-t»enzenetriol(phloro-

glucinol)/1,4-benzoquinone/phenoiyglutathionyl hydroquinone/uric acid (each 330 pM) or

dopamine/6-hydroxydopmaine (125 pM) in 0.033 M acetate buffer of pH 5.6. The reaction

was started by the addition of different reducing agent and the formation of

(Fe(ferrozine)J^* complex was monitored by recording the increase in absorbance at 562

nm with time and the amount of iron released from ferritin is expressed as pM Fe^Vmin.

The effect of oxyradical scavengers was studied by the addition of oxyradical

scavenger before the addition of polyphenol. The complete reaction mixture contained

in a final volume of 1.5 ml, femtin (100 pg), 1,2,4-benzenetriol (330 pM), ferrozine (500

pM), 0.033 M sodium acetate buffer of pH 5.6 and oxygen radical scavengers, such as,

urea and thiourea (5 mM), mannitol (50 mM), albumin, catalase and superoxide dismutase

(150 pg). The time course of formation of [Fe(ferrozine)3]^* release from bone marrow

Chapter - / / 30

cell lysate was assayed in a total volume of 1.5 ml containing 100 pi bone marrow cell

lysate, 500 |JM fen-ozine, 330 pM 1,2,4-benzenetriol in 0.1 M acetate buffer of pH 5.6 (0.5

ml). The amount of iron released is expressed as (JM Fe^Vmg protein.

Lipid peroxidation in brain homogenate

Lipid peroxidation in rat brain homogenate (2% in 0.15 M KCI) was assayed

according to the method of Bemheim et a/., (1948).

(a) In presence of iron or Jron:polyphenol

The assay mixture was incubated either with 20 pM iron, iron-polyphenol (20 pM :

40 pM) or none and 2.0 ml of brain homogenate (2%) in a metabolic shaker at 37°C,

Aliquots (1.0 ml) were taken at intervals and the reaction was terminated by the addition

of 1.0 ml of 10% ice-cold TCA.

(b) In presence of ferritin and 1,2,4-benzenetriol

The assay system (total volume 1.5 ml) contained 100 pg ferritin and 1,2,4-

benzenetriol (0-330 pM) in 0.033 M acetate buffer of pH 5.6. The contents were

incubated for 30 min, after which 2.0 ml of 2% brain homogenate was added and

incubated for 1 hr at 37°C. After 1 hr incubation, aliquots (1.0 ml) were taken out and the

reaction was terminated by the addition of 1.0 ml of 10% ice-cold TCA.

The contents in the above experiments were centrifuged and to the clear

supematants, an equal volume of 0.67% (w/v) TBA was added and heated for 15 min in

a boiling waterbath. The resultant TBA chromogen was read at 532 nm using a

Spectronic-2001 spectrophotometer. The values are expressed as nmoles of malon-

aldehyde equivalents generated by using E^^ = 1.56 x 10^ cm' M'.

Bleomycin-dependent degradation of DNA by 1,2,4-benzenetriol in presence of

ferritin

The effect of increasing concentrations of 1,2,4-benzenetriol on iron catalyzed

bleomycin-dependent degradation of calf thymus DNA in the presence of ferritin was

Chapter -II 31

evaluated. Briefly, the assay system (total volume 1.5 ml) contained 100 Jg ferritin and

1,2,4-benzenetriol (0.165 pM) in 0.033 M acetate buffer of pH 5.6. The contents were

incubated for 30 min, after v\/hich 250 |jg calf thymus DNA and 50 MQ bleomycin-

hydrochloride in 0.3 ml was added and incubated for additional 30 min at 37°C. The

reaction was tenninated by addition of 10% ice-cold TCA and to a dear supernatant add

an equal volume TBA. The resultant TBA chromogen formed from DNA was measured

and the values are expressed as described eariier.

Isolation of ferritin from rat liver and mobilization of Iron by 1,2,4-benzenetriol

Female albino rats (Swiss Wistar strain) were treated with FeSO .THzO (100 mg/kg

body weight, intraperitoneally) for three days consecutively. Rats were sacrificed on 4th

day and ferritin was isolated from liver by the method of Granick (1942) with some minor

modifications.

Liver from two rats was homogenized using Ultra Tun ax T-25 for 30 sec. in distilled

water. Dilute the suspension to 1 gm liver per 4 ml distilled water and heat rapidly with

stirring to 70-80°C for 5 min. The suspension was filtered while hot through a muslin

cloth. Adjust the pH of the filtrate to pH 4.6 with acetate buffer (0.5 M) and centrifuged

at 1500 rpm for 30 min. After discarding the precipitate, ammonium sulphate (30 gm/100

ml) was direclty added to clear red brown supernatant and kept at 4°C for overnight and

the resultant precipitate was centrifuged and dissolved in measured volume of distilled

water. Cadmium acetate (4 gms) was added per 100 ml suspension and centrifuged at

1000 rpm for 10 min to remove any impurity. A red brown supernatant containing

noncrystalline ferritin was obtained and this suspension was used to study the

mobilization of iron from ferritin by 1,2,4-benzenetriol. Release of iron was measured in

a total volume of 1.5 ml contained, 05 ml of sodium acetate buffer (0.1 M, pH 5.6), 330

MM 1.2,4-benzenetriol, 0.5 mM ferrozine and 50 or 100 pi ferritin isolated from rat liver

(0.4 and 0.8 mg equivalent protein). The formation of [Fe(ferrozine)3f* was monitored

(Kadir et al., 1992) by recording the increase in absorbance at 562 nm with time and

amount of iron released from ferritin is expressed as pM Fe^Vmin/mg protein.

Chapter - II 32

Protein estimation

Protein was estimated by the method of Lowry et al., (1951). Protein was

precipitated with 10% ice-cold TCA. The precipitated protein was centrifuged at 3000

rpm for 15 min and the protein pellet was dissolved in 1N NaOH. Finally suitable amount

of dissolved protein was diluted to 1.0 ml with distilled water and 5.0 ml of alkaline

copper reagent was added. After 10 min, 0.5 ml of Folin-Ciocalteu reagent was added.

Absorbance was read at 750 nm after 20 min. Bovine serum albumin (BSA) was used

as standard.

To prepare alkaline copper reagent, solution (A) and (B) were mixed in 1:1 ratio just

prior to use of the reagent. Solution (A) containing 8 gm sodium carbonate anhydrous

dissolved in 100 ml of double distilled water, and (B) containing 120 mg sodium

potassium tartrate and 60 mg of CUSO4.5H2O dissolved in 100 ml of double distilled

water.

Recovery of iron:polyphenol complex

Recovery of iron:polyphenol complex from bone marrow lysate (1 mg protein

equivalent) was monitored by assaying iron: polyphenol complex catalyzed bleomycin-

dependent degradation of DNA as compared to that without bone marrow lysate. Briefly,

the assay system in total volume of 1.0 ml with or without bone marrow suspension (1

mg protein) in phosphate buffer saline (0.1 M, pH 7.4) contained 250 yg calf thymus

DNA, 50 |jg bleomycin and iron: hydroquinone or iron: 1,2,4-benzenetriol (24 |JM:48 pM)

complex. The total reaction mixture was incubated for 30 min at 37°C and the reaction

was terminated by adding 1.0 ml of 10% ice-cold TCA. The contents were centrifuged

at 3000 rpm for 15 min and TBA colour was developed as described eariier. FeS04.7H20

was used to prepare iron and iron:polyphenols by the addition of iron and polyphenol in

1:2 ratio. Iron and iron: polyphenol solutions were prepared just before use.

Optical spectra

The optical spectra of polyphenols with or without the addition of Fe ^ were recorded

on Perkin-Elmer Lambda 15 UVA/IS spectrophotometer in freshly distilled water. The

Chapter - / / 33

incubation mixtures contained 100 pM hydroquinone alone or 1,2,4-benzenetriol alone

or with 200 pM Fe^* and scans were recorded every minute upto 4 min.

Lipid peroxidation in liver homogenate

Lipid peroxidation was estimated in 10% liver homogenate by the method described

by Bemheim e al. (1948). Liver homogenate (10%) was prepared in 0.15 M KCI. Liver

homogenate (10 ml) was taken in 25 ml conical flask and placed in metabolic shaker

waterbath at 37°C. An aliquot of 1.0 ml of homogenate was taken out at different time

interval (i.e. 0 hr and 1 hr) and precipitated by adding 1.0 ml of ice-cold 10% TCA. After

waiting for few min the tubes were centrifuged at 3000 rpm for 15 min. Finally, 1.0 ml

of clear supernatant was taken out and 1.0 ml of 0.67% (w/v) TBA was added. TBA

chromogen colour was developed by boiling the tubes on water bath for 15 min and read

at 532 nm. Results are expressed in terms of malonaldehyde equivalents nmoles/hr/gm

liver using molar extinction coefficient 1.56x10* cm' M' at 532 nm.

Sulphahydryl estimation

Total and free sulphahydryl (glutathione) were estimated using the standard

procedure described by Sedlak and Lindsay (1968).

(a) Total sulphahydryl

Liver homogenate in 0.15 M KCI containing 0.02 M EDTA (0.5 ml of 2%) was mixed

with 0.2M tris buffer containing 0.02 M EDTA (pH 8.2), 0.1 ml of 5,5'-dithiobis-

(2-nitroben2oic acid) (DTNB) (99 mg/25 ml absolute methanol) and kept the reaction

mixture at room temperature for 15 min. After centrifugation at 3000 rpm for 15 min,

absorbance of the supernatant nitromercaptobenzoic acid anion was read at 412 nm.

(b) Free sulphahydryl

To 1.0 ml of 2% liver homogenate (in 0.15 M KCI containing 0.02 M EDTA) was

added 1.0 ml of 10% ice-cold TCA. The test tubes were shaken intermittantly for 15 min

at room temperature and centrifuged at 3000 rpm for 15 min. Supernatant (1.0 ml) was

Chapter - / / 34

mixed with 1.4 ml of 0.4 M tris buffer containing 0.02 M EDTA (pH 8.9). Finally, 0.1 ml

of DTNB was added and mixed well. The absorbance of the nitromercaptobenzoic acid

anion formed was taken at 412 nm.

Sulphahydryl content was calculated from the standard plot of cysteine prepared in

the same manner and expressed in terms oi mmoles/100 gm liver

Haematological parameters

Haemoglobin (Hb) content in male mice was estimated by cyanomethaemoglobin

method (Drabkin and Austin, 1932). The basis of the method is dilution of blood in a

solution containing potassium cyanide and potassium ferricyanide. Haemoglobin is

converted into cyanomethaemoglobin in presence of Drabkin's cyanide - ferricyanide

solution. The absorbance of the solution is then measured in a spectrophotometer at a

wavelength of 540 nm.

Drabkin's reagent was prepared by dissolving 100 mg potassium ferricyanide and

25 mg potassium cyanide in 500 ml of distilled water. The reagent should be clear and

pale yellow in colour and can be stored at room temperature in a brown glass bottle.

When measured against water as blank in a spectrophotometer at wavelength 540 nm,

the absorbance must read zero.

Add 20 pi of blood into 4.0 ml of Drabkin's reagent and mixed well by inverting the

tubes several times. After mixing, the tubes were allowed to stand at room temperature

for atleast 10-20 min to ensure the completion of the reaction. Finally, the tubes were

read at 540 nm against Drabkin's reagent as a blank using Spectronic-2001

spectrophotometer. Cyanomethaemoglobin solution was used as a standard to calculate

the haemoglobin content.

Calculation

A54o0f test sample Cone std. (mg/dl) x dilution factor Hb(g/dl)= X

A540 of standard 1000

Other haematological parameters, such as white blood cell count, differential

leukocyte count and hematocrit value (%) were evaluated using standard procedures.

Chapter - II 35

Packing of Sephadex G-10 column and elution profile of iron alone, 1,2,4-benzenetriol alone and 1,2,4-benzenetriol:iron mixture

Sephadex G-10 (4.0 gm/200 ml) in 0.1 M sodium acetate buffer saline, pH 5.6 was

incubated overnight at room temperature. It was loaded to the column and allowed to

settle down and the gel column volume was 12 ml (12 cm x 1 cm). The column was

washed three times with acetate buffer and iron (as ferrous sulphate) (1 mM in 1.0 ml)

was passed through the column and eluted with 0.1 M acetate buffer of pH 5.6. Flow rate

of 2.5 ml/5 min was maintained and 1 to 30th fractions were collected at every 5 min.

Similarly, ImM in 1.0 ml of 1,2,4-ben2enetriol and 1.0 ml of 1,2,4-benzenetriol : iron

mixture (1 mM : 1 mM) were also passed through the column and 1 to 30th fractions were

collected.

In another set of experiment 1.0 ml of reaction mixture contained 100 pg horse

spleen ferritin, 2mM of 1,2,4-benzenetriol and with or without 1 mM ethylene-

diaminetetraacetic acid (EDTA) were passed through column and fractions were collected

at every 5 min.

Estimation of 1,2,4-benzenetriol and iron

1,2,4-Benzenetriol estimation was followed spectrophotometrically by recording the

optical density at 287 nm using a Spectronic-2001 spectrophotometer and iron estimation

was done according to the method of Peters et al., (1956) by using bathophenanthroline

sulphonic acid. Briefly, 2.0 ml of reaction mixture contained, 0.5 ml of aliquot from each

fraction, 1.5 ml of sodium acetate buffer (0.1 M, pH 5.6), 200 pg ascorbic acid and 50

pg bathophenanthroline sulphonic acid. Reaction mixture was kept at room temperature

for 5 min and absorbance was taken at 535 nm.

Preparation of glutathionyl hydroquinone

Equimolar concentration (200 pM) of 1,4-benzoquinone and glutathione reduced

(GSH) were mixed in distilled water in 1:1 proportion by volume. The nucleophilic

addition of GSH to 1,4-benzoquinone yields glutathionyl hydroquinone and its authenticity

was confirmed by the absorption maximum at 303 nm. No absorption was observed at

Chapter - / / ^

248 nm which is the absorption maximum for 1,4-benzoquinone. Free GSH was also not

demonstrable by Ellman's reagent.

Glutathlonyl hydroquinone and iron dependent TBAR formation from DNA or glutamate or deoxyuridine

of Iron catalyzed bleomycin-dependent release of TBAR from DNA in presence iron

or iron + GSH or iron + GSH + 1,4-ben2oquinone was performed according to the method

of Gutteridge (1987). Briefly, the assay system in a total volume of 1.5 ml consisted of

0.033 M phosphate buffer (pH 7.4), 250 pg calf thymus DNA, 50 jg bleomycin

hydrochloride and with or without oxyradical scavengers, viz. thiourea (5mM), mannitol

(50 mM), dimethylsulfoxide (33 mM), albumin, SOD and catalase (100 pg/ml). The

reaction was initiated by the addition of 15 pM iron (as FeS04.7H20) or 15 pM iron + 15

pM GSH or 15 pM iron + 15 pM GSH + 15 pM 1,4-t>enzoquinone. All these reactants

were dissolved immediately before use. The contents were incubated at 37°C for 30 min

and the reaction was terminated by the addition of 0.75 ml of 10% (w/v) ice-cold TCA.

The contents were centrifuged at 3000 rpm for 15 min and to the clear supernatants an

equal volume of 0.67% (w/v) TBA was added. TBA colour was developed by heating the

tubes for 15 min in a boiling water bath. The resultant TBA chromogen was read at 532

nm using a Spectronic-2001 spectrophotometer. The values are expressed as nmoles

of malonaldehyde equivalents generated by using £532=1.56x10^ cm' MV

Release of TBAR from L-glutamate or deoxyuridine was followed according to the

method of Gutteridge (1981). Briefly, the reaction mixture (total volume 1.5 ml)

contained, 0.033 M sodium phosphate buffer (pH 7.4)in 0.15 M NaCI, 4 mM glutamate

monosodium or 3.2 mM deoxyuridine and the reaction was started by the addition of

glutathlonyl hydroquinone (5.0 pM - 25.0 pM). The contents were incubated for 2 hr at

37°C and the reaction was terminated by the addition of 1.0 ml of 10% (w/v) ice-cold

TCA. TBAR was estimated as described in the case of release of TBAR from DNA.

Recovery of glutathionyl hydroquinone

Recovery of glutathionyl hydroquinone from bone marrow lysate (1 mg protein

Chapter - / / 37

equivalent) was monitored by assaying Iron- catalyzed bleomycin-dependent degradation

of calf thymus DNA as compared to that without bone marrow lysate.

Measurement of Oj'

Release of Oj' from glutathionyl hydroquinone (30 \M) in a total volume of 1.5 ml

was monitored by the superoxide dismutase inhibitable reduction of cytochrome c (80

pM) at 550 nm at 25°C spectrophotometrically using the extinction coefficient value of

reduced cytochrome c as 29,500 cm'M"' (Cumutte et al., 1974). Appropriate blanks were

used to con"ect any cytochrome c reduction in the absence of glutathionyl hydroquinone.

Reaction of glutathionyl hydroquinone with plasmid pUC 18 DNA

Reaction mixture in 50 pi, contained 20 pM phosphate buffered saline (pH 7.4), 300

ng plasmid pUC 18 DNA, 200 pM 1,4-benzoquinone alone or GSH alone or glutathionyl

hydroquinone and with or without oxyradical scavengers, viz. superoxide dismutase,

catalase (54 pg), mannitol, thiourea and sodium benzoate (2 mM). The contents were

mixed well and incubated for 3 hr at 37°C. After 3 hr incubation, 5 pi of solution

containing 40 pM EDTA, 0.05% bromophenol blue tracking dye and 50% (w/v) sucrose

was added and the solution was subjected to electrophoresis on 1% agarose gels in TBE

buffer (89 mM tris base, 89 mM boric acid and 2 mM EDTA, pH 8.0) for 3 hr at 60 mA

current. The gels were stained with ethidium bromide (0.5 pg/ml) viewed and photo

graphed on an UV-transilluminator. One single strand break in supercoiled DNA into

relaxed open circular form or double strand break into linear form, respectively, run

slower than undamaged supercoiled DNA as evidenced by 1% agarose gel

electrophoresis (Toyokuni and Sapripanti, 1993).

Isolation of rat liver nuclei

Approximately 4 gm rat liver was homogenized in 30 ml of Tris-EDTA buffer (50 mM

tris, 10 mM EDTA, pH 8.0) and centrifuged for 10 min at 2000 rpm. Pellet was washed

twice in Tris-EDTA buffer and resuspended to 10 ml with 0.1 M phosphate buffered

saline (pH 7.4) (Mahmutoglu and Kappus, 1987),

Chapter - II 38

Release of TBAR from rat liver nuclei in the presence of different polyphenols and effects of different metal chelator and oxyradical scavengers

The reaction mixture in a total volume of 1.5 ml contained, 0.1 M phosphate buffered

saline (pH 7.4), 0.5 ml isolated rat liver nuclei suspension (12 mg protein), 330 JM

hydroquinone/1,2,4-benzenetriol/6-hydroxydopamine/dopamine and with or without

copper (0.08 and 0.167 mM) or EDTA/DTPA (5 pM) or bathocuproine (5-25 pM) or

different oxyradical scavengers, such as thiourea (2.5 and 5.0 mM)/mannitol (50 mM)/

catalase(150 pg/1.5 ml)/SOD (100 jg/1.5 ml). The contents were incubated for 2 hr

at 37°C with occasional shaking and the reaction was terminated by the addition of 0.5

ml of 10% ice cold TCA. The contents were centrifuged and to 1.0 ml of supematant 0.5

ml of 0.67% thiobarbituhc acid (TBA) was added and heated in a boiling water bath for

15 min and the resultant malonaldehyde: TBA chromogen was read at 532 nm. The

results were expressed as nmoles of TBAR formed per hour per gm protein by using

extinction coefficient 1.56 x 10^ cm' M (Rao and Pandya, 1989; Quinlan and

Gutteridge, 1987).

Metal analysis

Metals were analyzed in liver tissue and isolated rat liver nuclei of rats of benzene

exposed and control group by the method of Berman (1980). Equal weight of liver tissue

or 3.5 ml of isolated nuclei was added into the digestion flask containing 20 ml of

digestion mixture (1:6:: Perchloric acid (60%): nitric acid). The flasks were boiled in the

fuming chamber on hot plate till dry. The dry ash was dissolved in 5.0 ml of 0.1 N nitric

acid and transfered into the test tube. The contents of iron and copper were read using

atomic absorption spectrophotometer. Results were expressed in terms of pg metal/gm

liver tissue or [jg metal/gm protein in liver nuclei.

Nuclear DNA damage by hydroquinone or 1,2,4-benzenetriol

Isolated rat liver nuclei were incubated with hydroquinone or 1,2,4-benzenetriol and

DNA damage was evaluated by agarose gel electrophoresis. Briefly, 50 1 of total

reaction mixture contained, 20 fj| of isolated rat liver nuclei suspension (350 pg protein).

Chapter - II 39

10 tJl of phosphate buffered saline (0.1 M, pH 7.4) and hydroquinone orl ,2,4-benzenetriol

(0.5 mM and 1.0 mM). Reaction mixture was incubated for 4 hr at 37°C and finally mixed

with 5 \i\ of solution containing 40 pM EDTA, 0.05% bromophenol blue tracking dye, 1%

sodium dodecyl sulphate and 50% (w/v) suaose. Total reaction mixture was loaded on

1% agarose gel for electrophoresis in TBE buffer (89 mM tris base, 89 mM boric acid

and 2 mM EDTA, pH 8.0) for 3 hr at 75 mA current. The gels were stained with ethidium

bromide (0.5 pg/ml), viewed and photographed on an UV-transilluminator.

STATISTICAL ANALYSIS

The statistical values and level of significance between the experimental and control

groups of animals were computed by the following formulas :

Standard deviation (SD)

(X, - X)^ SD =

n-1

Where,

X, = individual values X = arithmatic mean of individual values n = sample size

Standard error

SE

t value

t

(SE)

SD

{T

^A • '^B

/ (SEJ^MSEB)^

Where, x^ and Xg are two sample means and SE^ and SEg are the standard

errors.

Chapter • / / 40

Level of significance

After calculating the 't' value the level of significance (p value) was found out from

the table. The p values less than 0.05, 0.02, 0.01 and 0.001 were considered as just

significant, significant, more significant and highly significant respectively.

Chapter - IB Release of Iron from Ferritin

by 1,2,4-benzenetriol Introduction

Results

Discussion

Chapter - III 41

INTRODUCTION

Iron (Fe) is an essential element for living organisms because of its role in major

biological reactions such as the tricarboxylic acid cycle, electron transport, nitrogen

fixation, DMA synthesis and toxication and detoxication reactions (Theil, 1987). Iron in

aqueous solution has ready access to two stable oxidation states, the ferrous, Fe(ll) and

the ferric, Fe(lll). This property underlies the participation of iron in reactions spanning

all of biochemistry including those controlling the flow of electrons through bioenergetic

pathways, the activation of molecular oxygen, the decomposition of noxious derivatives

of oxygen such as peroxide and superoxide and binding of oxygen by haemoglobin,

myoglobin and haemerythrins (Aisen and Listowsky, 1980)

But, the same redox properties that make iron so useful in biocatalysis and oxygen

transport present hazards to the organism. Throughout the biological universe, therefore,

organisms have been compelled to evolve specific iron sequestering molecules to

maintain the element in soluble form available for transport and for biosynthesis to

essential iron proteins and enzymes. By suppressing the catalytic activity of iron in

Chapter - III 42

one-electron redox reactions, such iron-binders may also reduce exposure of cells to

potentially damaging species such as peroxide, superoxide and hydroxyl radicals

(Willson, 1977). In the vertebrate world, these iron-binding functions are managed by

transferrin and ferritin, the transport and storage protein of iron metabolism respectively.

Each of these proteins bind iron sufficiently tightly to resist formation of insoluble

hydroxylitic products. The binding of iron to these proteins is reversible so that the metal

is available upon demand to cells with a specific need so that it is incorporated into its

final biochemical form. The binding of iron by transferrin is to two specific sites with

well-defined stoichiometry, but in fenritin iron exists as polynuclear aggregates of variable

size and compositions (Aisen and Listowsky, 1980).

Storage of iron occurs in two forms; as a soluble protein enveloped core of ferritin,

and as a part of the nondescript insoluble hemosiderin. The latter consists of inorganic

iron associated with protein and other substances in variable proportions, and in fact,

may originate from degraded ferritin (Vidnes and Helgeland, 1973). Mammalian liver,

spleen and bone marrow are especially rich in ferritin but the protein is found in most

other tissues, in serum and in circulating blood cells. Ferritin is synthesized in response

to iron, which it sequesters in accessible form to provide a mobilizable reserve. Thus, the

protein can serve as a source or sink of iron to play a dynamic role in iron metabolism,

with regulation occuning at the level of protein synthesis and turnover, and iron deposition

and release (Aisen and Listowsky, 1980).

Ferritin is a large protein having molecular weight of about 450 kilodatton. Apoferritin

(protein moiety of ferritin only) consists of 24-polypeptide chains, made up of two types

of subunits, H and L, with molecular weight of 22,000 and 19,000 respectively (Biemond

et al.. 1988). The polypeptide chains are arranged in a spherical protein shell, with a

central cavity. X-ray and electron microscopic studies show that the protein shell is

nearly spherical with an external diameter of 120-130 A° and a central cavity of about 75

A° (Hoare et al., 1975). The iron is encapsulated in the central cavity which has eight

shallow pockets (Treffrye^ a/., 1977). The channels facilitate the movement of iron, and

this constitutes an important function of femtin. One molecule of ferritin can store as

many as 4,500 iron atoms (Harrison, 1977). Iron is present in ferritin largely in the form

Chapter - III 43

of ferric oxyhydroxide (FeOOH) together with some phosphate (Theil et al., 1979).

Clinicians monitor the level of ferritin in serum to obtain information about the state of the

reticuloendothelial iron stores, or the presence and progress of some malignant diseases

(Jacobs and Worwood, 1975; Worwood e a/., 1975). Several studies have indicated the

involvement of free radicals in initiation and promotion processess of carcinogenesis

(Wei and Frenkel, 1991). Large human prospective studies suggest that excess body

iron stores estimated by serum ferritin level, serum transferrin level, or transferrin

saturation are associated with carcinogenesis, such as primary hepatocellular carcinoma,

colon cancer, lung cancer, bladder cancer, acute lymphocytic leukemia and neuro

blastoma (Potaznic ef al., 1987; Evans ef a/., 1987). Thus, elucidation of the

mechanisms of iron-mediated oxidative damage may be important for the prevention of

carcinogenesis.

Redox cycling is a characteristic of transition metals such as iron and is closely

associated with the production of reactive oxygen species. In order to link the chemical

reaction with biological system the concept of the "catalytic" or "free" form of iron

(Gutteridge et al., 1982) is important. "Free" iron was originally measured as thiobarbitunc

acid reactive products (TBAR) formation in an in vitro system that contains sample fluid,

calf thymus DNA, bleomycin, ascorbate and HjOj. Bleomycin in the presence of Fe(ll)

degrade DNA to form TBAR. The mechanism of bleomycin-dependent DNA degradation

in presence of iron and reducing agent is shown in Figure-3.1. Although, other ions can

bind to bleomycin, they do not result in DNA degradation (Gutteridge et al., 1982).

Reactive oxygen species in biological system induce oxidative stress. Every cell,

whether using oxygen or not, has elaborate inducible protective mechanisms against

oxidative stress. There are almost always reducing agents, or enzymes associated with

reductants. Reducing molecules or enzymes include ascorbate, glutathione reduced

(GSH) and thiol-specific antioxidant (Chae et al., 1993; Yim et al., 1994). "Free" iron, if

it exists in vivo, can, therefore, t>e quite harmful because there are many reducing agents

mentioned above that can initiate the Fenton reaction via the production of H O as

follows:

O

10

.2 H o ^

5 CO

^ r O X 'J i: o w« QL 03

< + Q 5

< Q 5

<

V I t

< • m

O I

e g o

u > E o

OQ

c (1) O) 03 cn c o 3 •o 0) k. •c c ro c o

0) o c 0)

c o TO

•D TO k_ D) 0)

< Z Q

0) •D C 0) Q. dJ

X)

E o

E w 'c

o

CO

3

iZ

W ^

Chapter - III 44


2Fe(ll) + O2 + 2H* > 2Fe(lll) + H A

Fe(ll) + H2O2 > Fe(lll) + OH + OH

The mechanism of iron uptake, mobilization, release and utilization are well

understood (Crichton and Ward, 1992). Low molecular weight iron chelates which

maintains a dynamic equilibrium fc>etween iron uptake, storage and utilization have been

descrit>ed (Jacobs, 1977). Iron may be mobilized from ferritin by reduction with agents

such as dithionite, thioglycolate, or other thiols (Crichton, 1973), with removal facilitated

by chelators. Without reductants, chelating agents are either ineffective for releasing iron

from ferritin or remove it only very slowly (Crichton and Roman, 1978). Although

ascorbate, cysteine and reduced glutathione may serve as reductants in vitro, but they

are not efficient enough to be considered possible physiological mediators of iron release

from femtin. However, recently Baaderef a/., (1996) clearty demonstrated that ascorbic

acid is capable of mobilizing iron from ferritin in the cellular system by reduction of the

ferric ion core in neuroblastoma SK-N-SH cells. Superoxide anion radical generated by

xanthine-xanthine oxidase system(Topham e a/., 1982) and many toxicological reductants

which mobilize ferritin iron have been reported. In contrast, ferritin was able to stimulate

free radical damage, possibly by the formation of hydroxyl radicals, in many test systems.

A bulk of evidence obtained in vitro confirms that superoxide radical is also capable of

mobilizing iron from ferritin (Reif, 1992; Monteiro and Winterbourn, 1989a).

Several endogenous and xenobiotic agents have been reported to release Fe(ll)

from ferritin Fe(lll) in vitro (Aust et al., 1985). Thomas et al.. (1985) demonstrated that

xanthine/xanthine oxidase mediated generation of superoxide radical is able to reduce

and release iron from ferritin and concomitantly promote liposome lipid peroxidation.

Redox cycling of paraquat (Thomas and Aust, 1986a), the semiquinone radical of

adriamycin (Thomas and Aust, 1986b), nitric oxide (Reif and Simmons, 1990), 6-

hydroxydopamine (Monteiro and Winterixium, 1989b) and therapeutic and physiological

iron chelators (O'Connell et al., 1989) have been shown to promote mobilization and

reductive release of iron from ferntin. Certain diphenols in the presence of a reductant

Chapter - III 45

have also been shown to release iron from ferritin (Kadir e a/., 1992). It has been

suggested in particular that iron is released from stores under conditions in which

oxidative stress is involved in both systems in vitro (Thomas e a/., 1985) and isolated

perfused organs (Healing et a/., 1990). Ferrali et al., (1992) reported that oxidizing

agents viz. phenyl hydrazine, divicine and isouramil induced iron release and membrane

damage in erythrocytes. The same oxidizing agents were also shown to release iron

from ferritin (Monteiro and Winterboum, 1989a).

Expression of benzene toxicity requires its metabolism to toxic metabolites and

follows similar pathways in humans and animals. It is widely believed that the

polyphenolic metabolites of benzene accumulate against the concentration gradient in

bone marrow (Greenlee e a/., 1981a) and are responsible in the formation of reactive

oxygen species which may play a role in benzene induced toxicity. Hydroquinone and

1,2,4-benzenetriol, the two principal polyphenolic metabolites of benzene have been

shown to be toxic due to their capability to undergo autooxidation (Rao and Pandya,

1989). The suggested mechanism includes free radical formation via superoxide and

covalent binding of semiquinones to DNA, RNA or other cellular macromolecules

(Greenlee et al., 1981b). Although, the mechanism how benzene interferes with iron

metabolism is not well known, but accumulation of bleomycin-detectable low molecular

weight iron (LMI) compound in bone marrow was observed in experimental rats dunng

benzene exposure (Rao et al.. 1990). Earlier studies showed that benzene inhibits heme

biosynthesis at the level of 6-AU\ dehydratase (Freedman et al.. 1977; Rao and Pandya,

1980) and increase in heme oxygenase activity, a rate-limiting enzyme for heme

degradation (Abraham et al., 1986). As a result, depletion in regulatory heme content

(Siddiqui etal., 1988) and accumulation of iron have been reported (Pandya etal., 1990).

Superoxide dependent reductive release of iron from ferritin may increase the LMI pool

capable of undergoing redox reactions (Topham et al.. 1982).

With this available information that accumulation of polyphenolic metabolites of

benzene and formation of reactive oxygen species due to autooxidation of polyphenolic

metabolites, we investigated the efficacy of polyphenols to release iron from horse

spleen ferritin in vitro, in the presence of hydroquinone or 1.2,4-benzenetriol and other

Chapter - III 46

endogenously formed polyhydroxy compounds, and whether the iron released can

catalyze oxidation reactions.

RESULTS Release of iron from ferritin in the presence of polyphenols (hydroquinone or

1,2,4-benzenetriol) and other reducing agents was recorded in Table-3.1. Addition of

hydroquinone (330 pM) did not result in the release of iron from ferritin. However, the

same concentration of 1,2,4-benzenetriol resulted in a significant release of iron (3.2

pM/min) from ferritin. The release of iron was found to be linear upto 15 min and about

20% of total ferritin iron was released. Presence of dopamine failed to release iron from

ferritin, whereas 6-hydroxydopamine released large amounts of iron (4.6 [jM/min) from

ferritin as was reported eariier (Monteiro and Winterbourn, 1989b). Significant amounts

of iron release from femtin was observed in the nitrogen atmosphere, although to a lesser

extent than in aerobic conditions (Table-3.1). The efficacy of other polyphenols such as

1,2,3-t>enzenetriol (Pyrogallol) and 1,3,5-benzenetriol (phloroglucinol) to release iron from

ferritin were studied. Except 1,2,4-benzenetriol, other tnhydroxy benzene derivatives

failed to release iron from ferritin (Table 3.2). The release of iron from ferritin was

concentration dependent on 1,2,4-benzenetriol as is shown in Figure-3.2, although the

increase was not linear with the concentration of 1,2,4-t>enzenetriol under aerobic and

anaerobic conditions.

Table-3.3, represents the effect of oxyradical scavengers on the release of iron from

ferritin in the presence of 1,2,4-benzenetriol The release of iron was substantially

inhibited as was evident from the decreased formation of [Fe(ferrozine)3]^' in the

presence of albumin, catalase and superoxide dismutase (SOD). Thiourea and mannitol

which are hydroxyl radical scavengers were able to inhibit iron release to an extent of

about 15%. This indicates that 1,2,4-benzenetno! mediated release of iron was not

completely due to the mediation of active oxygen species generated during autooxidation

of 1,2,4-benzenetriol.

The effect of increasing concentrations of 1,2,4-benzenetriol on lipid peroxidation of

2% rat brain homogenate in the presence of femtin is shown in Figure-3.3. The increase

47

Table 3.1

Effect of benzene metabolites and other reducing agents on the release of iron from ferritin.

Addition

1,2,4-Benzenetriol (330 pM)

Hydroquinone (330 (JM)

1,4-Benzoquinone (330 [iM)

Phenol (330 pM)

Glutathionyl hydroquinone (330 pM)

Uric acid (330 pM)

6-Hydroxydopamine (125 pM)

Dopamine (125 pM)

pM Fe * released/min

3.2+0.11

2.1±0.18 (anaerobic)

N.D.

N.D.

ND.

N.D.

N.D.

4.6±0.15

0.3+0.01

Reaction mixture contained in a final volume of 1.5 ml, ferritin (100 pg/1.5 ml), different polyphenols, and ferrozine (500 pM) in 0.1 M sodium acetate buffer of pH 5.6. Measurements of Fe^*- ferrozine complex followed at 562 nm due to iron mobilization from ferritin by different reducing agents.

All values are average + SE of two sets of experiments conducted in duplicate.

N.D. = None detectable

48

Table 3.2

Effect of 1,2,4-benzenetriol and other trihydroxy benzene derivatives on the release of iron from ferritin

Addition \iM Fe^* released/min

1,2,4-Benzenetriol 3.2±0.11

1,2,3-Benzenetriol N.D.

1,3,5-Benzenetriol N.D.

Reaction mixture contained in a final volume of 1.5 ml, fenitin (100 |jg/1.5 ml), polyphenols (330 pM) and ferozine (500 pM) in 0.1 M sodium acetate buffer of pH 5.6. Measurements of Fe^*- ferrozine complex followed at 562 nm. N.D. = None detectable

e ^1 + +

A —Aerobic atmosphere

B —Anaerobic atmosphere

330 B T ( M M )

Figure 3.2 : 1,2,4-Benzenetriol concentration dependent iron release from horse spleen ferritin under (A) Aerobic, and (B) Anaerobic conditions.

49

Table 3.3

Release of iron from ferritin by 1,2,4-benzenetriol and inhibition by oxygen radical scavengers

Reaction mixture pM Fe'* released/min

Complete reaction mixture 3.20±0.11

+ Urea(5mM) 2.88±0.24(10)

+ Thiourea (5 mM) 2.64+0.08(18)

+ Mannitol (50 mM) 2.72±0.10 (15)

+ Albumin (150 pg/l.5 ml) 2.32+0.02(28)

+ Catalase(150[jg/1.5ml) 2.00±0.05(38)

+ SOD (150 ^^g/^ .5 ml) 2.16±0.12(33)

Complete reaction mixture contained in a final volume of 1.5 ml, ferritin (100 |jg/1 5 ml), 1,2,4-benzenetriol (330 pM), fen-ozine (500 pM) and oxygen radical scavengers in 0.1M sodium acetate buffer of pH 5.6. All values are average ± S.E. of t\NO sets of experiments conducted in duplicate. Numbers in paraentheses are percent inhibition.

o en CO

U) •rN' ^ fNI

m CO

•

00

, . ^

2 H,

* - , _ i f r '

H-m

c d) o ^

1^ <U TO N c C m

to fNj x : T—

o ro

If 'c s^ O >»-o o

55 o E ^ m 0) >- i i >»-U X O ^ 2 a> o ^ ^

UJ — Q-

CO

o i . 3

( j q /sd|oaj u ) SHVai

in a?

CM CO

(T> CD

CD iD

ro en

' " • ^

2 d.

*-^ h -m

c s= o ni _ o o ^ c o

1 c N ro Si "D ^ 2

•^ H

o -o

- Q.

8 l o E (-> o

9 -Q ro T^ 2 S< e ro Q o S ^ o c £ 3z 2 ^ U J •

CO

iZ

(jq/Sdiouju)savai

50

Table 3.4

Release of iron from isolated rat liver ferritin

1,2,4-Benzenetriol (MM)

00

330

330

Ferritin (mg equivalent protein)

0.8

0.4

0.8

by 1,2,4-benzenetriol.

pM Fe * released/min/ mg protein

0.00

2.21

3.80

Complete reaction mixture contained in a final volume of 1.5 ml, isolated rat liver ferritin (0.4 and 0.8 mg equivalent protein), 1,2,4-t)enzenetriol and ferrozine (500 (JM) in 0.1 M sodium acetate buffer of pH 5.6. All values are average of two sets of experiments conducted in duplicate with variations of 10% or less.

c

o I—

o.

3

o

o L. U .

o E c o

E o

in O

c o

7.0-

Tfmc ( min)

Figure 3.5 : Time course of formation of [Fe(ferrozine)3p* followed at 562 nm due to iron mobilization from bone marrow cell lysate by 1,2,4-ben2enetriol.

Chapter - III 51

in lipid peroxidation as measured by TBAR formation was almost linear with the increase

in 1,2,4-benzenetriol concentration which shows that there is 1,2,4-benzenetriol

concentration dependent increase in iron release from ferritin. Similarly, the iron release

from ferritin in the presence of increasing concentrations of 1,2,4-ben2enetriol was tested

for the iron catalyzed bleomycin-dependent degradation of calf thymus DNA. There was

a linear increase in TBAR formation from the deoxyribose moiety of DNA with the

increase in concentration of 1,2,4-benzenetriol (0-165 pM) (Figure-3.4).

Ferritin was isolated from female rat liver after inducing the ferritin level by injecting

FeS04.7H20 intraperitoneally. The time course of formation of [Fe(ferrozine)3]^* complex

due to release of iron from isolated rat liver fenitin in presence of 1,2,4-benzenetriol was

recorded. The release of iron from isolated ferritin was found to be linear with increase

in the amount of ferritin (Table-3.4).

Release of iron from bone marrow cell lysate in the presence of hydroquinone and

1,2,4-benzenetriol showed that hydroquinone did not release any iron from bone marrow

cell lysate (results not shown), whereas, 1,2,4-benzenetriol (330 pM) resulted in the

release of iron from bone marrow cell lysate. The increase in the release of iron from

bone marrow lysate in the presence of 1,2,4-benzenetriol as a function of time was also

observed (Figure-3.5).

DISCUSSION

Benzene exposure interferes with iron metabolism which includes, decrease in

incorporation of *®Fe into circulating erythrocytes (Lee ef a/., 1974). Exposure to benzene

leads to decrease in heme biosynthesis enzyme particulariy. 6-AL.A dehydratase

resulting in accumulation of 6-ALA in bone marrow (Rao and Pandya, 1980, Freedman,

et al., 1977), Accumulation of low molecular weight iron (LMI) in bone marrow during

benzene exposure has been demonstrated and it was shown that LMI catalyze

bleomycin-dependent degradation of calf thymus DNA (Pandya ef a/., 1990). However,

the mechanism of how benzene interferes with iron metabolism is not known.

Polyphenolic metabolites of benzene have been shown to accumulate in bone marrow

against the concentration gradient (Greenlee et al., 1981a; Rickert et al., 1979).

Chapter - III 52

Autooxidation of 1,2,4-benzenetriol has been shown to generate superoxide radicals

(Lewis et al., 1988b). Superoxide radical is a potent reductant of ferritin iron and causes

release of iron from ferritin (Topham et al.. 1982).

In the present investigation it was observed that the presence of 1,2,4-benzenetriol

induced iron release from ferritin in a concentration dependent manner. The possible

mechanism of iron release from ferritin by 1,2,4-benzenetriol is shown in Figure-3.6. The

release of iron from ferritin during autooxidation of 1,2,4-benzenetriol in vivo may result

in increased intracellular concentrations of free iron. The inability of 1,2,3-benzenetriol,

1,3,5-benzenetriol, hydroquinone or dopamine to release iron in comparison to 1,2,4-

benzenetriol and 6-hydroxydopamine suggest that hydroxy hydroquinone moiety is

essential. Similariy, dialuric acid, a tetrahydroxypyrimidine and isouramil, a trihydroxy-

pyrimidine have been shown to release iron from ferritin (Monteiro and Winterbourn,

1989a). Although, the physiological mechanism of iron mobilization from ferritin is pooriy

understood, the observed iron release from ferritin by 1,2,4-benzenetriol could occur

through the direct reduction of iron by the hydroxyhydroquinone form or via its

autooxidation intermediates i.e., superoxide radical and semiquinone. The semiquinone

radicals of several antitumorigenic compounds have been shown to reduce and release

ferritin iron (Thomas and Aust, 1986b; Butler et al., 1985).

The presence of oxyradical scavengers i.e., albumin, catalase or superoxide

dismutase caused 28, 38 or 33% inhibition, respectively of 1,2,4-benzenetriol dependent

iron release from ferritin, with the enzymatic activities of catalase and superoxide

dismutase (SOD) being of little importance relative to the protein effect of albumin. The

oxyradical scavenger insensitive rate of iron release from ferritin by 1,2,4-benzenetriol

is probably due to direct reduction of ferritin iron by 1,2,4-benzenetriol, as substantial

amounts of iron were released in a nitrogen atmosphere also. Based on the present

investigation, it is proposed that 1,2,4-benzenetnol induced iron release from ferritin might

be due to direct reduction and the iron released may facilitate the reduction of O2 by

1,2,4-benzenetriol resulting in the generation of superoxide radical. The autooxidation

rate of 1,2,4-benzenetriol has been shown to be 92-fold higher than that of the parent

hydroquinone which lacks an -OH substituent (Brunmark and Cadenas, 1988). The

Ferritin shell

/ \ /

+ ^ 2e" ;+(2H ; OH r

I

I

I

Ferritin ' channel /

Figure 3.6 : The possible mechanism of iron release from ferntin by 1,2.4-benzenetnol

Chapter - HI 53

presence of hydroquinone or dopamine did not result in iron release from ferritin,

probably due to the lack of an -OH substituent and the resulting slow autooxidation.

The results of the present study demonstrate that iron released from ferritin in the

presence of 1,2,4-ben2enetriol is capable of inducing lipid peroxidation and catalyzing

bleomycin-dependent calf thymus DNA degradation. The mechanism by which iron is


toxicity is not known. However, earlier studies had demonstrated that enhanced lipid

peroxidation was observed in experimental animals exposed to benzene (Rao and

Pandya, 1978; Uysal at al., 1986). Polyphenolic metabolites of benzene have been

shown to accumulate in bone marrow in millimolar concentrations during benzene

exposure (Greenlee e/ al., 1981a; Rickert e/a/., 1979). The present study indicated that

a much lower concentration of 1,2,4-benzenetriol (66 pM) is able to release iron from

ferritin. In a recent study (Singh et al., 1994) it was shown that iron in the presence of

1,2,4- benzenetriol was a potent agent to catalyze bleomycin-dependent degradation of

calf thymus DNA. Similariy, the iron released from ferritin by 1,2,4-benzenetriol could

catalyze bleomycin-dependent degradation of calf thymus DNA.

Toyokuni (1996) hypothesized that increase in free iron in the cytoplasm or nucleus

may be associated with an increased risk of cancer and suggested the possible

mechanisms of iron-induced carcinogenesis (Figure-3.7 ). Our present study indicates

that 1,2,4-benzenetriol is a potent reductant of ferric iron of ferritin and also mobilize and

release iron from ferritin core. The release of iron from bone marrow lysate by

1,2,4-benzenetriol may be of toxicological significance as this could lead to disruption of

intracellular iron homeostasis in bone marrow cells. Similariy, iron release was noticed

from ferritin isolated from liver in the presence of 1,2,4-benzenetriol. The release of iron

from ferritin during autooxidation of 1,2,4-benzenetriol In vivo may result in increased

intracellular concentrations of free iron which may attain to genotoxic levels. Involvement

of similar mechanism In vivo as 1,2,4-benzenetriol dependent reduction and release of

iron from storage sites could result in increased generation of active oxygen species which

in turn damage cellular macromolecules and also can interfere in maturation and

differentiation of erythroblasts. In summary, the present observations offer a new

11 K U.

I M O

s l:Al! J O ^ Q

E o

T) B Q. O X3 ro CO (/) <u c QJ O) O c o o

• D (U

o "O

c c o

0) E 0) s: ^^ U CD W CD

ro •,— o

P Q- > . >- O I I-

rs. ro <u 3

Chapter - III ^^

mechanism that the iron released from ferritin by 1,2,4-benzenetriol could contribute to

the better understanding of the toxicity of benzene. In the light of the present results, it

is believed that iron release in bone marrow cells in vivo leads to generation of

superoxide radicals, peroxidation of lipids and probably affects maturation and

differentiation of bone marrow cells.

Chapter - IV Prooxidant and Antioxidant Properties

of Polyphenol - Iron Complex: an Implication for Benzene Toxicity

Introduction

Results

Discussion

Chapter • IV 55

INTRODUCTION

Chronic exposure to benzene is known to induce aplastic anemia, leukemia, and

other related blood disorders (kalf, 1987). However, the precise mechanism by which

benzene induces these effects are unknown. It has been shown that benzene itself is

not actual toxicant. Expression of benzene toxicity requires its metabolism to reactive

intermediate(s). The reactive intermediates responsible for benzene toxicity however,

have not been definitly identified (Snyder et al., 1993). It is believed that the effect of

benzene is likely to be exerted through the action of multiple metabolites on multiple

targets through multiple biological pathways (Goldstein, 1989).

Benzene is metabolized in liver as well as bone marrow by cytochrome P-450

dependent mixed function oxidase enzymes (Huff etal., 1989). Although, the metabolism

of benzene is complex but is well understood. Benzene is primarily metabolized to

phenol, catechol, hydroquinone, 1,2,4-benzenetrio( (Porteous and Williams, 1949) and

open ring products such as trans, trans-muconaldehyde and trans, trans-muconic acid

Chapter - IV 56

(Latriano ef a/., 1986). The pathway of benzene ring opening is not known, but a number

of possible mechanism for oxidative benzene ring opening have t>een postulated (Grotz

etal., 1994),

Earlier reports have shown that hepatic metabolism of benzene results in

accumulation of polyphenolic metabolites against the concentration gradient in bone

marrow and exerts its toxic effects (Smith et al., 1989). Male Fischer rats exposed to

benzene 500 ppm have been shown to accumulate about 1 mM polyphenolic metabolties

in bone man-ow (Rickert et al., 1979). Hydroquinone and 1,2,4-benzenetriol have been

shown to be potent toxic metabolite of t>enzene (Irons, 1985). In an in vitro study, Rao

and Pandya (1989) observed that polyphenolic metakx)lites o1 benzene are autooxidized

faster in the presence of copper ions and resulting in the formation of thiobarbituric acid

reactive products (TBAR) from target molecules such as glutamate and calf thymus

DNA. The suggested mechanism includes free radical formation via superoxide and

covalent binding of the semiquinone metabolites of polyphenol to such molecules.

Greenlee et al., (1981b) demonstrated that the triphenolic metabolite of benzene,

1,2,4-benzenetriol, is the most reactive toward molecular oxygen. Its strong chemical

reactivity toward O2 is consistent with a high biological potency. 1,2,4-Benzenetriol

induces both single and double strand breaks in DNA (Kawanishi et al., 1989), causes

sister chromatid exchange in human lymphocytes (Erexson et al., 1985) and gene

mutations (6-thioguanine resistance) in V79 cells (Glatt, ef al., 1989). Currently in a series

of studies by Zhang et al., (1994) reviewed the roles of quinone, oxygen and transition

metal ions in the genotoxicity of 1,2,4-benzenetriol. Zhang et al., (1996) described the

potential role of metal ions (iron and copper) in the oxidation of 1,2,4-benzenetriol and

may play an important role in benzene-induced genotoxicity. 1,2,4-Benzenetriol has

been shown to induce oxidative DNA damage (formation of 8- hydroxydeoxyguanosine)

both in cultured human cells in vitro (Zhang et al., 1993) and in mouse bone marrow in

vivo (Kolachana et al., 1993). Despite the evidence that 1,2,4-benzenetriol-induced

active oxygen species are cytotoxic and genotoxic, surprisingly little is known regarding

the mechanisms of its reaction with Oj.

Chapter -IV cy

Recent studies revealed that treatment of HL-60 cells with hydroquinone resulted

in the formation of 8-hydroxydeoxyguanosine (8-OHdG) formation, suggesting that,

hydroquinone can induce oxidative DNA base modification in cells (Kolachana et al.,

1993). Although, the mechanism by which hydroquinone causes the above effects,

remains unclear. It is likely that some of these effects, if not all could be mediated by

further oxidative activation of hydroquinone. Similar observation was also obtained in

different cell lines whose peroxidase activation of hydroquinone results in the formation

of DNA adducts (Levay et al,, 1993), Recently Pongracz and Boded (1996) reported the

DNA adduct formation in HL-60 cell culture treated with hydroquinone. The synergistic

interaction of benzene metabolites (i,e, hydroquinone and 1,2,4-ben2enetriol) in the

production of DNA adduct may also play an important role in the genotoxic effects of

benzene in vitro (Levay and Bodell, 1992),

It has been suggested that iron is released from ferritin under conditions in which

oxidative stress is involved in both in vivo and isolated perfused organs (Mazur and

Carleton, 1965; Healing ef a/,, 1990), We observed during an/n wfro study that 1,2,4-

benzenetriol, a trihydroxy metabolite of benzene, was capable of releasing iron from

horse spleen ferritin, which was mediated to a certain extent by superoxide radical

(Ahmad et al., 1995), Similar results were observed by Oteiza (1995) where 8 -amino

levulinic acid (a heme precursor) releases iron from horse spleen and rat liver ferritin in

vitro via free radical formation, and affect iron metabolism in vivo. Available evidence

indicates that benzene induced myelotoxicity and carcinogenicity involve superoxide

radical formation during autooxidation of polyphenolic metabolites of benzene viz

hydroquinone or 1,2,4-benzenetriol (Subrahmanyam et al., 1991b). A fraction of cellular

non haem iron consisting of chelatable low molecular weight iron represent iron species

catalytically active in initiating free radical reactions (Gutteridge, 1987). In various

pathological conditions this decompartmentalized iron is "malplaced" which when bound

to bleomycin and in the presence of oxygen can degrade DNA (Gutteridge et al., 1985).

Conditions inhibiting heme synthesis and uncoupling of oxidative phosphorylation have

been shown to cause a large increase in the low molecular weight iron (LMI) fraction in

the cytosol (Bakkeren e/a/., 1985), Human normal cerebrospinal fluid and synovial fluid

Chapter -IV 58

from rheumatoid patients contain micromolar concentrations of non-protein bound LMI

(<10,000) compounds which are active in catalyzing bleomycin dependent degradation

of DNA and superoxide and hydroxyl radical reactions (Gutteridge et al., 1982). The

presence of LMI pool in various cells and organs including intestinal mucosa, heart, bone

marrow, liver, kidney, spleen, brain and in cultured Chang liver cells have been reported

(Mulligan et al. 1986). LMI were isolated from guinea pig reticulocytes as AMP-Fe and

ATP-Fe complexes which might be involved in cellular iron transport (Weaver and

Pollack, 1989). The interaction between iron-bleomycin and different catecholamines

have been reported (Lovstad, 1990)

We propose in the present studies that the chelatable low molecular weight iron

accumulated during benzene toxicity (Pandya et al., 1990) is the chelate of polyphenolic

metabolites of benzene with iron. In the present study we investigated the efficacy of

hydroquinone or 1,2,4-t)enzenetriol: iron complex to catalyze bloemycin dependent DNA

degradation. We also studied the effect of polyphenol: iron complex to catalyze

glutamate degradation and lipid peroxidation in vitro.

RESULTS

Iron catalyzed and bleomycin dependent degradation of calf thymus DNA was

markedly greater in the presence of iron polyphenol complex than in the presence of iron

alone (Table-4.1). Further, calf thymus DNA degradation was faster with iron: 1,2,4-

benzenetriol complex (2.4 fold) than with iron:hydroquinone complex (1.5 fold). The

degradation of calf thymus DNA showed an increase with increasing concentration of iron

(0-20 JM) or iron:polyphenol complex (1:2 ratio) (Figure-4.1).

The iron-dependent release of TBAR from glutamate exhibited a decrease in the

presence of either hydroquinone or 1,2,4-benzenetriol. The inhibition of iron-dependent

release of TBAR from glutamate was 81% in the presence of hydroquinone and 66% in

the presence of 1,2,4-benzenetriol (Table-4.1). Similariy, the iron-dependent lipid

peroxidation in brain homogenates was substantially inhibited in the presence of

hydroquinone where it was 39% or 1,2,4-benzenetriol where it was 43% (Table-4.1).

59

Table 4.1

Comparison of TBAR formation from DNA by Iron (20 ^M) and Iron: polyphenol (20:40 \ifA) complex in the presence of bleomycin

Parameters Iron lron:HQ lron:BT

Bleomycin dependent degradation 1.52 2.23 3.58 of calf thymus DNA

Iron dependent degradation of 8.25 1.56 2.83 glutamate

Lipid peroxidation 67.14 41.13 38.05

Experimental conditions are as described in the Materials and Methods section. All values are represented as nmoles of TBAR formed and are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

< m »•-O

in CD

"o E c

( f jM)

Figure 4.1 : Linearity of nmoles of TBAR formation from calf thymus DNA with increase in concentration of iron (t •), iron: hydroquinone complex (o—o) or iron; 1,2,4-ben2enetriol complex (A—A). The reaction was initiated by the addition of iron (0-20 pM) or ironpolyphenol (12 ratio)

60

Table 4.2

Recovery of Iron : polyphenol complex (24:48 pM) added to bone marrow lysate by the bleomycin dependent degradation of calf thymus DNA.

Addition lron:HQ lron:BT

-Bone marrow lysate 2.71 5.73

+Bone marrow lysate 3.33 7.67

Bone marrow lysate (1 mg protein) was added to iron.polyphenol cx)mplex (for detail see Materials and Methods section). All values are expressed as nmoles of TBAR formed from DNA and are averages of two sets of expehments conducted in duplicate with variations of 10% or less.

61

Table 4.3

Formation of TBAR from calf thymus ONA by bleomycin in the presence of Iron (20 pM) or iron with the potential iron chelator (1:2 ratio)

Addition

Iron alone

Iron + HQ

+ BT

+ Citrate

+ Glucose

+ Fructose

+ Glycine

+ Glutamic acid

+ Cysteine

+ Aspartic acid

+ AMP

+ ADP

+ ATP

+ Dithiothreitol

Expenmental conditions are as described in the Materials and Methods section. All values are averages of two sets of experiments conducted in duplicate with vanations of 10% or less.

nmoles of TBAR formed/ 30 min

1.52

2.67

3.52

0.19

1.40

1.29

1.03

1.53

1.59

1.09

0.82

0.92

1.49

0.49

o o o o

O O o

E c

o o ^ . g

c 2 8 ^

^ ^

— o o * - <D Si •-00 OJ

0)

5 •D

c

c <u

T O O

'—•" O X o OJ h—

o

fS o

^ 3 .

o o (N x: 5 k -

o V c o nj

O X

"^5

<

u 3

E c

o o

T3 C

TO TJ-

C O

! = M

ll c £:> O 0) •s > n 0)

o

= w .« ro

(U (0 i : • C Q>

" ~ 11

t - O

CO t ;

to o

o a. OT (D

m S

O 1

CD CM "

0)

Chapter - IV 62

The iron:polyphenol complex added to bone marrow lysate was 100% recoverable

when assayed by the bleomycin-dependent degradation of calf thymus DNA (Table-4.2).

Formation of TBAR from calf thymus DNA by bleomycin in the presence of iron,

iron;polyphenol or iron with potential endogenous iron chelators is recorded in Table-4.3.

The endogenous chelators viz. glucose, fructose, glutamic acid, cysteine and adenosine

triphosphate (ATP) did not affect the iron catalyzed bleomycin-dependent TBAR

formation from calf thymus DNA. Whereas, glycine, aspartic acid, adenosine mono-

phospahte (AMP) and adenosine diphosphate (ADP) caused a significant inhibition. The

presence of citrate and dithiothreitol resulted in a drastic inhibition of bleomycin-

dependent iron catalyzed TBAR formation from calf thymus DNA (Table-4.3).

The addition of FeSO^./HjO to hydroquinone or 1,2,4-benzenetriol in freshly distilled

water revealed no change in the optical spectra of hydroquinone. However, that of 1,2,4-

benzenetnol revealed a new absorption peak at 259 nm (Figure-4.2A and 4.2B).

DISCUSSION

Benzene exposure has been shown to generate organic free radicals and superoxide

radicals (Lewis et al., 1988b) and it is generally accepted that benzene requires

metabolism to express its toxicity (Subrahmanyam et al., 1991a). Polyphenolic

metabolites of benzene viz., hydroquinone and 1,2,4-benzenetriol have Ijeen shown to

be toxic and the suggested mechanism includes free radical formation via superoxide

during their autooxidation and the covalent binding of semiquinone to DNA, RNA and

other cellular macromolecules (Wick, 1980; Rushmore et al., 1984). In addition, the

active oxygen radical formed during autooxidation of polyphenolic metabolites has been

shown to be involved in the cytotoxic effects of benzene (Irons, 1985; Karam and Simic,

1989) Exposure to benzene resulted in decrease in antioxidant potential in serum

(Ahmad et al., 1994) and may allow oxygen intermediates such as superoxide and

hydrogen peroxide to survive in plasma long enough. Benzene exposure also leads to

an accumulation of bleomycin sensitive iron in bone marrow (Pandya et al., 1990).

1,2,4-Benzenetriol, a trihydroxy metabolite of benzene was capable of releasing iron from


Chapter - IV 63

(Ahmad et al., 1995), probably, this may also be the reason of accumulation of iron in

bone marrow during benzene exposure, as the polyphenolic metalwlites are accumulated

against the concentration gradient in bone marrow.

Certain flavonoids aitin and quercetin as phenolic compounds have affinity for iron

ions and act as iron chelators (Afanas'ev eif al., 1989). For example, plant phenolics

quercetin and myricetin were found to enhance iron ion dependent damage to DNA by

bleomycin (Afanas'ev et al., 1989). This was assumed to be due to reduction of feme

bleomycin to the fen^ous form by the phenols and this ferrous bleomycin complex in the

presence of oxygen, degrade DNA (Laughton et al., 1991). On the other hand,

antioxidant effects of flavonoids rutin and quercetin to scavenge superoxide and form iron

complexes have been reported. The cytoprotective activity of flavonoids catechin,

quercetin and diosmetin have been attributed to their iron-chelating ability (Morel ef a/.,

1993). However,the cytotoxicity of some polyhydroxy pyrimidines viz. divicine, isouramil

and dialuric acid has been attributed to their ability to release iron from fenitin and this

iron was shown to promote lipid peroxidation (Monteiro and Winterbourn, 1989a).

During benzene exposure of experimental animals many oxidation-reduction

reactions have been shown to occur (Irons, 1985; Lewis et al., 1988b) which may release

ferrous iron from ferritin. The ferrous iron may t>e chelated to the polyhydroxy

metabolites of benzene, viz., hydroquinone and 1,2,4-t>en2enetriol. The tentative chemical

structure of polyphenol iron complex for 1,2,4-t)enzenetriol:Fe(lll) is diagrammatically

presented in Figure-4.3.

We propose that the complex of polyphenol with iron (Figure-4.3) may be the low

molecular weight iron in bone marrow which has been reported to accumulate selectively

during the exposure to benzene (Pandya et a!., 1990). In the present study we observed

increase in iron-catalyzed bleomycin-dependent degradation of calf thymus DNA in

presence of hydroquinone or 1,2,4-benzenetriol. The iron.polyphenol complex appears

to be stable and intact as the recovery from bone marrow lysate was complete. It is

also possible that the hydroquinone or 1,2,4-benzenetriol possess combined iron-

chelating and iron ion reducing properties to stimulate calf thymus DNA degradation in

the presence of bleomycin. Further, the iron polyphenol complexes have shown an

-f X

+

X

E O U

c o

m

0) LL

+

X

a E o o c o

CQ

o

0)

o

w "TO o E o

> ro c

CO

CD

Chapter - IV 64

antioxidant activity by inhibiting iron-dependent lipid peroxidation in brain homogenate and

glutamate degradation. This is similar to the antioxidant properties exhibited by

flavonoids due to their ability to chelate iron (Afanas'ev et ai, 1989; Laughton et al..

1991). Thus the flavonoids and polyphenolic chelates limit the availability of iron for lipid

peroxidation and at the same time facilitate bleomycin-dependent degradation of calf

thymus DNA.

It has been shown recently that upon mixing hydroquinone with Cu(ll) the

absorbance of hydroquinone at 289 nm decreased with corresponding increase in

absorbance at 247 nm due to the formation of 1,4-t)enzoquinone (Li and Trush, 1993b).

However, the optical spectra of hydroquinone did not show any change in the presence

of ferrous sulphate. This indicates that hydroquinone is not converted to 1,4-ben2o-

quinone and iron is kept in the ferous state to catalyze oxidation reactions. The addition

of ferrous sulphate to 1,2,4-benzenetriol, on the other hand, revealed a new absorbance

peak at 259 nm with a slight decrease at 286 nm. These observations suggest a

complex formation of polyphenol with iron rather than reduction of iron and oxidation of

polyphenol.

It is concluded from the present study that the phenolic metabolites of benzene

enhance the availability of Fe(ll) to bleomycin. Such Fe(ll) is possibly made available due

to reduction of Fe(lll) by polyphenols, thus protecting the Fe(ll) from hydrolysis to

hydroxides and consequent autooxidation. Recently, a few studies have explained the

mechanisms underlying the involvement of iron in benzene toxicity. Zhang e al., (1995b)

have suggested that benzene ring fission is stimulated by iron. Further, they demonstrated

the oxidant-mediated genotoxicity of 1,2,4-t>enzenetriol in presence of metal ions

(particulariy iron and copper) via production of oxygen-derived active species (Zhang et

al., 1996). Accumulation of LMI and polyphenolic metabolites of benzene in bone man-ow

are well established. Probably iron:polyphenol complex may be the potent toxic

intennediate during the benzene exposure in bone marrow for its genotoxicity. However,

the formation of such complex in vivo is yet to be characterized during benzene

exposure.

Chapter - V Polyphenol'iron Complex: A Probable

Toxic Metabolite of Benzene Introduction

Results

Discussion

Chapter - V 65

INTRODUCTION

The various facts and figures that has accrued so far from survey reports and

experimental evidences has established that exposure of animals and human to benzene

can result in damage to the haematopoietic system leading to leukemia (Snyder and

Kocsis, 1975). Acute exposure to benzene has a strong toxic effect on the

haematopoietic system of both laboratory animals (Tunek et al., 1982), and humans

(Cohen et al., 1978) probably via interaction w/ith undifferen-tiated bone marrow cells.

Chronic exposure is carcinogenic in rats and mice (Maltoni et al., 1989; Huff et al., 1989).

Several studies have shown that benzene must be metabolized to exert its toxic effects

and that the metabolites are responsible for the both its cytotoxic and genotoxic effects

(Kalf, 1987).

Benzene is metabolized in the liver mainly to one or more phenolic metabolites and

accumulates against the concentration gradient in bone marrow and exerts its toxic effects

(Smith ef al., 1989). Bioactivation of benzene in haematopoietic cells may be important

for its leukemogenic effects (Andrews et al., 1979). Also, bioactivation of benzene by

Chapter -V ^

bone marrow and liver microsomal enzyme system appears to be responsible for the

haematopoietic toxicity possibly by covalent binding of a metabolite(s) to DNA (Snyder et

al., 1981). Many studies have also shown that benzene produces both structural and

numerical chromo-somal aberrations in the peripheral blood lymphocytes of occupationally

exposed individuals (Ding et al., 1983). Because consistent chromosomal aberrations

have tjeen observed in human leukemias and lymphomas, the induction of chromo-somal

damage by benzene or its metabolites may be critical in the development of

benzene-induced leukemia (Rothman et al., 1995). Although, haematotoxic and

leukemogenic effects of benzene are generally believed to be caused by its metabolites

as opposed to the parent compound, the ultimate reactive inter-mediate(s) responsible

for benzene toxicity is not known (Goldstein and Witz, 1992).

Hydroquinone and 1,2,4-benzenetriol the two principal polyphenolic metabolites of

benzene have been shown to be toxic and the suggested mechanism include free radical

formation via superoxide and covalent binding of the semiquinone to DNA, RNA and other

cellular macromolecules (Irons, 1985). Eariier it was observed that polyphenolic

metabolites of benzene are autoxidized faster to generate free radicals in the presence

of transition metal ion and can release aldehydic products from target molecules such as

glutamate, calf thymus DNA and damage human DNA (Rao and Pandya, 1989; Kawanishi

etal., 1989; Singh e/a/., 1994).

Many investigators have shown that benzene exposure interferes with iron

metabolism and heme biosynthesis in experimental animals (Lee et al.. 1974; Freedman

et a!., 1977; Rao and Pandya, 1980). Benzene administration also led to an accumulation

of iron in bone marrow cell, which can catalyze the formation of aldehydic products (base

propenal) from DNA in the presence of bleomycin (Rao et al., 1990). In all probability

benzene alters heme metabolism of erythroid cells, resulting in the increased

internalization of iron transferrin receptor complex and uptake of internalized iron is almost

entirely restricted to erythroblasts. In the present studies it was observed (Chapter-I) that

1,2,4-benzenetriol, a trihydroxy metabolite of benzene was capable of releasing iron from


(Ahmad et al., 1995). We propose that the low molecular weight iron (LMI) accumulated

Chapter- V 57

during benzene exposure (Pandya et al., 1990) is the chelate of polyphenolic metabolites

of benzene with iron. We investigated the efficacy of hydroquinone and 1,2,4-benzenetriol

in the presence of iron to catalyze bleomycin-dependent calf thymus DNA degradation.

In the presence of hydroquinone or 1.2.4-benzenetriol, iron catalyzed bleomycin

dependent degradation of DNA was markedly enhanced in comparison to alone.

Available evidence indicates that benzene exposure leads to accumulation of LMI

as well as polyphenolic metabolites of benzene in the bone marrow against the

concentration gradient. In the present study we investigated the poly-phenol:iron complex

formation in vitro and methodology standardized to fractionate these complexes by

Sephadex G-10 column chromatography. To demonstrate the polyphenol:iron complex

formation we utilized the system of iron released from ferritin by 1,2,4-benzenetriol. We

further investigated the haematotoxicity of hydroquinone:iron mixture in male mice to

evaluate the possible role of hydroquinone:iron complex in benzene induced

haematotoxicity.

RESULTS

The elution profile of iron, 1,2,4-benzenetriol and 1,2,4-t>enzenetriol:iron mixture were

studied by Sephadex G-10 column chromatography. Fractions were collected at every

5 min. interval (flow rate 0.5 ml/min) and were scanned at 287 nm (the absorption

maximum of 1,2,4-benzenetriol) using Spectronic 2001 spectrophotometer (Figure-5.1).

When iron alone was subjected to Sephadex G-10 column chromatography, the eluted

fractions did not show any peak in any fractions (1-30) at 287 nm. Whereas

1,2,4-benzenetriol alone eluted fractions showed an absorption maximum at 287 nm in

fraction number 21. However, when 1,2,4-t)enzenetriol:iron mixture was subjected to

Sephadex G-10 column chromatography, fraction numt)er 3 showed absorbance

maximum at 287 nm and fraction number 21 did not show any absorbance (Figure-5.1).

Iron content was estimated using colouring reagent bathophenanthroline sulphonic acid

in each fraction and absorbance was followed at 535nm (Figure-5.2). The fractions

collected from 1,2,4-benzenetriol alone subjected to Sephadex G-10 column did not show

any absorbance at 535 nm in any fraction (1-30). But, in case of iron alone and

0.20-1 -O Fc:BT complex ( I ' D - • BT alone •* Fe alone

FRACTION No

Figure 5.1 : Elution profile of iron alone (x x), 1,2,4-benzenetnol alone (• •) and iron:1,2,4-benzenetriol mixture (O—0) subjected to Sephadex G-10 column Fractions were collected at every 5 min interval (flow rate 0.5 ml/mm) and were scanned at 287 nm

O.^-i -* FG alone -o Fc'.BT complex! l:1) - • BT alone

E c n in *^ o

ft

o o <

13 17 21

FRACTION No.

25 29

Figure 5.2 iron estimation using colounng reagent bathophenanthroline sulphonic acid in each fractions and absorbance was followed at 535 nm. Fe alone (x x), Fe:BT complex (1:1) (o o), and BT alone (• •).

Chapter - V 68

1,2,4-benzenetriol.iron mixture showed an absorbance peak at 535 nm in fraction

number 3.

Further, the elution profile of 1,2,4-t)enzenetriol:iron complex formation was also

studied in presence and absence of ethylenediaminetetraacetic acid (EDTA) during the

release of iron from ferritin by 1,2,4-t3enzenetriol. In the preceding chapter it was reported

that presence of 1,2,4-benzenetriol, a potent toxic metabolite of benzene, resulted in the

release of iron from horse spleen ferritin in 0.1 M acetate buffer at pH 5.6 (Ahmad et al.,

1995). In the absence of EDTA we observed an absorbance peak at 287 nm in fraction

number 3 during the iron release from femtin by 1,2,4-benzenetriol. In addition to

absorbance peak of 1,2,4-benzenetriol.iron complex in fraction number 3, an absorption

peak of low intensity may be due to unchelated 1,2,4-benzenetriol was also observed in

fraction number 21. But in presence of iron chelator (EDTA) the iron released from femtin

by 1,2,4-benzenetriol was chelated with EDTA and eluted in fraction number 3, whereas

an increase in 1,2,4-benzenetriol peak intensity was observed in fraction number 21 at

287 nm (Figure-5.3). This observation indicates that as iron is not available to 1,2,4-

benzenetriol due to the presence of EDTA, all the polyphenol was eluted in fraction

number 21. However, in the absence of EDTA, all the iron released was accessible to

1,2,4-benzenetriol and 1,2,4-benzenetriol eluted in fraction number 3 along with iron.

Studies were conducted to evaluate the haematotoxicity of hydroquinone (25 mg/kg

body weight), iron (25 mg/kg body weight) and hydroquinone:iron complex after exposure

to male mice at 25 mg hydroquinone:25 mg iron complex/kg body weight i.p. in normal

saline for 20 days (single exposure) and also 7 days (twice in a day). Normal saline

treated group served as control.

Enlargement in liver size was observed in hydroquinone:iron complex and iron alone

treated groups compared to the control group. Whereas, increase in spleen weight was

observed only in group treated with hydroquinone.iron complex for 20 days (Table-5.1),

Similariy, significant increase in haemoglobin content was observed in all the three groups

compared to the control group. But hematocrit (%) did not show any change in any group

after 20 days exposure (Table-5.1). The above haematological parameters were also

evaluated after 7 days of i.p. treatment of above mentioned groups, twice a day at the

O O - EDTA

• • +EDTA

E c

00 fM

d o

11 15 19 23

FRACTION No.

Figure 5.3 : Elution profile of iron:BT complex formation during the iron release from ferritin by BT with or without the presence of EDTA Fractions were analyzed at 287 nm

69

Table 5.1

Relative liver/spleen weight, haemoglobin and hematocrit (%) of male mice after 20 days treatment

Group

CONTROL

HQ:lron complex

Iron alone

HQ alone

Relative Liver wt. (per 100 gm)

5.40±0.18

6,84±0.34*

6.15±0.28=

5.02±0,16' s

Relative Spleen wt (per 100 gm)

0.58±0.06

0.81±0.07=

0.87±0.14^^

0.57±0.05'^'

Haemoglobin (gm/dl)

14.3+0.46

17.2±0.45'

16.2±0.25"

16.710.58"

Hematocrit (%)

40.6+1.54

44.2+1.33''5

42.9+1.27^^

40.9+1.56^5

Treatment through single injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for 20 days. Values are mean ± S.E. of six mice. Exposed groups were compared with their respective control. P values = a <0.01, b <0.02, c <0.05, NS = Not significant.

70

Table 6.2

Relative liver/spleen weight, haemoglobin and hematocrit (%) of male mice after 7 days exposure, twice in a day

Group Relative Relative Haemoglobin Hematocrit Liver wt Spleen wt (gm/dl) (%) (per 100 gm) (per 100 gm)

CONTROL

HQ.Iron complex

Iron alone

HQ alone

4.83±0.17 0.57±0.06 15.7±0.54

6.35±0.18^ 0.80±0.08^ 15.510.34^^

5.7110.23" 0.57±0.04' s 15.3+0.62^^

5.00±0.23''^ 0.66±0.08''s 14.810.31"

36.5±0.93

35.7±0.54^

35.3±0.83'«

34.7+0.89^

Treatment through double injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for 7 days. Values are mean ± S.E. of six mice. Exposed groups were compared with their respective control. P values = a <0.001, b <0.02, c <0.05, NS = Not significant.

71

Table 5.3

Total WBC and differential leukocyte count (OLC) of male mice after 20 days exposure.

CONTROL HQ:lron Complex

Iron alone HQ alone

WBC (per Cum)

DLC (%)

Neutrophil

Lymphocyte

Eosinophil

Monocyte

Basophil

8333±657

25.7+2.17

73.7+2.11

0.50±0.12

0.17±0.16

NIL

102861505" 10110±115r^ 9983+1174^

38.9+1.87^ 43.0±1.8r

NIL NIL

22.0+1.57'^

60.3±1.9r 56.211.83" 76.7±1.33'«

0.1710.16''^ 0.33±0.2r^ 0.83±0.48'«

0.83+0.17" 1.00+0.26" 0.50±0.22^

NIL

Treatment through single injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for 20 days. Values are mean ± S.E. of six mice. Exposed groups were compared with their respective control. P values = a <0.001, b <0.05, NS = Not significant.

72

Table 5.4

Total WBC and differential leukocyte count (DLC) of male mice after 7 days exposure twice in a day

WBC (per Cum)

DLC (%)

Neutrophil

Lymphocyte

Eosinophil

Monocyte

Basophil

CONTROL

7492±320

23.3±2.0

74.7+1.6

1.00±0.4

1.00±0.4

NIL

HQrIron Complex

127061626"

45.912.1"

52.8+1.9"

0.2510.3' ^

1.13±0.4' s

NIL

Iron alone

11717±1066'

43.0±2.5"

54.2±2.0"

0.70±0.4''s

2.20±0.7' s

NIL

HQ alone

6950+539^

25.7±2.3'^

70.7±2.2^

2.20±0.8'^

1.50±0.5^

NIL

Treatment through double injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml nonnal saline and control group received 0.1 ml normal saline only) for 7 days. Values are mean ± S.E. of six mice. Exposed groups were compared with their respective control. P values = a <0.001, b <0.01, NS = Not significant.

73

Table 5.5

Lipid peroxidation, total and free -SH content in liver of male mice after 20 days exposure

Assay CONTROL HQ:lron Iron Complex alone

HQ alone

Lipid peroxidation 19.3t1.47 46.3+2.44' 44.7±2.80' 19.6+1.72 (nmoles/hr/gm liver)

Total -SH content 2.75±0.07 (mmoles/100 gm liver)

2.2910.06' 2.3710.08" 283±0.W^

Free -SH content 0.74±0.05 (mmoles/100 gm liver)

0.69±0.05''5 0.57±0.07''s 0.9110.04"

Treatment through single injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for 20 days. Values are mean iS .E . of six mice. Exposed groups were compared with their respective control. P values = a <0.001, b <0.05, NS = Not significant.

http://19.3t1.47

74

Table 5.6

Lipid peroxidation, total and free -SH content in liver of male mice after 7 days exposure twice in a day

Assay CONTROL HQ.Iron Iron HO Complex alone alone

Lipid peroxidation 11.1+2.52 22.3±3.25' 30.013.76" 14,9±1.58'* (nmoles/hr/gm liver)

Total-SH content 3.27±0.07 3.01±0.08^ 2.9310.06" 3.18±0.09^ (mmoles/100 gm liver)

Free-SH content 0.84±0.06 0.75±0.06''^ 0.73±0.05''s 0.74±0.05^ (mmoles/100 gm liver)

Treatment through double injection daily (i.p. = 25 mg each/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for 7 days. Values are mean ± S.E. of six mice. Exposed groups were compared with their respective control. P values = a <0.001, b <0.02, c <0.05, NS = Not significant.

Chapter - V 75

same dose. Similar pattern of results were observed all the three groups when compared

to control group except the haemoglobin content (Table-5.2). The haemoglobin content

did not show any significant alteration after 7 days exposure twice a day.

Significant increase in WBC was observed in hydroquinone: iron complex treated

male mice after single injection for 20 days exposure (Table-5.3). Similarly, significant

increase in polymorph (%) and monocyte (%) in hydro-quinone:iron complex and iron

alone treated groups with simultaneous decrease in lymphocyte (%) (P<0.001) were

observed when compared to the control group after 20 days of exposure. Whereas,

eosinophil (%) and basophil (%) did not show any alteration after 20 days of exposure.

Further, after 7 days exposure, twice in a day, same pattern of results in WBC and DLC

were observed. Significant increase in WBC was observed in hydroquinone:iron complex

and iron alone treated groups compared to the control group. Similarly, significant

increase in polymorph (%) with simultaneous decrease in lymphocytes were observed in

the groups treated with hydroquinone:iron complex and iron alone. Whereas, eosinophil

(%), monocyte (%) and basophil (%) counts did not show any alteration in any group

(Table-5.4).

We also studied the lipid peroxidation, total and free sulphahydryl content ( -SH

group) in liver of both sets of experiments (Table-5.5 and 5.6). Significant increase in lipid

peroxidation was observed in hydroquinone:iron and iron alone group with simultaneous

decrease in total -SH content in both groups after 20 days (single injection daily) and 7

days (twice in a day). Whereas, no significant alteration in free -SH content was observed

in any group except for the group treated with hydroquinone alone for 20 days (single

injection daily), where a marginal increase in free -SH content was observed (Table-5.5).

DISCUSSION

The haematotoxic effect of benzene in both humans and animals have been well

documented (Snyder, 1987). Chronic exposure of humans to benzene is associated with

vanous types of haematopoietic disorders including several types of leukemia. In

laboratory animals particularly rodents, benzene exposure reduces the number of various

bone marrow cells including the haematopoietic stem cells. The intensity of

Chapter - V 76

haematotoxicity produced by benzene exposure differ between species and even between

strains of the same species. There is abundant evidence that these differences in

response are due to difference in benzene metabolism (Nakajima e a/., 1993). Neun et

al.. (1992) suggested that differences in host target ceil susceptibility may also play a role

in the differential haematotoxic responses to benzene.

It was observed that chronic exposure to several strains of mice to high

concentrations of benzene (100 or 300 ppm) produced a rapid decline in the number of

circulating red cells, whereas the number of circulating granulocytes rose above the

control levels (Snyder et al., 1982). Exposure to t»enzene resulted in a variety of change

in the number of polymorph, lymphocytes, basophil, eosinophil and other bone marrow

cell fractions. The longer exposure to a lower concentration of benzene results in a much

greater haematotoxic effect (Cronkite et al., 1989). Exposure to 300 ppm benzene

caused high mortality, lymphocyto-penia, anemia, neutrophilia, and reticulocytosis in the

mice but only lymphocyto-penia in rats (Snyder et al., 1978). However, the metabolites

of benzene responsible for these effects are not known.

Earlier Greenlee et al., (1981 a) have shown that polyphenolic metabolites of benzene

accumulated in bone marrow against the concentration gradient. We propose that the

complex of polyphenol with iron may be the low molecular weight iron (LMI) in bone

marrow which has been reported to accumulate selectively during the exposure to

benzene (Pandya et al., 1990). In the present studies, we observed that the presence of

polyphenolic metabolites of benzene enhanced iron-catalyzed bleomycin-dependent

degradation of DNA. The polyphenoUron complex appears to be stable and intact as the

recovery from bone marrow lysate was complete (Singh et al., 1994). Although, the

benzene exposure lead to accumulation of iron as well as polyphenol metabolites of

benzene in the bone marrow against the concentration gradient but formation of such

complex in vivo is not known. In the present study, we standardized a methodology to

fractionate these complexes by Sephadex G-10 column chromatography. Results clearly

show the formation of polyphenoMron complex in in vitro condition.

In order to evaluate the haematotoxicity of hydroquinone:iron complex we studied the

effect of 25 mg hydroquinone; 25 mg iron or 25 mg hydroquinone or 25 mg iron/kg body

Chapter - V 77

weight and normal saline in male mice administered i.p. daily for 20 days (single

exposure) and significant alteration in various haematological parameters were observed.

Increase in haemoglobin content, hematocrit and WBC were noticed whereas DLC

showed increase in polymorphs and decrease in lymphocytes in hydroquinone:iron

complex and iron treated groups in comparison to normal saline administered group.

However, the effects were highly significant in hydroquinone:iron treated group and a

marked alterations were also noticed in this group when compared to iron treated group

only. However, no significant changes were observed in hydroquinone alone treated

group m compahson to norma] saline treated group.

The mortality rate in the case of hydroquinone;iron complex treated group was very

high at the dose 50 mg/kg b.w. This shows that the complex formation some how

increase the toxicity. Both absolute and relative organ weights of liver and spleen showed

a significant increase in hydroquinone.iron and iron treated groups, whereas hydroquinone

alone treated group did not show any change. Enlargement in liver and spleen size is one

of the important symptom of blood related disorder. An increase in spleen weight in CD-1

male mice exposed to benzene has also previously been reported (Green e a/., 1981a)

with a concomitant increase in granulocyte count as a consistent finding (Green et al.,

1981b). Similar observations of increased spleen weight and granulocyte number were

noticed in the group of mice treated with hydroquinone:iron complex. Total sulphahydryl

content showed significant decrease, whereas increase in lipid peroxidation were noticed

in both the groups without any change in hydroquinone treated group. This shows the

formation of free radical in vivo during the exposure of such complex and may have

toxicological implication.

Eastmond et al., (1987) have demonstrated that repeated combined exposure of

mice with phenol and hydroquinone led to decrease in bone marrow ceilularity in a

manner similar to the haematotoxic effects expressed by benzene. Following similar

experimental protocol (Guy et al., 1990), it was demonstrated that the radio iron uptake

into erythrocytes was significantly decreased in the group of mice coadministered with

phenol and hydroquinone. The combination of phenol and hydroquinone produced a

decrease in bone marrow ceilularity similar to that produced by benzene. Further Guy et

Chapter - V 78

a/., (1991) extended these observations to other metabolites of benzene, p-l)enzoquinone,

muconaldehyde and hydroqui-none, that may affect haematopoiesis either singly or in

combination. It has been suggested that reduced bone marrow cellulahty during the

coadministration of phenol and hydroquinone might be due to phenol-induced stimulation

of hydroquinone via the peroxidase-dependent metabolic activation in bone marrow and

increased covalent binding of hydroquinone to tissue macromolecules of blood, bone

marrow, liver and kidney (Smith et al., 1989; Subrahmanyam et al., 1990, 1991a).

Muconaldehyde (Witz et al., 1985) and 6-hydroxy-trans, trans- 2,4-hexadienol (Zhang et

al.. 1995b), the putative toxic intermediates of benzene have been shown to cause

decrease in sulphahydryl content, ^'Fe incorporation into circulating erythrocytes. Rao

and Pandya (1978) have shown that benzene increases lipid peroxidation 3 hr after

exposure and it has been reported that lipid peroxidation leads to loss of heme from

microsomes (De Matties and Sparks, 1973). Several fold increase in hepatic lipid

peroxidation was also observed in albino rats of either sex after benzene exposure

intrapehtoneally (0.5 ml/kg body weight) or subcutaneously (1 ml/kg body weight) for 10

days (Ahmad ef a/., 1994).

Similar observations comparable to above observations were noticed in mice treated

with the same dose for 7 days daily i.p. twice in a day. Present studies clearly indicate

that hydroquinone iron chelate showed marginal increase in haematotoxicity in

experimental animals. But the precise mechanism of marginal, yet significant increase

in haematotoxicity is not clear. Atleast some of the effects observed in hydroquinone.iron


following exposure of mice to benzene. Further studies are required to identify the

complex formation in vivo dunng benzene exposure and to explain the mechanism of

toxicity of such complexes and a possible role of such complexes in benzene toxicity.

Chapter - VI Glutathionyl Hydroquinone: A Possible

Toxic Metabolite of Benzene Introduction

Results\

Discussion

Chapter - VI -o

INTRODUCTION

Benzene is a well known leukemogenic and carcinogenic agent. Benzene induced

cytogenetic effects including chromosome and chromatid aberration, sister chromatid

exchanges and induction in micronuclei have been consistently found in in wVo animal

studies (Erexson et al., 1986). Benzene exposure has also been associated with

myelodysplasia and acute myelogenous leukemia (Snyder, et al.. 1993). Rodents

chronically exposed to benzene display similar effects except that a wider range of

carcinogenic responses have been reported (Maltoni et al.. 1985). Evidences for the

genotoxicity of benzene in human comes from studies of occupationally exposed

populations (Yardley-Jones et al., 1990) Since consistent chromosomal aberrations are

often observed in human leukemias, the ability of phenolic metabolites of benzene to

induce chromosomal damage in human implicates the benzene induced leukemia (Yager

et al., 1990). The evidence linking benzene exposure with leukemia was considered

sufficient proof of human carcinogenicity (Dosemeci ef a/., 1994). Despite extensive

research, the molecular mechanisms responsible for the induction of bone marrow

Chapter - VI gQ

toxicity and leukemia have yet to be fully elucidated. However, there is general

agreement that expression of benzene toxicity requires metabolism and follows similar

pathways in humans and animals exposed to benzene (Snyder e a/., 1993).

Recent report indicates that several metabolites of benzene are capable of reacting

with DNA (Levay and Bodell, 1992). Administration of metabolites of benzene induce

sister chromatid exchanges, micronuclei induction (Zhang et al., 1994), oxidative DNA

damage (Kolachana, 1993), and DNA strand breaks (Li and Trush, 1993a). One

mechanism by which benzene induces these genotoxic effects may be mediated by the

generation of one or more active oxygen species, such as superoxide anion radical

(O2 ) hydrogen peroxide (4 Q). hydroxy! radical ('OH) and singlet oxygen (^t^Q^

(Subrahmanyam et al., 1991b). Damage to DNA is perhaps one of the most crucial

events in cytotoxicity of reactive oxygen species (Halliwell and Aruoma, 1991). DNA

lesions resulting from exposure to reactive oxygen species includes modified bases,

abasic sites, single and double strand breaks and DNA protein crosslinks (Aruoma et al.,

1989a; Aruoma et al., 1989b), although, repair enzymes remove most but not all, of the

lesions formed during endogenous DNA damaged by reactive oxidant (Ames et al..

1993).

The majority of benzene metabolism occurs in liver to its major hydroxylated

metabolites (Subrahmanyam et al., 1991a) and has been reported to accumulate against

concentration gradient in bone marrow (Greenlee e al., 1981a). High levels of

myeloperoxidase in the bone marrow facilitate autooxidation of the polyphenols to quinoid

derivatives (Eastmond et al., 1986). The toxicity of quinoid metabolites of benzene has

been associated with their ability to form adducts with thiols and undergo faster

autooxidation to generate oxygen centered radicals (Subrahmanyam and O'Brien, 1985).

The formation of polyphenolic metabolites of benzene in rabbit exposed to benzene

and their accumulation in bone marrow against the concentration gradient and suggested

mechanism of toxicity has been described earlier (Singh et al., 1994). However, the

reactive intermediates responsible for benzene toxicity have not been definitly identified

(Snyder ef a/., 1993). The toxic effect of benzene is likely to be exerted by multiple of

metabolites through multiple of biological pathways (Goldstein, 1989).

Chapter - VI 81

Li and Trush (1993a) observed that hydroquinone/Cu(ll) and 1,2,4-benzenetriol/

Cu(ll), chemical metal redox were the two most efficient systems which induced strand

breakage in X-174 RF1 plasmid DMA. Recent studies revealed that treatment of HL-60

cells with hydroquinone resulted in the formation of 8-hydroxyguanosine (8-OHdG)

formation, suggesting that hydroquinone can induce oxidative DNA base modification in

cells (Kolachana et al.. 1993), although, the mechanism by which it causes these effects

remain unclear. It is likely that some of these effects, if not all could be mediated by

further oxidative activation of hydroquinone. Similar observation was also obtained in

different cell lines whose peroxidase activation of hydroquinone results in the formation

of DNA adducts (Levay et al., 1993).

The formation of 1,4-benzoquinone has been demonstrated from the oxidation of

hydroquinone by horse radish peroxidase and H2O2 (Yamazaki et al., 1959). Formation

of 1,4-benzoquinone by human myeloperoxidase and HjO; was also observed during the

oxidation of hydroquinone (Smith et al., 1989). 1,4-Benzoquinone has been shown to

induce single strand breaks in DNA of cultured cells (Pellak-Walker and Blumer, 1986),

to decrease the ability of stromal cells to support granulocyte/monocyte colony formation

(Gaido and Wierda, 1984), to form DNA adducts in all cell types tested (Levay et al.,

1993) and to inhibit DNA and RNA synthesis (Kalf, 1987), microtubule polymerization

(Irons and Neptun, 1980) and mitogen stimulation of lymphocyte growth (Wierda and

Irons, 1982).

1,4-Benzoquinone reacts with glutathione (GSH) to form a conjugate, glutathionyl

hydroquinone (Lunte and Kissinger, 1983). Macrophages from bone marrow have been

shown to be capable of peroxidative bioactivation of hydroquinone to 1,4-benzoquinone

which can either form a GSH-conjugate and/or covalently bind to protein or DNA (Thomas

et al., 1990), 1,4-Benzoquinone is a direct-acting alkylating agent. It readily reacts with

sulphahydryls and has been shown to inhibit microtubule assembly by blocking the

thiol-sensitive GTP binding site (Irons et al. 1981). However, the DNA adducts formed

in HL-60 cells treated with 1,4-benzoquinone were shown to be different from those

formed on reaction of purified DNA with 1,4-benzoquinone (Levay e al., 1991),

suggesting that the reactive intermediate in cells is the metabolite of 1,4-benzoquinone

c o

ro

X o o 3 ra

c 00

c g ro E

0) c o c D CT O

c o

u c CD O

O D

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Chapter - VI ^^

rather than 1,4-benzoquinone itself. Glutathionyl hydroquinone has been isolated as one

of the urinary metabolites of hydroquinone (Figure-6.1) indicating that glutathionyl

hydroquinone formation occurs in vivo (Nerland and Pierce, 1990). Glutathionyl

hydroquinone has been shown to autooxidize at several fold higher than the parent

hydroquinone to glutathionyl benzoquinone (Brunmark and Cadenas, 1988).

In the present study iron catalyzed bleomycin-dependent degradation of DNA was

investigated in the presence of glutathionyl hydroquinone. We also investigated the

potential of glutathionyl hydroquinone to damage supercoiled DNA in plasmid pUC 18.

Glutathionyl-hydroquinone complex concentration dependent release of aldehydic

products from glutamate or deoxyuridine was also observed. We further investigated the

haematotoxicity of glutathionyl hydroquinone in male mice to evaluate the possible role

of glutathionyl hydroquinone in benzene induced haematotoxicity. The studies revealed

that glutathionyl hydroquinone is a potent prooxidant and this observation may be of

toxicological importance in relation to benzene induced toxicity.

RESULTS

Glutathionyl hydroquinone conjugate was prepared by mixing 1,4-benzoquinone

(BQ) and GSH in distilled water in 1:1 ratio (equimolar concentrations). The nucleophilic

addition of GSH to 1,4-benzoquinone yields glutathionyl hydroquinone and its authenticity

was confirmed by the absorption maximum at 303 nm. No absorption was observed at

248 nm which is the absorption maximum for 1,4-benzoquinone (Figure-6.2).

Iron-catalyzed and bleomycin-dependent degradation of calf thymus DNA was

markedly enhanced (12 fold) in the presence of glutathionyl hydroquinone as compared

to the other experiment in the presence of iron alone (Table-6.1). Presence of 1,4-

benzoquinone did not influence the iron-catalyzed bleomycin- dependent degradation of

calf thymus DNA, however, presence of hydroquinone (HQ) or GSH influenced iron-

catalyzed bleomycin dependent degradation by 1 9 and 3.2 fold respectively (Table- 6.1).

Glutathionyl hydroquinone (GHQ) concentration dependent release of TBAR from target

molecules viz. L-glutamate or deoxyuridine was also observed (Table-6.2). The

o o

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83

Table 6.1

Iron-catalyzed bleomycin-dependent release of TBAR from calf thymus DNA in the presence of 1,4-benzoquinone (BQ), hydroquinone (HQ),

glutathione (GSH) or glutathionyl hydroquinone (GHQ).

Addition nmoles of TBAR formed per 30 min

Iron 1.38

Iron + BQ 1.38

Iron + HQ 2.67

Iron + GSH 4.42

GSH 0.00

Iron + GHQ 16.20

GHQ 0.00

Experimental conditions are as described in Materials and Methods section. All values are averages of two sets of experiments conducted in duplicate with variations 10% or less.

84

Table 6.2

Effect of varying concentration of glutathionyl hydroquinone on the release of TBAR from L-glutamate or deoxyuridine

Glutathionyl hydroquinone (pM)

5.0

10.0

15.0

20.0

25.0

nmoles of TBAR formed per 2 hr

L-glutamate

0.27

0.56

0.81

1.15

1.21

Deoxyuridine

0.23

0.46

0.90

1.17

1.40

Experimental conditions are as described in Materials and Methods section.

All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

Chapter - VI oc

superoxide anion released during autooxidation of glutathionyl hydroquinone (30 JM)

was found to reduce 2.4 nmoles of cytochrome c per min.

Glutathionyl hydroquinone dependent release of TBAR during iron-bleomycin

degradation of calf thymus DNA did not show any significant increase with the increase

in iron concentration (5-15 [JM) (Table-6.3). Similarly, varying the concentration of 1,4-

benzoquinone did not influence the release of TBAR from calf thymus DNA. However,

a linear increase in the release of TBAR from calf thymus DNA was observed with

increase in GSH concentration (5-15 \M) (Table-6.4). Release of TBAR from DNA in

the presence oi glutathionyl hydroquinone, iron and bleomycin was significantly inhibited

(43-51%) on addtion of oxyradical scavengers, such as thiourea (49%), mannitol (46%),

dimethylsulfoxide (43%), albumin (49%), superoxide dismutase (51%) and catalase

(46%) (Table-6.5). The glutathionyl hydroquinone and iron added to bone marrow cell

lysate was 100% recoverable when assayed by the bleomycin dependent release of

TBAR from calf thymus DNA (Table-6.6).

When the supercoiled plasmid pUC 18 DNA (300 ng) was incubated with 200 (JM

of 1,4-benzoquinone, GSH or glutathionyl hydroquinone for 3 hr at 3 7 ^ , cleavage of

plasmid DNA was observed only with glutathionyl hydroquinone resulting in the formation

of both relaxed open circular and linear form of DNA respectively, which run significantly

slower than supercoiled DNA on 1% agarose gels (Figure-6.3). Presence of DNA alone

(lane a), 1,4-benzoquinone alone (lane b), or GSH alone (lane c) did not result in the

nicking of pUC 18 DNA, Whereas, after 3 hr incubation at 37'C in the presence of

glutathionyl hydroquinone (200 pM), all the supercoiled pUC 18 DNA showed a major

damage in the form of single strand nick to give rise to open circular and a minor damage

in the form of double cut to give rise to linear form of DNA (lane d). The single and

double strand cleavage of pUC 18 DNA by glutathionyl hydroquinone (lane d) is

compared with linear form of pUC 18 DNA obtained by double strand single cut after

EcoRI digestion (lane e). Further, a linear increase in DNA damage was observed in the

presence of increasing concentrations of glutathionyl hydroquinone. After 3 hr incubation

at 37X, the DNA damage was increased with increasing concentrations of glutathionyl

hydroquinone (0-200 \M) (Figure-6.4).

86

Table 6.3

Effect of varying concentration of iron on the iron catalyzed bleomycin dependent release of TBAR from DNA In the

presence of GHQ (15 pM)

Concentration of Iron nmotes of TBAR formed ( pM) per 30 min.

5.0 13.24

7,5 15,27

10.0 16.00

12.5 16.17

15.0 16.20

Experimental conditions are as described in Materials and Methods section. All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

87

Table 6.4

Effect of varying concentration of GSH on iron-catalyzed bieomycin-dependent release of TBAR from calf thymus DNA in the

presence of BQ (15 pM) and iron (15 pM).

Concentration of GSH nmoles of TBAR formed (pM) per 30 min.

5.0 4.49

7.5 5.66

10.0 9.66

12.5 12.36

15.0 16.20

Experimental conditions are as described in Materials and Methods section. All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less.

88

Table 6.5

Effect of oxyradical scavengers on iron-catalyzed bleomycin-dependent release of T6AR from calf thymus DNA in the

presence of Iron (5 pM) and GHQ (S pM).

Reaction mixture

Complete reaction mixture

+ Thiourea (5 mM)

+ Mannitol (50 mM)

+ Dimethylsulfoxide (33 mM)

+ Albumin (100 MQ/ml)

+ Catalase(100Mg/ml)

+ SOD (100pg/ml)

Experimental conditions are as described in Materials and Methods section.

All values are averages of two sets of experiments conducted in duplicate with variations of 10% or less. *

Number in parentheses are percent inhibition.

nmoles of TBAR formed per 30 min.

6.73

3.46

3.65

3.85

3.46

3.27

3.65

* (49)

(46)

(43)

(49)

(51)

(46)

89

Table 6.6

Recovery of iron and glutathionyl hydroquinone added to bone marrow cell lysate for the bleomycin-dependent degradation of calf thymus DNA

Addition nmoles of TBAR formed per 30 min.

- Bone marrow lysate 5.38

+ Bone marrow lysate 9.04

Experimental conditions are as described in Materials and Methods section. All values are averages of two sets of expenments conducted in duplicate with variations of 10% or less.

a b

Figure 6.3 : Agarose gel electrophoresis of ethidium bromide stained pUC 18 DNA after treatment with BQ, GSH or GHQ (200 |JM each). Lane (a) DNA alone, lane (b) DNA + BQ, lane (c) DNA+GSH, lane (d) DNA+GHQ, and lane (e) pUC 18 DNA digested with EcoRI. Reaction mixtures were incubated at 37°C for 3 hr. The positions of supercoiled (SC), linear (LIN) and open circular relaxed (OC) DNA are indicated.

Figure 6.4 : Cleavage of pUC 18 DNA by increasing concentrations of GHQ. Lane (a) DNA only, lane (b-f) DNA+GHQ (25, 50, 75, 100 and 200 |iM), and lane (g) EcoRI digested ? DNA molecular marker (3530-21226 bp). Reaction mixtures were incubated at 37°C for 3 hr.

a b

Figure 6.5 : Cleavage of pUC 18 DNA by GHQ in presence of oxyradical scavengers. Lane (a) DNA only, lane (b) DNA+GHQ, and lane (c-g) DNA + GHQ+ SOD/catalase /mannitol/thiourea/sodium benzoate at 10 fold molar excess concentration. Reaction mixtures were incubated at 37°C for 3 hr.

90

Table 6.7

Relative liver/spleen weight, haemoglobin, and hematocrit (%) of male mice after glutathionyl hydroquinone treatment

Parameters Control Treated

Relative liver weight 4.73±0.36 6.00+0.15' (per 100 gm)

Relative spleen weight 0.62+0.08 O.SO+O.IO'' (per 100 gm)

Haemoglobin (gm/dl) 13.9 ±0.65 14.4 ±0.31^^

Hematocrit (%) 41.8 ±1.07 40.5 ±1.10^^

Treatment through single injection daily (i.p. = 100 mg/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for one month. Values are mean ± S.E. of six mice. Exposed group were compared with respective control. P values; a <0.01, NS = Not significant.

91

Table 6.8

Total WBC and differential leukocyte count (DLC) of male mice after glutathionyl hydroquinone treatment


WBC (per Cum)

DLC (%)

Neutrophil

Lymphocyte

Eosinophil

Monocyte

Basophil

8000±582

24.0±1.9

71.5±1,9

2.3+0.6

1.3+0.4

NIL

9117±209''^

27.0±3.4^^

71.0±3.3^'

1.8+0.4' ^

0.3+0.2^^

NIL

Treatment through single injection daily (i.p. = 100 mg/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for one month. Values are mean + S.E. of six mice. Exposed group were compared with respective control. P values; NS = Not significant.

92

Table 6.9

Lipid peroxidation, total and free -SH content in liver of mate mice after glutathlonyl hydroquinone treatment


Lipid peroxidation 25.0±4.6 33.8±4.5'^^ (nmoles/hr/gm liver)

Total-SH content 3.25±0.13 3.27±0.05''^ (mmoles/100 gm liver)

Free -SH content 0.73±0.07 0.85±0.05' ^ (mmoies/100 gm liver)

Treatment through single injection daily (i.p. = 100 mg/kg b.w. in 0.1 ml normal saline and control group received 0.1 ml normal saline only) for one month. Values are mean ± S.E. of six mice. Exposed group were compared with respective control. P values; NS = Not significant.

Chapter - VI 93

The effect of oxyradical scavengers, such as superoxide dismutase (250 units) and

catalase (1000 units), mannitol, thiourea and sodium t>enzoate (2mM) on pUC 18 DMA

strand cleavage were investigated to evaluate the role of reactive oxygen species. The

results shovi/ed that incubation of pUC 18 DNA with glutathionyl hydroquinone in the

presence of superoxide dismutase (SOD), catalase, mannitol, thiourea and sodium

benzoate showed a differential inhibition of cleavage of pUC 18 DNA (Figure-6.5).

Protection offered by catalase was maximum (more than 80%) (lane, d), whereas

protection by superoxide dismutase was only 30% (lane c) at 10-fold molar higher

concentrations. Mannitol (lane e), thiourea (lane f) and sodium benzoate (lane g) were

able to protect marginal at 10-fold molar excess concentrations to glutathionyl

hydroquinone.

Studies were also conducted to evaluate the haematotoxicity of glutathionyl

hydroquinone after exposure of male mice at 100 mg glutathionyl hydroquinone/kg body

weight i.p. in normal saline for one month.

Significant increase in relative liver weight (P<0.01) was observed in glutathionyl

hydroquinone treated mice compared to control group. Further, increased pattern of

relative spleen weight was observed which was however not significant (Table- 6.7).

Haemoglobin and hematocrit(%) values did not show any significant alteration (Table-6.7).

Slight increase in WBC count was observed in glutathionyl hydroquinone treated mice but

increase was not significant (Table-6.8). Neutrophil, lymphocyte, eosinophil, monocyte

and basophil did not show any significant alteration in treated group (Table-6.9). Further,

lipid peroxidation, total and iree -SH content in rat liver also did not show any significant

alteration in treated group (Table-€.9).

DISCUSSION

Although, the metabolism of benzene is very well understood the metabolite(s)

responsible for myelotoxicity of benzene is not known (Kalf, 1987). Benzene exposure

has been shown to generate organic Uee radicals and superoxide radical and it is

generally accepted that benzene requires metabolism to express its toxicity

(Subrahmanyam ei a/., 1991b). In our earlier studies we proposed that hydroquinone:

Chapter-VI ,4

iron complex may be the toxic intemfiediate of benzene metabolism (Singh et al., 1994).

Recent studies have emphasized on 1,4-benzoquinone as toxic metabolite of benzene

as it has been shown to fomi DMA adduds both in vitro and in vivo (Levay et a!., 1991;

Levay et al., 1993). The additon of GSH to 1,4-ben2oquinone has been shown to catalyze

1,4-reductive addition reaction of the Michael type involving a two-electron transfer to

yield glutathionyl hydroquinone (Brunmark and Cadenas, 1988). It was also observed

that dunng benzene exposure incorporation of iron into circulating erythrocytes was

inhibited (Lee et al., 1974) which might lead to accumulation of LMI components in bone

man-ow (Pandya et al., 1990). Since excess of both glutathionyl hydroquinone and iron

are available in bone marrow cells during benzene exposure, the potential of glutathionyl

hydroquinone to undergo autooxidation and promote iron catalyzed bleomycin dependent

degradation of DNA was studied. Several fold increase in the release of TBAR from DMA

indicates that glutathionyl hydroquinone autooxidation enhances the availability of Fe(ll)

to bleomycin by reducing Fe(lll), thus limiting the Fe(ll) from hydrolysis to hydroxides and

further oxidation. Superoxide release during autooxidation of glutathionyl hydroquinone

can reduce feme iron to ferrous iron (Halliwell et al.. 1992). Earlier we have also shown

that 1,2,4-benzenetriol was capable of releasing iron from horse spleen ferritin, in vitro,

which was mediated to a certain extent by superoxide radical (Ahmad et al., 1995). Iron

mediated generaion of reactive oxygen species have been implicated in the toxicity of

2-bromo-(glutathion-S-yl) hydroquinone to LLC-PK1 cells due to DNA fragmentation and

resultant cytotoxicity (Mertens et al., 1995). Recently, Lee et al., (1995) investigated

that 1,2,4-benzenetriol even in the absence of metal ion resulted in double strand

cleavage of pBR 322 plasmid DNA via active oxygen species formation.

The GSH concentration dependent increase in TBAR release from calf thymus DNA

in the presence of 1,4-benzoquinone and iron shows that the reaction is dependent on

the concentration of reducing equivalents. This particular observation may be of

toxicological importance as GSH is an important intracellular thiol present in millimolar

concentrations (Meister, 1988). GSH is also present in the nucleus and 1,4-benzoquinone

has been shown to form adducts with DNA, it is quite likely that 1,4-benzoquinone may

conjugate with GSH and form adducts with DNA. Probably, for this reason the DNA

Chapter- VI 95

adducts, formed in 1,4— benzoquinone-treated HL-60 cells were different from those

formed with purified DNA on reaction with 1,4-l)enzoquinone. As the autooxidation of

glutathionyl hydroquinone is eight-fold faster than hydroquinone, it is quite likely that

glutathionyl hydroquinone would cause DNA damage including chromosomal breaks

leading to genotoxic and carcinogenic effects which have been reported to occur during

benzene exposure.

The complete recovery of glutathionyl hydroquinone and iron added to bone marrow

lysate further indicates that both iron and glutathionyl hydroquinone are available to

bleomycin to release TBAR from calf thymus DNA. Previous studies have suggested the

involvement of iron or copper in benzene metabolites induced DNA damage through a

mechanism involving the generation of reactive oxygen species (Rao and Pandya, 1989).

Breakage in supercoiled pUC 18 plasmid DNA in the presence of glutathionyl hydro

quinone was concentration dependent. This may have toxicological importance during

benzene exposure where the metabolites of benzene specially 1,4-benzoquinone is

accumulated in bone marrow cells. Since GSH is present at a relatively high

concentrations in mammalian cells nuclei (Ca 19.2 mM) (Bellomo et al., 1992), it might

be expected that the thiol can perform a similar function in v/vo during t)enzene exposure

where 1,4-benzoquinone is converted to glutathionyl hydroquinone after reaction with

GSH and resultant intermediate interact with nuclear DNA. The exact mechanism of

DNA damage by glutathionyl hydroquinone is not known. The DNA damage may be due

to free radicals generation in the reaction mixture or due to covalent modification of DNA

bases by glutathionyl hydroquinone. Quinone-thioethers are known to exhibit redox

cycle with the concomitant release of oxyradicals (Rao et al., 1988; Brown et al.. 1991).

For this we investigated the protection offered by different oxyradical scavengers to DNA

breakage. More than 80% protection was observed in the case of catalase and about

30% by superoxide dismutase which selectively scavenges superoxide radical and

hydrogen peroxide. However, mannitol, thiourea and sodium benzoate which selectively

scavenge the hydroxy! radicals offered marginal protection (10-15%) even at 10-fold

molar excess concentration with respect to glutathionyl hydroquinone (200 \M). This

suggests that glutathionyl hydroquinone mediated strand scission in pUC 18 DNA is

Chapter - VI 96

mediated via superoxide radical and hydrogen peroxide rather than the involvement of

iron-catalyzed Haber-Weiss reaction. These observations indicate that pUC 18 DNA

damage by glutathionyl hydroquinone was not only mediated by free radical but some

other mechanism might also he responsible for plasmid DNA damage in the presence of

glutathionyl hydroquinone.

To establish, glutathionyl hydroquinone as haematotoxin, we exposed male mice at

dose 100 mg/kg body weight, intraperitoneally for one month. No mortality was observed

at this dose in glutathionyl hydroquinone treated group. Inaeased pattern of relative liver

weight (P<0.01) and spleen weight and WBC count were noticed, but these results were

not conclusive to say glutathionyl hydroquinone as potent haematotoxic metabolite of

benzene. The pharmacokinetics and distribution of administered glutathionyl hydro

quinone is not known. Probably, it does not reach the target organ, bone marrow as

glutathionyl hydroquinone form. Other reason may be due to difference in sensitivity of

species towards different benzene metabolites. Chronic toxicity studies by the National

Toxicology Program (Huff, 1983) have shown that B6C3F1 mice are more sensitive to

the haematotoxic and carcinogenic effects to benzene than F344/N rats. The greater

sensitivity of mice compared with rats to benzene toxicity may be related to a greater

metabolism of benzene to toxic intermediates, differences in detoxification of toxic

metabolites or greater inherent susceptibility of target tissues to the action of toxic

metabolites. Earlier,Longacre et al., (1981) showed that there are strain differences in

mice in the metabolism and toxicity of benzene and that saturation of metabolism may

play a role in producing the differences in toxicity.

However, it is concluded from the present in vitro studies that glutathionyl

hydroquinone is a potent prooxidant and due to its faster autooxidation to glutathionyl

t)enzosemiquinone may result in DNA damage. Our experiments showed that under mild

conditions at 37°C with the presence of 200 pM concentration of glutathionyl

hydroquinone, within 3 hr, all the supercoiled pUC 18 DNA has been converted to open

circular relaxed and linear form. Interaction of glutathionyl hydroquinone with DNA may

facilitate the formation of adducts with DNA which is one of the prerequisites of benzene

toxicity and carcinogenicity. Anderson et al., (1995) reported the DNA-damaging ability

Chapter - M 97

of benzene and its metabolites in human lymphocytes using the comet assay. Zhang

et al., (1995) pointed out that interest has focused on the interactions of benzene

metabolites and the toxicological significance of free radicals as ultimate toxic species

mediating benzene toxicity since no single ring-hydroxylated metabolite of benzene can

express all the known haematololgical effects of benzene. Further, experiments are

needed to explain significance and the precise mechanism of DNA damage by

glutathionyl hydroquinone, and the oxyradicals and semiquinone formed during its

autooxidation, in the expression of benzene induced haematotoxicity.

Chapter - VII Role of Transition Metal Ions (Fe, Cu)

During Benzene Toxicity Introduction

Results

Discussion

Chapter - VII 98

INTRODUCTION

Benzene exposure has been associated with many types of genetic damage

including chromosomal aberrations in animals and man, sister chromatid exchanges, and

micronuclei induction both in vitro and in vivo as well as aneuploidy in dividing cells.

However, it has not generally produced point mutations in genotoxicity test system

(Snyder and Kalf, 1994). Benzene is a substrate of cytochrome P-450 IIE1 (CYP2E1)

(Seaton et a/., 1994) and metabolized to phenolic intermediates to exert its toxic effect in

biological system (Kalf, 1987). Hydroquinone and 1,2,4-benzenetriol, the two principal

polyphenolic metabolites of benzene have been shown to be toxic and the suggested

mechanisms include free radical formation via superoxide and covalent binding of the

semiquinone to DNA, RNA and other cellular macromolecules.

Li and Trush (1993a) observed that hydroquinone/Cu(ll) and 1,2,4-benzenethol/

Cu(ll), chemical metal redox were the two most efficient systems which induced strand

breakage in (t)X-174 RF1 plasmid DNA. Studies revealed that treatment of HL-60 cells

Chapter - VII

with hydroquinone resulted in the formation of 8-hydroxydeoxyguanosine (8-OHdG),

suggesting that hydroquinone can induce oxidative DNA base modification in cells

(Kolachana et ai. 1993). The role of DNA adduct formation in the myelotoxic effects of

benzene exposure has not been defined. Levay and Bodell (1992) studies have

suggested that the myeloperoxidase in HL-60 cells can activate hydroquinone to form

DNA adducts. Later on Levay et ai, (1993) demonstrated that peroxidase activation of

hydroquinone results in the formation of DNA adducts in HL-60 cells, mouse bone

marrow, macrophages and human bone marrow. Recently, Pathak et ai, (1995)

demonstrated the formation of DNA adducts in the tx)ne man-ow of B6C3F1 mice treated

with benzene and later on Levay et ai, (1996) hypothesized that the adduct formation

might depend on both the dose and duration of treatment. Low level of benzene

administration failed to adduct fonnation in experimental animals. DNA adduct formation

was also observed in HL-60 cell culture treated with hydroquinone for 16 hr at 250 \M

concentration (Pongracz and Bodell, 1996). Zhang et ai, (1993) demonstrated the

potential role of copper in micronuclei induction and oxidation of DNA damage in human

lymphocyte and HL-60 cell by 1,2,4-benzenetriol.

There are a number of both enzymatic and non-enzymatic mechanisms by which

xenobiotic can be activated to reactive intermediates (Tnjsh and Kensler, 1991). Evidence

is emerging that the generation of active oxygen species in biological systems, either

through normal metabolic pathways or as a consequence of exposure to xenobiotics, such

as chemical carcinogens, is involved in the multistage process of carcinogenesis,

especially the promotion stage (Witz, 1991). Studies have provided evidence that

transition metals including iron and copper are capable of directly mediating the activation

or metabolism of several xenobiotics via a metal redox mechanism, which leads to the

formation of more reactive oxygen and other organic free radicals (Ahmad et ai, 1992;

Yamamoto and Kawanishi, 1992).

Trace amount of the metal ions are present in biological system. An estimated, one

third of all enzymes require a metal ion in one or more phases for their catalytic activity.

Transition metal ions, most notably iron and copper can facilitate the transfer of electrons

to biological macromolecules such as lipid, protein and DNA. In addition to their

Chapter - VII lOo

biological function, metal ions are responsible for tissue damage. In the presence of trace

metal ions the mutagenicity of polyphenolic metabolites of benzene showed an increase

to the tester strain Salmonella typhimurium (Lee and Lin, 1994). Earlier Krsek-Staples

and Webster, (1993) observed the iron-catalyzed oxygen resulting in the formation of

carbonyl derivatives with concomitant loss of enzyme function.

Maintenance of cellular iron homeostasis is a prerequisite for many essentia!

biological processes and for the growth of organisms, and is also a central element in the

regulation of immune function (Weiss et a!., 1995). Over the past decade, free

radical-mediated processes have been established in the pathology of hepatic iron

overload (Bacon and Britton, 1990), and unwinding of hepatic double-strand DNA in rats,

with chronic dietary iron overload was reported (Ediing et al., 1990). Further, Toyokuni

and Sagripanti (1993) demonstrated that Fe-citrate can constitute a powerful DNA

damaging system under physiological conditions.

The biological significance of copper has recently attracted with interest in

connection with carcinogenicity and mutagenicity (Agarwal et a!., 1989). Copper is an

essential component of chromatin (Saucier et al., 1991) and is known to accumulate

preferentially in the heterochromatic regions (Bryan e al., 1976). Copper sulphate

(CUSO4) showed clastogenic effects on the bone marrow cell chromosomes of mice in

vivo (Agarwal et al., 1990). Li et al., (1991) reported that copper accumulated in liver

tissues of LEC (Long-Evans Cinnamon) rats that has spontaneously developed

hepatocellular carcinomas, suggesting that the abnormal copper metabolism is involved

in hepatic carcinogenesis in LEC rats. The exposure of DNA to hydrogen peroxide in the

presence of Cu(l) or Cu(ll) is known to result in the induction of a variety of oxidative

lesions, including DNA strand breaks and base modifications (Kawanishi and Yamamoto,

1991;ltoefa/., 1992).

Previous studies have suggested the involvement of iron or copper in benzene

metabolites induced DNA damage through a mechanism, involving the generation of

reactive oxygen species (Kawanishi etal., 1989; Rao and Pandya, 1989). Although, iron

is known to be capable of catalyzing oxidative damage to DNA, copper has been shown

to be more potent. Copper is distributed throughout the body with liver and bone man-ow

Chapter - VII 101

being the two major storage organs. It is also closely associated with chromosomes and

DNA bases (particularly guanine) and the possible role of copper as a mediator of reactive

oxygen induced cytotoxicity as well as genotoxicity have been proposed (Li and Trush,

1993b). In this respect, the present studies were conducted to quantitate the metal ion

concentrations (iron and coper) in rat liver and isolated liver nuclei in benzene exposed rV

albino rats. Further, we studied the potential role of polyphenolic metabolites of benzene

to release thiobartDituric acid reactive products (TBAR) from isolated liver nuclei with or

without the presence of metal chelators and oxygen radical scavengers to explain the

possible role of metal ions in benzene induced genotoxicity and chromosomal aberration.

RESULTS Total iron and copper contents were estimated in liver tissue of male and female rats

exposed to benzene intraperitoneally (0.5 ml/kg body weight) for 10 days. The iron

content only showed significant increase in male and female rats (P<0.02; P<0.05), which

received benzene and no significant change in copper content was observed in either sex

when compared to respective control groups (Table-7.1). Iron and copper contents were

also analyzed in isolated rat liver nuclei of benzene exposed female rats (i.p). In isolated

rat liver nuclei also only iron content showed significant increase (P<0.05), but no change

was observed in copper content when compared to the respective control groups

(Table-7,2).

Formation of TBAR was observed from isolated rat liver nuclei in presence of

polyphenolic metabolites of benzene, such as hydroquinone and 1,2,4-benzenetriol and

other polyphenols, such as 6-hydroxydopamine and dopamine at 330 pM concentration.

Significant release in TBAR was observed in all cases but the release was maximum in

the presence of hydroquinone (34.6 nmoles TBAR/hr/gm protein) (Table-7.3). Increased

pattern of TBAR formation was observed with increase in hydroquinone concentrations

(0-330 pM), but the release was not linear with increase in hydroquinone concentrations

(Table-7.4).

Further, nuclear DNA damage was evaluated by agarose gel electrophoresis. Rat

liver nuclei were incubated with hydroquinone/1,2,4-benzenetriol (0.5 or 1.0 mM) and

102

Table 7.1

Effect of benzene* administration on total iron and copper metal contents in liver of albino rats

Iron (pg/gm tissue)

Copper (pg/gm tissue)

Male rat

Control

i.p.

Female rat

Control

i.p.

91.50±1.90

118.00+7.85^

115.00±8.58

143.0018.10"

3.24±0.12

3.67+0.19' ^

4.86±0.68

4.86±0.38''5

^Benzene was administered through single injection daily (i.p. = 0.5 ml/kg b.w.) for 10 days. Values are mean ± SE of five rats. Exposed groups were compared with their respective control. P values : a < 0.02; b < 0.05; NS = Not significant.

103

Table 7.2

Effect of benzene* administration on total iron and copper metal content in isolated liver nuclei of albino rats.

Iron (\iglgm protein) Copper (pg/gm protein)

Female rat

Control

l.p.

41.28+4.20

61.10+6.15*

36.70±3.28

42.40±3.9r^

*Benzene was administered through single injection daily (i.p. = 0.5 ml/kg b.w.) for 10 days. Values are mean ± S.E. of five rats. Exposed groups were compared with their respective control P values: a < 0.05; NS = Not significant

104

Table 7.3

Effect of polyphenolic metabolites of benzene and other polyphenols (330 pM) on release of TBAR from isolated rat liver nuclei.

Addition nmoles of TBAR formed/ hr/gm protein

None 0.0

Hydroquinone 34.6

1,2,4-Benzenetriol 19.8

6-Hydroxydopamine 27.2

Dopamine 18,4

Experimental conditions as described in Materials and Methods section. All values are average of two sets of experiments conducted in duplicate with variations 10% or less.

105

Table 7.4

Release of TBAR from isolated rat liver nuclei in the presence of increasing concentration of hydroquinone

Hydroquinone { pM) nmoles of TBAR formed/ hr/gm protein

33 10.9

107 14.1

182 18.9

256 27.0

330 34.6

Experimental conditions as described in Materials and Methods section. All values are average of two sets of experiments conducted in duplicate with variations 10% or less.

Figure 7.1 : Agarose gel electrophoresis of ethidium bromide stained rat liver nuclei treated with HQ/BT (0.5 or 1.0 mM). Lane (a) liver nuclei only, lane (b and c) liver nuclei + HQ (0.5 or LQinM), lane (d-e) liver nuclei + BT (0.5 or 1.0 mM) respectively, and lane (f) EcoRI digested ^DNA molecular marker (3530-21226 bp). Reaction mixtures were incubated at 37°C for 3 hr.

106

Table 7.5

Effect of externally added copper on TBAR release from Isolated rat liver in presence of hydroquinone

Addition nmoles of TBAR formed/hr/gm protein

Hydroquinone alone 34.6

+ Copper (0.08 mM) 37.9

+ Copper (0.167 mM) 36.7

Experimental conditions as described in Materials and Methods section. All values are average of two sets of experiments conducted in duplicate with vanations 10% or less.

107

Table 7.6

Release of TBAR from rat liver nuclei in the presence of hydroquinone and increasing concentration of bathocuproine (0-25 pM)

Components nmoles of TBAR formed/ hr/gm protein


+ 5 pM Bathocuproine 16.5 (52%)

+ 10 " " 15.8(54%)

+ 15 " " 14.9(57%)

+ 20 " " 12.9(63%)

+ 25 " " 11.5(67%)

Experimental conditions as described in Materials and Methods section. All values are average of two sets of experiments conducted in duplicate with variations 10% or less. Number in parentheses are percent inhibition.

108

Table 7.7

Release of TBAR from isolated rat liver nuclei in presence of hydroquinone with or without the addition of EDTA and DTPA (5 pM)



+ EDTA 17.3 (50%)

+ DTPA 15.5 (55%)

Experimental conditions as described in Materials and Methods section. All values are average of two sets of experiments conducted in duplicate with vanations 10% or less. Number in parentheses are percent inhibition.

109

Table 7.8

Effect of oxyradical scavengers on release of TBAR from isolated rat liver nuclei in presence of hydroquinone



+ Thiourea (2.5 mM) 6.4(81%)'

+ Thiourea (5.0 mM) 3.2(91%)

+ Mannitol (50 mM) 25.1(27%)

+ SOD(100Mg/1.5ml) 28.8(15%)

+ Catalase(150Mg/1.5ml) 34.5(0%)

Expenmental conditions as described in Matenals and Methods section. All values are average of two sets of experiments conducted in duplicate with variations 10% or less. 'Number in parentheses are percent inhibition.

Chapter - VII ^^^

electrophoresis was done on 1% agarose gel (Figure-7.1). When isolated rat liver nuclei

were incubated with hydroquinone for 3 hr at 37°C, nuclear DMA damage was observed

(lane b and c) at 0.5 or 1.0 mM concentration compared to the control nuclei (lane a). Gel

pattern also indicates some damage in nuclear DNA in presence of 1,2,4-benzenetriol

(lane d and e), however hydroquinone is more potent to cause damage to rat liver nuclear

DNA. Lane (f) is EcoRI digested DNA marker (3530-21226 bp) (Figure-7.1).

Externally added copper (0.08 and 0.167 mM) did not show significant increase in

TBAR formation when isolated rat liver nuclei incubated together with hydroquinone (330

pM) at 37°C (Table-7.5). However, inhibition in TBAR fromation was observed in presence

of bathocuproine (5-25 pM) in hydroquinone induced TBAR fonnation from rat liver nuclei.

At 5 pM concentration of bathocuproine, 52% inhibition was observed. Increase in the

concentration of bathocuproine did not show linear decrease in TBAR formation and only

about 67% inhibition was observed at 25 pM concentration of bathocuproine (Table-7.6).

Further, only 50-55% inhibition was observed in TBAR release in the presence of

EDTA/DTPA (5 pM), when isolated rat liver nuclei incubated with hydroquinone (330 pM)

at 37°C (Table-7.7).

Inhibition of TBAR formation from isolated rat liver nuclei was also studied in the

presence of different oxyradical scavengers, such as thiourea (2.5 and 5.0 mM), mannitol

(50 mM) catalase (150 pg/1.5 ml) and SOD (100 pg/1.5 ml). Presence of thiourea 2.5

mM and 5.0 mM resulted in the inhibition of TBAR release from rat liver nuclei to the

extent of 81% and 91% respectively (Table-7.8). Inhibition by mannitol and SOD were

only 27% and 15% respectively. However, presence of catalase failed to exhibit any

inhibition of TBAR formation from rat liver nuclei in the presence of hydroquinone

(Table-7.8).

DISCUSSION

studies related to the effect of hydroquinone or 1,2,4-benzenetriol on isolated rat liver

nuclei were conducted to monitor the damage to DNA, as copper ion is associated with

the heterochromatin. This particular study would help in better understanding of the

chromosomal aberrations/abnormalities associated with benzene exposure. Exposure to

Chapter - VII ^ 111

benzene resulted in significant increase in total iron content in liver tissue, however copper

content did not shov\/ any alteration. Earlier it was observed that benzene exposure

resulted in alteration of heme metabolism (Rao and Pandya, 1980) and accumulation of

low molecular weight iron components (LMI) in bone marrow (Pandya et al., 1990). The

elevated level of LMI in the bone marrow during benzene toxicity may lead to formation

of tissue damaging species like lipid peroxide radical, superoxide anion (Oj), oxyferryl

species and hydroxyl radical which may form reactive metabolite(s) and may react

immediately if they occur in the vicinity of vital molecules like nucleic acids (Lown and Sim,

1977) and polymerase enzymes (Rushmore et al., 1984) which may be responsible for

the ultimate cytotoxicity. During in vitro study Rao and Pandya, (1989) observed that

polyphenolic metabolites of benzene are autooxidized faster in the presence of metal ions

and resulting in the formation of TBAR from target molecules such as glutamate and calf

thymus DNA. The suggested mechanism includes free radical formation via superoxide

and the covalent binding of the semiquinone metabolites of polyphenols to such

molecules. Significant increase in metal ions in rat liver and rat liver nuclei during

benzene exposure may have toxicological implications for cytotoxicity of liver cells and

liver nuclear DNA damage in vivo.

Further in vitro studies revealed that hydroquinone or 1,2,4-benzenetriol at 330 pM

concentrations, released significant amount of TBAR from rat liver nuclei and only 50-55%

inhibition was observed in the presence of iron chelator such as EDTA or DTPA at 5 pM

concentration. Iron overload affects hepatic heme metabolism. However, the biochemical

mechanisms whereby iron overload affects heme metabolism are not completely

understood. Earlier Mahmutoglu and Kappus (1987) demonstrated the DNA damage in

isolated rat liver nuclei in the presence of redox cycling of bleomycin-Fe(lll) complex by

NADH dependent enzyme and suggesed that the enzymatic reduction of a

bleomycin-metal complex in the cell nucleus may be an essential step in the cytotoxic

activity of bleomycin.

Earlier, we demonstrated the prooxidant and antioxidant properties of iron: hydro

quinone and iron: 1,2,4-benzenetriol complex and suggested that iron catalyzed and

bleomycin-dependent degradation of DNA was markedly enhanced in the presence of

Chapter - VII ^ 112

hydroquinone or 1,2,4-benzenetriol than in iron alone (Singh et al.. 1994). We

demonstrated the formation of iron: polyphenol complex in vitro and fractionated on

Sephadex G-10 column (Chapter-Ill) and proposed that iron; hydroquinone complex may

play role in haematotoxicity of benzene. However, formation of such complex in vivo is

not known.

To demonstrate the possible role of copper in TBAR formation in presence of

hydroquinone, the isolated rat liver nuclei were incubated with or without the presence of

bathocuproine, a specific copper ion chelator. In the presence of bathocuproine, about

67% inhibition was observed. Increase in the concentration of bathocuproine (5-25 pM)

resulted in only marginal increase in inhibition of TBAR formation (52-67%). This may be

due to permeability of bathocuproine to cross the nuclear membrane is limiting with

increasing concentrations of bathocuproine as it contains two methyl groups (Mellow-Filho

and Meneghini, 1991). Copper, a natural constituent of cell nuclei has been suggested

to play a key role in structural organization and function of chromosomes (Hu et a/., 1990).

In this context, it appears important to focus attention also on the redox activity of copper

in the vicinity of DNA. Li and Trush, (1993a) demonstrated that Cu(ll) strongly induces

the oxidation of hydroquinone as such may be factor involved in the oxidative action and

toxic to primary bone marrow stromal cells at DBA/2J mice. They further observed that

Cu(ll) is capable of directly reacting with hydroquinone, causing the one electron oxidation

of hydroquinone to semiquinone radical (SQ) and reactive oxygen species generated in

chemical metal redox system were responsible for in vitro plasmid DNA damage (Li ef al..

1995). The oxidative activation of hydroquinone to the electrophilic 1,4-benzoquinone

through a peroxidase/H202 system has been suggested to play an important role in the

hydroquinone-induced bone marrow target cell injury (Smith ef al.. 1989).

Earlier, Pnjtz (1993) has shown that Cu(ll)-induced oxidation of certain compounds

such as ascorbate, catechol and o-phenylenediamine was enhanced by orders of

magnitude in the presence of DNA. DNA in combination with copper could promote the

bioreductive activation of genotoxic compounds and their progeny in the most vulnerable

cell compartment, the nucleus. Recently, Zhang et al., (1996) demonstrated the important

role of Cu^* in 1,2,4-benzenetriol-induced genotoxicity.

Chapter - VII 113

Endogenously generated oxidants that could lead to cellular DMA damage Include

superoxide radicals and H2O2. There have been many reports to indicate that the human

genome is potentially vulnerable to damage (or mutation) by H2O2 (Meneghini and Martin,

1993). Furthermore, with formation of DNA-Cu(l), the DNA becomes predisposed to

oxidative damage by H^Oj via Fenton type reaction that generate strongly oxidizing

radicals such as hydroxyl radical or CuOzH (Masarwa et al., 1988) in situ, i.e. near the site

of Cu(l) fixation within the DNA (Aruoma et al., 1991);

DNA + Cu(l) + H2O2 > DNA:OH + Cu(ll) + OH" I 1

DNA damage

Copper ions are much more efficient at promoting DNA damage by H2O2 than iron ions

(Aruoma et al., 1991), and this is probably due to the high affinity of DNA for Cu (I).

Presence of thiourea, 2.5 mM and 5.0 mM resulted in the inhibition of TBAR release

from rat liver nuclei to the extent of 81% and 91% respectively. Studies by Hanna and

Mason (1992) have shown that thiourea acts as a copper chelator. This further indicates

the possible role of copper in TBAR formation from rat liver nuclei incubated in the

presence of hydroquinone. Further, externally added copper (upto 0.176 mM) to the

reaction mixture did not result in any significant increase in the release of TBAR from rat

liver nuclei by hydroquinone. This particular observation indicates that the copper

associated with nuclear DNA was only responsible to catalyze autooxidation of

hydroquinone or 1,2,4-benzenetriol, during which oxyradicals are released and caused

degradation of the deoxyribose moiety of DNA, resulting in the formation of TBAR

products. Marginal inhibition in TBAR release was observed in the presence of other

oxyradical scavengers, such as mannitol (27%) and SOD (15%) However, catalase did

not show any inhibition in TBAR formation from rat liver nuclei in the presence of

hydroquinone. Probably, nuclear membrane selectively prevent these oxyradical

scavengers to reach the target site. The present in vitro studies, clearly demonstrate that

benzene metabolites (particularly hydroquinone) induced genotoxic effect in presence of

copper which is associated with the nuclear DNA. However, further studies are needed

to explain the precise mechanism of DNA damage and chromosomal aberration in vivo

Chapter - VII ,,.

during benzene exposure and effect of transition metal ions in benzene induced

genotoxicity and human leukemia.

Siimmar)' 115

Small amounts of a large variety of substances normally regarded as foreign to the

body are always existent in the environment. These are often toxic and occur frequently

In the modem world, man is increasingly dependent upon the use of synthetic chemicals

and other domestic products in larger number.

Benzene is frequently used as an industrial solvent of the modem time. Its excellent

solvent properties and its use as a starting material for wide ranging chemicals and

domestic products of commercial importance has made it indispensable. Its value in our

modem society is great, but due to its toxic nature, the use of benzene would undoubtedly

have an impact on our way of life. The use of benzene has been on the rise and

obviously the risk of exposure is also increasing. Though the exposure levels to benzene

can be brought down to acceptable levels but can not be eliminated.

Benzene is a well known genotoxic and carcinogenic agent. But the precise

mechanism of its genotoxicity and carcinogenicity are yet to be known. Therefore, in this

study an attempt has been made to explain the probable metabol(te(s) of benzene to

induce haematotoxicity and role of transition metal ions (iron and copper) to explain the

Summary 116

possible biochemical mechanisms of genotoxicity and carcinogenicity expressed during

benzene exposure.

Release of iron from ferritin by 1,2,4-benzenetriol

Iron is a vital metallic cofactor of several important biomolecules. It is normally

localized in ferritin (main iron storage protein) or transferrin (iron transport protein). But

this iron, when it gets decompartmentalized manifests lethally by triggering off free radical

reactions by means of Fenton and Haber-Weiss type of reactions:


2Fe(ll) + O2+ 2H* > 2Fe(lll) + H A

Fe(ll) + H2O2 > Fe(lll) +-0H + OH

Large human prospective studies suggest that excess body free iron is associated

with carcinogenesis, such as primary hepatocellular carcinoma, colon cancer, lung cancer,

bladder cancer, acute lymphocytic leukemia and neuroblastoma. Bleomycin detectable

iron has been shown to accumulate in significant concentrations in the bone marrow of

benzene exposed rats. Polyphenolic metabolite of benzene, particulariy 1,2,4-

benzenetriol (a trihydroxy benzene) was found to release significant amount of iron from

horse spleen ferritin under aerobic and anaerobic conditions. The release of iron from

ferritin during autooxidation of 1,2,4-benzenetriol in vivo may result in increased

intracellular concentrations of free iron. Although, the physiological mechanism of iron

mobilization from ferritin is pooriy understood, the observed iron release from ferritin by

1,2,4-benzenetriol could occur through the direct reduction of iron by the hydroxy

hydroquinone form or via its autooxidation intermediates i.e., superoxide radical and

semiquinone. It was observed that the iron released from ferritin in the presence of

1.2,4-benzenetriol was capable of inducing lipid peroxidation and catalyzing

bleomycin-dependent calf thymus DNA degradation. The mechanism by which iron is


toxicity is not known. However, the present observation offer a new mechanism that the

iron released from ferritin by 1,2,4-benzenetnol could contribute to the better

Summary Hy

understanding of the toxicity of benzene. Further, elucidation of the mechanisms of

iron-mediated oxidative damage may be important for the prevention of carcinogenesis.

Prooxidant and antioxidant properties of iron: polyphenol complex

Polyphenolic metabolites of benzene viz. hydroquinone and 1,2,4-benzenetriol have

been shown to be toxic and the suggested mechanism includes free radical formation via

superoxide during their autooxidation and the covalent binding of semiquinone to DNA,

RNA and other cellular macromolecules. Benzene exposure also leads to an

accumulation of bleomycin sensitive iron in bone marrow. 1,2,4-Benzenetriol, a tnhydroxy

metabolite of benzene was capable of releasing iron from horse spleen ferritin, which may

lead to accumulation of iron in bone marrow during benzene exposure, as the

polyphenolic metabolites are accumulated against the concentration gradient in the bone

man^ow. The ferrous iron may be chelated to the polyhydroxy metabolites of benzene viz.

hydroquinone and 1,2,4-benzenetriol.

Studies carried out with hydroquinone/1,2,4-benzenetriol and iron showed several

fold increase in iron-catalyzed bleomycin- dependent degradation of calf thymus DNA as

compared to iron alone. Complex formation some how increase the availability of iron to

degrade DNA. It is also possible that the hydroquinone and 1,2,4-benzenetnol possess

combined iron-chelating and iron ion reducing properties to stimulate DNA degradation

in the presence of bleomycin. Further, iron:polyphenol complex appears to be stable and

intact as the recovery from bone marrow lysate was complete. On the other hand,

iron:polyphenol complexes have shown an antioxidant activity by inhibiting of

iron-dependent lipid peroxidation in rat brain homogenate and glutamate degradation.

This is similar to the antioxidant properties exhibited by flavonoids due to their ability to

chelate iron. Thus the polyphenolic chelates limiting the availability of iron for lipid

peroxidation and at the same time facilitate bleomycin- dependent degradation of DNA.

Summary 118

Polyphenol-iron complex: A probable toxic metabolite of benzene

An attempt was made to examine in vitro formation of polyphenol:iron complex.

Complex formation was fractionated on Sephadex G-10 column chromatography. Results

clearly show the formation of polyphenoUron complex under in vitro condition. Polyphenol

iron complex formation was also observed during the release of iron from ferritin in the

presence of 1,2,4-benzenetriol. Although, benzene exposure leads to accumulation of

iron as well as polyphenoiic metabolites of benzene in the bone marrow against the

concentration gradient, the formation of such complex in vivo is yet to be identified.

We evaluated the haematotoxicity of hydroquinone.iron complex as a possible

haematotoxic intenmediate during benzene exposure. Results indicate that hydroquinone

iron chelate showed marginal increase in haematological parameters in experimental

animals. But the precise mechanism of marginal, yet significant increase in haema

tological indices is not clear. Atleast, some of the effects observed in hydroquinone:iron


following exposure of benzene to mice.

Glutathionyl hydroquinone: A possible toxic metabolite of benzene

Glutathionyl hydroquinone has been isolated as one of the urinary metabolites of

hydroquinone, indicating that glutathionyl hydroquinone formation occurs in vivo and

autooxidized at several fold higher than the parent hydroquinone to glutathionyl

benzoquinone. We noticed that glutathionyl hydroquinone is a potent prooxidant to

damage both calf thymus as well as plasmid pUC 18 DNA and this observation may be

of toxicological importance in relation to benzene induced genotoxicity.

Haematotoxicity of glutathionyl hydroquinone was studied in male mice at dose 100

mg/kg body weight, intraperitoneally for one month. Increased pattern of relative liver and

spleen weight and WBC were noticed, but these results were not conclusive to explain

GHQ as potent haematotoxic metabolite of benzene. The pharmacokinetics and

distnbution of administered GHQ is not known. Whether GHQ administered i.p. reaches

Sujiimary

the target organ i.e. bone marrow is yet to be evaluated. Other reason may be due to

difference in sensitivity of species towards different t enzene metabolites.

Role of transition metal ions (Fe,Cu) during benzene toxicity

Earlier studies have provided enough evidence that transition metals including iron

and copper are capable of directly mediating the activation or metabolism of several

xenobiotics via a metal redox mechanism, which leads to the formation of more reactive

oxygen and other organic free radicals. We observed that exposure to benzene leads to

increase in total iron content in liver and isolated rat liver nuclei. Benzene metabolites

induced the DNA damage particularly in the presence of iron and copper. This study

would help in better understanding of the chromosomal aberrations/abnormalities

associated with benzene exposure. Copper which is associated with DNA, may play a

potential role in nuclear DNA damage in presence of hydroquinone. However, the

molecular mechanism of its damage is not well understood. Significant increase in iron

content in liver and in isolated liver nuclei during benzene exposure and the copper which

IS associated with DNA may have toxicoiogical implications for cytotoxicity of liver cells

and liver nuclear DNA damage in vivo.

In the light of our investigations following conclusions can be made:

• 1,2,4-Benzenetriol is a potent reductant of feme iron of ferritin and releases iron from

ferritin core. The release of iron from bone marrow lysate by 1,2.4-benzenetriol may

be of toxicoiogical significance during benzene exposure as this could lead to

disruption of intracellular iron homeostasis in bone marrow cells

• The iron:polyphenol chelate, on one hand, is a more potent DNA cleaving agent in

the presence of bleomycin, and on the other hand, it is a less effective free radical

generator as compared to iron alone. However, the formation of such a complex in

vivo is not known. Further studies are needed to characterize such complexes in

vivo and evaluate their toxicoiogical significance during benzene exposure.

• Polyphenol forms complex with iron under in vitro condition and may be a possible

haematotoxic intermediate during benzene exposure

Summary ,_. IZO

The present in vitro studies revealed that glutathionyl hydrcK|uinone is a potent toxic

metabolite of benzene and acts as a prooxidant due to its faster autooxidation to

glutathionyl benzoquinone and oxyradicals which may induce DNA damage.

Exposure to benzene resulted in significant increase in total iron content in liver

tissue and in isolated liver nuclei.

Benzene metabolites (particularly hydroquinone) induced genotoxic effect in

presence of copper which is associated with the nuclear DNA. However, further

studies are needed to explain the precise mechanism of DNA damage and

chromosomal aberration in vivo during benzene exposure and effect of transition

metal ions in benzene induced genotoxicity and human leukemia.

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1. Sarfaraz Ahmad, Vinita Singh and G.S. Rao (1994) "Antioxidant Potential in serum & liver of albino rats exposed to benzene". Indian J. of Experimental Biology: Vol. 32, 203-206.

2. Vinita Singh, Sarfaraz Ahmad and G.S. Rao (1994) "Prooxidant and antioxidant properties of iron: hydroquinone and iron: 1,2,4- benzenetnol complex Implication for benzene toxicity". Toxicology, Vol. 89, 25-33.

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4. Vinita Singh, Sarfaraz Ahmad and G.S. Rao(1996) "Release of aldehydic product from glutamate or deoxyuridine by S-aminolevu\inic acid in presence of copper ions". Indian J. of Expenmental Biology. Vol. 34, 208-210.

5. Sarfaraz Ahmad and G.S. Rao. "Cleavage of pUC 18 DNA by glutathionyl hydroquinone." Chemico-Biological Interaction, (Communicated in September, 1996).

6. Sarfaraz Ahmad and G.S. Rao. "EDTA: An alternate sp€;ctrophotometric reagent for iron estimation". Current Science (Communicated in September, 1996).

7. Sarfaraz Ahmad and G.S. Rao. "In vitro identification and characterization of polyphenol-iron complex: A possible toxic metatx)lite of benzene", (manuscript under preparation).

8. Sarfaraz Ahmad, R.C. Murty and G.S. Rao. "Role of transition metal ions (iron, Copper) in relation to genetic toxicity dunng benzene exposure." (manuscript under preparation).

studies related to identification of toxic metabolites of

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