specific dna adducts induced by some mono-substitued epoxides in vitro and in vivo

Chemico-Biological Interactions 129 (2000) 209–229

Mini-Review

Specific DNA adducts induced by somemono-substitued epoxides in vitro and in vivo

Mikko Koskinen *, Kamila PlnaDepartment of Biosciences at No6um, Center for Nutrition and Toxicology, Karolinska Institute,

S-141 57 Huddinge, Sweden

Received 1 August 2000; accepted 6 September 2000

Abstract

Alkyl epoxides are important intermediates in the chemical industry. They are also formedin vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in manytoxicology assays which has been associated with their covalent binding to DNA. Hereaspects of the formation and properties of DNA adducts, induced by some industriallyimportant alkenes and mono-substituted epoxides are discussed. These include propyleneoxide, epichlorohydrin, allyl glycidyl ether and the epoxy metabolites of styrene andbutadiene. The major DNA adducts formed by epoxides are 7-substituted guanines, 1- and3-substituted adenines and 3-substituted cytosines. In addition, styrene oxide and butadienemonoepoxide are able to modify exocyclic sites in the DNA bases, the sites being in the caseof styrene oxide N2- and O6-positions of guanine, N6-adenine as well as N4-and O2-cytosine.In vivo the main adduct is the 7-substituted guanines. The 1-substituted adenines have alsoshown marked levels, and these adducts should also be targets in biomonitoring of humanexposures. Due to its low mutagenicity, 7-substituted guanines are considered as a surrogatemarker for other mutagenic lesions, e.g. those of 1-adenine or 3-uracil adducts. © 2000Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Biomarkers; DNA adducts; Epoxides; Mutagenicity

www.elsevier.com/locate/chembiont

Abbre6iations: AGE, allyl glycidyl ether; BD, 1,3-butadiene; BMO, butadiene monoepoxide; CBI,covalent binding index; CYP, cytochrome P450; DEB, diepoxybutane; EBD, epoxybutanediol; ECH,epichlorohydrin; EO, ethylene oxide; HP, 2-hydroxypropyl; PO, propylene oxide; SO, styrene 7,8-oxide.

* Corresponding author. Tel.: +46-8-6089245; fax: +46-8-6081501.E-mail address: [email protected] (M. Koskinen).

0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (00 )00206 -4

M. Koskinen, K. Plna / Chemico-Biological Interactions 129 (2000) 209–229210

1. Introduction

Epoxides, or oxiranes, are oxygen containing heterocyclic compounds. Due to thelarge ring strain associated with the three-membered ring they are reactivemolecules. Because of their reactivity they are important intermediates in chemicalindustry, especially in polymer production. Wide-ranging industrial applications ofepoxides have resulted also in considerable human exposure. In addition to directexposure, humans are exposed to epoxides via metabolism of different alkenes.Compounds such as ethylene, propylene, butadiene (BD), styrene, vinyl chlorideand acrylamide are metabolised to 1,2-epoxides by cytochrome P450-dependent(CYP) monooxygenases. The human exposure is of concern because the epoxidesare alkylating agents in vivo being able to react with different nucleophilic centersof cellular macromolecules including proteins and DNA. DNA adducts in turnhave shown considerable association with carcinogenic processes in vivo [1,2].

The adduct formation of the simple epoxides have been reviewed previously[3–5]. This minireview updates the previous ones by focusing to some of the morerecent data on the range and properties of these DNA adducts in vitro and in vivo.The article covers some important mono-substituted epoxides to which humans aredirectly exposed or those that are formed by metabolism after exposure to corre-sponding alkenes (Table 1). Ethylene oxide (EO), a non-substituted epoxide, isincluded in some cases for comparison. All the epoxides, or the alkenes, are agentswith considerable occupational exposures. Some of them like BD are also environ-mental pollutants to which humans are exposed via tobacco smoke or car exhaust.The most likely route of exposure to these agents is by inhalation, although thepossibility of dermal and oral absorption should also be considered [6–11].

2. Metabolism

The formation of epoxides from alkenes, including, e.g. ethylene and propylene,is mainly mediated by CYP-dependent monooxygenases. This occurs by incorporat-

Table 1The epoxides included in this study, and the agents to which humans are exposed occupationally or inthe environment, resulting in the epoxide exposure

Epoxide Substituent to the epoxy moiety Exposure

–HEthylene oxide (EO) Ethene, EO–CH3Propylene oxide (PO) Propene, PO

Butadiene monoepoxide (BMO) –CH�CH2 Butadiene, BDEpoxybutane diol (EBD) –CH(OH)–CH2(OH) BDDiepoxybutane (DEB) –CH(O)CH2 BD

Styrene–C6H5Styrene 7,8-oxide (SO)Epichlorohydrin (ECH) ECH–CH2Cl

AGE–CH2–O–CH2–CH2�CH2Allyl glycidyl ether (AGE)

M. Koskinen, K. Plna / Chemico-Biological Interactions 129 (2000) 209–229 211

ing an atom of molecular oxygen into the substrate [12]. Even though this processis the first step in transforming lipophilic chemicals to excretable form, certainchemicals are activated by this to their ultimate carcinogenic form. Styrene ismetabolized to styrene oxide (SO), mainly by CYP2B6 followed by CYP1A2,CYP2E1 and CYP2C8 [13]. Some of the styrene metabolizing cytochromes havebeen found to be polymorphic [14]. BD, with two double-bonds in its structure,shows a rather complex metabolic pathway. It is mainly oxidized by CYP 2E1 and2A6 primarily to 3,4-epoxy-1-butene enantiomers (butadiene monoepoxide, BMO)[15], which is subjected to further metabolism. It can be oxidized to diastereomericdiepoxybutane (DEB) or hydrolyzed to 3-butene-1,2-diol via epoxide hydrolase.3,4-Epoxy-1,2-butanediol (EBD) may be formed by hydrolysis of DEB or byoxidation of 3-butene-1,2-diol [16–19].

1,2-Epoxides are relatively long-lived in aqueous solution at neutral pH, thehalf-lives of hydrolysis at 37°C being of the order of 50–200 h [3,20]. The half-livesin vivo are some 100 times shorter, showing that the major detoxification pathwaysare enzymatic. Metabolism of epoxides generally leads to intermediates that areconsiderably less reactive than the parent compound.

One detoxifying pathway of epoxides is the addition of water to form 1,2-diols,which are of low reactivity, the reaction being catalysed by epoxide hydrolase[21,22]. Several distinct forms of epoxide hydrolase have been identified, whichdiffer in physical properties and substrate preferences. The extent to which epoxidehydrolase is involved in human metabolism of the studied epoxides is not clear,neither is the possible effect of the polymorphism of this gene. Still, this pathwaywas shown to be the major detoxification pathway of SO in humans [23,24].

Another possible pathway is inactivation by glutathione S-transferases, formingglutathione S-conjugates [25,26]. These conjugates are generally degraded in liverand kidney and excreted in urine as the corresponding mercapturic acids (N-acetyl-S-cysteine conjugates) [22]. The importance of this pathway in humans is indicatedby the presence of S-hydroxyethylcysteine in the urine of EO exposed hospitalworkers [27]. Genetic polymorphism in several glutathione S-transferase genes hasbeen found, which may cause differences between individuals in this metabolicpathway [28].

3. Reactions with nucleic acid constituents

Mono-substituted epoxides have two electrophilic carbons, i.e. a- and b-carbons.The site of nucleophilic attack on alkyl epoxides under physiological conditions isprimarily at the less substituted, sterically more accessible, b-carbon. The substi-tuted a-carbon is expected to be less reactive, owing to steric hindrance and theelectronic effects of the substituent. Epoxides with a vicinal aromatic (SO) or vinylgroup (BMO) can, however, react through the both carbons because the substituentincrease the positive charge at the a-carbon. Because a-carbon is asymmetric twodiastereomeric products are therefore formed through nucleophilic attack on eithercarbon [3,5].


The site of alkylation of the DNA constituents is mainly determined by the ioniccharacter of the substrate [29]. Thus, alkyl epoxides that are not able to stabilise anionic charge to any great extent, like aliphatic alkyl epoxides, react predominantlyat ring nitrogen positions in DNA bases. Under physiological conditions the mainalkylation sites are 7-guanine, 1- and 3-adenine, and 3-cytosine (Table 2). Incontrast, SO and BMO modify also exocyclic groups [30–34]. The reaction mecha-nisms of nucleoside alkylation have been studied using optically active epoxides. Inthe case of ring-nitrogen substitution, the reaction through the b-carbon has beenfound to follow direct displacement by SN2 type of reaction mechanism[30,31,33,48]. In contrast, under neutral conditions the exocyclic sites open theepoxide in SO only at the a-carbon, resulting in both inverted and retainedstereochemistry, indicating prominent SN1 type of nucleophilic attack [30,31,33]. Ithas been concluded that the exocyclic amino groups involve substrate ionisationthat decreases in the order guanine\adenine\cytosine, correlating inversely withthe pKa values of those nucleic acid bases [33].

Mainly mono-substituted products are obtained by the treatment of nucleic acidconstituents with epoxides. Bis-substituted deoxyguanosines have been detected bySO alkylation involving N2- and O6-positions or N2-and 1-positions [54]. Alsobis-alkylation involving base and phosphate has been detected [55]. Phosphatealkylation in nucleotides has been detected for AGE, PO and SO [38,41,55]. Whena dinucleotide dGpdT was treated with SO no phosphotriesters were detected in theintervening phosphate suggesting that they are not expected to be formed in DNA[55].

Epichlorohydrin (ECH) differs from the other epoxides under study by being abi-functional alkylating agent [39,40]. It is susceptible to a second SN2 displacementreaction via the loss of chlorine. For adenine alkylation, it is proposed that theepoxide first undergoes ring opening by the 1-position, since it is the mostnucleophilic nitrogen. Cyclisation and loss of HCl, via the attack of N6 on thecarbon carrying the chlorine results in formation of the 1,N6-2-hydroxypropano-adenine adduct [39]. A similar product has been found after reaction of de-oxyadenosine with cyanoethylene oxide, another bi-functional agent [56].

For the adduct studies, properly characterised standard compounds are needed.To prepare different standards in sufficient amounts, some adjustments of thereaction conditions are needed. The reaction under neutral pH usually leads mainlyto 7-guanine substitution through the b-carbon in relatively high yields [5]. Reac-tion at glacial acetic acid shows also preferential 7-alkylation, however, in the caseof SO favouring reaction at a-carbon [30]. When the reaction is performed underalkaline conditions, i.e. above the pKa-value of the 1-position of guanine, N2- and1-alkylated products are obtained in higher yields [55,57]. The O6-alkylated stan-dards are best obtained by reaction of 2-amino-6-chloropurine with the propersodium alkoxide [5]. The optimisation of reaction conditions for preparation of thestandards of the other nucleobases than guanine is rather little examined with thecurrently studied epoxides. In the case of adenosine, glacial acetic acid has shownto favour the ring-nitrogen alkylation through the a-carbon of SO, similarly as for7-guanosine alkylation [31]. The 3-alkylated thymidine monophosphates can be


Tab

le2

Bin

ding

site

sof

the

epox

ides

innu

cleo

base

sfo

rmed

byth

ere

acti

onof

the

epox

ide

and

the

nucl

eic

acid

cons

titu

ent

invi

tro

unde

rph

ysio

logi

cal

pHan

dte

mpe

ratu

re

Gua

nine

Ura

cil

Ref

eren

ces

Ade

nine

Cyt

osin

eT

hym

ine

N3

N3

O2

N4

N3

N7

N3

N2

O6

N1

N6

bb

––

bb

bcE

O[3

5,36

]bb

b–

ba bbb

bb

––

bbc

[37,

38]

––

PO

b–

b–

–b

bcE

CH

[39,

40]

b–

–bd

bd

bb

––

–bc

bb[4

1]A

GE

b–

–b

a,b

a,b

a,b

aa,

bac ,b

cSO

[30,

31,3

3,34

,42]

a,b

aa,

ba,

ba,

bb

a,b

a,b

b–

a,b

ac ,bc

a,bb

[32,

43–4

9]B

MO

a,b

–a

a,b

bbb

b–

––

––

[50–

52]

bD

EB

––

b–

––

––

bb[5

0–53

]b

–E

BD

b–

ab,

reac

tion

thro

ugh

b-ca

rbon

ofth

eep

oxid

e;a,

reac

tion

thro

ugh

the

a-ca

rbon

.In

the

case

ofE

Oa-

and

b-ca

rbon

sca

nno

tbe

dist

ingu

ishe

d.b

Seco

ndar

ypr

oduc

tsfo

rmed

from

byth

eD

imro

thre

arra

ngem

ent

from

the

1-al

kyla

ted

aden

ine.

cSe

cond

ary

prod

ucts

form

edby

deam

inat

ion

from

the

3-al

kyla

ted

cyto

sine

s.d

Dat

aon

1-an

dN

6-a

deni

neof

EC

Hre

pres

ent

form

atio

nof

the

1,N

6-2

-hyd

roxy

prop

ano

addu

ct.


Table 3In vitro stabilities (half-lives) of the different adducts in double-stranded DNA, at neutral pH and37°C

7-Guanine (h) 3-Adenine (h) 1- to N6-adenine 3-Cytosine Referencesrearrangement (h) deamination (h)

–EO –75 [36]10–15221 53– [38]120PO– –ECH [64]72 –150 �4820 [40,41]AGE 38– –SO (a/b) [65]51/51 10/21– –– [48]48BMO

–30 – – [52]BDEEBD – – – [52]31

obtained in high yields at alkaline conditions, as well as, 3-alkylated uridinemonophosphates [34,46,55].

4. Reactions with DNA in vitro

In DNA, EO and the mono-substituted alkyl epoxides all modify preferentiallythe 7-position of guanine mainly due to the high nucleophilicity and stericalavailability of the position [3,5]. Other primary adducts of alkyl-epoxides are thoseat 1- and 3-position of adenine and 3-position of cytosine [35,38,41,49,51,58]. In thecase of SO, the spectrum is wider due to the reactive a-carbon, including also N2-and O6-positions in guanine, N6-adenine and N4-cytosine [34,59–61].

4.1. Stabilities of the adducts

Ring nitrogen-substituted products are all unstable in some fashion; thus, theirpossible biological effects might be influenced by chemical transformations sec-ondary to the initial DNA alkylation. The secondary transformations are generallyqualitatively similar in nucleosides and nucleotides as in DNA, even though therates may differ considerably. Examples of such instabilities are the facile depurina-tion of 7-alkyl-deoxyguanosine and 3-alkyl-deoxyadenosine, and the imidazole ringopening of 7-alkyl-deoxyguanosines [62]. These reactions are acid- and alkali-catalysed, respectively, but occur even at physiological conditions. The depurina-tion is due to the quaternary nitrogen sites, which lead to cleavage of the glycosylbond, to regain the aromaticity. The 3-alkylated adenine adducts are considerablymore labile than those of the 7-guanines (Table 3). Since both of these secondarylesions can persist in DNA for extended periods, they may contribute to inductionof biological consequences. On the other hand, the lability of the 7-guanine and3-adenine adducts can also be useful in applying them as noninvansive biomarkersof exposure by monitoring their amounts in urine [63].


Other secondary lesions are products at the 3-position of uracil, which have beenshown to originate from deamination of the corresponding 3-cytosine adduct[34,38,41,58,66]. Furthermore, alkylations of N6-adenine have been shown tooriginate from a Dimroth rearrangement of 1-substituted adenine, facilitated by theadjacent hydroxyl group [37]. Direct alkylation at N6-adenine occurs primarily aftersubstitution at the a-carbon of epoxides containing electron-withdrawing sub-stituents, such as SO and BMO [47,61,66]. In addition to the Dimroth rearrange-ment, 1-alkyl-adenines, especially in the case of SO-adducts, can undergodeamination to the corresponding hypoxanthine adducts [61].

4.2. Relati6e amounts of the adducts

The adducts are generally formed in a time- and concentration-dependentmanner. However, because of the differing stabilities of the adducts, the relativeproportions of the adducts at different sites may depend on the length of exposure.7-Guanine and 3-adenine adducts reach an apparent steady-state level while thelevel of the chemically more stable adducts continue to increase. Table 4 shows theamounts of other adducts relative to that of the 7-position of guanine. In double-stranded DNA, 75–90% of total alkylation takes place at the 7-position of guanine,and 4–8% at the 3-adenine. Thus, a vast majority of the alkylation occupies labilesites that lead to apurinic sites.

The 1-position of adenine and 3-cytosine are highly nucleophilic sites that do notreact extensively in DNA because of steric hindrance. The sum of 1- and N6-substi-tuted adenines have been found in the range 4–14% for aliphatic alkyl-epoxidesrelative to the 7-alkylation. The sum of 3-substituted cytosines/uracils has beenfound in the range 2–6% of 7-substituted guanine (Table 4). The yields of1-AGE-adenine and 3-AGE-cytosine (including 3-AGE-uracil) in single-strandedDNA were 19 and 6 times higher, respectively, as compared to double-strandedDNA [38]. This difference is expected since the 1-position of adenine and the3-position of cytosine are involved in the Watson–Crick base pairing. The corre-sponding difference for the BMO adducts between double- and single-strandedDNA was reported to be in order of 10–20 times [58]. In the case of 1-adeninealkylation by SO, also the deaminated products are found to constitute a consider-able proportion of the total alkylation [61]. The distribution of the adduct originat-ing form those of 1-adenines has been found to differ in nucleosides, single-strandedDNA and double-stranded DNA in favour of formation of b1-SO-deoxyinosinewith increased structural complexity [61]. This indicates the importance of thedeaminated fraction in in vivo.

In the case of the SO- and BMO-alkylation different regioisomers are formed. InDNA, the ring-nitrogens open primarily the b-carbon of SO and BMO, being inaccordance with the alkylation of free nucleic acid constituents. For the 7-alkyl-guanines the reaction through the b-carbon predominates only slightly[34,49,58,65]. However, in the case of the 3-alkyl-adenines, the a-isomer predomi-nates, being in contrast to the alkylation of free adenine [49,65]. The reason for thereversed reactivity order for adenine within DNA was suggested to be related to


Tab

le4

Rel

ativ

eam

ount

sof

diff

eren

tad

duct

sfo

rmed

byth

ere

acti

onof

the

epox

ide

and

doub

le-s

tran

ded

DN

Ain

vitr

o,at

neut

ral

pHan

d37

°Ca

EC

H[4

0,64

]SO

b[6

7]B

MO

b[5

3,58

,68]

EB

D[6

8]P

O[3

8]E

O[3

5]A

GE

[41]

100

100

100

100

100

7-G

uani

ne10

010

0–

–4.

5–

N2-G

uani

ne–

––

1010

1511

1011

–3-

Ade

nine

25e /

0.5f

–16

d9c

141-

/N6-a

deni

ne10

3.5

51

1.5

–2

64

3-C

ytos

ine/

urac

il–

2–

––

–N

4-C

ytos

ine

–

aT

heva

lues

give

nar

ere

lati

veto

the

7-su

bsti

tute

dgu

anin

es.

bD

iffe

rent

geom

etri

cal

isom

ers

are

not

sepa

rate

din

the

case

ofSO

and

BM

O.

cD

ata

on1-

and

N6-a

deni

neof

EC

Hre

pres

ent

form

atio

nof

the

1,N

6-2

-hyd

roxy

prop

ano

addu

ct.

dIn

clud

esth

ede

amin

ated

1-ad

enin

ead

duct

.e

Onl

y1-

aden

ine

[53]

.fO

nly

N6-a

deni

ne[5

9].


steric constraints in the double-stranded DNA [49]. Moreover, the 3-alkylation ofadenine in DNA is in contrast to the corresponding reaction of free nucleosides inwhich no such products are observed. In DNA, the 3-position of adenine is notinvolved in hydrogen bonding and is sterically exposed in the minor groove ofa-helical DNA thus being accessible for alkylation [58]. In contrast, in freenucleosides the site may be blocked by intra- or intermolecular hydrogen-bonding[69].

Rate constants for reaction with DNA can be determined in in vitro experimentsto compare the reactivity of different epoxides [70,71]. In Table 5 are given thesecond-order rate constants for the reaction of some epoxides with 7-position ofguanine in DNA in vitro. The rate constant for the reaction of AGE with 7-guaninein DNA [41] is about twelve and three times lower than those for EO and PO,respectively. Thus, the rate of reaction seems to decrease with increasing length ofthe substituent on the epoxide, as it has been previously observed for other relatedepoxides [72,73]. The reactivity of ECH towards DNA is about twice that of PO.This is in accordance with the relative reactivity of the two compounds towardsmodel nucleophilic compounds such as water and ammonia [3].

Based on its relative stability in DNA and high concentration relative to theother adducts formed, 7-substituted guanine would be a suitable marker of epoxideexposure. Although 1-substituted adenine is present at lower levels than 7-substi-tuted guanine, it is considerably more stable and this adduct could therefore be asuitable alternative, particularly if the rearrangement product, N6-substitutedadenine, could be analysed simultaneously. The stable (and biologically probablymore important) 3-uracil adduct is yet another alternative biomarker, although itslevels are expected to be considerably lower. Products with thymidine are expectedto be present at very low levels [5,46].

Table 5Chemical reactivity of epoxides in vitro for the 7-position of guanine in DNA and corresponding CBIfor rat or mouse liver DNA after a single i.p. dose

CBI (nmol 7-guanine/mol dNp per mmol/kg body ReferencekDNA (7-guanine)×104a

weight)(l/g DNA per h)

EO 0.96 11 [36,74]0.25 0.6 [75]PO0.08 0.04b [76]AGE

[64,77]0.2ECH 0.6[78]Styrene 0.18c 0.2b

a The rate constants were calculated as in [70].b Calculated for mouse liver DNA.c The rate constant of SO, M. Koskinen, unpublished.


5. Specific DNA adducts in vivo

DNA adduct formation of chemical carcinogens or their reactive metabolites isconsidered to constitute an initial step of carcinogenesis [1]. The adduct levelrepresents an integration of exposure, absorption, distribution, metabolism andreactivity of carcinogen; chemical stability of adduct; the action of repair processes;and cell turnover. As long as the passive uptake, transport mechanisms or enzy-matic processes are not saturated, inhibited or induced a linear dose-responserelationship for adduct formation is expected after in vivo exposures. Consequently,saturation or induction of different processes involved in formation and repair ofDNA adducts are likely to have important implications for mutagenesis andcarcinogenesis, particularly when exposing experimental animals to high doses andusing data from such studies in cancer risk assessment [79,80]. In addition,deviation from linearity could take place if the exposure is causing toxicity orproliferation to the studied cells. The net result of various toxicokinetic parametershas been termed biologically effective dose (molecular dose) — that is, theinteraction of the carcinogen with critical cellular targets [81]. Recent studies inanimals [82] and humans [83] suggest that levels of DNA adducts in tissues areindeed reflective of carcinogen doses.

5.1. Adducts in experimental animals

5.1.1. 7-Substituted guaninesThe formation and stabilities of the alkyl epoxide-induced DNA adducts have

been studied in rodents in order to understand the role of specific adducts in in vivomodels. Following single and repeated exposures of rats and mice to EO demon-strated similar accumulations of 7-(2-hydroxyethyl)-guanines in both target andvarious non-target tissues, except in testis where lower levels were observed [84].This is obviously due to the rapid and even distribution of EO to all tissues excepttestis, and the ability of EO to act as a direct alkylating agent [84]. 7-(2-Hydrox-yethyl)-guanines were disappeared slowly from mouse kidney and rat brain andlung, being consistent with the loss of the adduct by chemical depurination.However, in the other organs studied more rapid removal was observed indicatingactive repair [84]. After exposure to PO, another directly alkylating agent, PO-sub-stituted 7-guanines were found in considerably higher levels in target tissue (nose)when compared to non-target tissues including lungs, lymphocytes, spleen, liver andtestis [85]. By exposure of 500 ppm of PO for 20 days the alkylation at therespiratory nasal epithelium was up to 98 PO-adducts per 106 nucleotides. The rateof elimination of the 7-substituted guanine was about the same in all examinedtissues, corresponding closely to the spontaneous rate of depurination of thisadduct, thus excluding the active repair of the 7-PO-guanine adducts [85]. Thehigher adduct level in the respiratory tissues is therefore due to a higher dose of POdeposited in this organ.

For epoxides that are formed by the CYP-systems from alkenes the adductformation is somewhat more complex event. It is expected that adduct formation


would be higher in the organs where the metabolic activation is taking place.Indeed, in mice liver where the CYP activity is highest, slightly higher 7-BMO-gua-nine levels were detected after BD inhalation, as compared to lung and kidney [86].But the difference was only slight, suggesting that the DNA-reactive metabolitescirculate in the blood and reach steady-state concentrations over time [86]. By theexposure of rats and mice to BD, the two isomers of the 7-BMO-guanines areformed to similar extent [68]. However, differences have been observed in the levelsof different enantiomers which may be related either to enantioselective repair orformation of the reactive metabolites [87]. The 7-trihydroxybutyl-guanine adductsformed from EBD (or DEB) constitute the highest proportion of the BD derivedadducts in vivo [52,86]. The different enantiomers of trihydroxybutyl-adductsvaried in the manner that adducts derived from RR and SS enantiomers of EBDwas twice the level from RS and SR enantiomers [88]. Contrasting results have beenreported, suggesting the main adduct being the RS isomer of EBD, which wasexplained by the authors to be due to the differences in the production rates of RR-or RS-BDE isomers among different mouse strains [52]. As in the case of PO, thepersistence of 7-trihydroxybutyl-guanine adducts was similar to the half-life ofspontaneous in vitro depurination [88]. Slight species differences were observedbetween mice and rats, the persistence of being somewhat greater in mice. Thissuggests more active repair of these adducts in rats [88].

After single i.p. injection of styrene, Pauwels et al. [78] determined the 7-guanineadducts of SO in various mice tissues. For doses up to 4.35 mmol/kg b.w., adductlevels up to 6.3 of 7-guanine adducts per 107 nucleotides were detected, with a cleardose-response relationship. The adducts were most abundant in the lungs, ca. 30%more than in liver and spleen. The higher adduct level in lungs could be a result ofhigh CYP-activity converting styrene to SO and for lack of epoxide hydrolaseactivity in that organ.

The comparison of the adduct levels induced by different epoxides is difficultmainly due to the large variation in the protocols used in animal studies. Thecovalent binding indices (CBI), adduct level per unit exposure dose, has been usedto compare chemical carcinogens with respect to their capacity to alkylate macro-molecules in vivo. The CBI of different epoxides at the 7-position of guanine inliver DNA of rodents exposed to single intra peritoneal dose (assuming lineardose–response relationship for adducts formation) is shown in Table 5. Differencesin adduct levels formed by the studied epoxides in vivo could be attributed, to acertain extent, to differences in rates of alkylation at the nucleophilic sites in DNA.The difference between EO and PO is higher than what can be explained solely bydifferent reactivity of these two compounds toward 7-guanine. A higher rate ofdetoxification of PO in rat liver is a likely contributing factor. The observeddifference in levels of PO versus EO adducts lasted even after repeated exposures byinhalation (20 days), at conditions when the steady-state level was most likelyachieved. Under such circumstances, the concentration of the 7-guanine adduct inlung and spleen from 500 ppm PO [85] was about 2 times lower than theconcentration of the 7-guanine adduct found in lung and spleen of rats exposed to100 ppm EO [89] for the same period of time, i.e. EO being 10 times more efficientthan PO.


Comparison of styrene to the epoxides in Table 5 is hampered by the need formetabolic activation to SO. SO and PO have similar reaction rates in vitro but POgives higher CBI value, which is probably related either to the slow formation ofSO or faster detoxification of SO compared to PO. Also, in spite of higher chemicalreactivity, the capacity of ECH to alkylate rat liver DNA [64] (Table 5) is lowerthan that of PO, indicating a faster detoxification of ECH compared to PO. Thedifference between ECH and PO in alkylating capacity in vivo was confirmed instudies of haemoglobin adducts in rats [77]. Among studied compounds, AGE hasthe lowest ability to alkylate 7-guanine in DNA in vivo [76], as indicated by itslowest in vitro reactivity towards this position in DNA [41]. In another study,binding of glycidyl ethers to haemoglobin of mice was studied [20]. Thehaemoglobin binding indices, 1.1–1.2 pmol/g globin for AGE and butyl glycidylether and 1.3 pmol/g globin for phenyl glycidyl ether and cresyl glycidyl ether perkg body weight, were similar to that of PO (1.4 pmol/g globin) and 5–6-fold lowerthan that of EO (7 pmol/g).

5.1.2. Substituted adenines and cytosinesBecause of the instability of the 7-substituted guanines, they are reflective of a

rather recent exposure. In the case of a chronic exposure, chemically more stable 1-or N6-substituted adenines and 3-substituted cytosines or uracils should be thetarget, since they should be accumulating in the absence of active repair. Morelow-dose long-term animal studies would be needed to better characterise theaccumulation of these adducts and thus to predict the adduct relevant for thehuman studies.

Recently, 1-alkylated adenines have been detected by the 32P-postlabelling assayin rodents after conversion to the N6-alkylated adenines by the Dimroth rearrange-ment [38,53,90]. In rats exposed to 500 ppm of PO by inhalation during 20 days,1-hydroxypropyl (HP)-adenine was detected in nasal epithelium, lung andlymphocytes [38]. N6-HP-adenine, on the other hand, was found only in the tissuesof the nasal cavities. The highest level of 1-HP-adenine (2.0 adducts per 106

nucleotides; i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium,which also represents the major target for tumour induction in the rat followinginhalation of PO. The levels of this adduct in the lung and in the lymphocytes wereconsiderably lower, amounting to 15 and 9%, respectively, of that of the respiratorynasal epithelium, corresponding with the formed adduct levels of 7-HP-guanine inthe same tissues [85]. In rats sacrificed 3 days after cessation of exposure, nosignificant decrease of 1-HP-adenine was observed, indicating a very slow repair ofthis adduct [38].

Also, the formation of the N6-adenine [87] and 1-adenine [53] adducts of BMOand the 1-trihydroxybutyl-adenine adducts [90] in rats exposed to BD by inhalationhave been identified. Similarly to 7-substituted-guanines and haemoglobin adductsthe 1-trihydroxybutyl-adenine is formed to higher levels than the correspondingBMO adducts [53,68,91]. 1-BMO-adenine adduct level was ca. 40% of the 7-BMO-guanine level being somewhat more than the BMO-proportion in the in vitrotreated DNA [53]. N6-BMO-guanine was ca. 0.5% of 7-substituted guanines [87]


corresponding to proportions in in vitro BMO-treated DNA. The amount ofN6-BMO-adenine was the same immediately after the exposure and 3 weeks laterindicating chemical stability and lack of efficient DNA repair machinery for theN6-adduct [87].

Recently, the 1-SO-adenine adduct has also been detected in mice lungs afterinhalation of styrene [67]. The b1-SO-adenine adducts levels were ca. 3% of the levelof the b7-SO-guanines, being slighly lower than the proportion in in vitro SO-treated DNA. Also the O6-SO-guanines have been identified by the 32P-postla-belling methods after i.p. injection of styrene [78] or by inhalation [92].

3-HP-uracil have been detected in the respiratory nasal epithelia of PO-exposedrats with concentration of 0.02 adducts per 106 nucleotides (0.02% of 7-HP-gua-nine), suggesting repair of the cytosine and/or uracil adducts. Further support forthis hypothesis was obtained by incubation of PO-treated DNA with a proteinextract from mammalian cells. In this experiment 3-HP-cytosine, but not 3-HP-uracil or 1- and N6-HP-adenine was repaired [38]. Furthermore, experiments with abacterial uracil glycosylase indicated that this enzyme was not involved confirmingthe presumption that 3-alkyl-cytosines are not expected to be repaired by thisenzyme [38].

5.2. Human adducts

The adduct studies in humans concerning the epoxides under study are still quitefew, but, especially due to the recent advances in the 32P-postlabelling assay, thedata are amounting. Particularly, DNA-adduct data relative to polymorphisms ofxenobiotics metabolising enzymes can be expected in the near future, revealing theindividual susceptibility to the DNA adduct formation after the exposure.

In populations exposed to relatively high concentrations of styrene significantincreases of levels of DNA adducts, that were assigned as O6-substituted guanines[93–95] and N2-substituted guanines [96], have been reported. In lymphocytes oflaminators the mean O6-guanine adduct level was 5.4/108 normal nucleotides ascompared to 1.0/108 in controls [94]. However, the identity of these adducts havelater raised some doubts [92]. The mean adduct level reported by Horvath et al. [96]for N2-guanine adduct was 16/108 with a linear relationship between dose of styreneexposure and the adduct level.

More recently, by using 32P-postlabelling/HPLC assay Zhao et al. [97] reportedthe 1-trihydroxybutyl-adenine adducts in lymphocytes of humans occupationallyexposed to BD. The mean adduct level in the exposed workers was 4.5 adducts/109

normal nucleotides as compared to 0.8/109 in controls.Even though being the main adduct in vitro and in animal studies, the 7-guanine

adducts of the epoxides have been difficult to detect in humans. 7-(3-Chloro-2-hy-droxypropyl)-guanine adducts after exposure to ECH has been reported, the adductlevels ranging from 1 to 7 adducts/109 nucleotides [64]. The levels of 7-(3-chloro-2-hydroxypropyl)-guanine detected were 1–2 orders of magnitude lower than the7-methyl- and 7-(2-hydroxyethyl)-guanine reported in white blood cells of bothnon-smokers and smokers [98,99].


6. Mutagenic aspects of specific adducts

Several studies have indicated that only certain DNA alkylation products con-tribute to the mutagenic or carcinogenic activity of alkylating agents. For instance,it has been found that O6-alkyl-guanines are quantitatively associated with carcino-genesis or mutagenesis [100,101], whereas low molecular weight adducts at 7-posi-tion of guanine are generally not promutagenic [102]. Secondarily formed apurinicsites and ring-opened products on the other hand are potentially mutagenic lesions[103].

Most epoxide-alkylation in DNA has been shown to take place at 7-guanine and3-adenine, leading to potentially mutagenic apurinic sites. The 7-guanine and3-adenine adducts are expected to result in GC�TA and AT�TA transversions,respectively, since DNA polymerase preferentially adds an adenine opposite to anapurinic site [103]. Such mutations have indeed been found in SO-treated hypoxan-thine-guanine phosphoribosyl transferase (hprt) mutant clones [104] and in PO-treated Salmonella hisG46 and hisG428 [105]. AT�TA transversions have also beenidentified at the hprt locus in mice splenic T cells exposed to BD, whereas exposureto BMO and DEB produced more GC�TA transversions [106]. It has been shownthat in genomic DNA the steady state of apurinic/apyrimidic sites is �1 lesion per105 nucleotides [107]. In humans, the adduct levels induced by the epoxides studiedcould be expected in level up to few adducts per 108 nucleotides and in experimentalanimals few adducts per 106 nucleotides. Therefore, the mutagenic role of theapurinic sites originating from the 7-guanine or 3-adenine adducts induced by theseepoxides can be considered rather small, especially because the apurinic/apyrimidicsites are constantly being repaired.

Even though formed to lower extent, substitution at a base-pairing sites of DNAcan be expected to be more mutagenic as compared to the 3-adenine or 7-guanineadducts. The dominating type of SO-induced hprt-mutation was the AT�GCtransition [104] and short term animal studies on BD have shown the mutations atthe AT base pairs to be the predominant ones [108–111]. These mutations are likelyrelated to 1- or N6-alkylation of adenine residues. The AT�GC transition wasobserved in a site-specific mutation study in which a SO adduct at N6-adenine wasinserted in N-ras gene codon 61 [112]. However, the N6-adenine adduct showed arather low miscoding potential [112], probably because the adduct has still thepossibility for base-pairing with thymine residues. The same transition was alsoobserved in a study by Carmical et al. [113] where RR enantiomer of BDE wasinserted at the N6-position of adenine within the N-ras codon 61. Interestingly, thecorresponding SS enantiomer yielded exclusively AT�CG mutations [113]. Itappears that the N6-adenine adducts are not responsible for the mutagenesisassociated with the exposure BD or styrene metabolites. More likely mutageniccandidates are the 1-adenine adducts, or the corresponding deaminated 1-hypoxan-thine adducts [61], since they occupy a central Watson–Crick base pairing sitedisrupting the normal hydrogen-bonding.

In the case of the BD metabolites, DEB is 100-fold more mutagenic than BMOand is probably involved in the BD-induced carcinogenicity [114]. This might be


related to the cross-linking ability of DEB [115,116], or to the high levels EBDadducts originating from DEB. N2-guanine adducts of BMO and BDE were foundto be an order of magnitude more mutagenic than the N6-adenine adducts in recenta site-directed mutagenesis study [117]. The different relative mutagenic potency ofvarious stereoisomers was evident, but no distinct mutational signature of theN2-guanine adducts were observed.

Deamination of 3-cytosine adducts may also have a marked role in the mutage-nesis induced by the epoxides studied. For propylene oxide it has been suggestedthat 3-HP-uracil is a mutagenic lesion [118] leading to GC�AT and to minorextent GC�TA and GC�CG mutations [119]. Recently it was shown that usingprotein extracts from mammalian cells that enzymatic repair exists for removal of3-HP-dCyd but not for the corresponding uracil adduct, and that the uracilglycosylase is not working on the adducts [38]. Furthermore, a mutagenicity studyapplying a site-specific incorporation of a 3-hydroxyethyluridine adduct in a55-nucleotide template suggested that the adduct may be a critical mutagenic lesioninduced by EO, leading to GC�AT and GC�TA mutations [120].

7. Conclusions

7-Guanine and 1-adenine adducts could be useful as biomarkers of exposure tothe studied epoxides. The major advantage of 7-substituted guanines is their highconcentration relative to the concentration of other adducts formed. But because oftheir lower mutagenicity they can be mainly used as a surrogate marker for otherpromutagenic adducts. Although the levels of 1-substituted adenines in DNA arelower than that of 7-substituted guanines, they represent a feasible alternative dueto persistence in vivo (shown for propylene oxide) and the high specificity andsensitivity of 32P-postlabelling analysis. Further, the 1-adenine adducts are interest-ing since they appear to have an important role in mutagenicity of the epoxides.Another lesion by the epoxides that should be considered in the adduct studies invivo is the 3-substituted uracil. This is mainly because of its persistence and obviousmutagenicity.

Acknowledgements

Thanks are due to Professor Kari Hemminki for critical reading of themanuscript. The work was supported by the Swedish Council for Work LifeResearch and by the European Communities, QLK4-1999-01368.

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specific dna adducts induced by some mono-substitued epoxides in vitro and in vivo

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