rf FAOIUNEPIUSSR

nternationai TraIning Course 'TRAlNINO ACTIVJTIES ON FOOD CONTAMINATION CONTROL -

AND MONITORING WITH SPECIAL REFERENCE TO MYCO1OXINS

L. V. KRAVCHENKO

AFLATOXINS AND THEIR BIOLOGICAL

ACTIVITY

Centre of International Projects, GKNT

Moscow, 1984

AFLATOXINS AND T1lIR BIOLOGICAL AC1IVITY

L. V • CRAVCHEM(0

The group of aflatoxine (secondary metaboittea of micro-

soopie fungi of the Aepergillus genus)includea more than 10

compounds of similar chemical structure and biological, action.

It in mostly aflatoxins 81, B, 1' and 02 that are found in

natural conditions as oontaminante of food products and feed..

Out of four main representatives of eflatoxine, B 1 is most to-

xic and, as a rule, U is ayntheeized in the largest amounts.

The results of studying aflatoxine over 20 years mince

their discovery have shown that most mammals (including pri-

uiatee), birds, some species of fish, insects, and aicroorga-

nines are susceptible, in verioua degrees, to the toxic action

of efistoxins.

g.tsboliea of aflatoxin.

The alimentary pathway is the main form of entry of nfl.-

toxins in the organism, the principal and, in most of cases,

the only damaged organ being the liver.

Aflatoxin B 1 is found in the liver of rats as early as

30 minutes after being administered, and its concentration in

liver reaches the maximum level within two hours. After single

intraperitonsal administration, approximately 20% of labelled

toxin is retain.d in the organism of rats within 24 hourej the

highest concentration of the toxin is found in the liver. Si-

ailer re ults were obtained in experiments on mice, hamsters,

sheep, pigs, and poultry.

gxperiiente with labelled nflatoxin B 1 have shown that

I-I

-2- the main pathway of withdrawal of the toxin (in an unaltered

form or as a etabolite) Is its excretion with bile and urine,

and only a small anount is isolated with the exhaled air in

the form of CO2 . Most of investigators, studying the rate of

5etBp,olizRtiOfl of aflatoxine in different species of animals,

have found that the half-life of aflatoxin B 1 in the organism

is 12-15 hours.

Fxperiaier,te conducted in recent years, both in vitro, and

in vivo, have shown that afletoxins are metabolized by the ma-me enzymatic systems as other xenobiotice. Aflatoxin B may

be subject to hydroxylation by zircosomal oxidaaee with a mi-

xed function o give lees toxic metabolites -- aflatoxins

Q 1 1 and P 1 . Af].atoxin M 1 , in many species of animals, is one

of the main metabolitee which are found in milk and urine.Tkiva,

it has been detected in milk of cows, sheep, and goats which

had consumed feed contaminated with aflatoxin B 1 ,

The second possible pathway of detoxication of aflatoxin

B 1 in the organism is the reduction of cyclopentenone to afla-

toxicol with the participation of soluble cytosol dehydrogena-

sea. This reaction is revereable and therefore many authors

consider aflatoxicol as a "reserve" toni of aflatoxin B the cell.

Finally, atlatoxin B 1 , with the engagement of the eaae

enzymatic ayotea of the liver aicrosoisea, can be "activated"

i.e. it can be turned into compounds with a more pronounced

toxicity. It is supposed that one of such active forms of afla-

toxin B Is its hesiacetal-aflatoxth B2.,the other Corm being

ito 2,3-epoxide. It is also believed that epoxidation affecte '-4

-3-

the double bond of the terminal furane ring of the molecules

of the most toxic representatives of the 8flatoxin family--

aflatoxins B 1 , G1. and M 1 , whereas aflatoxinu B 2 and 02 whose

molecules do not have that double bond, possess a ich lower

biological activity. It should be emphasized that acute toxic

effect (aflatoxin B 25 ) and carcinogenic activity (2,3-epoxide

of aflatoxin Bi) of aflatoxins are primarily aesociatad with

these ctiveCmataboljtes of aflatoxin B 1 .

The 2 9 3-dihydrodiol of aflatoxin B 1 which is formed from

the epoxide, just as other derivativea of aflatoxin B 1 , may

produce in liver cello conjugates with glutathione, cysteine,

gluouronic and sulphuric acids and in this form be excreted

from the organism with bile or urine.

Biological activity of aflatoxino

Acute aflatoxicosis

Acute alimentary toxicoses aesociated with the ingestion

of contaminated feeds have been described in 1960 almost con-

currently for turkey-poulto, ducklinga, pigs, and calves. The

most susceptibie among farm animals are 3-12 weeks old piglets,

pregnant saws, and 1-6 months old calves. As for puultry, high

susceptibility to aflatoxine is found in turkey-poults and

ducklings; lees susceptible are young pheasants while chicken

are characterized by a relative resiatanca.

The leading clinical symptoma of acute intoxication with

aflatoxins is the absence of appetite, loss of body mase, and

a reduction in weight gain. It is necessary to emphasiz, a

1-2

-4- rapid development of ayiaptouia of intoxication and a high death

rate anong animal.. Aflatoxiooees in bird, are di.tingut.hed

by eymptome of the damage to the flerVOUe eyetem, in calves by

the dieruption of the funotion of the ga.tro-int..tinal tract,

in pigs and dogs by the development of Jaundice. Characteristic

symptoms of aoute intoxication are multiple haesorrhagio and

old ema8.

Aflatoxine are hepatotropto toxin., the target organ in

all species of animals being the liver. Aflatoxina oauee dif-

ferently expressed and differently localized neoroses of the

liver parenchyma and also adipoge and albuminoue degeneration

of hepatocytes. A oharacteriatio feature of the cotton of afla-

toxins is rapid proliferation of the epitheliva of biliary

duct..

Table 1 sums up eome data on the action of aflatoxin-

contaminated f..d on farm sni.male and poultry.

Changes in the liver, .iailar to those observed in tarn

animal., are found in experimental condition, in most of test

animal,. The LD50 values for some species are shown in Table 2.

Depending on the susceptibility to aflatoxin B 1 , the animali

may be aorted into three groupsa 1) very eu.ceptible for which

LD50 4 1 wg/kg; 2) suaceptible forwhtoh LD in 1-10 mg/kg;

and 3) re.istant for which LI)50> 10 mg/kg.

It should be noted that aflotoxin B 1 in most active among

aflatoxine. The relation between the toxtcitu of individual

repreaentUt ives of this group may be demonstrated on an example

of LD50 values for one-day old dicklirigs, which are 0.36, 1.70,

0.18, and 2.83 mg/kg, for aflatoxin. B, B, G I and 02' zeap.o-

-5- lively. LD of aflatxtn B1 for rots of the Fischer line in

1.16 .g/kg,a*d that of aflatoxin 01 is 1.5-2.0 !*g/kg; aflato-

zinc B2 and 0 2 are weakly toxic at doses in excess of 200 mg/kr

Interaction on the toxic action of aflatoxina upon prian-

te• is of definite interest. It has been danonetrated in •xp-

rla.nts on Theses aonk.y* and czab.atsre that aflatoxin Si

in doses ranging from 62 1gAg of the body mans to 5 rr4/kg

induses changes in the liver which are cbaiaotertstic of afla-

toxicosis and rapid (at high doe..) death of animals. A ope-

del syndrome ha* bien d..cribsd in young 9acnca faac1culari

f.aale, otter the int.rnal ad.tni.trs*ion of aflatoxin

in a dose of 0.5 or 1.5 mg/kg. The characteristic clinical

symptoms were aaughtaig, vomiting, diarrhea, and coma. The

changes in liver included osetrilobular neorosin, moderate

proliferation of btliarj, ducts, and nasal?, adipose degenera-

tion which was also observed in the heart and kidneys. Osdema

of the brain and the degen.ret ive changes of nary, cells were

also observed. Soma of these change, were sintlar to symptoms

noted in chtldsn who suffered from Beja's syndrome which will

be described later on. Thug, the esnaitivity of monkeys to

acute action of aflatoxins in indubitable.

As seen from rable 1 and 2. there are con.id.rable inter-

specific differ.ncea in eusc.ptibtltty to e.flatxina. The rea-

son, according to anny tnvcctigatore, in due to the differences

in the rates of afletoxin metabolization between the epectea;

other authors believe it to be associated with different path-

ways of metabolism.

It is noteworthy that there are differences in euscepti-

1-3

Cattle

calves

bulls

adult oow•

: 08//day day

0.5 aflatoxin B 1] kg/day

0.7 mg/kg feed 1.0 mg/kg feed

2.0 ag/kg feed 16.0-46.0 rnpg/kg

0.6-0.9 mg/day/oow

iforses 0.015 aflatoxin B 1 kg/day

0.15 mg/kg/day 0.3 mg/kg/day

Piga mase 6.5 kg 0.62 mg/kg

mae. 20 kg 0.26 mg/kg feed 0.065 mg/kg/day

mass 22 kg

Poultry chicken

broilers

2 - 4 mg/kg feed

0.25 mg/kg feed i-i mg/kg f.ed

0.25 mg/kg feed

0.6 mg/kg feed 1.5 mg/kg feed 2.5 mg/kg feed 5 - 10 mg/kg feed

laying hens 2 - 8 mg/kg feed

20 mg/kg feed

-6-

Table I Toxic action of feeds contaminated with aflatozins on

farm animals and poultry

Animal

Doe. 1ff.ot

Drop in weight gain Drop in weight gain, ooagulopathy Coagulopathy. n.oroeee of liver, death

Drop in wght Death (59

eiday.)

gain

Drop in milk yield Deteotion of aftatoxin in milk Detection of aflatoxin in milk

Disruption of liv.r funo-tion, jaundice, death on the 37th day Ditto, death on 26th day Ditto, death on 12-15th day

Corresponds to LD 0 (internally) Growth retardation Suppression of i,mnuno-gene aim Acute toxicosis, death

Drop in weight gain Necrosis of the liver, death

3uppression of iiwnuno-genesis Drop in resistance Drop in weight gain Coagulopathy Necrosis of the liver, death Reduction in egg laying capacity Reduction in egg-laying capacity, detection of aflatoxin in eggs

'-I

-7- Table 2

Value. of LD50 of aflatoxin B 1 for some epecise of farm

and laboratory animal. (aingi. adasini.tration)

Animal LD50 , ag/kg of body mace

Duckling. 0.34-0.56

Rabbita 0.3 -0.5

Rainbow trout 0.5

Oat. 0.55

tnk 0.5 -0.6

Pig. 0.62

Dog. 1.0

Guinea pies 1.4 -2.0

3h..p 2.0

Monk.y. 2.2

Rates

newborn 0.56

w.enitnge 5.5

adult male. 7.2

adult female. 17.9

Chicken 6.5 -16.5

tc. 9.0

flasneter. 10.2

Chicken embryo. 0.025pgA,ggj

1-4

-8- bility to aflatoxina even among breede of one and the ammo

epectee. For inetance, the otudy of 18 etraine of chicken,

turkey-poulte and quiii indicated that only one breed -- New

Hampshire-- is diotinguishad by high euaceptibility to aflato-

un B 1 . A comparteon of the auoaptjbtiiti.e of the enbryoa

of different breede of hen to aflatoxin B revealed that most

remietnat to this aflatoxin are Rhode Inland enbryoe and the

leaet euoceptible are the enbryoe of the White I'iymouthrock

breed.

In vitro etudiem of different oyeteae have greatly faci-

litated the elucidation of the btoloical activity of efiato-

xina. Thum, toxic propertlee of aflatoxina have been proved in

relation to culturee of chick embryo lIver, baby rut liver,

lunje, and kidneya, calf and monkey kidneya,human liver, eta.

In theae eyatemo, the activity of aflatoxin 8 I was damonatrat-

ad at a done ranging from 0.01 to 10 jig/mi. Ito toxicity was

exprenoed in the changes in the morphology of cultivated cells

and in the dieruption of their functional activitys auppreasion

of the myntheota of nucleic ooida and protein.

The information about high eumoeptibUtty of tienue oalle

of man --liver, lunge, blood cello, and akin fibrobleete --to

aflatoxing is of intereet. Along with eflatoxin B 1 , toxic cotton

on the celia of hunan embryo liver is exerted by aflatoxia.

20 a 2 .and Gi. Numeroua obeervatiOne and experimento have indicated

that afletoxina are atrong immunodepreasante, which woetly

affect cellular immunity. The P ayetem of cellular immunity

is diotinguiehed by extremely hig eunceptibility to aflatoxine '

- 9- 9 1 and m i . Afletoxine also influence the meohanieme df non-

.p.oifio r..ietance of the orantme - the nyntheete of some

freotione of the ooepl.ment. the production of interferon.

etc.

Chronic afluitoxtoocie

Chronic intoxication with aflatoxins entails the develop-

meat of malignant tuui3ure of the liver. At prenent, aflatoxinu,

primarily eflatoxin B, are damned with the ntron.et chemical

ceneirog.na.

When adsinietered internally, aflatoxine induce hepatomna

in all thus far studied epeciee of animals, mp.cificeuly in

rat, of the Pincher, Wistar, and Porton eli-nine, ducklinpm,

chicken, rainbow trout, malson, guppi.e, pole cat., doge and

monkeys.

A linsar depndence of the frequency of h.patocellular

cercinoase upon the do.s of afla toxin BI in the ret ion was ob-

ssrv.d in rate of the Pincher strain: at aflatoxin concentra-

tion of 1 pg/kg the fr.qu.ncy of tumoure was 10%, at a conoen-

tretion of 100 pg/kg it was 100% (?abl. 3). The frequency mdi-

cen of cancer in the lifetim, of rate, theoretically calculat-

.d by the results of various experimental studien, were 240/10

at a concentration of aflatozin B 1 in the ration 0.1 pg/kg and

1100i105 rate at a concentration of 0.3pjkg of feed.

1-5

Tabi. 3

Dependence of carcinogenic activity of aflatoxin

in male rate of the Fischer strain upon its cont.nt

in the ration

Aflatoxin Duration of Frequency garliest time

concentration, feeding, weeks of liver of the develop-

g/kg carcinoma meat of the tumour, weeka

0 74-109 0/18 -

1 78 - 105 2/22 104

5 65-93 1/22 93

15 69-96 4/21 96

50 71 - 97 20/25 82

100 54 - 88 28/28 54

single intraperitoneal administration of aflatxin

in fewale rate at a does correepondSng to LD50 aloe led to

the development of h.pato.as in coven out of 13 rat. within

60-128 week..

Carcinogenic action of afletoxine may manifest itself

H1SO in the progenys the development of cholangiooarotaomae

has been observed in bady rate subjected to prenatal (intrau-

term.) or poet-natal (through mother's milk) exposure to afia-

toxin Da

There are reports about the development of tumour, out-

cide the liver as a reault of the attni.tratiOn of aflatoxin

carcinoama of the stomach, ad.nocaroinomas of the large

-11- intestine, kidnwys and lungs, tumoure or the salivary glands,

tDngue, and oeaophagus, The cases of the development of ear-

comes in the place of subcutaneous edainiatration of eflato-

un are also described.

0arcinogentc properties of other aflatoxine --B 20 0 1,

020 M i are lees pronounced. When afletoxin 01 was administered

to rete with dringkng water it induced not only tunoure of the

liver but also tumoure of the kidneys. Aflatoxin B2 in a total

do.s of 150 ag per animal induced hepatocellular carcinomas in

three out of nine rate within of 57-59 weeka. In the name as

rice of experiments, aflatoxin Bi administered in a done of

1.3 ag per animal induced hepatomas in 9 out of 9 rate within

46 weeks. These results indicate that the effective done of

afletoxin B2 in 115 times higher than the dose of qflatoxtn B 1

giving ris, to hapatoinae in ret..

Unlike rats, nice manifest wall-pronounced resistance to

the carcinogenic action of aflatoxine administered internally.

Prolonged consumption of aflatoxin Bi by aloe at a concentra-

tion up to 1000 pg/kg of the feed failed to induce any tum3urs.

At the seas time when aflatoxtn Bi was intraperitoneally admi-

nistered to baby mice in a doe, of 1,25 pg/kg during the first

7 days of life or in a dose of 6 pg/kg during three day., h.pa-

tome, were found within 80 weeks (Table 4).

Rainbow trout I. known for high susceptibility to aflato-

xinn. Th. inclusion of aflatoxin B 1 into its feed at a rate of

0.1 pg/kg induc.i the development of hepatoaae withIn 20 month..

Aflatoxi'i K 1 manifests Itself an a weaker hapatocaroinogen in

experiments i4th trout (Table 5).

1-6

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-13..

Table 5

H.pstocsroinog.n.city of aflatoxin 9 in rainbow

trout as ooapor.d to aflatoxin

Aflatoxin Liver tumzr frequence

1v.l in males females the ration,

4.0 4/28 (14%) 13/27 (48%)

16.0 22/21 (81%) 11/14 (79%)

i 32.0 24/25 (96%) 13/14 (93%)

K I 64.0 21/24 (88%) 9/10 (90%)

5 14.0 15/22 (68%) 18/23 (78%)

For a long time (up to 1971) primate, were believed to

be r..iutant to carcinogenic action of aflatozina. It was

only in 1972 that Gopsian and collaborators publiuhed a report

about the development of hepatooellular carcinoma in a male

rh.aus monkey which had been fed with partially purified afla-

toxin. B, and 01 for 5.5 y.ar.j the carcinoma was detected 8

years aft.r the comeenoement of the experiment. Purtheron,

the.ei re.ults were repeatedly confirmed by other authore. The

h.pathocarcinog.nio •ffect of aflatozine 8I and of a mixture

of aflatoxin. B 1 • 82 + 01 + 02 ha-s been deiaonatrated for

marmoaete and tupatae of both aexea. It ahould be nted that

attempts at determining the doac dependence of the effect have

not been undertaken for prinatem.

I-?

-14- Teratogenic and nutagento action of afintoxins

Teratogenic.properti.s of aflatoxin Bi have been deter-

mined in experimental conditions for hamsters, rate, mice,

chicken, and Jepaname smooth snake (Ory2iue latipee).

Atlatoxin administered to hamsters on the 8th day of pre-

gnanoy induced malformations in 29.4% of tootua.e. The add-

nietration of aflatoxin B 1 at a concentration of only 1.0

g/kg to female wiatar rate every other day for the firet 14

days of pregnancy entailed the death of 1% of embryos, resorp-

tion of 6.8% of embryoe, while 3.5% of fetuses had various mal-

formations of developments iicrooephalia, hernias, bradideoty-

lies.

In 11.5% of mice embryos exposed to aflatoxin B 1 , at the

8th day of intrauterine development various malformations were

observeds brain herniae, anomalies of the gastrointestinal

tract.

The administration of aflatoxin B 1 to the yolk sack of hen

eggs at the 6th day of incubation entailed the development of

malformations in 65-90% (depending on the doss) of embryo..

Aflatoxin 81 induces ohro.osoaal aberration, and breakups

of the DNA in plant and animal cell.. It has been likewise de-

monstrated that it produces mutationa of genes in bacterial

test-systems (the Ames test) after mstaboliaatton ("motivation")

by mioromomal preparations from the rat or buman liver.

Studies of recent years have demonstrated that autagenio

and txigenio propertise of the precursors of aflatoxin B 1

(during its biosynthesis) increase as their structure becomes

more complex and reach their peak in aflatoxin Hi. The dipsat-

-15-

.no. of mutagento prop.rtise of aflatoxin B 1 upon the presence of the laotons ring in its structure has been demonstrated.

Factors. influencing the biological activity

of aflatoxin.

A lerge amount of data nocumulated till now demonstrates

the possibility of modification of toxic, oaroinogenio, and

other manifeetationi of the biological activity of aflatoxine

within broad limit, by using different factors.

The toxic action of aflatoxine denda considerably upon

the age and sex of animale. The common feature for all species

is a decrease in their susceptibility be aflatozin.e with age.

A. was seen in Table 2, LD50 for newborn rate is almost 1/10

of that for weaned rate and is 1/13 of that for the adult male

rat..

Numerous studies have demonstrated that females are more

resistant both to the acute toxic and to the carcinogenia ac-

tion of atlatoxinu compared to males. Table 2 also shows that

adult mal, rats are approximately 2.5 times more susceptible

to aflatoxin B 1 than female. (LD50 7.2 and 17.9 mg/kg of body

mass, reepectively). It is not without interest that when afla-

toxin B 1 I. included in the ration, of rate of both sexes, the

frequency of pre-cancer changes in the liver is practically

the same for male, and females, but the period between the

appearance of these change, and the development of heatic

carcinoma in females in much longer than in males. Table 6

gives the estimated figures of the total amounts of aflatoxin

later I the organism of animals (male and female rats) in

the period preceding the appearance of the liver tumoure.

1-8

-16- Table 6

The dependence of carcinogenic activity of aflatoxin

upon eax of rate

Concentration Total dome of afla- Time of detection of the of eflatoxin Bi toxin B 1 , mg/kg liver tumoure, daym mg/kg of the anital

ration malem femalea malea fernalem

1.0 2.9 5.9 245 448

0.015 0.095 0.115 476 560

There are all grounde to believe that the revealed sex

distinctions in the eunceptibility of animals to aflatoxine

are determined by differencee in the hormonal background. The

adininiatration of diethyletilbeetrol concurrently with afla-

toxin 81 to male rate entatle coneiderable lowering in the

frequency of liver tumoura (8 out of 40 as against 25 out of

35 in the control). The development of tunoura in the liver

in mal, rate under the influence of aYlatoxin Bi was prev.nted

by a preliminary hypophyeotomy and also by oaatration of the

animala. The adminiatrntton of teetoeteron along with afletoxin

to omatrated rata entailed death of all experimental animale.

There is special interest in the information about the

influence of the factore of nutrition upon the biological ac-

tivity of tiflmtoxine. It has been demonstrated that under the

conditions of protein inaufficienoy the frequenoy of tumou.ra

of the liver under the action of aflatoxin 81 is much higher

than against the background of full-value nutrition and the •

tine of the development of tumours is conniderably ieee.

-17- Thu., in rate which were fed on rations containing 9% of pro-

tein, tumoure of the liver developed in 11 out of 15 animals

after the passage of 8 months whereas in rate which consumed

ratione with 22% of protein tumours developed in 7 out of 14

animal. within 10 months . In the case of protein insufficien-

cy, the acute toxic effect of aflatoxina is also more pronoun-

ced. At the same time there is a different information mdi-

onting a decrease in the carcinogenic effect of aflatoxine un-

der the conditions of protein deficiency in the rations. It

was shown that not only protein insufficiency but also the de-

ficiency of certain amino acids (tryptophan, specifically) may

aggravate aflatoxtoosie.

Speaking of other alimentary factors capable of modifying

the biological activity of aflatoxins, we should single out

lipotropic agents, some vitamins, and mioroelenants. The in-

sufficiency of .ethionine and choline in the rations has a

protective action in relation to the acute toxic effect of

aflatoxin B 1 , but reliably intensifies its carcinogenic acti-

vity in experiments on rate. At the e&ne ttae, a more pronounc-

ed deficiency of these lipotropto substances in the rations

rather decreases, than increases, the frequency of hapatocarci-

no.ae in rate which were given aflatoxin Bi. It should be lIk,e-

wise etr.seed that the effeot of lipotropto substance, which

modify carcinogenic activity depends largely upon the amount

and quality of fats in the ration of experimental animal..

The final biological effect of aflatoxine is strongly

influenced by the availability of vitamins. For instance, the

defl.cency of vitamin. A and C produces inhibition of the mete-

-18-

boltam of afletoxin B 1 . Som authors etrese that whenever the-

re is a deficiency of vitamin A, there is a greater frequency

of carcinomas of the large intestin, in rate.

Pronounced protective action in relation to aflatoxina

has been d.aonetrat.d by selenium and copper added at defini-

te oonc.ntratione to the rations of rate.

inally, attention should be drawn to the activation of

inicrosoinal oxidases with a mixed function under the influence

of ethanol which may intensify the formation of the waotiv.

form of aflatoxin B 1 its 2,3-spoxide-- and increase thereby

the hepatocarcJnogenio activity of atlatoxin B 1 .

It seems that most of factors modifying the aflatoxin

toxicity act by changing the activity of enzymatic systems

which metabolize aflatoxins. This supposition is confirmed by

the results of study of the influenc, of inductor. and Inhibi-

tors of rnicroeoaal oxidaaee with a mixed function upon the

biological activity of aflatoxins. Thus, when test animals

are given phenobarbital which inducee the activity of oxidas,.,

the toxic effect of aflatoxin B 1 decreases which manifests it-

self in the reduction of the inhibiting action of the toxin on

the iyntheaie of the protein, the prevention of the liver nec-

roses, the weakening of aflatoxin's carcinogenic properties.

At the same time, the administration of an inhibitor of microso-

mel oxidasee (5KP525A) was accompanied by an intensification

of the damaging action of afletoxin B1 upon the liver.

Thus, the results of eudes of aflatoxicosee in farm ani

male and the experimental findings enable us to class aflato-

xina with most potent hepatotoxic and hepatocarciaogenic to-

-19- xins. It should be stressed that the extent of biological acti-

vity of these microtoxins depede creatly upon the species

of animal., their age, sex, and the nature or nutrition.

Action of aflatoxina on man

Clinical observations

Since eflatoxine were found to possess strong hepatotdxio.

and hepatooaroinogenio propert tee, the relatively high frequen-

cy and level of the contamination of food products with atm-

toxins and the abundance of aflatoxin producer in natural con-

dit tons make us to olaee these aiorotoxine with biological pol-

lutants of the environment which are potentially dangerous for

man's health.

cut, aflatoxicoees in humane are rare and are associated

with high concentrations of aflatoxine in food (ranging from

0.2 to several mg/kg). All cases of poisonings occurred in

countries distinguished by a high level of contaia.nation of

food products with aflatoxina (Table 7). The sicknee of child-

ren in Senegal was caused by peanut flour containing aflatoxin

at a concentration of up to 1.0 mg/kg. In India, children in

the ae group from 1.5 to 5.0 years when treated for the e'n-

droms of protein insufficiency - kwashiorkor—were given pea-

mid flour which contained aflatoxin B 1 at a concentration of

0.3 mg/kg. The average daily dose of the toxin in this case

was 1.1 /kg of body mess. In on. of described case., the

liver of a 15 year-old boy which died of acute hepatitis de-

monstrated changes which are obaraotertotiâ of aflatoxiconia.

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-21-

The diaeaee was caused by maniac containing aflatoxin B,at a

concentration of 1.7 mg/kg.

A convincing instance of the association of aflatoxine

with acute hepatitis in people was the outbreak of toxic hepa-

titis in the North-West districts of India in 1974. The disea-

se was characterized by a subacute commencement with a fever an

a subsequent rapid development of jaundice (98% of cases) and

aecttis. The patients demonstrated hepatoeplenomengalia. Liver

section obtained by biopsy and autopsy demonstrated a charao-

t.riatio proliferation of biliary ducts. The death rate was

very high. The analysis of food products indicated that the

cause of the dioeaee aen maize which contained aflatoxin 8 1

at a concentration of up to 15.6 pg/kg. with this level of

contamination, the daily ingestion of aflatoxin ranged from

2 to 6 mg/per person which corresponds to daily doses of up

to 120g/kg of body mass.

Special mention should be made of information about the

influence of aflatoxin upon man in industry. In one of the

instance, 7 people out of 55 who were angaged in the process-

ing of peanuts and other oil-bearing crops, demonstrated the

development of cancer of varying localization (the period of

observation was 11 y.ars, the time of exposure was 2-3 years).

The concentration of aflatoxins in air in this case could have

been in a range of 0.87 to 72 ig/a3. Two oases of pulnonary

adenoizatosie with a lethal outoome have been described in pe-

ople handling Brazilian peanuts. Changes characteriutio of the

action of aflatoxin B 1 were found in their lungs. Finally, a

report is known about finding carcinoma of the large inteutine

-22- in two reeearch workers who for a nuaber of years were engaged

in the isolation and purification of afletoxine.

The study of patients suffering of cirrhosis of the liver,

residing in the regions of Iran where this disease is believed

to be frequent, revealed the presence of ntis toxin 9 in the

urine of six out of 26 patients. Aflatoxin was never found in

the urine of patients with other diagnoses. The patients came

to the clinic from villages which were noted for a high con-

centration of aflatoxin 9 in milk.

The poesible relation of Rays's syndrome with the oontamt-

nhtion of food by eflatoxtha is still debatable. Out of four

countries where this relationship was studied (Thailand, New

Zealand, USA, and Czechoslovakia) and where aflatoxin B 1 had

been found in the liver of patients, only Thailand is situated

in an area with a high frequency of contamination of food with

aflatoxime. The three following reporte are noteworthy.

In Thailand, the autopsy of 23 children deceased as a

result of Rays's syndrome revealed considerable amounts of

aflatoxin B 1 (up to 93/kg) in the liver, in the content of

the stomach and the intestines (up to 127)og/kg), and in bill.

Traces of aflatoxin B 1 have been also detected in other tis-

sues-brain, kidneys- and in the urine.

Reports from Czechoslovakia present results of clinical

observation of 27 children in tte age group from 3 days to 8

years who also died of Reye'F syndrome. The disease was cha-

racterized by an acute oneet. In some cases, brain ey.ptoas

prevailed and the disease lasted from 2 to 3 months. In suba-

cute canes, periportal fibrosis and the oroitferation of the

bile ducts were observed in the liver: when the disease con-

-23- ttnu.d for 4 month., there were syaptoma of cirrhosis. In all

cases, aflatoxin B 1 was found in liv.r tissues at a concentra-

tion of from 20 to 2760 /kg; in three paces aflatoxin U 1

was also found.

In 1979 Rajan and collaborators published results of an

anaijais of aflatoxine in the liver of 8 children with Raye'a

syndrome. In six cases, the concentration of aflatoxin 21

ranged from 2.23 to 17.33g/kg of the tissue. In two children 0

during the acute stage of the disease, the aflatoxin was found

also in blood at a concentration of 11.93 and 31.3 mg/aL There

are reports of other investigators about cases when aflatoxin

B 1 was found in blood serum of patients suffering from Reye's

syndrome.

Thus, available inforinatin makes it possible to conclude

that aflatoxins may play a definite part in the development of

Rays's syndrome in humans in some areas. At the same time we

cannot exclude the possibility that the pathological changes

peculiar to Rays's syndrome, may land to the disruption of

metabolism and elimination of aflatoxis, thereby retaining

them in Ih. organism.

Bpideniological studies

The results of studying a possible correlation between

the level of contamination of food products with aflatoxthe

and the frequency of primary cancer of the liver inhumans

are of considerable interest.

Primary cancer of the liver in guropsan countries compri-

ses 1.2 per cant of all cancer cases, in USIA 2.5-2.8 per cent,

in African countries 14%. The highest frequency of this disease

-24- in obnerved in Bantu male, in South Africa (Moznmbtque) up to

68-77% of all eancr case..

Epide,iologtcal studien indicate a correlation between the

level of daily ingestin of aflatoxine and the frequency of pri-

mai'y ennoer of the liver in some pert, of Ic.nya, Mosambiqus,

Swaziland, and Thailand (Table 8).

Most interesting are the date about the age distribution

of the frequency of prihery cancer of the liver among the p0-

pulatian of Nozaabique. It is noteworthy thr.t in areas with

a high frequency of this dinease the peak In found in the early

age period - 20-29 year., whereas in areas with a low frequency

of primary cancer of the liver, the morbidity increased with

age gradually. This pronounced "juvenation" of prinary cancer

of the liver may be explained by a greater susceptibility of a

growing organism to the carcinogenic action of aflatoxine.

Some authors believe that the viru, of hepatitie B which

in widely spread in countries with a high incidence of primary

cancer of the liver say also act as a cofactor in the etiology

of this disease.

In studies undertaken in Indoneeta on 71 patients euffer-

ing of primary cancer of the liver, aflatoxine were found in

biopsy samples of the liver in 57.7 case.. Anatnnesttc data in-

dicatod the consumption of contaminated food products, includ-

ing a prolonged daily consumption of peanuts. Aflatoxin B 1 was

found in eeapiee of food products at concentrations ranging

from 17 to 1190 pg/kg while afla toxin G, was found at concen-

trations ranging from 5 to 630 , g/kg.

In the United States, we know of a case when aflatoxin B1

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-26- at a ooneentratton of 520g/kg of wet weight was found in

the lifer ttseu• of a patient suffering of carcinoma of the

rectum and liver.

Published epideiologioal studies are limited in volume

end beidee, they evaluate but one of the possible etiol3gic-

.1 factorej the influence of aflatoxine. There are no doubts

that other factors • such as malnutrition, viruses, other my-

cotoxine, plant alkaloids, tielminthiasee, may also play an

etiological or a modifying part in the development of cancer

of the liver.

Conclusion

Thus, the observations of aliientary aflatoxicosee among

farn animals in many countries and the results of numerous

experimental studies demonstrated that alfetoxins are highly

toxic compounds which affect mainly the liver. Acute aflatoxi-

cosia to characterized by the development of necroees of h.pato-

cytea and the proliferation of the biliary ducts; chronic into-

xication may entail cirrhosis of the liver and the dsvelpment

of hops tomas.

Afletoxin B 1 induces chromosomal aberrations and ruptu-

res of the DNA in plant and animal cells, and in some baot.-

riml teat systems - gene mutations after activation with 'Uc-

rosoniel enzymatic systems. High concentrations of afletozias

have a teratoganio action upon some species.

The biological activity o' aflatoxins is derendent on the

age and sex of aniiale. The nature of nutrition, and priaarily

the amounts of protein, lipotropic substances, and some vita-

-27-

mina, may materially modify the course of aflatoxicoses.

A correlation has been found between the level of afla-

toxin B 1 arriving with food and the incidence of cancer of

the liver in man, in areas with high frequency and high level

of contamination of food produots with atlatoxine and high

frequency of the primary onnoer of the liver.

At the macne time, the number of trends in the study of

biologial aotivity of aflatoxine call for further develop-

ment. It is necessary to have a more detailed information about

the abaorpt ion of nflatoxina in the gastrointestinal tract and

about the rate of tJeir withdrawal from tissues. We find it

important to study the modify!ng role of alimentary factore

and of other biologically active substances in the manifesta-

tion of the toxicity of aflatoxina. This aspect should be ta-

ken into ooneideration also in epiderniological studies related

to the connection between primary oanoer of the liver in man

and intoxication with low concentrations of aflatoxins.

The suppooition about the casual relation between the

consumption of aflatoxina and primary Cancer of the liver in

man and also about the role of eflatoxins in the developaent

if Reye'e eundroae requires a further substantiation.

28

1. Aflatoxin. ScL.ntifio bcckgzound, control, and inpli.stioaa.

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Assoojation b.tws.n aflatozin contact of food and h.pato.n

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Anla I., 8bj*asl. K., 8r.entvanaaurthy V., JaysrsJ A.?.,

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3. 1230 flHK BHHHTFI