structure-activity relationships in toxicity and...

[CANCER RESEARCH 31, 1936-1942, December 19711

Structure-Activity Relationships in Toxicity andCarcinogenicity of Aflatoxins and Analogs1

G. N. Wogan, G. S. Edwards,2 and P. M. Newberne

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

SUMMARY

Aflatoxins B! and G,, in single doses, were lethal and hadsimilar relative potencies in the duckling (50% lethal dose,0.73 mg/kg versus 1.18 mg/kg) and in the rat (50% lethal dose,1.16 mg/kg versus 2 to 4 mg/kg). Aflatoxins B2 and G2 wereless potent in the duckling (50% lethal dose, 1.76 mg/kg versus2.83 mg/kg) and were nontoxic to rats at doses of 200 mg/kg.

Aflatoxin BI induced hepatocellular carcinomas in ratsdosed by stomach tube, the optimum carcinogenic regimenbeing 1.5 mg/rat given in 40 equal doses over 8 weeks. Withthis dosing regimen, aflatoxin GÃŒwas carcinogenic to rat liverat doses of 2.0 and 1.4 mg/animal and also inducedadenocarcinomas of the kidneys in 4/26 rats.

When given by i.p. injection, aflatoxin B2 at a total dose of150 mg/rat (40 equal doses over 8 weeks) inducedhepatocellular carcinomas in 4/9 rats. Aflatoxin BI dosedaccording to a comparable regimen induced liver tumors in 9/9animals at a total dose of 1.3 mg per rat.

Multiple s.c. injection of aflatoxin B, resulted in sarcomasat the injection site in 9/9 rats within 44 to 58 weeks after atotal dose of 0.4 mg/rat over 20 weeks. Aflatoxin B2 at a totaldose of 12 mg/rat according to the same treatment scheduleinduced no tumors in 10 rats killed after 78 to 86 weeks.

Tetrahydrodeoxyaflatoxin B( and 3 synthetic compoundscontaining the substituted coumarin portion of the aflatoxin Bconfiguration were nontoxic and noncarcinogenic at doses 100to 200 times higher than aflatoxin B! .

Collectively, these results indicate that the furofuran moietyof the aflatoxin structure is essential for toxic andcarcinogenic activity. Moreover, the presence of the doublebond in the terminal furan ring is an important determinantof potency, particularly for acute and chronic effects in rats.The importance of the substituents on the lactone portion ofthe molecule is also illustrated by the difference in potency ofaflatoxins B] and GI in all systems studied.

'This is Paper I of a series. Paper 2, "Structure-Activity

Relationships in DNA Binding and Nuclear Effects of Aflatoxin andAnalogs," follows. Financial support was provided by National CancerInstitute Contract PH-43-62-483.

Portions of this work formed the basis of a thesis submitted toMassachusetts Institute of Technology in partial fulfillment ofrequirements for the S.M. degree by Mr. Joseph B. Fashakin, whoseassistance we acknowledge.

'Present address: Department of Cell Biology, RockefellerUniversity, New York, N. Y. 10021.

Received April 20, 197 1; accepted July 20, 1971.

INTRODUCTION

The aflatoxins comprise a useful series of compounds forstructure-activity investigations concerning mechanisms oftoxicity and carcinogenicity. We report here and in thefollowing paper results of studies on the naturally occurringand synthetic aflatoxins and analogs shown in Chart 1. Thesecompounds were compared with respect to their biochemicalproperties, toxicity to ducklings and rats, and carcinogenicityto rats.

Structure-activity relationships among naturally occurringaflatoxins have previously been investigated in several systems.Aflatoxins B!, B2, GI, and G2 have been compared withrespect to acute and subacute toxicity to ducklings (7, 23, 24).The potency of various aflatoxins has also been evaluated invitro in cultures of liver cells (22, 26) and in rat liver slices (8,9).

Available data indicate that aflatoxin B,, B2, G,, and G2were all active in the test systems studied and further thatthere was a consistent pattern of activity, the order of potencybeing B, > G] > B2 > G2. On the basis of this evidence, it hasgenerally been assumed that the same structure-activityrelationships should apply to the in vivo toxic andcarcinogenic properties of these compounds in species otherthan the duckling. Systematic investigations into the validityof this assumption have, until recently, been impeded bylimited availability of aflatoxins other than BI and G, and ofderivatives or structural analogs. We have studied the lethalityand carcinogenicity of a series of such compounds in rats andducklings and report here evidence of differences in responsebetween the 2 species and also evidence concerning structuralrequirements for activity in the rat.

MATERIALS AND METHODS

Aflatoxins BI and G! were isolated from a mixture ofaflatoxins as described previously (2). The 2 compounds werepurified by column chromatography and crystallization, andpurity was established by thin-layer chromatography andspectral properties. Aflatoxins B2 and G2 were prepared bypartial hydrogÃ©nation of BI or G! andtetrahydrodeoxyaflatoxin B, was prepared by hydrogÃ©nationto completion of aflatoxin Bj (2). Because of the necessity ofavoiding contamination of the reduced derivatives byunreacted starting materials, the products (B2, G2, andtetrahydrodeoxy BI) were subjected to repeatedchromatography and crystallization. The products used for

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Aflatoxin Toxicity and CarcinogenicPotency

9 QÃŒT

AFLATOXIN B,

OCH,

AFLATOXIN B, AFLATOXIN G,

TE TRAHYDRODEOXY

AFLATOXIN B,

CHjO"

COMPOUND "Ã• COMPOUND '8' COMPOUND "11"

Chart 1. Structures of aflatoxins and synthetic analogs.

animal studies contained very low levels of aflatoxin BÂ¡or GÃŒas determined by thin-layer chromatography. The aflatoxin B,content of the B2 or tetrahydrodeoxy BI preparations did notexceed 1 part in 6 X IO4, and the aflatoxin G i content of theG2 preparation was not greater than 1 part in 2 X IO3 .

Three synthetic compounds were also investigated. Theseproducts contained the fused coumarin-cyclopentene systemsof the aflatoxin B molecule, substituted in differentconfigurations, but did not contain the dihydrofuro- ortetrahydrofurofuran rings of the aflatoxin molecule. These 3compounds, synthesized (2) in the course of the structureelucidation of aflatoxins were: 5,7-dimethoxycyclo-pentene[c]coumarin (Compound 2);5,7-dimethoxycyclopentenone[3,2-c]coumarin (Compound8); and 5,7-dimethoxycyclopentenone[2,3-c]coumarin (Compound 11). The trivial numbers refer to designations used inthe original description.

Vehicles used for administration to animals depended uponthe solubility characteristics of each substance and the doselevel required. Aflatoxins Bt and Gt were administered insolution in spectral grade DMSO3 (Burdick and Jackson

Laboratories, Muskegon, Mich.). Aflatoxins B2 and G2 at lowdose levels were also administered in DMSO solution. Eachanimal received a total volume of 0.05 ml of DMSO per dose.At higher dose levels, test substances were suspended in SSMcontaining, per ml: NaCl, 9 mg; sodiumcarboxymethylcellulose, 5 mg; polysorbate 80, 4 jul; benzylalcohol, 9 Â¡A;water to 1 ml (Cancer Chemotherapy NationalService Center, National Cancer Institute, Bethesda, Md.).Solutions or suspensions were always prepared immediatelybefore use.

Male Fischer rats (Charles River Breeding Laboratories,Wilmington, Mass.) of either 40 to 50 or 190 to 210 g of bodyweight (28 and 60 Â±3 days of age, respectively) were used.They were housed in individual cages and fed a purified diet(25) ad libitum. Male white PÃ©kinducklings were dosedimmediately upon receipt, generally within the 1st day of age(45 to 50 g), and were maintained thereafter in conventionalheated batteries. They were fed a commercial poultry feed.Rats were dosed by i.p. injection or by gastric intubation.Ducklings were dosed by intubation into the crop before theywere allowed access to food or water.

Single-dose lethality experiments in ducklings were carriedout on aflatoxins BI , B2, G,, and G2 with a total of 60

animals per compound. Mortality was recorded over a 14-dayperiod, and LD50 values were calculated by the method ofLitchfield and Wilcoxon (15).

Quantitative determination of LDSO values in rats wasprohibited by limitations in available quantities of somecompounds. Single-dose protocols were therefore designed toestimate the relative lethal potencies of aflatoxins andstructural analogs. DMSO solutions of aflatoxin BI and G,and suspensions of aflatoxins B2 and G2 in SSM wereadministered in single doses to 190- to 210-g rats. Theremaining compounds were administered as suspensions inSSM to 40- to 50-g rats.

Further data on subacute toxicity to rats were derived frommultiple-dosing procedures used in carcinogenesis evaluationsof the compounds. Experiments to evaluate carcinogenicpotency were extensions of the multiple-dosing protocols usedearlier in studies of aflatoxin B, carcinogenesis in rats (25).Aflatoxins BI , B2, and G, and Compounds 2, 8, and 11 wereadministered to rats by stomach tube. Dosing was either on 5consecutive days per week or 3 times per week (on alternatedays), continued for various lengths of time to give differenttotal doses. Aflatoxins B! and B2 were also compared by 2other routes of dosing. In 1 experiment, they wereadministered by i.p. injection, and in another they weresuspended in trioctanoin and administered to rats by repeateds.c. dosing (twice per week for 20 weeks) in order to comparetheir abilities to induce sarcomas at the injection site. Furtherdetails of the carcinogenesis protocols are given, together withresults obtained, in the following section.

Animals killed or dying during the course of chronic dosingexperiments were subjected to systematic necropsyprocedures, and histopathological examination was conductedon all tissues. Lesions of the liver appearing during the chronicdosing experiments were classified according to the schemedescribed earlier for the development of aflatoxinhepatocarcinogenesis (.17).

RESULTS

The 1st experiment consisted of single-dose, 7-day LDSOdeterminations for aflatoxins B,, B2 , Gj, and G2 in ducklings.These values were 0.73, 1.76, 1.18, and 2.83 mg/kg,

Table 1Single-dose lethality of aflatoxins and analogs to rats

3The abbreviations used are: DMSO, dimethyl sult'oxide; SSM,steroid-suspending medium; LD5â€ž,50% lethal dose.

CompoundAflatoxin

B,Aflatoxin

B2AflatoxinG,Aflatoxin

G2TetrahydrodeoxyB,Compound

11Compound2Compound

8Dose(mg/kg)2.0-3.01.0-2.00.7-0.912-2001.0-1.51.8-2.03.0-10.0170-20032-128100-200200200Mortality

at14days6/629/460/200/200/1911/2254/540/40/60/50/50/5

DECEMBER 1971 1937



G. N. Wogan, G. S. Edwards, and P. M. Newberne

respectively. The results reproduce earlier data, in that all 4aflatoxins were lethal to ducklings, with Bj most potent,followed in order by G], B2, and G2. The maximumdifference in potency was approximately l :4 (B^Gj).

The lethality of aflatoxins and related compoundsadministered in single doses to rats is summarized in Table 1,which indicates several differences from the duckling response.Aflatoxins B, and G, were lethal to rats, B, being morepotent than G,. The LDSO value for B, was 1.16 mg/kg(95%confidence interval, 0.91 to 1.48 mg/kg), whereas the LDSOforG, was between 1.5 and 2.0 mg/kg. Aflatoxins B2 andG2,and also tetrahydrodeoxy B! , and Compounds 2, 8, and 11 allfailed to show evidence of toxicity to rats at doses about 200times greater than an effective level of aflatoxin B] in thesame assay system.

Similar evidence was obtained when mortality was recordedin the course of multiple dosing of rats during carcinogenesisexperiments. The details and results are summarized in Table2, which presents the pattern of mortality in rats dosed for 4weeks with the compounds listed. Aflatoxins B! and GÃŒwerelethal, but much larger doses of aflatoxin B2 and Compounds2, 8, and 11 caused no mortality over the same dosing period.In fact, dosing with the latter compounds was continued untileach rat had received 150 to 156 mg of these compounds'in 50

weeks and no mortality occurred. These findings emphasizefurther the lack of response to B2 in the rat.

Experiments were conducted to evaluate the relativecarcinogenic potencies of aflatoxins B,, B2, and G! andCompounds 2, 8, and 11. Aflatoxin G2 and tetrahydrodeoxyB, were not included because they could not be prepared inadequate quantity from the limited amounts of starting

Table 2Mortality in rats following multiple dosing by stomach tube with

aflatoxins or analogs

CompoundAflatoxin

B,Aflatoxin

B2Aflatoxin

G,Compound

2Compound

8Compound

11Dosing

schedule25

Mg/rat 5days/wkfor4wk50g/rat

5days/wkfor4wk3.75mg/rat on alter

nate days for 4wk50Mg/rat 5days/wkfor

4wk100ng/rat 5days/wkfor4wk1

mg/rat 3times/wkfor4wk1

mg/rat 3times/wkfor4wk1

mg/rat 3times/wkfor4 wkTotal

dose(mg/animal)0.51.052.51.02.012.012.012.0Mortalityat 4weeks0/108/100/110/104/100/100/100/10

Table 3Carcinogenicity of aflatoxins B, and GÂ¡dosed to rats by stomach tube

Liver lesions

Compoundand dosing

regimen

Sacrificetime

(weeks)Focallipid

Hyper-plasia

Transitionalcells

Hepato-ceUular

carcinoma

Aflatoxin B,25 Mgin 20 doses over 4wk (total dose, 0.5 mg)

25 Mgin 40 doses over 8wk (total dose, 1.0 mg)

37.5 Mgin 40 doses over8 wk (total dose, 1.5 mg)

AflatoxinG,650 Mgin 14 doses over2.5 wk (total dose, 0.7 mg)

25-4374

48

1420

42-58

48

1420

42-461968

3/3

3/31/3

4/4

1/2

3/3

1/21/3

2/3

3/3

3/31/32/3

1/3

7/7

18/18 (2)a

2/31/3

17/17 (7)"

100 Mgin 14 dosesover2.5wk (total dose, 1.4mg)50

Mgin 40 doses over8wk(total dose, 2.0 mg)196848142045-641/12/52/3

1/33/33/32/33/51/318/18

(6)Â°

" Indicates occurrence of metastatic lesions in the lungs.b A total of 4/26 animals dosed with aflatoxin G, had renal adenocarcinomas within 68 wk.





Table 4CarcinogenicityofaflatoxinsB, and B? in rats

Liver lesions

CompoundanddosingregimenSacrificetime(weeks)FocallipidHyper-plasiaTransitionalcellsHepato-cellularcarcinoma

Aflatoxin B,70 Mgin 9 doses over 2wk (total dose, 0.63 mg)

Aflatoxin B,50 Mgin 10 doses over 2wk (total dose, 0.5 mg)100 /ng in 10 doses over2 wk (total dose, 1.0 mg)

DMSO control50 M!in 40 doses over8 wk

Atlatoxin B,32.5 Mgin 40 doses over8 wk (total dose, 1.3 mg)

Aflatoxin B,3.75 mg in 40 doses over8 wk (total dose, 150 mg)

DMSO50 M'in 40 doses over8wk

14374975

78

6278

78

Dosed by intubation

2/21/11/1

1/1

0/8

1/4

6/9

5/7

1/4 2/4

Dosed by i.p. injection

1746

1346

57-59

59

2/2

2/9

1/3I/I3/9

9/9 (5)Â°

3/9

0/10

0 Indicates occurrence of metastatic lesions in lungs.

materials available at the time the experiments wereconducted. Specific protocols used in each case are indicatedin the tables containing the results. In general, theexperimental design was intended to evaluate both total doselevel and duration of exposure with respect to their influenceson tumor incidence and length of induction period.

Table 3 compares the incidence of hepatocellular carcinomaand preneoplastic lesions in rats dosed with aflatoxin B! orGÃŒ. Both compounds were effective in inducing liver tumors inall animals surviving the highest dose level used. Several aspectsof these findings are noteworthy. Aflatoxin B! inducedhepatocellular carcinomas when 0.5 mg/rat was given in 20doses (over 4 weeks); by 43 weeks, the only lesion present wasfocal lipid accumulation, the earliest lesion consistently foundin aflatoxin hepatocarcinogenesis. Tumors developed muchmore rapidly at higher dose levels given over a longer period.At a total dose of 1.5 mg/rat given in 40 doses over 8 weeks,fully developed hepatocellular carcinomas were present within14 weeks (i.e., 6 weeks after termination of dosing). In ourexperience, this dose and schedule represent optimumconditions for liver tumor induction by aflatoxin B! . Higherdoses caused excess mortality.

In general, the same pattern of responses was obtained withaflatoxin G]. The lowest dose used induced only preneoplasticlesions. However, 2.0 mg/rat given in 40 doses inducedpreneoplastic lesions within 4 weeks and hepatocellularcarcinomas within 45 to 64 weeks. Although this experimental

design does not permit quantitative comparison, it appearsthat aflatoxin G, is less potent than B! in itshepatocarcinogenic action.

Other tumors found in rats treated with aflatoxins B] or G]included renal adenocarcinomas that appeared in 4/26 animalsdosed with GÃŒ.Testicular interstitial cell adenomas wereobserved with equal frequency in control and treated animals,as reported earlier (25). No liver lesions suggestive ofpreneoplastic changes were observed in control rats.

Data comparing the carcinogenic effects of aflatoxins B!and B2 are summarized in Table 4. In the 1st experiment, thetoxins were administered by stomach tube. Aflatoxin B), at atotal dose of 0.63 mg/animal, induced focal lipid accumulationwithin 14 weeks, but 75 weeks were required for the inductionof hepatic tumors. Aflatoxin B2, at doses of 0.5 and 1.0mg/animal, induced parenchymal cell hyperplasia but notumors in a similar length of time. This experiment wasinitiated at a time when our supply of aflatoxin B2 was verylimited. As larger amounts became available and indications ofits low order of acute toxicity were obtained, a 2ndexperiment was carried out, and the results are also presentedin Table 4.

The objective of this experiment was to determine whetheraflatoxin B2 would exhibit carcinogenic activity at a dose level100 times greater than the carcinogenic dose of aflatoxin B, inearlier studies. In order to minimize the possible influence ofabsorption from the gastrointestinal tract, dosing was done by

DECEMBER 1971 1939



G. N. Wogan, G. S. Edwards, and P. M. Newbeme

i.p. injection. The dosing regimen was the 40-dose protocolfound to be optimal in the earlier experiment (Table 3).Aflatoxin B,, given according to this protocol and at a totaldose of 1.3 mg/animal, caused liver carcinomas by 46 weeks.Aflatoxin B2, dosed according to the same schedule to a totaldose of 150 mg/rat, induced hepatocellular carcinomas in 3/9animals after 57 weeks. An additional 5/9 animals treated withB2 had advanced preneoplastic liver lesions at that time, whileevidence of early cancer was present as early as 13 weeks ofthe study.

Aflatoxins B! and B2 were also compared with respect totheir abilities to induce local sarcomas following multiple s.c.injections to rats. Suspensions of the toxins in trioctanoinwere injected s.c. at the base of the neck; doses of 0.2 ml weregiven twice per week for 20 weeks. Aflatoxin B|, at a totaldose of 0.4 mg/rat, induced tumors in 9/9 animals within 58weeks. Aflatoxin B2. at a total dose of 12 mg/rat. or thevehicle alone failed to induce tumors in 20 rats surviving to 78to 86 weeks. Thus, aflatoxin B2 showed no evidence ofcarcinogenic activity under these conditions.

In a final experiment, effects of chronic treatment withlarge doses of Compounds 2, 8, and 11 were evaluated. Thecompounds were administered as suspensions in SSM bystomach tube 3 times per week, at dose levels of 1.0mg/animal per dose. Treatment was continued for 52 weeks,each animal receiving a total of 156 mg of test compound.Results indicate that none of these 3 substances is carcinogenicto the liver under these conditions. The only positive findingsafter 72 to 87 weeks were mild focal lipid accumulationpresent in livers of 8/8 rats treated with Compound 2 and 1/9rats treated with Compound 8. Compound 11, which isstructurally most similar to aflatoxin B), caused no apparenteffects in 9 animals surviving for 74 weeks. No gross ormicroscopic lesions were observed in these rats or in 9 animalsdosed with the suspending vehicle alone.

DISCUSSION

Results of these experiments reveal several characteristics ofthe aflatoxin molecular configuration that are important to itsbiological activity. Aflatoxins B! and GÃŒwere active in everysystem in which they were tested. They were lethal toducklings and to rats when given in single or repeated dosesand also were carcinogenic to rats. In all of these systems, B,was more potent than GÃŒ. These data therefore indicate thataddition to the Bj molecule of the 2nd lactone ringÃG, versusB,) reduced the biological activity by a factor of about 2 inthose instances where quantitative comparisons are warranted(e.g., LDSO values).

Responses to aflatoxins B2 and G2, the dihydro derivativesof B] and G! , provide additional information on importanceof the terminal furan ring for biological activity. As regardstheir acute toxicity to ducklings, the LDSO values of B2 andG2 were 2.4 times higher than comparable values for B] andG! . The reduced derivatives were therefore active but were lesspotent than the parent compounds in the duckling.

These toxicity data indicate a structure-activity series withdecreasing potency in the order aflatoxins BI > Gj > B2 >G2 and are in agreement with many previous reports. Our

present data on lethality to ducklings confirm precisely theconclusions reached by Carnaghan (7) as well as our ownearlier observations (2, 24). Similar conclusions havepreviously been reported with respect to toxicity to rainbowtrout (5), zebra fish larvae (1), chicken embryos (21), as wellas to cultures of human embryonic lung cells (13) and humanembryonic liver cells (22, 26). A similar order of effectivenesshas also been described for the ability of aflatoxins to alterbiochemical processes in vitro (8, 9).

These studies on toxicity of aflatoxins B2 and G2 to rats arethe first such evaluations to be reported. Doses of thesecompounds as high as 200 mg/kg failed to elicit signs oftoxicity under conditions where the LDSO doses of aflatoxinsBI and G, were in the range of 1 to 2 mg/kg. The dihydroderivatives were thus inactive as compared to the parent toxinsby a factor of at least 100 to 200. Lack of activity was alsonoted with respect to subacute toxicity ; a dose of 52 mg of B2per rat caused no sign of toxicity. Aflatoxin B, was lethal at adose of 1 mg/rat under comparable conditions. The fact thatthese same batches of aflatoxins B2 and G2 were toxic toducklings but inactive in rats indicates a marked speciesdifference in response. Mechanisms responsible for thisdifference are unknown, but some possibilities are suggestedby subsequent findings, as described below.

Further information pertinent to thestructure-activity consideration is provided by the data onCompounds 2, 8, and 11. These synthetic analogs all containthe substituted coumarin portion but lack thedihydrofurofuran configuration of the aflatoxin B, molecule.All 3 compounds were inactive as regards acute and subacutetoxicity and were also noncarcinogenic at doses up to 200mg/animal. Compound 11 is of greatest interest, since itcontains all of the elements of the aflatoxin BI molecule, inthe appropriate configuration, except the dihydrofurofuranmoiety. Its lack of activity thus indicates the importance ofthis portion of the molecule for biological activity. Thesesubstances have not been studied previously in the rat, but wehad found earlier that they were nontoxic to ducklings (24),and, more recently, Compound 11 was shown to be nonlethalto chick embryos (21) and inactive as a carcinogen for rainbowtrout (4).

Collectively, this information indicates that 2 functionalitiesof the aflatoxin molecule are important determinants of itsbiological activity, as regards acute effects. Substituents fusedto the lactone portion of the coumarin nucleus determineactivity to the extent that the aflatoxin G configuration is lesspotent than that of aflatoxin B. Further indication of thespecific importance of this segment of the molecule isprovided by the recent finding that "aflatoxicol," the

hydroxylated derivative produced by reduction of thecarbonyl group of aflatoxin B! , is less toxic than B! toducklings (10). Considerably more evidence points to theimportance of the dihydrofurofuran portion of the aflatoxin Bmolecule. Compounds lacking this portion (e.g.. Compound11) are inactive in all systems tested. Moreover, reduction ofthe vinyl ether double bond in the terminal furan ring bringsabout significant reduction in potency (aflatoxin B2 versusBI) in most systems and nearly total loss of activity withrespect to acute toxicity to rats. Further evidence on theimportance of the specific configuration of the terminal furan




ring comes from studies on the hemiacetal (called aflatoxinB2a) produced by addition of water across the double bond ofaflatoxin B,. This derivative is inactive with respect to toxicityto ducklings (11, 14) and is not lethal to chick embryos (18).Aflatoxin B2 is active in both systems.

On the other hand, structural changes at another positionappear to have little if any influence on biological potency ofaflatoxin B|. Hydroxylation at the bridge carbon of the furanrings occurs as a metabolic transformation of aflatoxin BÂ¡,producing aflatoxin M, (12, 16). Available informationindicates that aflatoxins M! and BI are of approximatelyequal potency with respect to toxicity to ducklings (20) andthe induction of biochemical and ultrastructural changes in ratliver (19).

In many respects, our findings on carcinogenic properties ofthese compounds correspond with their acute effects. Thus, asexpected, aflatoxins Bj and Gt both proved to be activecarcinogens to rats when administered in multiple doses bystomach tube. Although precise quantitative comparisons ofpotency are not possible from the data, aflatoxin Bj wasclearly more potent than G! in inducing liver cell carcinomas.In the only other instance in which pure aflatoxin GÃŒwasstudied in the rat, Butler et al. (6) also demonstrated it to beof lesser potency than B! . The same relationship holds withrespect to tumor induction in rainbow trout by these 2aflatoxins (3, 4). The induction of significant numbers ofkidney tumors by aflatoxin G! in these experiments agreeswith the earlier findings of Butler et al. (6).

Compounds 2, 8, and 11 were inactive as carcinogens forthe rat when given in divided doses over 1 year. A similar lackof carcinogenic activity of Compound 11 for the rainbowtrout has been reported (3). These compounds therefore werewithout demonstrable activity in any of the systems tested,indicating further the importance of the furan moiety indetermining biological activity.

Comparisons of aflatoxins B, and B2 in the carcinogenesisexperiments yielded interesting information. Whenadministered at a total dose of 150 mg/rat, aflatoxin B2induced hepatocellular carcinomas in 3/9 rats killed after 57to 59 weeks; advanced preneoplastic lesions were present inthe livers of 3/9 additional animals killed at the same time.These may have progressed to carcinomas had the animalsbeen held for longer periods. By comparison, aflatoxin B! at adose of 1.3 mg/rat induced carcinomas in 9/9 animals within46 weeks.

Thus, aflatoxin B2 was active as a carcinogen for rat liverwhen given parenterally at a dose level 115 times greater thana more effective dose of aflatoxin B,. This finding wasunexpected in view of the lack of acute toxicity of B2 atcomparable or greater dose levels. Although a definitiveexplanation for this difference in acute and chronic responsecannot yet be provided, several points are relevant. We hadanticipated the potential importance of contamination of theB2 preparation by aflatoxin B) and therefore purified thematerial used to the point where maximum B, levels were notgreater than 1 part in 60,000. Therefore, under the conditionsof the experiment, the B] received by the rats throughcontamination would have been far below the minimum levelknown to be effective in this strain of rats (25).

A plausible explanation may lie in the manner in which


aflatoxin B2 is metabolized. Conversion of B2 into B! (or intosome other common active metabolite) may be responsible forthe generation of active material sufficient to induce thenumber of tumors observed. On the basis of our earlierexperience (25), conversion of only 0.1 to 1.0% of the totaldose would generate an effective carcinogenic dose of BI.Direct evidence for this postulate is not yet at hand. Incontrast to its carcinogenic action to rat liver followingparenteral dosing, aflatoxin B2 showed no evidence ofcarcinogenicity to s.c. tissues following multiple injection.

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1971;31:1936-1942. Cancer Res G. N. Wogan, G. S. Edwards and P. M. Newberne of Aflatoxins and AnalogsStructure-Activity Relationships in Toxicity and Carcinogenicity

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