Aflatoxin-DNA adduct formation in chronically dosed rats fed a choline-deficient diet

Nutritional modulation of male Fischer rats by a choline-deficient/methionine-low diet dramatically increases hepatocarcinogenesis and reduces time to first tumors induced by aflatoxin B1 (AFB1). The effect of this diet on hepatic aflatoxin-DNA adduct burden in male Fischer rats dosed with a carcinogenic regimen of AFB1 was examined in this study. After 3 weeks of ingestion of a choline-deficient/methionine-low diet or control semi-purified diet, rats were administered a carcinogenic regimen of 25 micrograms [3H]AFB1 for 5 days a week over 2 weeks. Six choline-deficient and four control diet rats were killed 2 h after each dose, and liver DNA isolated. In addition, hepatic DNA was isolated from animals 1, 2, 3, and 11 days after the last [3H]AFB1 administration. At all time points HPLC analysis of aflatoxin-DNA adducts was performed to confirm radiometric determinations of DNA binding levels. No significant quantitative differences in AFB1-DNA adduct formation between the dietary groups were observed following the first exposure to [3H]AFB1; however, total aflatoxin-DNA adduct levels in the choline-deficient animals were significantly increased during the multiple dose schedule. When total aflatoxin-DNA adduct levels were integrated over the 10 day dose period, a 41% increase in adduct burden was determined for the choline-deficient animals. While this increase in DNA damage is consistent with the hypothesis that DNA damage is related to tumor outcome, the biochemical basis for this effect still needs to be elucidated.     

Comparative effect of dietary butylated hydroxyanisole and {beta}-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation, and carcinogenesis in rainbow trout

Butylated hydroxyanisole (BHA) and ß-naphthoflavone(BNF), both chemicals with anti-carcinogeneic properties insome experimental animals, were compared for effects on afiatoxinB1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesisin the rainbow trout. Dietary BHA had no effect on the hepatictumor incidence when fed at 0.03 or 0.3% 4 weeks prior to andduring a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, whenfed at 0.005 or 0.05% under similar conditions, significantlyreduced tumor response, which confirms previous results in trout(Nixon et al.9 Carcinogenesis, 5, 615–619, 1984). BHAfed at either 0.03 or 0.3% for 8 weeks had no post-initiationeffect on the 52 week hepatic tumor incidence of trout exposedto a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiationexposure to 0.05% BNF significantly enhanced AFB1 tumor response.The influence of dietary BHA and BNF on AFB1 metabolism andDNA adduct formation and persistence in trout were examined.A 3 week pre-treatment with 0.3% dietary BHA had no effect onin vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1,2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietaryBNF reduced hepatic AFB1-DNA adducts to 33–60% of controllevels at 0.5, 1, 2 and 4 days after AFB1 exposure. This wasaccompanied by significantly lower blood and liver levels ofAFB1 during the first 24 h after i.p. injection. Livers of BNFtrout also contained 4-fold more of the less carcinogenic metabolite,aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite withsimilar carcinogenicity as AFB1. Bile AFL-glucuronide levelswere significantly decreased in BNF-fed trout, but total bileglucuronides were significantly increased due to a 15-fold increasein aflatoxicol-M1 glucuronide. Freshly isolated hepatocytesfrom BHA-fed fish, when incubated with AFB1 for 1 h, showedno difference in levels of AFB1-DNA adducts or ratios of AFB1metabolites when compared to hepatocytes isolated from fishfed a control diet only. By contrast, dietary BNF has been previouslyshown to greatly enhance AFM1 production, reduce AFL production,and significantly reduce AFB1-DNA adduct formation in isolatedtrout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph,65, 379–385, 1984). These results indicate that dietaryBHA up to 0.3% does not alter AFB1 metabolism or DNA adductionin trout, nor does it inhibit or promote AFB1 hepatocarcinogenesisin this species. This is in contrast to anti-oxidant enhancementof AFB1-glutathione conjugation, reduction of AFB1-DNA binding,and consequent reduction of tumor response in rats. The nullresults in trout thus support enhanced glutathione conjugationas the major mechanism for BHA inhibition of AFB1 cardnogenesisin mammalian models. By contrast, BNF dietary pre-treatmentappears to inhibit AFB1 carcinogenicity in trout by enchancingglucuronide formation and elimination of the carcinogen, leadingto reduced DNA adduct formation in target tissue.     

The inhibitory effects of ethoxyquin on the carcinogenic action of aflatoxin B1 in rats

Ethoxyquin (EQ), a widely used antioxidant, inhibits the carcinogenic effects of polycyclic aromatic hydrocarbons. The aim of the present study was to determine whether EQ modifies the hepatocarcinogenic effects of aflatoxin B1 (AFB1) in rats. Both compounds were administered in the diet. Rats were fed either EQ for 2 weeks and then AFB1 for 6 weeks, EQ and AFB1 simultaneously or EQ following the cessation of AFB1 treatment. The results indicate that EQ can inhibit the hepatocarcinogenic effects of AFB1 and that the most effective inhibition is obtained when EQ and AFB1 are given simultaneously.     

The influence of partial hepatectomy on the biphasic response of gamma glutamyl transpeptidase to aflatoxin B1

We previously observed a biphasic response in rat hepatic gamma glutamyl transpeptidase (GGT) activity to aflatoxin B1 (AFB1) feeding [9]. We have extended this observation to examine the effect of partial hepatectomy (PH) on the activity and distribution of GGT at different stages of the feeding regime. In control-fed animals GGT levels were elevated 3-7 days after PH with increased activity in periportal hepatocytes. In animals fed a sub-carcinogenic dose of AFB1 (up to 4 weeks) the effect of PH on GGT activity was similar to that in control animals, but increased activity was mainly due to biliary hyperplasia. There was no obvious difference between animals returned to control diet after PH and those returned to toxic diet. In animals fed 4-15 weeks the percentage increase in GGT activity 1 week after PH correlated with length of time on AFB1 diet before operation, with an increase in number and size of altered foci. These results further support the idea that there is a preliminary toxic response in GGT activity followed by a secondary response more closely related to the carcinogenic process.     

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.     

Aflatoxin B1 induction of hepatocellular carcinoma in the embryos of rainbow trout (Salmo gairdneri).

Liver cancer in rainbow trout was induced by exposure of fertile eggs to an aqueous, 0.5 ppm (microgram/ml) solution of aflatoxin B1 (AFB1) for 1 hour. Single treatments, given on alternate days during the embryonic period, produced a low cancer incidence (less than 20%) prior to formation of the embryonic liver on day 14, but a steadily increasing incidence from day 15 (31.7%) until day 23 (58.3%), in fish examined 1 year later. Treatment of trout eggs with [14C]AFB1 was used to quantitate the amount of AFB1 absorbed by the eggs. Twenty-one-day-old rainbow trout eggs absorbed approximately 30 ng of [14C]AFB1 during a 1-hour exposure to 0.5 ppm aqueous [14C]AFB1. After 1 day 85-90% of the [14C]AFB1 was either metabolized and excreted or leached from the egg. The residual [14C]AFB1 remained constant until hatching when an additional 50% was lost. Comparison of the amount of AFB1 absorbed by eggs with the amount of AFB1 consumed per fish during a 1-year feeding trial at 4 ppb in the diet indicates that the trout embryo is even more sensitive than juvenile trout to the carcinogenic properties of AFB1.     

Effect of dietary protein level on aflatoxin B1 actions in the liver of weanling rats.

The hepatocarcinogenic responses of rats to aflatoxin B1 (AFB1) are believed to depend on microsomal activation of the toxin, followed by macromolecular binding. Dietary protein insufficiency is reported to reduce the level of microsomal metabolism, and therefore would be expected to reduce the AFB1-induced carcinogenicity. Indeed, diminished hepatocarcinogenicity in low-protein diet fed weanling rats that had received AFB1 has been reported. In the present study, carcinogenicity and other toxic effects of AFB1 (0.5 p.p.m.) fed to weanling male Fischer F344 rats on a low-protein diet (5%) or normal-protein (20%) diet for up to 8 weeks were examined. In our study, in contrast with the previous report, all animals that had survived some initial toxicity were found to have developed hepatic tumors or hyperplastic gamma-glutamyltransferase-positive foci a year later. The low-protein diet also produced sub-acute toxicity after AFB1 exposure in the weanling rats, leading to severe histological changes, and the death of about half the animals after 3-4 weeks of exposure. Animals fed an AFB1-containing normal-protein diet also exhibited AFB1-induced hepatocarcinogenicity, but not the sub-acute toxicity. The levels of hepatic enzymes involved in AFB1 metabolism were examined in animals fed the low- or normal-protein diets in the absence of AFB1. The low-protein diet, fed to 3 week weanlings for the subsequent 5 weeks, decreased hepatic cytochrome P450 levels, as well as the in vitro capacity of microsomal fractions to form AFB1-8,9-dihydrodiol, an index of AFB1-8,9-epoxide formation. Rats on a normal-protein diet did not show these changes. This discrepancy between the observed increase in sub-acute toxicity and decrease in microsomal activities in the low-protein fed animals implies that the toxic effects observed in these rats were not directly related to metabolic activation of the toxin. In contrast to the diminished microsomal in vitro AFB1 activation, however, in vivo AFB1-DNA adduct formation ability in rats receiving the low-protein diet in the absence of AFB1 was found to become elevated more rapidly during the 5 week experimental feeding period, compared with animals receiving the normal-protein diet. This was accompanied by a more rapid fall in the levels of AFB1-glutathione S-transferase isozyme activity in the low-protein fed animals. The results of this study on weanling rats support the importance of AFB1-GSH in protecting against the carcinogenic responses to AFB1, and probably also the sub-acute toxicity of the latter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carcinogenicity of dietary aflatoxin M1 in male Fischer rats compared to aflatoxin B1

Aflatoxin M1 (AFM), an hydroxy metabolite of the potent carcinogenic mycotoxin aflatoxin B1 (AFB) is frequently found in milk and other dairy products. Sufficient amounts of AFM were produced to study the carcinogenicity of this compound. AFM was fed to male Fischer rats starting at 7 weeks up to 21 months of age. Agar-based semisynthetic diets contained 0.0, 0.5, 5.0, and 50.0 micrograms/kg of AFM or 50 micrograms/kg of AFB. Hepatocellular carcinomas were detected in two of 37 rats and neoplastic nodules were found in six of 37 rats fed 50 micrograms/kg AFM between 19 and 21 months. No nodules or carcinomas were observed in the lower AFM dose groups. Nineteen of 20 rats fed a diet containing 50 micrograms/kg of AFB developed hepatocellular carcinomas by 19 months of age. Carcinogenic potency of the aflatoxins was reflected by morphometric quantitation of foci detected in hematoxylin and eosin stained sections. Three rats fed the diet containing 50 micrograms/kg AFM developed intestinal carcinomas. None were observed in other groups. Under the conditions of this experiment AFM was found to be a weak hepatic carcinogen compared to AFB and to possess intestinal carcinogenicity.     

Dietary lipotropes, hepatic microsomal mixed-function oxidase activities, and in vivo covalent binding of aflatoxin B1 in rats.

Weanling male Sprague-Dawley rats were fed either a nutritionally complete synthetic diet (Diet 1) or a diet marginally deficient in choline and methionine, and lacking folacin (lipotrope deficient, Diet 2) to determine the role of hepatic mixed-function oxidase metabolism of aflatoxin B1 (AFB1) in the Diet 2-induced enhancement of AFB1 hepatocarcinogenesis previously reported. Hepatic microsomal mixed-function oxidase activities, as assayed by ethylmorphine N-demethylation, ethoxycoumarin O-dealkylation, cytochrome c reduction, AFB1 metabolism, and cytochrome P-450 content, were all depressed by Diet 2. Furthermore, the proportion of an i.p. dose of AFB (1 mg/kg) that became covalently bonded to DNA and RNA was similarly reduced when measured 6 hr after administration. The formation of AFB1-protein adducts was not influenced by dietary treatment. The depression of DNA and RNA adduct formation in the Diet 2 animals was probably related to the lower mixed-function oxidase activities and not to an alteration of glutathione levels, which remained unchanged by dietary treatment. These results suggest that the marginally lipotrope-deficient diet does not enhance tumor formation through an increased microsomal activation of AFB1. Alternative hypotheses without data are suggested.     

Quantitative inter-relationships between aflatoxin B1 carcinogen dose, indole-3-carbinol anti-carcinogen dose, target organ DNA adduction and final tumor response

A number of recent studies have described inhibitor-mediated reductions in the covalent DNA binding and tumorigenicity of various carcinogens, in species such as rats, mice and rainbow trout (Salmo gairdneri). Since inhibitory effects have, in most cases, been reported after testing at one carcinogen and one inhibitor level only, the detailed relationships between carcinogen dose, inhibitor dose, in vivo DNA binding and final tumor response are not well understood in any species. To determine these relationships we have employed the trout model in a combined DNA binding/tumor dose-response protocol using approximately 10,000 animals. Trout were pretreated with one of five different dose-levels of indole-3-carbinol (I3C), a naturally occurring anti-carcinogen found in cruciferous vegetables such as broccoli and cabbage. After 4 weeks, animals received the same dietary level of I3C for a further 2 weeks together with [3H]aflatoxin B1 (AFB1) in the dose-range 10-320 p.p.b. From tanks containing 150 animals (three tanks per I3C-AFB1 dose-point), 15 fish were selected at random in order to assess hepatic AFB1-DNA binding levels. Remaining animals were returned to control diet for determination of tumor response at 12 months. Linear increases in DNA binding occurred with dose of AFB1 at each I3C dose-level. Successive increases in I3C dose gave dose-related decreases in AFB1-DNA binding, resulting in a series of curves of decreasing slope. Shifts in DNA-binding slopes were compared quantitatively with horizontal displacements towards higher carcinogen dose in corresponding tumor dose-response curves. At I3C doses of less than or equal to 2000 p.p.m., the inhibitor-altered tumor response was predicted precisely by changes in dose received (DNA adducts formed) in the target organ. These data constitute the first direct evidence of pure anti-initiating activity by a natural anti-carcinogen found in human diet, where all animals were treated at the same time and under identical conditions of exposure in both DNA binding and tumor studies. The data are discussed further in view of (i) their implications for DNA binding-carcinogenicity correlations and the concept of 'molecular dosimetry', and (ii) limitations in the current database on anti-carcinogenesis as regards in vivo potency information, particularly for 'ambivalent modulators' which exhibit both inhibitory and promotional activity.     

Effect of dietary protein on the response of rainbow trout (Salmo gairdneri) to aflatoxin B1.

Diets containing either 49.5% or 32% casein or fish protein concentrate (FPC) were fed to young rainbow trout (Salmo gairdneri) for 12 months. Five levels [0, 2, 6, 18, and 54 parts per billion (ppb)] of aflatoxin B1 (AFB1) were given in each of four different diets. A 30-fish sample was taken at 6, 9, and 12 months to determine the influence of diet on the carcinogenicity of AFB1. Both levels of casein produced similar hepatoma incidences at each level of AFB1. The diet high in FPC produced more tumours than did the casein diets at 2, 6, and 18 ppb AFB1, whereas fish fed the diet low in FPC had a significantly (P less than 0.05) lower hepatoma incidence than did the other three groups. The liver size (percent body wt) was smaller at higher toxin levels in all instances. The growth of fish given 32% casein was less than that of the other groups.     

Inhibitory effect of a polychlorinated biphenyl (Aroclor 1254) on aflatoxin B1 carcinogenesis in rainbow trout (Salmo gairdneri).

Duplicate lots of 120 rainbow trout (Salmo gairdneri) fingerlings were fed for 1 year semipurified diets containing 6 ppb aflatoxin B1 (AFB1), 100 ppm Aroclor 1254 (a polychlorinated biphenyl), and 6 ppb AFB1 plus 100 ppm Aroclor 1254. Appropriate controls were also maintained. Samples were taken at 1, 2, 4, 6, 9, and 12 months to monitor tumor incidence, Aroclor 1254 accumulation, and histopathology of liver, spleen, and kidney tissues. At the end of the year, 26 of 37 (70.3%) trout fed 6 ppb AFB1 had hepatocellular carcinomas, compared to 14 of 46 (30.4%) trout fed 6 ppb AFB1 plus 100 ppm Aroclor 1254, a highly significant reduction in tumor incidence in the trout on the Aroclor 1254-containing diet. None of the control or Aroclor 1254-fed fish had liver tumors. Levels of Aroclor 1254 increased rapidly during the first 6 months, then plateaued at approximately 80 ppm on a whole-fish basis. AFB1 inhibited growth but Aroclor 1254 did not. Glycogen depletion of hepatocytes and hyperemia, and white pulp depletion of the spleen were the only changes induced by Aroclor 1254.     

Comparative metabolism and DNA binding of aflatoxin B1, aflatoxin M1, aflatoxicol and aflatoxicol-M1 in hepatocytes from rainbow trout (Salmo gairdneri)

DNA binding and metabolism patterns of ³H-labeled aflatoxinB₁ (AFB₁) and its phase I metabolites, aflatoxicol (AFL), aflatoxinM₁ (AFM₁) and aflatoxlcol-M₁ (AFL-M₁), were compared in freshlyprepared rainbow trout (Salmo gairdneri) hepatocytes. Aflatoxinswere incubated with hepatocytes for periods up to 1 h, cellularDNA was isolated and specific activities determined by scintillationcounting and Burton analysis. Data for (pmol bound aflatoxin/µgDNA)/(µmol dose) versus time fit a linear function (P< 0.002)passing nearly through the origin for each aflatoxin.DNA binding at 1 h relative to AFB was: AFL, 0.53 ? 0.07; AFM0.81 ? 0.20 AFL-M₁ 0.83 ? 0.24. Statistical analysis indicatedthat binding of AFL, AFM₁ and AFL-M₁ were significantly lessthan that of AFB HPLC analysis of the cellular supernatantsindicated that the major metabolites were AFL, AFB₁ AFL-M₁ andAFM₁ from AFB₁ AFL, AFM and AFL-M₁ substrates, respectively.Small quantities of hydroxylated metabolites and glucuronidesalso were detected in some of the incubations. The time-coursedata suggested that initial formation of major metabolites wasrapid and that, by 20–30 min, net changes in metabolitelevels decreased or approached zero. Because the four compoundspossessa 8,9-double bond, DNA binding could be due to activationof the parent substrates as well as of their phase I metaholites.Based on current mutagenicity data and limited carcinogenicitystudies, AFM₁, and AFL-M₁ have binding levels which are higherthan expectedcompared to AFB₁ and AFL.     

Chemopreventive properties of chlorophyllin: inhibition of aflatoxin B1 (AFB1)-DNA binding in vivo and anti-mutagenic activity against AFB1 and two heterocyclic amines in the Salmonella mutagenicity assay

Chlorophyllin (CHL), a sodium/copper derivative of chlorophyll, has been used to treat a number of human conditions with no toxic effects being reported. Recent studies have described the anti-mutagenic activity of CHL in several short-term genotoxicity assays; however, this compound has not been reported to inhibit carcinogen--DNA binding in vivo, and it has yet to be evaluated as an anti-carcinogen in any species. The chemopreventive properties of CHL were studied in trout using inhibition of aflatoxin B1 (AFB1)--DNA binding as an end-point. Chlorophyllin and AFB1 were coadministered in the diet, and carcinogen--DNA binding levels were determined in liver after 1, 3, 5 and 7 days. Linear increases in AFB1--DNA binding occurred with time of treatment at each CHL dose level (0, 500, 1000 and 2000 p.p.m.). Each increase in CHL dose produced a concomitant decrease in AFB1--DNA binding, resulting in a series of curves of decreasing slope. At the highest CHL dose level of 2000 p.p.m., AFB1--DNA binding was inhibited by 70%. These results suggest that CHL should be a potent inhibitor of AFB1-induced hepatocarcinogenesis in this model. In the Salmonella assay, CHL exhibited potent anti-mutagenic activity against AFB1 and two heterocyclic amines when incubated in the presence of trout liver activation systems. CHL also inhibited the mutagenic activity of AFB1-8,9-epoxide in the absence of a metabolic activation system. Dietary CHL substantially inhibited liver AFB1-DNA binding in vivo, even when AFB1 was given by i.p. injection to avoid direct AFB1--CHL interaction in the diet or gut. Collectively, these studies support a CHL inhibitory mechanism involving complex formation with the carcinogen in the gut coupled with electrophile scavenging or further complexing in the target organ.     

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of riboflavin on the catalytic activity of liver fractions

Weanling rats were kept on a synthetic riboflavin-free diet for 4 weeks, and subsequently on the same diet but supplemented with riboflavin for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. A decrease in both activities was evident during riboflavin deficiency, and this could be restored after normal supply of the vitamin. The decrease was attributed to a fall in the endogenous flavin content, specifically the coenzyme flavin adenine dinucleotide which forms an integral part of the microsomal monooxygenase that catalyzes the activation reactions. The vitamin and its coenzymes, however, inhibit the microsomal enzyme activity when added in excess in the in vitro system. It is envisaged that riboflavin may play a role in regulating the carcinogenic activity of AFB.     

Effect of long-term feeding of nivalenol on aflatoxin B1-initiated hepatocarcinogenesis in mice

Nivalenol, a trichothecene, occurs widely in cereals and foods; our current two-year feeding trial has revealed no tumorigenic activity in female mice. To investigate whether dietary nivalenol modulates the development of aflatoxin B1 (AFB1)-initiated hepatocarcinogenesis, one-week old C57Bl/6 x C3H F1 mice were injected intraperitoneally with 6 mg/kg bw AFB1 and six weeks later fed diets containing 0, 6 or 12 ppm nivalenol for one year. Male mice in all three groups developed hepatocellular carcinomas and adenomas, while the incidences in females were 31% in those given AFB1 alone and 20% and 0 in those given AFB1 with 6 and 12 ppm nivalenol, respectively. These findings indicate that dietary nivalenol suppresses AFB1-initiated hepatocarcinogenesis in female mice, presumably by acting on the promotion step.     

Mechanisms of anti-carcinogenesis by indole-3-carbinol: detailed in vivo DNA binding dose-response studies after dietary administration with aflatoxin B1

Several recent reports have described inhibitor-mediated reductionsin the covalent binding of various carcinogens to DNA in vivo.The majority of these studies show inhibitory effects aftertesting at one inhibitor and one carcinogen dose level only.Consequently,the detailed relationships between inhibitor dose,carcinogen dose, and in vivo inhibitory potency have not beenclearly delineated in any species. To systematically determinethese relationships in vivo, rainbow trout (Salmo gairdneri)were exposed to a range of carcinogen (aflatoxin B1, AFB1) andinhibitor (indole-3-carbinol, I3C) doses by concomitant dietaryexposure. Inhibitory potencies were then assessed using in vivocovalent binding of AFB1 to hepatic DNA as an end-point. Linearincreases in DNA binding occurred with increasing dose of AFB1and with time of inhibitor/carcinogen co-treatment, at eachI3C dose level. Successive increases in inhibitor dose resultedin corresponding dose-related decreases in AFB1-DNA bindingsuch that a series of curves of decreasing slope was produced.AFB1-DNA binding was suppressed by almost 95% at the highestI3C dose tested. Thesestudies describe for the first time sucha degree of inhibition by I3C on covalent binding of AFB1 toDNA in vivo, where inhibitor and carcinogen are covariablesadministered repeatedly in the diet. Moreover, the linear inhibitoryresponse observed at low I3C doses indicates the possible absenceof any significant threshold for I3C protection against AFB1-DNAbinding. Thus, even at low levels I3C may offer some protectionagainst chemically-induced neoplasia.     

Relative contribution of dietary protein level and aflatoxin B1 dose in generation of presumptive preneoplastic foci in rat liver

Male weanling F344 rats were orally gavaged with aflatoxin B1 (AFB1) in daily doses of 200, 235, 270, 300, and 350 micrograms/kg/day for a total of 10 doses over a 12-day period, and then 1 week after the last dose they were fed diets of varying protein (casein) content to compare the contribution of AFB1 dose and dietary protein level on the development of presumptive preneoplastic gamma-glutamyltransferase-positive (GGT+) foci in rat liver. All animals were fed the same 20% dietary casein level during the dosing period. One week after the end of the dosing period, one-half of the animals in each dose group were then continued on the 20% casein diet for the entire 12-week foci-development period; the remaining half in each dose group were fed lower levels of dietary casein during the foci-development period for the increasing AFB1 dose groups (20, 16, 12, 8, and 4% casein for the 235-, 250-, 270-, 300-, and 350-micrograms/kg/day groups, respectively). The AFB1 dose groups used were determined in a preliminary experiment. In this previous experiment, a clearly discernible threshold dose at about 100-150 micrograms AFB1/kg/day (below which no GGT+ foci were observed) and a steep slope between 150 and 400 micrograms/kg/day were produced. In the second experiment, while the expected positive slope of (AFB1) dose versus (GGT+ foci) response relationship was found for animals fed the 20% casein diet, the dose response for the animals fed the lower levels of casein was eliminated, providing evidence that nutrient intake during the postdosing foci development is more rate limiting toward the development of these preneoplastic lesions than is the carcinogen dose.     

Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver

The involvement of tumor promotion in the hepatocarcinogenic action of peroxisome proliferators has not been generally accepted. We studied the effect of nafenopin (NAF) as a model compound in a two-stage initiation-promotion protocol. Carcinogenesis was initiated by a single dose of aflatoxin B1 (AFB1) in female (AFB1, 5 mg/kg) and male (AFB1, 2 mg/kg) Wistar rats. After recovery NAF was fed via the diet, providing a daily dose of 100 mg/kg body weight. Phenobarbital (PB) (50 mg/kg body weight) was fed to female rats as a positive control. The following results were obtained. (a) At weeks 40, 55, 59, and 70, significantly more and larger liver tumors were present in AFB1-NAF-treated rats than in rats receiving either compound alone, and the effect of the combined treatment was clearly more than additive, in three independent experiments including both sexes. This suggests tumor promotion by NAF. Male rats responded more strongly than females. Similarly, PB enhanced the yield of liver tumors. Histologically, tumors were hepatocellular adenoma or carcinoma. In group AFB1-PB the majority consisted of eosinophilic and glycogenstoring cells. However, adenoma and carcinoma of groups AFB1-NAF and O-NAF consisted of weakly basophilic cells. (b) Phenotypically altered foci were evaluated in hematoxylin- and eosin-stained liver sections from the female rats. NAF treatment after AFB1 had little effect on number and size of eosinophilic-clear cell foci and decreased the number of trigroid foci. However, it led to a dramatic increase (20-fold after 70 weeks of NAF treatment) in number and size of foci of a special phenotype that was extremely rare after AFB1 alone and virtually absent in group AFB1-PB. Hepatocytes in these foci are characterized by weak diffuse basophilia and some eosinophilia, similar to the phenotype in adenoma and carcinoma, and by absence of gamma-glutamyltranspeptidase (GGT) expression. Based on these findings, we propose the hypothesis that NAF promotes the development of liver tumors via a mechanism involving amplification of a specific subtype of altered hepatic foci.     

Species susceptibility to aflatoxin B1 carcinogenesis: comparative kinetics of microsomal biotransformation

The biotransformation of the potential human carcinogen aflatoxin B1 (AFB1) was studied using hepatic microsomes from the rat, mouse, monkey, and human. Initial rates of AFB1 oxidation to aflatoxins Q1, M1, and P1, as well as the reactive intermediate AFB1-8,9-epoxide, were determined using a high performance liquid chromatography assay. The rates of generation of these AFB1 metabolites were investigated at low substrate concentrations (more representative of environmental exposures) and also at high ("saturating") concentrations commonly utilized in studies in vitro. Striking differences in ratios of the metabolites were observed. At an AFB1 concentration of 124 microM, mouse and monkey microsomes had the highest rates of AFB1-8,9-epoxide formation. Primate liver microsomes formed aflatoxin Q1 in large amounts but failed to produce detectable aflatoxin P1. Determination of the rates of formation over initial AFB1 concentrations ranging from 15 to 475 microM revealed that the proportion converted to AFB1-8,9-epoxide increased at lower substrate concentrations in the case of the rat and human microsomes but not with mouse or monkey microsomes. The differences in patterns of metabolite formation with varying concentrations have implications for interspecies comparisons of carcinogenic potency of AFB1.