Minimal dose and time protection by lindane (gamma-isomer of 1,2,3,4,5,6 hexachlorocyclohexane) against liver tumors induced by aflatoxin B1

This study was carried out in order to investigate the minimal exposure to lindane (LD, 99.72% gamma isomer of 1,2,3,4,5,6 hexachlorocyclohexane), a chlorinated hydrocarbon insecticide, required to protect against liver tumor induced by aflatoxin B1 (AFB1). Materials fed to Buffalo strain rats were as follows: LD 100 ppm; AFB1 1 ppm, LD 100 ppm plus AFB1 1 ppm; and control basal diet. The experimental animals were clinically observed and then serially killed at 1, 3, 5, 10, 15 and 82 weeks. Concurrent administration of LD with AFB1 to rats for more than 3 weeks totally inhibited the incidence of AFB1-induced hepatocellular carcinomas by week 82. Only 1 of 20 rats (5%) fed the same regimen for 1 week developed liver tumors. Animals given 1 ppm AFB1 singly for 15 weeks had a high liver tumor incidence (31.5%). No animals developed liver tumors in LD-treated and control groups. LD may inhibit AFB1-induced liver tumors by stimulating hepatic metabolism and excretion of AFB1 so that less carcinogen is available to liver tissue.     

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.     

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.     

Promotion of aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency

Indole-3-carbinol (13C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout (Bailey et al., J. Natl. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick et al., unpublished results) when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human 13C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed 13C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, 13C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of 13C treatment. Promotional enhancement of tumor incidence by 13C was found to be significant when 13C treatment was delayed for several weeks or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to 13C treatment and to be decreased but still evident when 13C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify 13C promotional potency in terms of its dietary concentration, a series of AFB1 tumor dose-response curves was established, each with a different level of 13C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing 13C concentration. The level of 13C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm 13C, fed continuously, with no substantial threshold for promotion. By comparison, 13, when fed before and with AFB1, shows a 50% inhibitory value (13C concentration that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm 13C [Dashwood et al., Carcinogenesis (Lond.), 10: 175-181, 1989]. Thus the potential for 13C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.     

Modulation of aflatoxin-B1 hepatocarcinogenesis in trout by dehydroepiandrosterone: initiation/post-initiation and latency effects

Previously, we demonstrated that dehydroepiandrosterone (DHEA) enhances aflatoxin B1 (AFB1) hepatocarcinogenesis in trout when administered following AFB1 exposure. This paper examines the effect of DHEA on tumor latency and the comparative potency for DHEA to modulate AFB1 carcinogenesis when administered prior to and concurrent with AFB1 compared with a post-initiation exposure. Trout were initiated by a 30 min water bath exposure to 10 p.p.b. AFB1. At 3 months post-initiation, animals were started on either control diet or a diet containing 444 p.p.m. dehydroepiandrosterone (DHEA). Fifty trout per treatment were sampled prior to the start of experimental diets, and then at monthly intervals for the next 7 months and examined for the presence of tumors. Tumors were not detected in initiated controls until 7 months after initiation. In initiated trout fed DHEA, the first tumor was detected 5 months after initiation (after just 2 months of dietary DHEA). At 6 months post-initiation, 20% of the AFB1-initiated trout fed DHEA had tumors, while no tumors were visible in either AFB1-initiated controls or noninitiated trout fed DHEA. A second experiment was designed to determine if the enhancing effect of DHEA on AFB1 carcinogenesis is dependent on the time of DHEA administration relative to the time of AFB1 exposure, and if DHEA could be chemopreventive if administered prior to and concurrent with AFB1. Trout were fed one of two levels of DHEA (888 or 1776 p.p.m.) either prior to and during a 4- week initiation period of dietary AFB1 administration, or for 8 weeks following initiation with AFB1. At 9 months after initiation the livers were examined for tumors. Neither exposure protocol provided protection towards AFB1 hepatocarcinogenesis. The strongest enhancement occurred when DHEA was fed during the post-initiation period. Levels of p53 and p34cdc2 were decreased by DHEA treatment, indicating that DHEA may act through alterations in cell-cycle control.      

Aflatoxin B1 carcinogenesis and its relation to DNA adduct formation and adduct persistence in sensitive and resistant salmonid fish

Rainbow trout (Salmo gairdneri)and coho salmon (Oncorhyn-chuskisutch) were exposed to aflatoxin B1(AFB1) either by passiveembryo uptake or by dietary treatment after hatching and feedingonset. Trout exposed as embryos to an aqueous solution of 0.5p.p.m. AFB1 for 15 min showed a 62% tumor incidence 12 monthslater, whereas coho salmon exposed to a similar solution for30 min showed only a 9% incidence. The difference between salmonand trout response was even greater by dietary AFB1 treatment.Trout exposed for 4 weeks to 20 p.p.b. dietary AFB1 had a 62%tumor response 12 months later, whereas salmon exposed to 40p.p.b. dietary AFB1 for 4 weeks failed to develop tumors. A5% tumor incidence was observed in salmon 12 months after 3weeks exposure to 5000 p.p.b. dietary AFB1, a lethal dose fortrout. In addition to a lower tumor incidence when comparedto trout, the neoplastic response of salmon to AFB1 is to producebenign hepatic adenomas in contrast to the malignant hepatocellularcarcinomas seen in trout. AFB1 metabolism, DNA adduct formation,adduct persistence in vivo and in vitro and cytochrome P-450isozyme composition were compared in livers of trout and salmonto understand the role of metabolism and initiation in thisspecies difference. AFB1-DNA binding was 7–56 times greaterin trout than salmon liver at various times after AFB1 injection,20 times greater in embryos or in freshly isolated trout hepatocytepreparations after a 1 h incubation with aflatoxin Bl, and 18times greater in trout liver after a three week dietary (80p.p.b.) exposure. The major AFB1-DNA adduct was 8, 9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB1 in both species. Persistence of AFB1-DNA adducts in vivoin liver was high compared to mamalian systems, implying thatactive enzymatic removal of bulky DNA adducts is low in bothspecies and probably not a factor in their differential responseto aflatoxin. Species differences in other phase I and phaseII metabolism pathways and in AFB1 elimination were, overall,much less striking than those previously observed for troutfed inhibitors of aflatoxin carcinogenesis. Rates of bileeliminationof AFB1 detoxication products, and total excretion of aflatoxinsinto water after AFB1 exposure, were not significantly differentbetween trout and salmon. Since detoxication differences werenot observed, the species difference in AFB1-DNA binding appearsto reflect less efficient cytochrome P-450 metabolism of aflatoxinto the reactive 8, 9-epoxide in salmon, compared to trout. Insupport of this hypothesis, trout liver microsomes displayeda K_m (7.5 µM)for AFB1-DNA adduction in vitro that was7-fold lower than salmon (52 µM). Furthermore, immunoquantitationof various P-450 isozymes suggest that salmon liver microsomeshave much lower amounts of an isozyme immunochemically relatedto trout P-450 LM₂ which has previously been shown to be themajor isozyme catalyzing AFB1 8, 9-epoxidation. Other, post-initiationdifferences were not ruled out by these studies and may contributeto the differential response of rainbow trout and coho salmonto AFB1 hepatocarcino-genesis.     

Induction of liver tumor in Sherman strain female rats by polychlorinated biphenyl aroclor 1260.

Sherman strain female rats (200) were fed 100 ppm of polychlorinated biphenyl (Aroclor 1260) for apporximately 21 months, and 200 female rats were kept as controls. The rats were killed when 23 months old. Twenty-six of 184 experimental animals and 1 of 173 controls had hepatocellular carcinomas. None of the controls but 146 of 184 experimental rats had neoplastic nodules in their livers, and areas of hepatocellular alteration were noted in 28 of 173 controls and 182 of 184 experimental animals. Thus the polychlorinated biphenyl Aroclor 1260, when fed in the diet, had a hepatocarcinogenic effect in these rats. The incidence of tumors in other organs did not differ appreciably between the experimental and control groups.     

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.     

Comparison of transplacental and neonatal initiation of mouse lung and liver tumors by N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and promotability by a polychlorinated biphenyls mixture (Aroclor 1254)

We have previously shown a positive tumor-promoting effect ofa single dose of Aroclor 1254 on lung and liver tumors initiatedneonatally in the mouse by N-nitrosodimethylamine (NDMA). Inthis study, we have confirmed and extended this observationwith NDMA and the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone(NNK) given either transplacentally or postnatally, followedby a single dose of Aroclor 1254 on day 56. This polychlorinatedbiphenyl (PCB) mixture was an effective promoter of both lungand liver tumors; however, there were specific initiator andsex-related differences in this response. Aroclor administrationsignificantly increased the incidence of lung tumors initiatedtransplacentally by NDMA or NNK in male mice. Neither nitrosamineinitiated tumors transplacentally in females, but lung tumorsinitiated with NNK and liver tumors caused by NDMA in neonatalfemales were promoted by PCBs. Both liver and lung tumors initiatedneonatally by NDMA in male animals, but not NNK-initiated tumors,were promoted by PCBs. These data confirm that PCBs are ableto promote both NDMA- and NNK-initiated tumors, but with chemical-,sex- and age-dependent difference; this suggests influencesof both quantitative and qualitative factors in susceptibilityto tumor promotion.     

Inhibition of aflatoxin B1-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic preneoplastic foci and tumors by low protein diets: evidence that altered GGT+ foci indicate neoplastic potential.

Previous studies in this laboratory with young Fischer 344 male rats have shown that the post-initiation development of aflatoxin B1 (AFB1)-induced gamma-glutamyltranspeptidase positive (GGT+) hepatic foci was markedly inhibited by low protein feeding, even though the energy intake was greater. This dietary effect, however, did not necessarily apply to hepatic tumor development. Thus, the present investigation was undertaken to examine this dietary effect upon the development of hepatic tumors and, is so doing, to determine the correlation of foci development with tumor development. Following AFB1 dosing (15 daily doses of 0.3 mg/kg each), animals were fed diets containing 6, 14 or 22% casein (5.2, 12.2, 19.1% protein) for 6, 12, 40, 58 and 100 weeks. Foci at 12 weeks and tumors at 40, 58 and 100 weeks developed dose-dependently to protein intake. Foci development, tumor incidence, tumor size and the number of tumors per animal were markedly reduced while the time to tumor emergence was increased with low protein feeding. Non-hepatic tumor incidence also was lower in the animals fed the lowest protein diet. Foci development indices (foci number, per cent liver volume occupied) were highly correlated with tumor incidence at 58 and 100 weeks (r = 0.90-1.00). Tumor and foci inhibition occurred in spite of the greater energy intake.     

Iron as a synergist for hepatocellular carcinoma induced by polychlorinated biphenyls in Ah-responsive C57BL/10ScSn mice

Male Ah-responsive C57BL/10ScSn mice received a single dose of iron-dextran (600 mg Fe/kg) and were fed a diet containing 0.01% of the PCBs mixture Aroclor 1254 for up to 12 months. Iron caused a marked synergistic increase in liver size from 2 months with greatly elevated mitotic rates, numbers of basophilic foci and incidences of bile duct and oval cell proliferation. Although at 4 months much of the liver enlargement was due to iron-depleted hyperplastic regions and lipid accumulation, by 8 months seven out of nine mice had nodules (hepatocellular adenomas) whereas none were observed in the Aroclor alone group. After 12 months, 16 out of 18 mice had multiple nodules and/or hepatocellular carcinomas whereas only one of 16 mice was positive in a group not given iron. Basophilic nodules were more common than clear cell nodules in those mice with carcinomas than in those animals without. Preloading with iron also greatly enhanced the development of cholangiofibrosis at 8 and 12 months. Preliminary experiments with the polybrominated biphenyl mixture Firemaster BP-6 indicated a similar synergistic interaction with iron. No effects of iron and Aroclor 1254 on the liver were observed in the Ah-nonresponsive strain DBA/2. Iron potentiated the development of uroporphyria after exposure to those chemicals in C57BL/10ScSn mice but not in the DBA/2 mice. Therefore in C57BL/10ScSn mice the carcinogenicity of PCBs and possibly PBBs, is modulated by iron status and probably not at a late stage where these chemicals may act in a promotional manner.     

Perturbation of hepatocyte nuclear populations induced by iron and polychlorinated biphenyls in C57BL/10ScSn mice during carcinogenesis

The induction of hepatocarcinogenesis by polychlorinated biphenyls(PCBs) in C57BL/10ScSn mice is markedly potentiated by iron.To investigate the effects of iron and PCBs on nuclear populations,C57BL/10ScSn mice received a single dose of iron—dextran(600 mg Fe/kg) and were fed a diet containing 0.01% of the PCBsmixture Aroclor 1254 for up to 6 months. DNA content of isolatednuclei and hepatocytes was estimated by flow cytometry. Cellsuspensions and nuclei isolated from Aroclor treated mice after6 months contained increased diploid (2N) populations comparedto controls. In contrast, iron treatment of mice markedly enhancedfractions of octoploid (8N) nuclei by 2 weeks and this effectpersisted over the 6 month period. When Aroclor 1254 and ironwere administered together there was a synergistic increasein the mononucleated diploid fraction which was significantat 2 weeks and highly significant at 6 months. This became thepredominant nuclear effect. At six months, Aroclor 1254 andiron, both alone and in combination, also increased the rateof DNA synthesis in hepatocytes as measured by bromodeoxyuridine(BrdU) incorporation. The chronic polyploidizing effect of ironoverload alone was investigated further and shown to be proportionalto the dose and was detectable as early as 2 days after 600mg Fe/kg and 1 week after 150 mg Fe/kg. Polyploidization ofnuclei was inhibited by the oral iron chelator CP94. Iron alsoinduced a prolonged reduction in the incidence of binucleatedcells. Histologically, nuclear enlargement due to iron was confinedto the midzonal region of the liver lobule, whereas iron depositionwas greatest in the periportal region. Iron (600 mg/kg) alsocaused increased nuclear polyploid states in hepatocytes ofadult rats and gerbils. Similarly, weanling mice with a dominantlydiploid cell population, when treated with iron (300 mg/kg),exhibited a significant shift to a tetraploid (4N) populationand a marked increase in proliferation as measured by BrdU incorporationand proliferative cell nuclear antigen (PCNA) detection. Theseresults indicate that Aroclor 1254 and iron induce changes inthe mouse hepatocyte population that involve 2N and 8N nucleirespectively. The combination treatment leads to the emergenceand proliferation of a mononucleated, diploid population asobserved frequently in chemical hepatocarcinogenesis. The reasonfor the chronic polyploidizing effect of iron is unknown, butmay imply both increased DNA synthesis and impairment of nucleardivision with implications in human conditions of iron overload.     

Promoting effects of polychlorinated biphenyls (Aroclor 1254) and polychlorinated dibenzofuran-free Aroclor 1254 on diethylnitrosamine-induced tumorigenesis in the rat

The hepatic tumor-promoting activity of a commercial polychlorinated biphenyl mixture, Aroclor 1254 (AR 1254), with and without its intrinsic polychlorinated dibenzofuran (PCDF) impurities, was investigated. Male Sprague-Dawley non-inbred albino rats were treated with 66 microgram diethylnitrosamine (DENA)/ml drinking water for 5 weeks and subsequently given a control diet or a diet supplemented (100 ppm for 18 wk) with either AR 1254 or AR 1254 from which the PCDF moieties were removed (AR 1254-PCDF). Of those animals receiving DENA alone, 16% exhibited hepatocellular carcinomas. Of those rats treated with DENA followed by administration of AR 1254 or AR 1254-PCDF, 64 or 84%, respectively, developed hepatocellular carcinomas. Thus promotion with either AR 1254 or AR 1254-PCDF significantly (P less than 0.05) increased the incidence of DENA-initiated hepatocellular carcinomas. Administration of AR 1254 or AR 1254-PCDF alone did not induce hepatic tumors. Therefore, PCDF impurities were not necessary for the promoting activity of AR 1254.     

Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver

Aroclor 1254 is a complex mixture of polychlorinated biphenyls (PCB) that upon prolonged administration has been reported to produce hepatic tumors in mice and rats. The ability of Aroclor 1254 to promote enzyme-altered foci was determined in an initiation/promotion bioassay in rat liver. Initiation was accomplished in rats that received a 2/3 partial hepatectomy followed in 24 h by diethylnitrosamine (DENA). Aroclor 1254 was administered to each rat 7, 28 and 49 days after the DENA and some of the rats were killed 21 days after each dose of Aroclor. The liver of rats that received Aroclor 1254 on either day 7 or on day 7 and 28 contained an increased incidence of gamma-glutamyltranspeptidase (GGTase)-positive foci compared to partial hepatectomized and DENA treated rats given tricaprylin (the solvent for Aroclor 1254). Therefore, Aroclor 1254 was demonstrated to enhance the appearance of enzyme-altered foci after only a single oral dose.     

Induction of liver tumors in male Wistar rats by feeding polychlorinated biphenyls (Aroclor 1260)

Two groups of 32 male Wistar rats, each 5 weeks of age, were fed on protein diet containing polychlorinated biphenyl (Aroclor 1260), at 50 ppm and 100 ppm levels, respectively, for 120 days. This not only brought about gross hepatic changes but induced neoplastic nodules with adenofibrosis in 75% and 50% of the rats of the respective groups. None of the control animals showed such changes. The study revealed that feeding of the PCBs can not only induce liver adenofibrosis in young male Wistar rats in a short duration of time, but also showed that the carcinogenic potentiality in male rats fed Aroclor 1260 is greater when fed at a lower dose.     

Analysis of ras gene mutations in rainbow trout liver tumors initiated by aflatoxin B1.

The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c-ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA----GTA transversions, two were codon 13 GGT----GTT transversions, and one was codon 12 GGA----AGA transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA----GTA mutations, one codon 12 GGA----AGA mutation, and one codon 13 GGT----GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.     

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.     

Promotion by polychlorinated biphenyls of lung and liver tumors in mice

Polychlorinated biphenyls (PCB), which are tumor promoters,have been found in human tissues for decades. Their contributionto cancer risk may only now start to appear, due to long humancancer latency and the nature of tumor promotion. Epidemiologicalassociations have been seen between PCB exposure or tissue contentand cancer at several sites. In rodents, tumor promotion byPCBs has been little studied in tissues other than liver. Previously,in an experiment modeling infant carcinogen exposure followingPCBs received in milk, lung and liver tumors, initiated neonatallyin mice by the environmental nitrosamine N-nitrosodimethylamine(NDMA), were promoted by later treatment with Aroclor 1254.The present study was undertaken to confirm and characterizethe effects of Aroclor 1254 on tumor number, latency, size andmalignancy. Male Swiss mice were given NDMA on postnatal day4 and Aroclor 1254 (250 mg/kg) on day 8, and killed at intervals.Eight PCB congeners were quantified in the carcasses. Incidencesof mice with NDMA-initiated lung tumors at 28 weeks of age wereincreased 2.5-fold by PCBs. Multiplicities of lung tumors wereenhanced four-fold by PCBs at 28 and 52 weeks. By 72 weeks tumornumbers were similar in the NDMA-only and NDMA–PCB groups.Liver tumors first occurred in significant numbers at 52 weeksand only in mice receiving both NDMA and PCBs. As for the lung,at 72 weeks the incidence was high in both the NDMA-only andNDMA–PCB groups. Sizes of tumors and liver carcinoma incidencewere not altered by PCB treatment. Carcass analysis revealeda significant positive association between lung tumor numbersat 28 weeks and relative percentage of 2,2',4,4',5-pentachlorobiphenyl,with no other correlations. The results confirm that PCBs promotelung as well as liver tumors, by triggering the early appearanceof latent initiated tumors otherwise presenting in old age.     

Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss).

The expression of immunoreactive glutathione S-transferase (GST) was examined in hepatic neoplasms induced in rainbow trout by aflatoxin B1 (AFB) or 1,2-dimethylbenzanthracene (DMBA). Tumors were induced in adult trout by continuous dietary exposure to 8 p.p.b. AFB1 for 12 months or embryo bath exposure to DMBA (5 p.p.m. for 24 h, 3 times with 12 h intervals between exposures). Polyclonal antiserum specific for the two major trout hepatic GST subunits in trout liver was produced by immunizing rabbits with affinity-purified trout GST. Hepatocellular, cholangiolar and mixed neoplasms as well as foci of hepatocellular alteration were examined for GST immunoreactivity by the PAP technique. The majority of lesions were GST-deficient (AFB treated, 67%; DMBA treated, 54%), whereas GST expression was induced in 21% (AFB treated) and 31% (DMBA treated) of altered hepatic foci. The GST-induced foci were consistently small (AFB treated, 0.07 +/- 0.05 mm2; DMBA treated, 0.02 +/- 0.01 mm2) and none had progressed beyond the altered focus stage. The majority of larger advanced lesions (adenomas and carcinomas) were GST deficient (AFB treated, 2.33 +/- 0.35 mm2; DMBA treated, 2.95 +/- 0.59 mm2). These studies demonstrate that induced GST expression occurs in some small populations of hepatocytes, but not in larger advanced stages of malignant progression of aflatoxin- or PAH-induced hepatic neoplasms in rainbow trout.