Hepatocarcinogenicity of glandless cottonseeds and cottonseed oil to rainbow trout (Salmo gairdnerii).

Glandless cottonseed kernels are available for purchase and consumption by the general public. These kernels contain no gossypol but still have a full complement of naturally occurring cyclopropenoid fatty acids, which in rainbow trout are active as synergists with aflatoxins and primary liver carcinogens. Diets containing glandless cottonseed kernels or a lightly processes cottonseed oil produced significant numbers of hepatocellular carcinomas in rainbow trout after 1 year. The much greater incidence of cancer induced by the kernel than by the oil indicates that synergists or other carcinogens may be present in the kernel in addition to the cyclopropenoid fatty acids.

A limited epizootic of neuroblastoma in coho salmon reared in chlorinated-dechlorinated water.

During the 1976-77 brood year, approximately 12 cases of neuroblastoma were observed in a captive group of 100,000 fingerling coho salmon (Oncorhynchus kisutch) reared in a commercial hatchery. The tumors were large, occurring in the skeletal muscle near the dorsal fin causing conspicuous bulging of the overlying integument. Tumors examined from 3 fish each consisted of neuroblasts in trabecular patterns interspersed by glial fibrillar material and linear cavities resembling central neural canals lined by ependyma-like cells. Ganglion-like cells also were apparent morphologically and by special stain. Cancer of the tumor was characterized by an abundance of mitotic figures with occasional abnormal divisions, local invasion of normal tissues, and potentially metastatic tumor cell aggregates in organ vasculature. The etiology of this tumor may have been related to mutagenic-carcinogenic halogenated compounds possibly formed in the hatchery water supply during continuous chlorination of incoming river water.

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.

Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol.

Indole-3-carbinol (I3C), a natural constituent of cruciferous vegetables, is an inhibitor in several experimental animal models of carcinogenesis by polynuclear aromatic hydrocarbons or aflatoxin B1 (AFB1) when administered prior to or during carcinogen exposure. For assessment of the postinitiation effects of I3C, rainbow trout were exposed to dietary I3C in two different protocols--before and during AFB1 exposure or after AFB1 exposure only. Preinitiation exposure to I3C reduced AFB1-initiated hepatocellular carcinomas in trout as previously reported, but post-initiation I3C exposure strongly enhanced the tumor incidence above the positive AFB1 control. These results reveal the need for additional research to elucidate the overall effect of I3C on chemical carcinogenesis.

Hepatocarcinogenicity of sterigmatocystin and versicolorin A to rainbow trout (Salmo gairdneri) embryos.

Versicolorin A (VA) and sterigmatocystin (ST) are biosynthetic precursors of aflatoxin B1 (AFB1). The carcinogenicity of these compounds relative to AFB1 was determined with the use of rainbow trout (Salmo gairdneri) embryo exposure. Exposure of 14-day rainbow trout embryos to a 0.5-ppm aqueous suspension of ST for 1 hour produced a 13% incidence of hepatocellular carcinomas among survivors 1 year later. Similar exposure of trout eggs to a 0.5-ppm solution of AFB1 produced a 53% incidence among survivors. Subsequent exposure of 21-day rainbow trout embryos to 5- and 25-ppm solutions of VA resulted in hepatocellular carcinoma incidences among survivors of 42 and 68%, respectively, at 12 months. A 0.5-ppm AFB1 positive control group had a 68% incidence among survivors of hepatocellular carcinomas at 1 year. These results established the carcinogenicity of VA for the first time and confirmed previous reports of ST carcinogenicity. Both compounds were of sufficient potencies to warrant caution as possible human health hazards.

Carcinogenicity and acute toxicity of dimethylnitrosamine in rainbow trout (Salmo gairdneri).

Four-week-old rainbow trout (Salmo gairdneri) were fed diets containing 0, 3, 50, 200, 400, and 800 ppm dimethylnitrosamine (DMN) for 52 weeks. At the end of 52 weeks, the fish were fed a control diet without DMN for an additional 26 weeks. Samples were taken at 26, 52, and 78 weeks to determine tumor incidence. A dose-related carcinogenic response was established from these results, and an equation was derived to relate the level of the carcinogen to the hepatocellular carcinoma incidence. From a published dose-response study that used outbred Porton rats, a second equation was derived for comparison. Rats and trout were approximately equal in their sensitivity to DMN carcinogenesis. The median lethal dose after ip injection of DMN was 1,770 mg/kg body weight in rainbow trout. Relative to the range of 15-50 mg/kg body weight reported for several mammalian species, trout were resistant to the acute toxicity of DMN.

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.

Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection.

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

Carcinogenicity of aflatoxicol in Fischer 344 rats.

Aflatoxicol (AFL), a metabolite of aflatoxin B1 (AFB1), is formed in vitro by liver preparations from several species including humans. A positive correlation appears to exist between the sensitivity of a species to AFB1 and the species ability to metabolize AFB1 to AFL. Conversion of AFB1 to AFL is, therefore, a questionable detoxification step. The carcinogenicity of a diastereoisomeric mixture of AFL, prepared chemically from AFB1, was compared to AFB1 by tumor incidences being determined in 4 groups of 20 weanling male F344 rats fed either a negative control diet with no aflatoxin, a positive 50-ppb AFB1 control diet, a 50-ppb AFL diet, or a 200-ppb AFL diet for 1 year and then killed at the end of the 2d year. The respective hepatocellular carcinoma incidences were 0, 40, 20, and 70%, demonstrating that AFL is carcinogenic in the rat. The data show that a diastereoisomeric mixture of AFL is one-half as carcinogenic as AFB1, and the dose response appeared nearly linear in that a fourfold increase in dose produced a 3.5-fold increase in tumor incidence. The data did not establish unequivocally that AFL is a proximate carcinogen, but metabolism of AFB1 to AFL should not be considered an efficient detoxification reaction.

Carcinogenic response of rainbow trout (Salmo gairdneri) to aflatoxin Q1 and synergistic effect of cyclopropenoid fatty acids.

Aflatoxin Q1 (AFQ1), the major microsomal biotransformation product of aflatoxin B1 (AFB1) formed in vitro by monkey and human liver preparations, was fed to rainbow trout (Salmo gairdneri) in a semipurified diet at levels of 20 ppb for 1 year and 100 ppb for 10 months. As a test for carcinogenicity in hatched fish, it was also used at 1 ppm in a water solution and exposed for 1 hour to fertile trout eggs. The 20-ppb dietary exposure and 1-ppm egg exposure failed to elicit a carcinogenic response; however, the 100-ppb dietary exposure produced a 10.6% incidence of hepatocellular carcinomas at the end of 1 year. Administration of 50 ppm methyl sterculate, a cyclopropenoid fatty acid, in combination with 100 ppb AFQ1 resulted in a synergistic tumor response similar to that previously noted with administration of AFB1 plus aflatoxin M1. The tumor incidence was 89.1% in the fish on the combined diet. These results indicated that AFQ1 was approximately 100 times less carcinogenic than was AFB1. This comparison of carcinogenic potencies was very similar to the comparison of the relative mutagenicities of the two compounds in the Ames bacterial mutagen assay system.

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.

Anticarcinogenic activity of indole-3-carbinol acid products: ultrasensitive bioassay by trout embryo microinjection.

The relative contribution of indole-3-carbinol (I3C) and its acid condensation products to the anticarcinogenic activity of this crucifer phytochemical has been studied using trout embryo microinjection. I3C was treated with 0.07 N HCl to give a reaction mixture (RXM) comprising < 0.5% parent compound and over 20 products, the most prevalent being the dimer 3,3'-diindolylmethane (I33') and a related cyclic trimer (CT). RXM, I33' or CT was injected into embryos with [3H]aflatoxin B1 (AFB1) and total embryonic DNA was isolated 1, 3, or 10 days postinjection. Compared with controls given AFB1 alone, I3C failed to inhibit carcinogen-DNA binding at any time point. In contrast I33', CT, and RXM inhibited AFB1-DNA binding by an average of 37, 51, and 65%, respectively. Coinjection of AFB1 and 350 microM I3C, RXM, or I33' into trout embryos reduced AFB1-induced hepatocarcinogenesis after 1 year from 43.4% in positive controls to 36.0, 12.2 (P < 0.05), and 24.6% (P < 0.05), respectively. No tumor data were obtained in the AFB1 plus CT group due to poor survival of the embryos posthatching. These results indicate that acid condensation products, not the parent compound, represent the anticarcinogenic species in trout and that their formation in the stomach is a likely prerequisite for I3C anticarcinogenesis.

Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Salmo gairdneri) and the synergistic effects of cyclopropenoid fatty acids.

Aflatoxicol (AFL), a major metabolite of aflatoxin B1 (AFB1) in the Mt. Shasta rainbow trout (Salmo gairdneri), was found to produce hepatocellular carcinoma in trout. It was administered in a casein diet to duplicate groups of 120 fingering trout. In the same manner, additional duplicate groups received one of the following: no toxicant; AFB1; the diastereomer of AFL (AFL'); cyclopropenoid fatty acids (CPFA); and CPFA plus AFB1, AFL, and AFL'. Eight months after the initiation of the study, the following incidences of carcinoma were observed: control (0%); 20 ppb AFB1 (56%); 29 ppb AFL (26%); 61 ppb AFL' (0%); 50 ppm CPFA (3%); 20 ppb AFB1 plus 50 ppm CPFA (96%); 29 ppb AFL plus 50 ppm CPFA (94%); and 61 ppb AFL' plus 50 ppm CPFA (55%), showing both the carcinogenicity of AFL and the synergistic effects of CPFA. Twelve-month incidences were correspondingly higher in all cases. Aflatoxin M1, another metabolite of AFB1 in rainbow trout, was reported previously to be carcinogenic in trout. These results support the hypothesis that metabolism in rainbow trout does not effectively detoxify AFB1, but rather the formation of AFL extends the carcinogenicity of AFB1 and may contribute to the high sensitivity of rainbow trout to AFB.

Long term dietary indole-3-carbinol inhibits diethylnitrosamine-initiated hepatocarcinogenesis in the infant mouse model.

Indole-3-carbinol (I3C), a natural component from cruciferous vegetables, has been demonstrated to be a modulator of carcinogenesis in various animal models. Along with the promising perspectives of I3C as a possible chemopreventive agent for human breast cancer, some concerns have been raised regarding the tumor-promotional potency of this compound in other target organs. In this study we examined the hepatic tumor-modulatory properties of I3C fed to C57BL/6J mice, initiated with diethylnitrosamine (DEN). Infant male mice were initiated with 0, 2 or 5 mg/kg DEN (i.p. injection) at 15 days of age. Mice were weaned 9 days later and immediately put on AIN76A semipurified diet (with no antioxidants) containing 0 or 0.15% (1500 ppm) I3C. In addition, at the age of 2 months, one group of mice initiated with 2 mg/kg DEN was injected i.p. with a single dose (20 mg/kg) of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), to serve as a positive control group for promotion. Mice were sampled for hepatic tumors at the age of 6 or 8 months. Each sampled group contained 11-12 mice except the HCB group (nine animals). After 8 months, there was a statistically significant (P < 0.0005) inhibition of hepatocarcinogenesis observed for I3C-fed animals initiated with the high dose of DEN. A single injection of HCB at 2 months of age significantly (P = 0.0003) enhanced hepatocarcinogenesis in mice initiated with 2 mg/kg DEN. There was no statistically significant difference between groups sampled at 6 months of age. Our observations indicate that long term administration of I3C in the diet inhibits DEN-initiated hepatocarcinogenesis in the infant mouse model.

Hepatocarcinogenic potency of mixed and pure enantiomers of trans-7,8-dihydrobenzo[a]pyrene-7,8-diol in trout.

The hepatocarcinogenic potency of pure and racemic trans-7,8-dihydrobenzo[a]pyrene-7,8-diol was investigated in embryos and sac-fry rainbow trout. Embryos microinjected with (+/-)-trans-7,8-dihydrobenzo[a]pyrene-7,8-diol ((+/-) BP-7,8-DHD) developed liver tumors 9 months after hatching. However, this exposure protocol resulted in high mortalities. Microinjection of newly hatched sac-fry with 0.01-1.0 microgram of (+/-) BP-7,8-DHD resulted in a dose-dependent production of liver tumors (0-13%) similar to the results with embryos but without the problem of high mortalities. Co-injection of sac-fry with (+/-) BP-7,8-DHD and either beta-naphthoflavone or carbon tetrachloride significantly enhanced the tumor response (approx. 3-fold). The relative carcinogenic potencies of the pure (+) and (-) enantiomers of BP-7,8-DHD were evaluated by microinjection into sac-fry at doses of 0.5-5.0 micrograms. The results demonstrated that the (-) enantiomer was 4-18 times more potent than the (+). Mixed carcinomas were the most prevalent liver tumors observed. These results demonstrate that trout embryos and sac-fry are both responsive to hepatocarcinogenesis initiation by injection with BP-7,8-DHD. The marked enhancement seen with co-injection of sac-fry with beta-naphthoflavone or carbon tetrachloride suggests that both cytochrome P-450-dependent and lipid peroxidation-dependent pathways could be involved in bioactivation of this compound, presumably through epoxidation at the 9,10-position. As is the case with mammals, the (-) enantiomer of BP-7,8-DHD is a more potent carcinogen than the (+) enantiomer.

Covalent binding of (+) 7S-trans-7,8-dihydrobenzo [a]pyrene-7,8-diol to trout DNA: P-450- and peroxidation-dependent pathways.

Bioactivation in vivo of pure (+) 7S-trans-7,8-dihydrobenzo[a]pyrene- 7,8-diol ((+) BP-7,8-DHD) was investigated in rainbow trout. Embryos, microinjected with 0.01-1.0 microgram of [3H]-(-)-7S-trans-7,8-dihydrobenzo[a]-pyrene-7,8-diol-anti-9,10-epoxide ((-) anti-BPDE), exhibited a dose-dependent increase in DNA adduction. Subsequently, microinjection of trout embryos with [14C] (+) BP-7,8-DHD also demonstrated a dose-dependent increase in DNA adduction. To determine the relative contribution of P-450-dependent versus peroxidation-dependent epoxygenation of (+)-BP-7,8-DHD, trout embryos were co-injected with [14C]-(+)-BP-7,8-DHD and either beta-naphthoflavone (BNF) (CYP1A1 inducer) or carbon tetrachloride (CCl4) (lipid peroxidation enhancer). Co-injection with BNF tended to enhance covalent binding to DNA, which was consistent with rapid induction of CYP1A1. Co-injection with CCl4, significantly increased covalent binding of [14C]-(+)-BP-7,8-DHD to DNA, suggesting a contribution from non-enzymic cooxidation. 32P-Postlabeling analysis of liver DNA adducts following i.p. injections of (+) BP-7,8-DHD did not detect appreciable amounts of (-) anti-BPDE-dG from juvenile trout fed control diets or diets containing hydrogen peroxide or BNF. On the contrary, BNF pre-feeding markedly enhanced the levels of an adduct which co-chromatographed with authentic (+) syn-BPDE-dG. These results confirm that trout are capable of metabolically activating BP-DHD to the ultimate carcinogen BPDE and that BNF stimulates CYP1A1-dependent epoxygenation, but peroxidation-dependent activation may not contribute significantly to the bioactivation of BP-7,8-DHD in vivo.

Long-term, high-dose dietary exposure of rainbow trout to butylated hydroxyanisole is non-carcinogenic.

The hepatocarcinogenic and/or promotional properties of butylated hydroxyanisole (BHA) were tested in rainbow trout. Four groups of 100, 21-day-old trout embryos were exposed to 0.5 ppm aqueous aflatoxin B1 (AFB1) for 30 min, and four similar groups were sham treated. After hatching, swim-up, and the onset of vigorous feeding behavior (2 weeks pest swim-up), duplicate groups of 60 AFB1-treated and sham-treated fry were started on a test diet containing 0.6% (6000 ppm) BHA, and the other duplicate groups of treated and untreated fry were fed the control Oregon Test Diet (OTD). After 8 months of feeding BHA or OTD, the fish were necropsied for tumor detection, with particular attention given to the stomach and liver. No tumors were seen in the livers or stomachs of the sham-treated fish fed OTD or BHA, showing that BHA is not carcinogenic to rainbow trout under the conditions of this experiment. Promotional results were equivocal, with one tank of fish having a higher hepatic tumor incidence, but the other the same as the positive AFB1 control. When the tanks were combined, however, there was no statistical difference between the two groups.

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Fish models for environmental carcinogenesis: the rainbow trout.

Progress over the past 30 years has revealed many strengths of the rainbow trout as an alternative model for environmental carcinogenesis research. These include low rearing costs, an early life-stage ultrasensitive bioassay, sensitivity to many classes of carcinogen, a well-described tumor pathology, responsiveness to tumor promoters and inhibitors, and a mechanistically informative nonmammalian comparative status. Low-cost husbandry, for example, has permitted statistically challenging tumor study designs with up to 10,000 trout to investigate the quantitative interrelationships among carcinogen dose, anticarcinogen dose, DNA adduct formation, and final tumor outcome. The basic elements of the trout carcinogen bioassay include multiple exposure routes, carcinogen response, husbandry requirements, and pathology. The principal known neoplasms occur in liver (mixed hepatocellular/cholangiocellular adenoma and carcinoma, hepatocellular carcinoma), kidney (nephroblastoma), swim bladder (adenopapilloma), and stomach (adenopapilloma). Trout possess a complex but incompletely characterized array of cytochromes P450, transferases, and other enzymic systems for phase I and phase II procarcinogen metabolism. In general, trout exhibit only limited capacity for DNA repair, especially for removal of bulky DNA adducts. This factor, together with a high capacity for P450 bioactivation and negligible glutathione transferase-mediated detoxication of the epoxide, accounts for the exceptional sensitivity of trout to aflatoxin B1 carcinogenesis. At the gene level, all trout tumors except nephroblastoma exhibit variable and often high incidences of oncogenic Ki-ras gene mutations. Mutations in the trout p53 tumor suppressor gene have yet to be described. There are many aspects of the trout model, especially the lack of complete organ homology, that limit its application as a surrogate for human cancer research. Within these limitations, however, it is apparent that trout and other fish models can serve as highly useful adjuncts to conventional rodent models in the study of environmental carcinogenesis and its modulation. For some problems, fish models can provide wholly unique approaches.

The hepatocarcinogenicity of diethylnitrosamine to rainbow trout and its enhancement by Aroclors 1242 and 1254.

Rainbow trout (Salmo gairdneri) were fed diets containing 100 ppm Aroclor 1242 (AC42) or Aroclor 1254 (AC54) in combination with 1100 ppm diethylnitrosamine (DEN) for one year. The incidence of hepatocarcinomas was determined and compared with the incidence in trout fed 1100 ppm DEN alone. The two Aroclors dramatically enhanced tumor incidence from 10.2% in the positive controls (DEN alone) to 40.2% for AC42 and 21.6% for AC54. This is in contrast to previous results obtained when AC54 was fed concomitantly with aflatoxin B1 (AFB1), where a substantial inhibition of carcinogenesis was observed. The alteration of chemical carcinogenesis in trout by PCB, therefore, depends upon the carcinogen involved and is not a generalized effect.