Dennis W. Shelton

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.

Inhibitory Effect of Aroclor 1254 on Aflatoxin-Initiated Carcinogenesis in Rainbow Trout and Mutagenesis Using a Salmonella/Trout Hepatic Activation System

Abstract Duplicate lots of 140 rainbow trout ( Salmo gairdneri ) were fed either control diet (CD) or 100 ppm Aroclor 1254 (a polychlorinated biphenyl—PCB) for 3 mth followed by initiation of liver carcinomas with 20 ppb dietary aflatoxin B 1 (AFB 1 ) for 2 wk. At 8 and 12 mth after AFB 1 treatment, fish were sampled and tumor incidence determined. Trout that were prefed PCB showed a 45% inhibition in tumor incidence at 12 mth, when compared to those prefed CD. Throughout the experiment fish were sampled to determine the time relationship of PCB bioaccumulation. A rapid uptake of PCB into total body fat was seen, with concentration of 594 ppm at the time of AFB 1 exposure. Liver benzo[ a ]pyrehe monooxygenase (B[ a ]PM) activity was also induced at the time AFB 1 exposure began. The Ames mutagenesis assay was used to determine the effects of in vitro Aroclor 1254 on AFB 1 -induced mutagenesis using a trout liver preparation. A PCB dose-related inhibition was observed with a 67% inhibition at the highest dose tested (500 μg Aroclor 1254/plate). Proposed mechanisms of the inhibition of AFB 1 -induced carcinogenesis/mutagenesis are discussed.

Effect of dose on the inhibition of carcinogenesis/mutagenesis by aroclor 1254 in rainbow trout fed aflatoxin B1

Prior studies have shown that Aroclor 1254 (PCB) differentially alters the incidence of aflatoxin B1 (AFB1) induced hepatocellular carcinomas in trout, depending upon the time of PCB administration relative to AFB1 exposure (Shelton et al., 1983). When fed simultaneously with AFB1, PCB inhibits carcinoma incidence. We investigated the effect of AFB1 and PCB dose on this inhibition. Duplicate tanks of 100 rainbow trout were fed AFB1 at concentrations of 1, 4, or 8 ppb, either with or without the addition of 50 ppm PCB. Other groups were fed 4 ppb AFB1 + 5 ppm PCB, 50 ppm PCB alone, or control diet alone. After 9 and 12 mo, 40 and 60 fish per tank, respectively, were sampled to determine the incidence of liver tumors. The results show a parallel inhibition of the AFB1-tumor dose-response curve by the presence of 50 ppm PCB. Fish fed 4 ppb AFB1 + 5 ppm PCB showed slight inhibition in response when compared with 4ppb AFB1 alone. Also, livers from fish fed 50 ppm PCB were used to prepare S20 for use in the Salmonella mutagenesis assay. These livers were less efficient in converting AFB1 to a mutagen, when compared to control S20. The AFB1-mutagenesis dose-response curve was again shifted parallel to the right of the curve generated using control S20. These results suggest that the inhibitory action is at least partly at the level of carcinogen activation. The finding of parallel, as opposed to proportional, inhibition with varying carcinogen exposure for certain classes of inhibitors may have important implications for inhibition of environmental carcinogenesis at low levels of carcinogen exposure.

Time- and dose-dependent effects of dietary cyclopropenoid fatty acids on the hepatic microsomal mixed function oxidase system of rainbow trout

Abstract Rainbow trout ( Salmo gairdneri ) were fed diets containing various levels of cyclopropenoid fatty acids (CPFAs) and sampled at several time intervals to measure the effects on the mixed function oxidase (MFO) system. Feeding rainbow trout a diet containing 300 ppm CPFAs for 1 wk was sufficient to cause measurable changes in hepatic cytochrome P -450 content, microsomal protein content, NADPH—cytochrome creductase activity, and aryl hydrocarbon hydroxylase activity. After 3 wk, diets containing 50, 150 or 300 ppm CPFAs caused variable effects on mixed function oxidase (MFO) enzyme activities; however, diets containing 450 and 600 ppm CPFAs suppressed NADPH—cytochrome c reductase, ethoxycoumarin- O -deethylase, aryl hydrocarbon hydroxylase, p -nitroanisole- O -demethylase, epoxide hydrase, and glutathione transferase activities and decreased cytochrome P -450 and microsomal protein content compared to controls. The suppressive effects of dietary CPFAs on the trout MFO system is consistent with the decreased ability of liver from trout fed CPFAs to biotransform aflatoxin B 1 (AFB 1 ) to less active derivatives. The S20 liver fraction from trout fed diets containing CPFAs however, did not enhance the mutagenic activity of AFB 1 using Salmonella typhinurium TA 98. The results indicate that dietary CPFAs in trout do not induce the cytochrome P -450 dependent microsomal MFO system which converts AFB 1 to the active 2,3-oxide. The mechanism by which dietary CPFAs act as synergists with AFB 1 seems to be through the post-initiation promotion of AFB 1 formed lesions rather than through alteration of the MFO system or AFB 1 metabolism.

Effects of dietary aroclor 1254 and cyclopropene fatty acids on hepatic enzymes in rainbow trout.

Diets containging Aroclor® 1254 (PCB); cyclopropene fatty acids (CPFA), and PCB plus CPFA were fed to rainbow trout (Salmo gairdneri) for 15 weeks to determine the effects on hepatic microsomal activities of ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and benzo(a)pyrene monooxygenase. Ethoxyresorufin-O-deethylase activity continued to increase to a level 77-fold higher than control at week 15. Ethoxycoumarin O-deethylase and benzo(a)pyrene monooxygenase activities increased to 7.1-fold and 47-fold over control at week 9, respectively. Cytochrome P-450 values remained approximately 2-fold above controls from week 5 through week 15. At weeks 1 and 3, cytochrome P-450 levels were not significantly different from control.Dietary CPFA significantly depressed ethoresor-ufin-O-deethylase and ethoxycoumarin-O-deethylase activities, but had no effect on benzo(a)pyrene monooxygenase activity. Ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and benzo(a)pyrene monooxygenase activities in the combined PCB-CPFA-fed trout were significantly higher than in control- or CPFA-fed trout, and significantly lower than in PCB-fed trout. This is the first time dietary PCB s have been shown to induce the MFO system in rainbow trout. These results provide a possible explanation for the effects of dietary PCB s on the metabolism and expression of other chemical carcinogens.

Quantification of the joint effects of mixtures of hepatotoxic agents: Evaluation of a theoretical model in mice

Abstract An approach previously developed for studying the effects of toxic mixtures on whole organism performances (i.e., growth, mortality) was evaluated to determine its applicability and limitations at the organ system level. The approach was tested by quantifying the hepatotoxic effects of carbon tetrachloride (CCl 4 ), monochlorobenzene (MCB), acetaminophen (ACET), and selected mixtures of these compounds in male albino CF-1 mice. Based upon parallel dose-response curves, concentration addition was predicted for the mixtures of both CCl 4 + MCB and CCl 4 + ACET. The actual dose-response relationship for each mixture was empirically determined and compared to the predicted curves. In the case of the CCl 4 + MCB mixture, the observed dose-response curve did not deviate from that predicted on the basis of concentration addition. However, a similar comparison for the CCl 4 + ACET mixture revealed a statistical difference between the observed and predicted curves. The joint effects for the mixture of CCl 4 + MCB is thus classified as concentration additive. The model proves to be adequate in predicting, classifying, and describing the joint effects of these hepatotoxicants.