J. E. Nixon

The Sensitivity of Rainbow Trout and Other Fish to Carcinogens

Systematic design of replacement chemicals with reduced toxicities will require knowledge of mechanisms of action of parent compounds, especially in species which occupy the environment of most likely exposure. For aquatic systems, the rainbow trout has proven a valuable model for studying mechanisms of carcinogenicity. By comparison, small aquarium species show great potential as in situ field monitors of aquatic contamination by toxic chemicals but are less developed for mechanism studies. Fish species, especially rainbow trout, have also proven useful alternatives to traditional rodent models for comparative studies on mechanisms of action of nonaquatic carcinogens. These kinds of comparative studies form an essential basis for extrapolation of animal studies to man. Carcinogenicity testing of individual compounds and their replacements can provide only limited information on the expected impact of such chemicals on natural populations, since these populations are unavoidably exposed to potent modulators of the carcinogenic response. Hence any program which aims at redesign of commercial chemicals with reduced toxicities must have as a prior aim the full understanding of the mechanisms of joint carcinogen-inhibitor-promotor interactions. Because of their high sensitivity, low cost per individual, and low background tumor incidences, fish models such as the rainbow trout may be the only vertebrate models in which it is economically practical to initiate such complex studies.

Glucuronides in bile of rainbow trout (Salmo gairdneri) injected with [3H]aflatoxin B1 and the effects of dietary β-naphthoflavone

Rainbow trout Salmo gairdneri were fed a diet containing the mixed-function oxidase system inducer, beta-naphthoflavone or were fed a control diet. For the two respective diets, as much as 50 and 12% of an i.p.-injected dose of [3H]aflatoxin B1 was recovered in the bile. The major product in the bile of beta-naphthoflavone-fed trout was an aflatoxicol-M1 glucuronide, whereas the major product in the control bile was an aflatoxicol glucuronide.

Assay of cyclopropenoid lipids by nuclear magnetic resonance

Nuclear magnetic resonance (NMR) method for the quantitative analysis of cyclopropenoid fatty acids (CPFA) in lipids is described. Good accuracy is obtained at CPFA concentrations of 1% to 100%. At a CPFA concentration of 10%, NMR is accurate to 0.5%. The position of absorption of the two ring methylene hydrogens is solvent dependent.

Effect of subacute toxic levels of dietary cyclopropenoid fatty acids upon membrane function and fatty acid composition in the rat.

The effect of subacute toxicity levels of dietary cyclopropenoid fatty acids upon several physiological parameters was determined in the rat. Diets containing 2% corn oil, 2%Sterculia foetida oil or 2% hydrogenatedSterculia foetida oil were fed.Sterculia foetida oil (50% cyclopropenoid fatty acids) fed rats exhibited retarded growth, elevated organ to body wt ratios, increased saturation of tissue lipid, and abnormal histopathology when compared to corn oil and hydrogenatedSterculia foetida oil fed rats. Growth was retarded 50%, liver/body wt doubled, and the percentage of saturated fatty acids in adipose tissue increased 2.5-fold forSterculia foetida oil vs. corn oil comparisons. Three membrane systems were examined in corn oil andSterculia foetida oil fed rats. Erythrocyte hemolysis rate in 0.3 M glycerol was increased by 30%; induction of mitochondrial swelling by reduced glutathione was inhibited completely and microsomal codeine demethylase activity was depressed nearly 50% inSterculia foetida oil fed rats. The ability of cyclopropenoid fatty acids to inhibit fatty acyl desaturase and influence tissue and membrane lipid composition is discussed. Most of the detrimental effects observed in cyclopropenoid fatty acids fed rats may be associated with alteration of normal lipid metabolism and membrane function.

Mechanisms of Dietary Modification of Aflatoxin B1 Carcinogenesis

Trout were fed a range of dietary components which altered their carcinogenic response to aflatoxin B1 (AFB1). Dietary protein at levels substantially exceeding nutritional requirements were synergistic with AFB1. Cyclopropene fatty acids (CPFA) were carcinogenic when fed alone at 20 or 55 ppm, and synergistic when fed with AFB1. In contrast, several flavonoid and indole compounds, especially beta-naphthoflavone (beta-NF) and indole-3-carbinol, inhibited the carcinogenic response when fed prior to and along with AFB1. The mechanisms by which some of these dietary factors modulate AFB1 carcinogenesis were investigated. Dietary beta-naphthoflavone was shown to substantially induce the levels of mixed function oxidase (MFO) activities assayed in vitro. These changes were accompanied by alterations in AFB1 metabolism and binding in freshly isolated hepatocytes. AFB1 incubated in hepatocytes freshly isolated from fish fed beta-NF diet was metabolized more rapidly, showed enhanced rates of detoxication reactions, and decreased accumulation of AFB1-DNA adducts compared to control hepatocytes. These results suggest that beta-NF inhibits AFB1 carcinogenesis at least in part by altering MFO activities such that detoxication is enhanced and initial DNA damage by AFB1 is reduced. In contrast, high dietary protein is a synergist for AFB1 carcinogenesis, and this appears to occur primarily by enhancing the transformation probability for AFB1-initiated genome damage. Fish treated with AFB1 as embryos and then reared on high protein diets had substantially higher incidences of hepatocellular carcinoma (86%) than similarly treated fish fed normal protein diet (44%) or high protein controls without AFB1 exposure (0-2%). The synergistic behavior of dietary CPFAs also appears to partially involve enhanced transformation following DNA damage by AFB1. Fish exposed as embryos to AFB1 and then fed CPFA-containing diets are known to show promotion effects similar to the high protein results (Hendricks, J.D., Proc. 11th Int. Symp. of the Princess Takamatsu Cancer Research Fund, in press.) However, factors other than promotion are involved in the synergism between CPFA and AFB1. Preliminary studies indicate that dietary CPFAs repress MFO activities and depress DNA damage by AFB1 in vitro. If this occurs in vivo, then the net synergistic effect of dietary CPFAs would involve depression of initial AFB1-induced DNA damage, but highly efficient promotion of transformation from the remaining lesions.

Identification and mutagenicity of aflatoxicol-M1 produced by metabolism of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout (Salmo gairdneri) fed β-naphthoflavone

beta-Naphthoflavone (beta NF) fed to rainbow trout (Salmo gairdneri) at 50 or 500 ppm in the diet, modified the in vitro metabolism of aflatoxin B1 (AFB1) by the postmitochondrial fraction (PMF) of the liver. Production of aflatoxicol (AFL) was significantly less in the 500 ppm beta NF-fed group (33.9 ng/mg protein) than in the control group (45.7 ng/mg protein), aflatoxin M1 production was dependent on the dose of beta NF, being greatest in the 500 ppm beta NF-fed group (48.9 ng/mg protein), intermediate in the 50 ppm beta NF-fed group (3.7 ng/mg protein), and was not detected in controls. A new trout metabolite, 4-hydroxyaflatoxicol (aflatoxicol M1, AFLM1) was also detected in small amounts from in vitro metabolism by liver PMF from beta NF-fed trout. Sufficient quantities of AFLM1 for confirmation of identity by ultraviolet spectra, mass spectra and nuclear magnetic resonance spectra were prepared by biotransformation of AFL using liver microsomes and isolation by HPLC. In a modified Ames mutagen assay with Salmonella typhimurium TA98, ALFM1 was 4.1% as mutagenic as AFB1 in a previous determination. The carcinogenicity of AFLM1 to rainbow trout is expected to be considerably less than that of AFB1.

The effect of indole-3-carbinol, an aflatoxin B1 hepatocarcinoma inhibitor, and other indole analogs on the rainbow trout hepatic mixed function oxidase system

Rainbow trout were fed diets containing 3.4 mmol of indole-3-carbinol, indole-3-ethanol, indole-3-aldehyde, or indole-3-acetic acid in 100 g salmon oil/kg diet for 3 weeks. The indoles did not increase hepatic microsomal cytochrome(s) P-450 or P-448 nor induce the associated mixed function oxidase enzyme activities as measured by ethoxyresorufin-O-deethylase (EROD) and benzphetamine-N-demethylase activities. Indole-3-carbinol did not alter the in vitro metabolism of aflatoxin B1 to aflatoxicol and aflatoxin M1; but the other indoles did suppress the formation of aflatoxin M1 from aflatoxin B1. The results suggest that the mechanism by which indole-3-carbinol protected rainbow trout from aflatoxin B1 (AFB1) hepatocarcinogenesis was not via the alteration of the mixed function oxidase system.

Null carcinogenic effect of large doses of nitrosoproline and nitrosohydroxyproline in Wistar rats.

Summary The nitrosamino acids, nitrosoproline or nitrosohydroxyproline, were given orally to weanling rats in four weekly doses totalling 290 mg/animal and the rats were observed for their lifetime. Negative controls received water and positive controls were given diethylnitrosamine. The rats were maintained on a basal semi-purified diet or a basal diet containing cyclopropenoid fatty acids. No tumorigenic effect was seen in rats given nitrosoproline or nitrosohydroxyproline. Diethylnitrosamine produced a high incidence of hepatomas, as expected. Cyclopropenoid fatty acids had no significant effect on the carcinogenicity of the nitrosamino acids.

The effects of route of exposure and combined exposure of mixed function oxidase inducers and suppressors on hepatic parameters in rainbow trout (Salmo gairdneri)

Abstract Hepatic microsomes obtained from rainbow trout (Salmo gairdneri) administered β-naphthoflavone (βNF) either by a single intraperitoneally (IP) injected dose of 100 mg/kg body wt. or by feeding 500 ppm for 1 wk showed increased cytochrome(s) P-450 content and increased aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECOD) activities compared to controls. The microsomal maximum absorbance wavelength of carboxyferrocytochrome P-450 was 450 nm for the control trout, 448 nm for the trout administered βNF by diet, and 449 nm for the trout administered βNF by IP. Hepatic microsomes from trout fed 500 ppm phenobarbital (PB) for 3 wk showed no induction as demonstrated by cytochrome(s) P-450 content, NADPH-cytochrome c reductase, ethoxyresorufin-O-deethylase (EROD), ECOD, and benzphetamine-N-demethylase (BND) activities. The S20 liver fractions from trout fed βNF were less efficient in converting aflatoxin B1 to a mutagen to Salmonella typhimurium TA 98 than were the S20 liver fractions from control trout. This suggests that dietary βNF may suppress the microsomal conversion of aflatoxin B1 to the active 2,3-oxide in trout. Trout fed 500 ppm βNF for 3 wk followed by 3 more wk of βNF plus 0, 50, 150, 300, 450, or 600 ppm cyclopropenoid fatty acids (CPFAs) exhibited a significant decrease in the following parameters with an increase in dietary CPFAs: liver wt./body wt. percent, microsomal protein and cytochrome(s) P-450 content, and NADPH-cytochrome c reductase, ECOD, AHH, and glutathione transferase activities. Feeding 300 ppm CPFAs with 500 ppm βNF caused a shift in the maximum absorbance wavelength from 448 nm to 449 nm and decreased cytochrome(s) P-450 content by 71% whereas 450 ppm CPFAs caused a further shift to 450 nm and a corresponding 77% decrease in cytochrome(s) P-450 content. βNF induced and CPFAs suppressed the trout hepatic mixed function oxidase system.

Mass spectrometry of the silver nitrate derivatives of cyclopropenoid compounds

Abstract 1,2-dihexylcyclopropene, 1,2-diheptylcyclopropene, 1,2-dioctylcyclopropene, methyl malvalate, and methyl sterculate have been reacted with silver nitrate in methanol to form the corresponding methoxy and ketone derivatives. GLC, IR, and MS data are presented. The mass spectra and a possible method for determining the cyclopropene ring position are discussed.