Norman E. Pawlowski

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.

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.

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.

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.

Mass spectra of methyl sterculate and malvalate and 1,2-dialkylcyclopropenes☆

Abstract The mass spectra of 1,2-dipropyl-, 1,2-dipentyl-, 1,2-dihexyl-, 1,2-diheptyl-, and 1,2-dioctyl-cyclopropene, methyl malvalate, methyl sterculate, malvalyl alcohol, 1,2-dipropyl-, 1,2-dipentyl-, and 1,2-dihexylcyclopropene-3-carboxylic acid, and methyl-9,10-(carbethoxymethano)-9-octadecenoate are presented. A noticable feature of the 1,2-disubstituted cyclopropene spectra is the total absence of a cyclopropenium ion. The cyclopropenes with a carboxyl group in the 3-position yield cyclopropenium ions in the mass spectra. β-Cleavage to a allylic ion appears to be important.

Metabolism and tissue distribution of label from [9,10-methylene-14C]sterculic acid in the rat.

The metabolism of14C-sterculic acid, labeled in the methylene carbon of the cyclopropene ring, was investigated in Wistar rats. Comparison of the distribution of radioactivity in tissue and excreta as a function of time showed that the free sterculic acid was metabolized faster than the methyl ester and that the sterculic acid administered by intragastric intubation was absorbed and metabolized at a faster rate than that administered by intraperitoneal injection. The concentration of label in blood serum reached a maximum 2 hr after intubation and then rapidly declined. Incorporation of radioactivity into most organs peaked at 4 hr with liver peaking at a maximum of 11% of the administered dose and other organs at less than 1%. Label in depot fat steadily increased to 8% at 26 hr. Less than 1% of the administered dose was expired in CO2 in the same time period. Excretion of label reached a maximum of 48% in urine and 11% in feces by 16 hr. The majority of the label in liver was in the fatty acid portion of the lipid fraction. The relative amount of label in microsomal and mitochondrial subcellular fractions of liver changed with time suggesting that these organelles may be involved in the metabolism of sterculic acid. Rats fed control diets appeared to metabolize sterculic acid in the same manner, but at a slower rate than rats acclimated to dietary cyclopropene fatty acids. Low recovery of label in expired air showed that the methylene carbon of the cyclopropene ring was not oxidized to CO2. These data suggest that rats readily absorb sterculic acid and excrete labeled compounds primarily in the urine.

HPLC Analysis of cyclopropenoid fatty acids

Cyclopropenoid fatty acid methyl esters have been analyzed by high pressure liquid chromatography (HPLC). The refractive index detector’s linear response to various lipids was studied. Verification of peak identity was by spectroscopy of collected peaks and cochromatography with authentic samples. The HPLC method is simple, convenient and gives precision and absolute, values which are consistent with those from traditional methods.

Rat urinary metabolites of [9,10-methylene-14C]sterculic acid☆

1. The metabolism of [9,10-methylene-14C] sterculic acid was studied in corn oil and Stercula foetida oil fed rats. The majority of the radioactivity was excreted into the urine as short chain dicarboxylic acids. The main urinary metabolites were cis-3,4-methylene adipic acid, cis-3,4-methylene suberic acid, trans-3,4-methylene adipic acid, cis-3,4-methylene pimelic acid, and cis-3,4-methylene azelic acid. 2. Formation of these urinary metabolites requires alpha-, beta-, and omega-oxidation plus reduction of the cyclopropene ring to a cyclopropane ring. Sterculic acid must be transported through both mitochondrial and microsomal systems. 3. Other non-radioactive urinary compounds were also identified. A proposed pathway for the metabolism of sterculic acid and possible detrimental effects caused by these metabolites is discussed.

Incorporation of label from [9, 10-methylene-14C] sterculic acid in rainbow trout,Salmo gairdneri

The distribution of radioactivity from sterculic acid, labeled on the 9,10-methylene carbon of the cyclopropene ring, was investigated in trout,Salmo gairdneri. Fifty percent of the administered dose was excreted in feces and urine by 168 hr, but less than 1% of the dose was expired as carbon dioxide during the same time period. Incorporation of radioactivity into most organs peaked at 119 hr, and the majority of the label in the liver was in the fatty acid portion of the lipid fraction. Total lipid radioactivity in liver was higher in trout conditioned to cyclopropene lipids, and a substantial amount of label was found in phosphatidylcholine and ethanolamine phospholipids as well as neutral lipid. The data demonstrate that rainbow trout readily absorb, transport and incorporate sterculic acid into tissue lipid, including membrane lipid, but cannot oxidize the methylene carbon of the cyclopropene ring to carbon dioxide.