Liver injury and expression of cytochromes P450: evidence that regulation of CYP2A5 is different from that of other major xenobiotic metabolizing CYP enzymes.

The purpose of this study was to find out how liver injury caused by two well-known hepatotoxins, chloroform and thioacetamide, alters the expression of hepatic xenobiotic metabolizing cytochrome P450 (CYP) enzymes of DBA/2N mice. Dose-dependent toxic effects of the two hepatotoxins were verified by histological examination. Along with the toxicity, intense staining of immunoreactive material was detected in the centrilobular zone, with anti-CYP2A5 antibody in hepatic tissue. This apparent increase in the expression of Cyp2a-5 was verified by Northern blot and Western blot analyses and by determining the enzymatic activity, coumarin 7-hydroxylase, in hepatic tissue. The results suggest that liver injury due to these hepatotoxins increases the expression of Cyp2a-5 and that the expression is pretranslationally regulated. The increased expression of Cyp2a-5 is in contrast with that of other xenobiotic metabolizing CYPs because a dose-[dose-dependent] dependent decrease of the total hepatic P450 content and either a decrease or no change in the levels of CYP1A, 2B, 2C, 2E1, and 3A4 were observed. The results suggest that essential differences exist in the regulation of CYP2A5 and other major xenobiotic metabolizing CYP enzymes and that in a damaged liver CYP2A5 may be a major catalyst of xenobiotic metabolism.

Cytochrome P450 2A of nasal epithelium: regulation and role in carcinogen metabolism.

In this study, we found that rat nasal coumarin-7-hydroxylase (COH) activity was two orders of magnitude higher than rat hepatic COH activity and could be induced by adding coumarin to the rats' drinking water. In western blot analysis, an anti-cytochrome P450 (Cyp) 2a-5 (mouse liver COH) antibody recognized a sharp band in the microsomal fraction of rat nasal epithelium but not of the liver; the band comigrated with Cyp2a-5. The intensity of the band was increased by the coumarin treatment. Similarly, in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5 mRNA; however, no hybridizable mRNA was recognized in liver preparations. Unlike the protein level, the level of the mRNA was not increased by coumarin. When northern blot analyses were performed with two oligoprobes specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited rat nasal COH activity and aflatoxin B1 (AFB1) metabolism completely. It inhibited N-nitrosodiethylamine (NDEA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by 80-90%. In contrast, the hepatic metabolism of the four compounds was not affected by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a strong but variable inhibition of the nasal metabolism of AFB1, NDEA, and NNK was seen. The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2a-5. The increase probably occurs by protein stabilization or stimulation of translation. The results also show that the enzyme has a key role in the nasal metabolism of three well-known carcinogens, AFB1, NDEA, and NNK and may therefore be an important contributing factor in nasal carcinogenesis.

Diet and oxidative stress in breast, colon and prostate cancer patients: a case-control study.

OBJECTIVE: To study the changes in pro-oxidant-antioxidant status in breast, colon and prostate cancer patients as compared to respective controls. DESIGN: Cross-sectional case-control study. The pro-oxidant status was measured by analysing alkanes (ethane and pentane) in exhaled air and lipid peroxidation (as malonaldehyde) in blood samples. The antioxidant capacity was measured by studying blood glutathione concentration, vitamin concentrations and serum antioxidant capacity in liposomes in vitro. SETTING: Aberdeen hospitals. SUBJECTS: Breast, prostate and colon cancer cases, and age- and sex-matched control patients (hospitalized for a benign disease). Breast cancer patients were females, prostate cancer patients were males and colon cancer patients were both males and females. Controls were age-matched to within 5 years, sex-matched and matched for smoking habits. RESULTS: The dietary study suggested a higher monoene and polyene fat intake in prostate cancer than in controls while in other cancer patients no significant differences were found. Breast and colon cancer patients tended to have lower vitamin intakes than controls. Pentane concentration in exhaled air increased in breast cancer patients as compared to respective controls. In serum total antioxidant capacity no significant differences were found. Both breast and colon cancer patients showed decreased C18:2 and C20:4 fatty acid concentrations in red blood cells while C22:6 concentration was elevated in breast cancer patients. CONCLUSIONS: Oxidative stress may be associated with malignant diseases, suggesting the importance of simultaneous analysis of pro- and antioxidation in the search of mechanistic parameters leading to the tumour formation.

Dietary fat- and phenobarbital-induced alterations in hepatic antioxidant functions of mice.

Inbred strains of mice with differential response to known tumor promoters were compared with respect to their susceptibility to modulation of hepatic antioxidant enzymes by long-term treatment with high fat diet (HF) and phenobarbital (PB). Mice of the C57BL/6J (C57), C3H/HeOuJ (C3H) and DBA/2J (DBA) strains were fed diets containing low (5%) or high (15%) amounts of fat (sunflower oil) for 26 weeks from the age of 6 weeks onwards. Groups of mice on the 5% fat diet received 0.05% PB in their drinking water from 12 to 22 weeks of age. Mice of the C57 strain are known to be refractory to promotion of hepatocarcinogenesis, the C3H strain has a high incidence of spontaneous tumors and is sensitive to promotion by HF and PB, and the DBA strain is especially sensitive to promotion by PB. Within all strains of mice, and in both dietary groups, the degree of oxidative stress in the liver was found to increase with age, as was indicated by the increased amounts of TBA reactive material (lipid peroxidation) and decreased glutathione (GSH) and phospholipid contents of the tissue. HF elevated the amount of TBA reactive material in the liver of C57 and C3H mice, induced GSH-peroxidase and Mn-superoxide dismutase activities in the C3H strain, and depressed the hexose monophosphate shunt activity within all mouse strains. PB drastically decreased the amount of TBA reactive material in the liver in all mouse strains, increased catalase activity in all strains and the activity of GSH-peroxidase in the C3H and DBA strains. The above strain differences in responses of hepatic antioxidant functions to HF and PB parallel the differential responsiveness of these mouse strains to promotion of hepatocarcinogenesis by these agents, and the increased antioxidant capacity was proportional to susceptibility to tumor promotion.

Gas chromatography-mass spectrometry analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and feces.

A method has been developed to measure levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) excreted in urine and feces. The method involves organic solvent extraction, derivatization to form electron-capturing bis-pentafluorobenzyl derivatives, and analysis by gas chromatography-negative ion chemical ionization mass spectrometry using a deuterium-labeled internal standard. The method can detect PhIP at levels of less than 1 ng/g in rat urine (5 ng/24 hr) and 5 ng/g (wet weight) in rat feces (50 ng/24 hr). Sprague-Dawley rats given a single 50 micrograms dose of PhIP by gavage excreted an average of 0.6% of the dose in the urine and 25% of the dose in the feces as unchanged PhIP, in the first 4 days after treatment. To make this method applicable for the analyses of biological fluids of PhIP-exposed human subjects, it is now being improved by using immunoaffinity chromatography.

High variability of nitrosamine metabolism among individuals: role of cytochromes P450 2A6 and 2E1 in the dealkylation of N-nitrosodimethylamine and N-nitrosodiethylamine in mice and humans.

We undertook this study to answer several questions regarding nitrosamine metabolism. Kinetics of nitrosamine metabolism showed the involvement of at least two enzymes in the dealkylation of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in mouse liver microsomes. Coumarin inhibited both reactions competitively. On the other hand, microsomal coumarin 7-hydroxylase was inhibited by NDMA (Ki 2.7 mM) and NDEA (Ki 0.013 mM). The big difference in the Ki values suggests a higher affinity of NDEA than NDMA to Cyp2a-5 (mouse cytochrome P450coh). A specific antibody against Cyp2a-5 inhibited more of the microsomal NDEA (up to 90%) than NDMA (up to 40%) dealkylation. The converse was true with anti-Cyp2e-1 antibody. These results suggest that the primary substrate for Cyp2a-5 is NDEA and for Cyp2e-1, NDMA. Western blot analysis of human liver microsomes showed a great interindividual variation in the amounts of CYP2A6 (human cytochrome P450coh) and CYP2E1. Also, coumarin 7-hydroxylation and nitrosamine dealkylation varied greatly among individuals. A high correlation (r = 0.93, P < 0.001) was found between NDEA and coumarin metabolism. Both activities were associated with CYP2A6. On the other hand, little or no correlation was found between microsomal CYP2A6 and CYP2E1 or between CYP2E1 and NDEA dealkylation. Immunoinhibition of human microsomal NDEA metabolism by CYP2a-5 antibody varied greatly among individuals (10-90%), suggesting, as in the case of mice, that NDEA is metabolized primarily by CYP2A6, at least in some individuals. Taken together the data suggest that (1) the metabolic activation of nitrosamines in humans varies greatly among individuals; (2) different nitrosamines may partially be metabolized by different cytochrome P450 isozymes; and (3) because of similarities between nitrosamine metabolism in mice and humans, inbred strains of mice would be relevant experimental models for studying nitrosamine activation.

Drug metabolism in rats with cancer induced by N-nitrosodiethylamine and phenobarbital.

The metabolism of R- and S-warfarin in vivo and in vitro, bufuralol in vitro, and antipyrine and debrisoquine in vivo were studied in rats with cancer induced by N-nitrosodiethylamine and phenobarbital treatment. Microsomal cytochrome P-450 content was greatly reduced in both healthy and cancerous parts of the livers of tumour-bearing animals. The specific activities of R-warfarin and bufuralol 1'-hydroxylases were significantly elevated in rats with cancer. The activities of S-warfarin hydroxylases expressed per mg microsomal protein were reduced in animals with cancer, whereas those of R-warfarin and bufuralol 1'-hydroxylases were not. The urinary excretion of R-7-hydroxywarfarin was increased and those of S-6- and S-4'-hydroxywarfarin decreased in rats with cancer. The correlations between microsomal formation and urinary excretion of all warfarin metabolites were poor, except for R-7-hydroxywarfarin. Antipyrine oxidation was increased in the cancerous state but the urinary metabolic profiles were similar in rats with cancer and in controls. The metabolism of debrisoquine was decreased in tumour-bearing animals. Antipyrine metabolism did not show any correlation with either warfarin or debrisoquine metabolism, whereas several relationships were observed between warfarin and debrisoquine metabolism and between warfarin and bufuralol metabolism.

Mechanisms of fat-related modulation of N-nitrosodiethylamine-induced tumors in rats: organ distribution, blood lipids, enzymes and pro-oxidant state.

Groups of rats, either dosed with N-nitrosodiethylamine (NDEA) for 10 weeks (from the age of 7 to 17 weeks) or untreated, were fed diets containing either 2% (low fat, LF) or 30% polyunsaturated fat (high fat, HF) on an equicaloric basis from 5 weeks until rats were 43 weeks old. Biochemical parameters were measured during and at the end of the experiment in various organs, blood, urine and exhaled air, for correlation with the presence or absence of tumors. The HF diet tended to increase the number of hepatic tumors induced by NDEA, while the number of extrahepatic tumors was higher in rats fed on the LF diet; also the overall tumor incidence was higher in the LF group. In the HF/NDEA group, only two benign extrahepatic tumors were found. Plasma total and free cholesterol and triglyceride concentrations were lower in the HF than the LF group without NDEA treatment. In animals bearing liver and/or extrahepatic tumors all plasma lipid concentrations were lower than in tumor-free animals. Only minor or no changes were detected in blood catalase activity, malondialdehyde level, reduced glutathione (GSH) level or GSH-related enzymes and excretion of thioethers in the urine due to dietary modulation or NDEA. Changes in the liver that were associated with the HF diet were: (i) increased amounts of some polyunsaturated fatty acids and of total phospholipids in liver microsomes; (ii) an enhanced level of lipid peroxidation in liver; (iii) a decrease in liver glutathione levels during NDEA treatment, with a simultaneous adaptive increase in superoxide dismutase levels, and a decrease in renal glutathione levels in both treated and untreated groups; (iv) enhanced microsomal induction of aminopyrine N-demethylase and epoxide hydrolase activities by NDEA, and (v) decreased hexose monophosphate shunt (HMS) activity. All mono-oxygenase activities were lower, and the activities of epoxide hydrolase, UDP-glucuronosyltransferase and HMS were higher, in liver tumors than in non-tumorous liver of similarly-treated rats. Neither diet nor NDEA had a major effect on drug-metabolizing enzyme activities in lung and kidney. HF diet significantly increased ethane exhalation (an indicator of the whole-body pro-oxidant state) over those on the LF diet: in rats on either diet, it was further increased when NDEA was given. Ethane exhalation was still elevated 30 weeks after the cessation of NDEA treatment. Our results suggest an association between the observed changes in biochemical parameters, notably oxidative stress, due to dietary modulation and the altered tumor incidence and organ distribution of tumors induced by NDEA.

Bacterial formation of N-nitroso compounds from administered precursors in the rat stomach after omeprazole-induced achlorhydria.

The role of bacteria in catalysing intragastric formation of N-nitrosothiazolidine-4-carboxylic acid and N-nitrosomorpholine was investigated in a rat model of omeprazole-induced achlorhydria. Omeprazole-treated rats gavaged with nitrosation-proficient bacteria were treated with nitrosamines and/or precursors and compared to control animals that received no omeprazole treatment/no bacteria. Rats given thiazolidine-4-carboxylic acid, nitrate and 10(11) cells of Escherichia coli, had a five times higher endogenous formation of N-nitrosothiazolidine-4-carboxylic acid as compared to controls. Endogenous formation of N-nitrosomorpholine was quantified by measuring its urinary metabolite N-nitroso-(2-hydroxyethyl)glycine; when rats were given morpholine and nitrite together with E. coli or Pseudomonas aeruginosa endogenous N-nitrosomorpholine formation was increased approximately 2.5-fold as compared to controls. In the same experiment, a higher excretion of unchanged N-nitrosomorpholine was also observed in omeprazole-treated rats receiving bacteria as compared to controls. Rats given morpholine, nitrate and E. coli or P. aeruginosa, excreted three times higher levels of N-nitrosomorpholine as compared to controls. These results conclusively demonstrate that nitrosation-proficient bacteria are capable of increasing intragastric formation of N-nitrosothiazolidine-4-carboxylic acid and N-nitrosomorpholine. These N-nitrosamines are formed from nitrate (or nitrite) and the respective amino precursor via reduction of nitrate into nitrite and bacterial nitrosation catalysis.

Bacterial formation of N-nitroso compounds in the rat stomach after omeprazole-induced achlorhydria.

N-Nitrosamine formation by bacteria in the achlorhydric stomach has been proposed as an important factor in the development of gastric cancer. Thus, the effect of the presence of bacteria in the stomach on endogenous nitrosation was investigated in rats given omeprazole (an inhibitor of gastric H+, K((+)-ATPase) which reduces gastric secretion sufficiently to allow survival of a bacterial suspension of Escherichia coli or Pseudomonas. When rats were given both thiazolidine 4-carboxylic acid and nitrate, greater endogenous nitrosamine formation was observed in rats receiving omeprazole and an E. coli suspension than in control or omeprazole-treated rats. A similar result was obtained when rats were given morpholine and nitrate. Since the endogenous formation of N-nitrosomorpholine (NMOR) can be evaluated more precisely from the levels of its urinary metabolites, N-nitrosohydroxyethylglycine (NHEG), the metabolism of NMOR was studied in omeprazole-treated rats. In this preliminary study, we showed that 60% of an oral dose of NMOR was excreted as NHEG, while in rats with a higher gastric pH 20% was excreted as NHEG. The amount of endogenously formed NMOR was increased in omeprazole-treated rats given morpholine and nitrite together with bacteria, and greater excretion of unchanged urinary NMOR was observed. Thus, as shown in this in-vivo model, bacteria efficiently reduce nitrate to nitrite and catalyse nitrosation, resulting in increased endogenous formation of N-nitroso compounds in the achlorhydric stomach.

Characterization of the cytochrome P450 isozyme that metabolizes ochratoxin A, using metabolic inducers, inhibitors and antibodies.

The phenotypic pattern of drug biotransformation is determined by both host and environmental factors. Debrisoquine is a good probe for phenotyping individuals, as its metabolism is not affected by age, gender, smoking habits or alcohol intake. People with Balkan endemic nephropathy or with urinary tract tumours in endemic areas are more frequently extensive metabolizers of debrisoquine than are healthy people. This finding has led to studies of the possible relationship between the metabolism of ochratoxin A and its toxicity and carcinogenicity on experimental models. Ochratoxin A is metabolized mainly in the liver into R- and S-isomers of 4- and 10-hydroxyochratoxin A, and the reaction is catalysed by cytochrome P450 haemoprotein. Animal species that are genetically different in their capacity to metabolize debrisoquine differ similarly in their capacity to metabolize ochratoxin A: female DA rats that are poor metabolizers of debrisoquine also poorly metabolize ochratoxin A, as assayed by urinary excretion of both the parent compound and of 4-hydroxyochratoxin A. Ochratoxin A hydroxylase activity is low in DA rat liver (and kidney) but is inducible by phenobarbital and 3-methylcholanthrene; debrisoquine hydroxylase is not known to be inducible by enzyme inducers. The reaction of ochratoxin A hydroxylase thus resembles those induced by 3-methyl-cholanthrene and catalysed by cytochrome P450IA. Ochratoxin A hydroxylase activity was further characterized in the livers of B6 and D2 mice that had been treated with typical enzyme inducers. Ochratoxin A hydroxylase was weakly inducible by phenobarbital, 3-methyl-cholanthrene and 2,4,7,8-tetrachlorodibenzodioxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a high fat diet on liver DNA methylation in rats exposed to N-nitrosodimethylamine.

Previous experiments have shown that a high fat diet changes incidence and tumour sites by N-nitroso-dialkylamines. The purpose of this study was to examine the effect of high and low fat diet on DNA methylation 6 weeks after the end of a chronic N-nitrosodimethylamine (NDMA) exposure (total dose 150 mg/kg). The concentration of O6-methyldeoxyguanosine (O6-MedG) in liver DNA was measured by immunoassays. The level of O6-MedG persisted 6 weeks after the last dose of NDMA and was 6-fold higher (P less than 0.05) in animals on high fat as compared to low fat diet. In another experiment, in which rats on a low and high fat diet received a single NDMA dose (2 mg/kg), the time-dependent removal of O6-MedG from liver and the hepatic O6-methylguanine DNA-alkyltransferase activity was not modified by the type of diet. These results indicate that a high fat diet enhances DNA methylation in the liver, after chronic treatment by NDMA, and that this effect is likely to be responsible for an increased incidence of liver haemangiosarcomas.

Quantity and saturation degree of dietary fats as modulators of oxidative stress and chemically-induced liver tumours in rats.

Male rats were fed, from weaning onwards, either 2, 12.5 or 25% sunflower seed oil (polyunsaturated fatty acids, PSA) or lard (saturated fatty acids, SFA) and from the age of 15 weeks subgroups were given N-nitrosodimethylamine (NDMA) for 30 weeks. Blood levels of lipids were assayed and during the study exhaled ethane was measured as an index of in vivo lipid peroxidation (LPO). At the age of 50 weeks, rats were killed and livers were analysed for tumours. PSA diets decreased plasma cholesterol and triglyceride concentrations vs. respective SFA diet; NDMA administration did not affect plasma cholesterol but enhanced triglyceride concentration. NDMA markedly enhanced LPO. An increase in dietary fat content from 2 to 25% enhanced ethane exhalation, more in rats fed PSA than the SFA diet. In the 25% PSA group, indomethacin in the diet strongly inhibited LPO. Prevalence of liver haemangiosarcomas increased from 42% to 80% (p less than 0.05) in NDMA-treated animals when PSA increased from 2 to 25%; in the group having a 25% PSA diet containing indomethacin, the NDMA-induced tumour incidence was reduced to 64%. In NDMA-treated rats fed SFA diets the prevalence of haemangiosarcoma increased from 43% (2% fat) to 67% (25% fat). The data show that NDMA modifies plasma lipids and increases LPO. The quantity and saturation degree of fats altered the frequency of chemically-induced tumours and modified LPO. As an index of free radical reactions, LPO may have an important role in carcinogenesis. Dietary fat thus appears to promote carcinogenesis through mechanisms that involve LPO.

Modulating effects in human diets of dietary fibre and beef, and of time and dose on the reactive microcapsule trapping of benzo[a]pyrene metabolites in the rat gastrointestinal tract.

Trapping by magnetic polyethyleneimine (PEI) microcapsules was utilized to investigate the influence in male rats of dose, human dietary composition and time-dependence on reactive metabolites of benzo[a]pyrene (B[a]P) in the gastrointestinal (GI) tract; also, PEI microcapsules modified with copper phthalocyanine tetrasulphonic acid (CPTS) were tested in vivo for trapping of endogenous mutagens having planar molecular structure. In a preliminary experiment the PEI microcapsules were administered by gavage at 0, 24 and 48 h, with [14C]B[a]P at 2 h to chow-fed BDVI rats; microcapsules were recovered from faeces collected at 24, 48 and 72 h, and then subjected to an extraction sequence showing that the trapped B[a]P metabolites were inconsistent with B[a]P diol epoxide trapping (as previously found) and unaltered by elapsed time or 5-fold dose alteration of B[a]P. Then five groups of F344 rats were fed isocalorically either one of four low-fat human diets or rat chow; in order to investigate influences of diet both on B[a]P and endogenous mutagens, half of each group was tested at 2 weeks with this PEI microcapsule/[14C]B[a]P protocol and then at 3 weeks, PEI-CPTS microcapsules (two gavages). So as to provide a cross-over comparison, the other half of each group was first tested with PEI-CPTS microcapsules followed by PEI microcapsules/[14C]B[a]P 1 week later. The human diets were prepared from cooked British foods so as to simulate the adequate intake of all nutrients required by humans; but with 3-fold differences in intake levels of beef and dietary fibre non-starch polysaccharide (NSP), while ensuring the same intake of available energy, protein, fat and calcium. They gave very similar body-weight gains in the four groups but greatly reduced faecal weight, protein and total faecal enzyme activity compared with chow; the extraction pattern of microcapsule-trapped B[a]P metabolite radioactivity was not significantly altered. However, human diet consumption caused a 2- to 6-fold increase in B[a]P metabolite binding to microcapsules and reductions in microcapsule recovery, net 70-h B[a]P excretion, faecal protein and total activities for beta-glucuronidase and beta-galactosidase; these effects were more pronounced after 3 weeks, presumably due to prolonged dietary adaptation. Increased NSP in human diets significantly increased the B[a]P metabolite excretion and marginally reduced the microcapsule binding. The increase in microcapsule binding of B[a]P metabolites, interpreted as reflecting an increased amount of reactive metabolites encountered, was related to the dietary intake weight ratio of beef/NSP.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleophilic selectivity and reaction kinetics of chloroethylene oxide assessed by the 4-(p-nitrobenzyl)pyridine assay and proton nuclear magnetic resonance spectroscopy.

The nucleophilic selectivity (Swain-Scott's constant s) of chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, was determined to be 0.71 using the 4-(p-nitrobenzyl)pyridine (NBP) assay (Spears method). The molar extinction coefficient of the adduct formed between NBP and CEO was measured; and the second-order rate constants for the reactions of CEO with NBP and with thiosulfate were estimated at three temperatures. The disappearance of CEO and the formation of chloroacetaldehyde (CAA) and glycolaldehyde (GCA) were followed in D2O or a mixture of D2O/hexadeuterated acetone (acetone-d6), using Fourier transform proton nuclear magnetic resonance spectroscopy (1H-FTNMR). Evidence was obtained that CEO reacts with chloride ions to yield CAA at a rate constant of about 17 M-1 h-1 in D2O/acetone-d6 (1 : 1, v/v) at 280 K. Under the same conditions, the first-order rate constant kr for the thermal rearrangement of CEO into CAA was estimated to be approximately 0.41 h-1. These data suggest that the isomerization of CEO may be a minor reaction in physiological saline. These chemical properties of CEO are discussed in relation to the mechanism of vinyl chloride-induced carcinogenesis.

Polymorphic ochratoxin A hydroxylation in rat strains phenotyped as poor and extensive metabolizers of debrisoquine.

1. Dark agouti (DA) and Lewis rat strains, which show a genetic polymorphism for debrisoquine-4-hydroxylation, were treated either with a single dose of ochratoxin A (OA) or for 8 weeks with 5 doses per week. Levels of OA and its 4-hydroxy metabolite (4-hydroxy-OA) excreted in urine were determined. 2. At all doses, the metabolic ratio of OA:4-hydroxy-OA was two to five times greater in DA than in Lewis rats, as was the metabolic ratio of debrisoquine:4-hydroxy-debrisoquine. These results are consistent with our previous findings in vitro that hepatic and renal OA 4-hydroxylase activity is three to four times lower in DA than in Lewis rats. These data give further support to the possible co-segregation of genes regulating OA and debrisoquine 4-hydroxylation.

Lipid peroxidation as a possible cause of ochratoxin A toxicity.

Addition of the mycotoxin ochratoxin A (OA), a nephrotoxic carcinogen, to rat liver microsomes greatly enhanced the rate of NADPH or ascorbate-dependent lipid peroxidation as measured by malondialdehyde formation. NADPH-dependent lipid peroxidation in kidney microsomes was similarly enhanced by OA. The process required the presence of trace amounts of iron but cytochrome P-450 and free active oxygen species appeared not to be involved. The efficiency of several ochratoxins (ochratoxins A, B, C, alpha and O-methyl-ochratoxin C) to enhance lipid peroxidation was related to the presence and reactivity of the phenolic hydroxyl group. Furthermore, the ability of these ochratoxins to enhance lipid peroxidation in microsomes correlated precisely with their known toxicities in chicks. Administration of ochratoxin A to rats also resulted in enhanced lipid peroxidation in vivo as evidenced by a seven-fold increase in the rate of ethane exhalation. These results suggest that lipid peroxidation may play a role in the observed toxicity of ochratoxin A in animals; a mechanism is proposed. (Formula: see text). Ochratoxin A: X = Cl; R1 = R2 = R3 = R4 = H Ochratoxin B: X = H; R1 = R2 = R3 = R4 = H Ochratoxin C: X = Cl; R1 = R2 = R3 = H; = R4 = CH3 O-Methyl-ochratoxin C: X = Cl; R2 = R3 = H; R1 = R4 = CH3 (4R)-4-hydroxyochratoxin A: X = Cl; R1 = R3 = R4 = H; R2 = OH (4S)-4-hydroxyochratoxin A: X = Cl; R1 = R2 = R4 = H; R3 = OH Fig. 1. Chemical structures of the various ochratoxins.

Recoverable, semipermeable, microencapsulated DNA surrogates for monitoring the colorectal cavity; in-situ effects of fibre and meat in human diets on benzo[a]pyrene and possible endogenous cross-linking agents.

Semipermeable magnetic microcapsules containing polyethyleneimine (PEI) as a DNA surrogate are shown to trap 14C-benzo[a]pyrene and hitherto unknown, endogenous, putative cross-linking agent(s) within the gut of male Fischer rats. Trapping is substantially modulated by complete, cooked human diets fed isocalorically and varied three-fold in either beef, fat or bran fibre nonstarch polysaccharide within the normal human intake levels. Preliminary results indicate that the crosslinking agent(s) are derived from microflora. Using metabolized benzo[a]pyrene as a model DNA damaging agent within the gut, beef and decreased bran fibre were found to increase its availability, paralleling risk alterations found in nutritional epidemiology. These novel microcapsules are capable of intercepting a range of substances relevant to DNA damage.

Monoclonal antibody characterization of hepatic and extrahepatic cytochrome P-450 activities in rats treated with phenobarbital or methylcholanthrene and fed various cholesterol diets.

Monoclonal antibodies (MAb) against 3-methylcholanthrene (MC)- and phenobarbital (PB)-inducible forms of cytochrome P-450 isozyme were used to characterize changes in aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECDE) activities modulated by dietary cholesterol. Rats were induced by MC or PB, and immunochemical inhibition of AHH and ECDE activities was studied as an indication of changes in cytochrome P-450 isozyme patterns. Feeding of a cholesterol-free diet markedly decreased enzyme activities both in liver and in small intestinal mucosa, and the highest activities were observed after feeding rats a high (2%)-cholesterol diet for one month. As a control, a normal pelleted diet (0.1% cholesterol) was used; in rats fed this diet, intermediate levels of monooxygenase activities were present. Although no diet-dependent change in total AHH and ECDE activities was observed in kidneys and lungs, diet apparently modulated isozyme composition in the lungs, as indicated by a change in the immunochemical inhibition pattern with MAb; no such shift was observed in the kidneys. In liver and intestine, in addition to changes in total activity, isozyme composition was also altered, as indicated by inhibition of the catalytic activities of cytochrome P-450 by MAb. Our data infer that dietary cholesterol can: (i) modulate total monooxygenase activities, especially in the intestine; (ii) change the cytochrome P-450 isozyme composition in liver and intestine; (iii) change isozyme composition without changing overall enzyme activity, e.g. in lungs; and (iv) have no effect in a tissue (e.g. kidney) that lacks constitutionally the P-450 isozyme responsive to cholesterol.

Rapid oxidative stress induced by N-nitrosamines.

We have investigated the generation of prooxidant state shortly after administration of N-nitrosamines (NA) to rats. N-Nitrosodimethylamine (NDMA) was found to increase ethane exhalation (EE) rapidly in a dose-related manner. EE remained elevated for several days after single doses of NDMA. Similarly, lipid peroxidation (LP) in the liver (measured by four methods) increased rapidly showing a peak 20 min after NDMA dose. The increase of LP was preceded by a decrease in retinol concentration in the liver. N-Nitrosodiethanolamine, too, increased EE and LP in the liver, whereas N-nitrosomethylbenzylamine had no effect. Thus, hepatocarcinogenic NA induced LP in their target tissue, and the LP enhancing effects of NA were not related to their acute toxic effects.