Jean-Claude Béréziat

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.

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.

Influence of ethyl alcohol on carcinogenesis with N-nitrosodimethylamine

This paper presents the results of an experiment on the combined action of nitrosodimethylamine and ethyl alcohol in C57BL mice. As shown, alcohol can act to change the target organ of that liver carcinogen by favouring development of olfactory neuroepitheliomas, which infiltrate the frontal lobe of the brain.

Occurrence in human urine of new sulphur-containing N-nitrosamino acids N-nitrosothiazolidine 4-carboxylic acid and its 2-methyl derivative, and their formation

SummaryTo quantitate endogenous nitrosation reactions in man, the quantity of N-nitrosoproline (NPRO) excreted in the urine after ingestion of proline and/or nitrate was estimated. When this monitoring method (NPRO test) was applied in clinical and field studies, several hitherto unidentified N-nitroso compounds were frequently detected. These were recently identified as sulphur-containing N-nitrosamino acids, N-nitrosothiazolidine 4-carboxylic acid (NTCA), and trans- and cis-isomers of N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA).NTCA and NMTCA were readily formed in vitro following nitrosation at acidic pH of the respective precursor, thiazolidine 4-carboxylic acid (TCA) or of 2-methylthiazolidine 4-carboxylic acid (MTCA). As the latter compounds can be formed by reaction of l-cysteine with formaldehyde or acetaldehyde, respectively, NTCA and NMTCA were also formed by reacting l-cysteine with the respective aldehyde and with nitrite at optimal pH (2.5 for NTCA and 4.5 for NMTCA).Up to 95% of NTCA and NMTCA given orally to fasted rats was recovered as such in urine and faeces within 2 days. Administration of TCA or MTCA, together with nitrite increased the urinary excretion of NTCA and NMTCA, as did co-administration of l-cysteine, nitrite, and the respective aldehyde.NTCA and NMTCA were also detected in the 24-h urine of human volunteers, and smokers tended to excrete higher levels than nonsmokers. Daily excretion levels varied, however, and a diet supplemented with ascorbic acid significantly descreased the total amount of nitrosamino acids. NTCA and NMTCA may occur in human urine as a result of (i) intake of preformed N-nitroso compounds; (ii) intake of thiazolidine 4-carboxylic acid or its 2-methyl derivative and subsequent nitrisation in vivo; (iii) endogenous two-step synthesis by the reaction of l-cysteine with the respective aldehyde and a nitrosating agent.Thus, measurement of NTCA and NMTCA together with NPRO in urine may provide an index for the exposure of human subjects to nitrosamines or their precursors, i. e., nitrosating agents, certain aldehydes, or aldehyde-generating compounds. Our data demonstrate unequivocally that N-nitroso compounds are formed in the human body, as suggested previously by Druckrey. Their relevance to human cancer at specific sites should now be investigated.

Induction of specific base-pair substitutions in E. coli trpA mutants by chloroenthylene oxide, a carcinogenic vinyl chloride metabolite

Chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, induces base-pair substitution mutations but not frameshift mutations in bacteria. The mutational specificity of CEO was investigated in Escherichia coli, using the trpA mutants developed by Yanofsky. Reversion frequencies to tryptophan prototrophy were analysed, and CEO was found to induce more GC----AT transitions than AT----TA transversions, in addition to a low frequency of other types of substitution. This specificity indicates that CEO is mutagenic through a miscoding DNA adduct. The results are discussed in relation to the various CEO-DNA adducts formed and to their reported or expected mispairing properties.

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.

Influence of ethyl alcohol on the carcinogenic activity of N-nitrosonornicotine.

The present paper describes an experiment designed to investigate the effects of the combined action of different doses of N-nitrosonornicotine (NNN) and ethyl alcohol in BDVI rats. Dose-response relationships of NNN was clearly shown. Ethyl alcohol did not appear to increase, to a great degree, the tumour incidence of NNN. However, ethyl alcohol did shorten the tumour latency period in the groups given NNN in alcoholic solution. In addition, an infiltration of the olfactory tumours to the brain was observed more frequently in both males and females given the high dose of NNN in alcoholic solution.

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.

Elevated Lipid Peroxidation in Rats Induced by Dietary Lipids and N-Nitrosodimethylamine and its Inhibition by Indomethacin Monitored Via Ethane Exhalation

The effect of dietary lipids alone or in combination with an administered carcinogen, N-nitrosodimethylamine (NDMA), on whole body lipid peroxidation was studied in rats in vivo. Groups of rats were fed diets containing 2%, 12.5%, or 25% of either saturated or polyunsaturated fat. Lipid peroxidation in individual animals was determined by measuring the concentration of ethane in exhaled air. Increased ethane exhalation was found in rats when the amount of dietary fat was increased from 2% to 12.5%, but animals receiving 12.5% or 25% fat in the diet exhaled ethane at similar rates. Rats consuming polyunsaturated fat exhaled more ethane than those eating saturated fat. In all groups, NDMA administration drastically increased ethane exhalation. Indomethacin completely blocked the increase in ethane exhalation caused by dietary lipids.

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-Scotts 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.