Assessment of the bioavailability of PAHs in rats exposed to a polluted soil by natural routes: induction of EROD activity and DNA adducts and PAH burden in both liver and lung.

In order to assess bioavailability of polycyclic aromatic hydrocarbons (PAHs) present in soils, male laboratory rats were exposed to litters of control and polluted soils. After 88+/-2 h of exposure, several biomarkers were measured in both liver and lung. When rats were exposed to SIV soil, contaminated by a mixture of at least 13 PAHs, (1) only 2 or 3 PAH compounds were detected in liver and lung; (2) cytochrome P450-dependent monooxygenase activity, followed by 7-ethoxyresorufin O-deethylase (EROD) activity measurement, was highly induced in liver (13-fold-induction) and lung (up to 78-fold); and (3) DNA adducts were significantly increased. For what concerns soil artificially contaminated by only one PAH (phenanthrene or B[a]P), EROD activity was not or fully induced, respectively. These results demonstrate the occurrence of a high bioavailability of PAHs to mammals in natural conditions of exposure. First results concerning DNA adducts must be profound, but they already show that a short exposure of mammals to PAH-polluted soils can lead to potential genotoxic effects. EROD activity can be used as a sensitive biomarker in both liver and lung of rats maintained on litters of soils in the laboratory, and such a test can be used routinely to contribute to risk assessment.

Bioavailability of PCBs to male laboratory rats maintained on litters of contaminated soils: PCB burden and induction of alkoxyresorufin O-dealkylase activities in liver and lung.

Male rats from the Sprague-Dawley laboratory strain were maintained in the laboratory during 3 days and 1 night on litters containing a reference soil and different amounts of a soil, mainly polluted by PCBs (207 ppm expressed in Aroclor(R) 1254; SIII soil). Two categories of biomarkers of exposure were measured in both liver and lung of these rats: PCB burdens and activities of microsomal liver and lung cytochrome P450-dependent mono-oxygenases, namely ethoxy-, pentoxy-, and benzoxy-resorufin O-dealkylase activities (EROD, PROD, and BROD, respectively). PCB burdens in liver and lung of rats exposed to SIII soil were 1,845 and 241 ppb, respectively (expressed in Aroclor(R) 1254 equivalents). EROD, PROD, and BROD were significantly induced in the liver of rats exposed to SIII soil, while only EROD activity was induced in the lung. Induction of hepatic EROD activity was approximately 3- to 5.4-fold; pulmonary EROD activity was induced by 9- to 12-fold. In the lung, PROD and BROD activities were inhibited. When rats were exposed to SIII soil diluted with various amounts of standard ISO soil, a nearly linear dose-response relationship was found between the level of PCBs in the litter and EROD activity in both liver and lung. A nonlinear dose-response relationship exists with hepatic BROD activity; no dose-response relationship was observed with hepatic PROD and pulmonary PROD and BROD activities. EROD activity measurement in both liver and lung of rats maintained on a litter of PCB polluted soil was used to assess the bioavailability to mammals of PCBs.

Activities of liver and lung cytochrome P450-dependent monooxygenases and antioxidant enzymes in laboratory and wild Norway rats exposed to reference and contaminated soils.

Laboratory and wild Norway rats were exposed in the laboratory to an uncontaminated soil and to a soil from a site contaminated with petrochemical waste. Activities of microsomal lung and liver cytochrome P450-dependent monooxygenases, including 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD) and 7-benzoxyresorufin O-debenzylase (BROD) were measured at selected times during the course of the study. The highest degree of induction of hepatic EROD (7-fold) was shown after 3 days of exposure to the contaminated soil. However, two months later, the EROD activity declined to fourfold increase over the control. The PROD and BROD activities displayed a similar time course of induction, but the degree of induction was lower. The induction of hepatic monooxygenase activities was observed in both laboratory and wild rats. Lung monooxygenase EROD was highly induced (up to 28-fold) after 3 days of exposure, and the activity remained elevated throughout the two-month experiment. BROD and PROD activities were not induced. The activities of three antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase (Se- and non-Se- dependent) and catalase also were measured in lung and liver cytosol, but no significant changes were observed after two months of exposure to contaminated soil.

Activities of cytochrome P450-dependent monooxygenases and antioxidant enzymes in different organs of Norway rats (Rattus norvegicus) inhabiting reference and contaminated sites.

Wild Norway rats (Rattus norvegicus) were collected from a site contaminated by a range of polycyclic aromatic hydrocarbons (PAHs), mineral oils, polychlorobiphenyls (PCBs) and heavy metals. Activities of cytochrome P450 monooxygenases (ethoxy-, pentoxy- and benzoxy-resorufin O-dealkylases, and 4-nitrophenol hydroxylase) were measured in microsomal fractions from liver and lung. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and non-selenium-dependent glutathione peroxidases) were also measured in cytosolic fractions from lung and liver, and in erythrocytes. The levels of activities were compared with those found in control laboratory rats and in wild Norway rats reared in a terrarium. Results show that rats living in a polluted environment have monooxygenase activities higher than that of control animals in both liver and lung. Some modifications of antioxidant enzyme activities were also found in these animals.

Stereoselective sulfoxidation of the pesticide methiocarb by flavin-containing monooxygenase and cytochrome P450-dependent monooxygenases of rat liver microsomes. Anticholinesterase activity of the two sulfoxide enantiomers.

Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 microM-1.min-1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 microM-1.min-1 for the (A) and (B) enantiomers, respectively).

In vivo metabolism of aminopyrine by the larvae of the helminth Heligmosomoides polygyrus.

The in vivo N-dealkylation of [13C-2]-labeled aminopyrine by the L1-L2 larvae of Heligmosomoides polygyrus was demonstrated by the use of a sensitive gas chromatography-mass spectrometry method. This is the first evidence for the possible existence of a cytochrome P-450-dependent activity in helminths.

Action of methotrexate on cytochrome P-450 monooxygenases in rats. Study performed with [13C]-aminopyrine micro breath test.

The aim of this work was to study the action of methotrexate on the cytochrome P-450 hepatic monooxygenases and to follow the evolution of the metabolic processes with time, after oral and intra-peritoneal administration of these drugs to rats. In order to perform this study, we used a new technique the [13C]-aminopyrine breath test (ABT). At the end of the in vivo study, the rats were sacrificed and the cytochrome P-450 concentration as well as microsomal enzymatic activities of aminopyrine N-demethylase, 7-ethoxyresorufin desalkylase, 7-ethoxycoumarin desalkylase, 7-pentoxyresorufin desalkylase were determined. A histological study of the liver was also carried out. Finally, haematological and biochemical parameters were also determined. In this study, 3 series of 6 rats were used: a control group, a group receiving 70 mg/kg of methotrexate by intra-peritoneal route, and a group of rats treated orally with 1 mg/kg of methotrexate every 2 days. For each observation, the variation of the ABT scores and of the cytochrome P-450 amounts as well as the microsomal aminopyrine N-demethylase activity were in good agreement.

Stereoselective S-oxygenation of an aryl-trifluoromethyl sulfoxide to the corresponding sulfone by rat liver cytochromes P450.

Toltrazuril sulfoxide (TZR.SO) is the metabolite of the antiparasitic drug toltrazuril (TZR; 1-methyl-3-[3-methyl-4-[4-[trifluoromethyl]thio]phenoxy]phenyl- 1,3,5-triazine-2,4,6(1H,3H,5H)-trione). The results of the present paper demonstrate that TZR.SO was metabolized by rat liver microsomes to the corresponding sulfone (TZR.SO2). The reaction was mediated almost exclusively by different cytochromes P450, the most active being cytochromes P450 3A. TZR.SO exists as a racemic mixture; when each enantiomer was incubated separately in the presence of untreated rat liver microsomes, a 7.3-fold difference in the rate of S-oxygenation was found, indicating a marked substrate enantioselectivity for the reaction.

Specific and enantioselective sulfoxidation of an aryl-trifluoromethyl sulfide by rat liver cytochromes P-450.

Evidence based on thermal stability and enzyme inhibition data suggests that the sulfoxidation of the drug toltrazuril by rat liver microsomes is catalyzed by different cytochromes P-450. Pretreatment of rats by different inducers--phenobarbital, 3-methylcholanthrene, dexamethasone, and triacetyloleandomycin--results in a 2.1-, 2.6-, 2.9-, and 1.8-fold increase, respectively, in the rate of sulfoxidation. The highest increase (8.4-fold) was observed after treatment of microsomes from triacetyloleandomycin-treated animals by potassium ferricyanide. Castration and aging also modify the sulfoxidase activity. The relative rate of formation of the two toltrazuril enantiomers [(A)- and (B)-sulfoxides] depends on the source of the microsomes, suggesting that different cytochromes P-450 have different stereoselectivities.

Induction and characterization of cytochromes P450IA and -IIB in the newt, Pleurodeles waltl.

The newt (Pleurodeles waltl) is an amphibian species used in a mutagenicity test (micronucleus). This study was carried out to establish if the inducibility of hepatic cytochromes P450 of this species is similar to that of the rat. Our results showed that the newt is characterized by a lower level of hepatic cytochrome P450-dependent activities than the rat. Variations of enzymatic activities according to sex and season were observed. Specific activities in newt were characterized by an almost complete insensitivity to induction by phenobarbital pretreatment. On the other hand, pretreatment by 3-methylcholanthrene resulted in an increase in the metabolism of several hydroxycoumarin and resorufin derivatives, similar to the effects observed in rat liver. Occurrence of specific forms of cytochromes P450 was assessed by specific antibodies.

Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration.

The metabolic fate and pathway of the imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) were investigated in the rat after administration of oral single doses with radiolabelled molecules. At both dose levels (50 and 250 mg/kg body weight), virtually all of the ingested [14C-phenyl]prochloraz was excreted in the urine or faeces within 96 hr, the bulk of excretion occurring between 24 and 48 hr after dosing. Urinary elimination accounted for 61 and 68% of the respective initial doses. Urinary metabolic products were isolated and identified by thin-layer chromatography, gas chromatography or gas chromatography coupled with mass spectrometry analysis. Prochloraz was completely metabolized with no unchanged compound being excreted in the urine. The main biotransformation products in rat urine were 2,4,6-trichlorophenoxyacetic acid and its corresponding alcohol, the latter as a glucuronic acid conjugate. Ring hydroxylation also occurred, with the hydroxy-2,4,6-trichlorophenoxyethanol and hydroxy-2,4,6-trichlorophenoxyacetic acid metabolites excreted in small amounts in the urine. 2,4,6-Trichlorophenol and unconjugated 2,4,6-trichlorophenoxyethanol were identified as minor urinary metabolites.

Hepatic microsomal monooxygenase activities in natural populations of the Mallard DuckAnas platyrhynchos, the Tufted DuckAythya fuligula and the Great Crested GrebePodiceps cristatus.

Birds from three waterfowl species were collected over a period of three years, mainly near the river Rhine in France. Cytochrome P-450 content and monooxygenase activities were measured in liver microsomes of 71 Mallard DucksAnas platyrhynchos, 57 Tufted DucksAythya fuligula and 64 Great Crested GrebesPodiceps cristatus. The monooxygenase activities were estimated by measuring the dealkylation of substituted alkoxycoumarins, substituted alkylresorufins, and the metabolism of the insecticide parathion to its neurotoxic metabolite, paraoxon. A very high dispersion of values was shown, with no indication of season- or sex-related differences. It was concluded that the cytochromes P-450 in the studied populations of these waterfowl species are not induced, even if some individuals exhibit elevated cytochrome P-450 and enzymatic activities. Correlations were found between some enzymatic activities which suggested that the regulation of monooxygenase activities is widely different between birds and rodent laboratory species.

Effect of beta-naphthoflavone and MCPA on liver and kidney drug-metabolizing enzymes from the carp, Cyprinus carpio.

The effects of beta-naphthoflavone (beta-NF) and a chlorophenoxyacetic acid herbicide (MCPA) on hepatic and renal monooxygenase activities and conjugating enzymes from immature carp (Cyprinus carpio) were studied. beta-NF increased hepatic monooxygenase activities but the patterns of differential induction generally obtained in rat liver microsomes with two series of homologous substrates, alkoxycoumarins and alkylresorufins, were not found to be similar in carp liver microsomes. On the other hand, MCPA caused no changes in oxidative metabolism, with the exception of decreased aryl hydrocarbon hydroxylase activity. Renal activities were not modified by MCPA, while beta-NF treatment resulted in marked increases in monooxygenase activities with alkylresorufins as substrates. No changes were found in conjugation activities after treatment with MCPA or beta-NF. These results indicate that (a) the herbicide MCPA should have no effect on drug-metabolizing enzymes from carp, and (b) the hepatic and renal monoxygenase activities of carp are responsive to beta-NF, allowing their use in monitoring water pollution.

Induction and inhibition of rat liver cytochrome(s) P-450 by an imidazole fungicide (prochloraz).

Prochloraz (1-[N-propyl-N-2(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) is an imidazole molecule widely used as a fungicide. This study reports the in vivo and in vitro effects of this compound on microsomal drug metabolising enzymes from rat liver. In vivo pretreatment of animals (250 mg/kg body wt for 3 days) with prochloraz elicited complex modifications. When animals were sacrificed 24 h after the last dose, an increase in total cytochrome P-450 was observed as well as an increase in catalytic activities towards benzphetamine, alkoxyresorufins and alkoxycoumarins. However, when animals were sacrificed 48 h after the last dose, a lower induction of 7-ethoxyresorufin O-deethylase and a higher induction of 7-pentoxyresorufin O-depentylase and 7-benzoxyresorufin O-debenzylase were found. Such results lead us to consider prochloraz as a "mixed inducer" of the hepatic cytochromes P-450. In vitro experiments were indicating a strong inhibition of 7-alkoxyresorufin O-dealkylase activities by prochloraz. The analysis of the CO-difference spectrum of cytochrome P-450 showed also tight binding of prochloraz to the haemoprotein in animals sacrificed 24 h but not 48 h after the last dose. Furthermore, prochloraz did not induce significantly the microsomal cytochrome P-450 IVA1-dependent 12-hydroxylation of lauric acid.

In vivo and in vitro metabolism of benzo(a)pyrene by the larva of the newt, Pleurodeles waltl.

1. The larva of the amphibian species, Pleurodeles waltl was shown to metabolize benzo(a)pyrene in vivo into a variety of oxidized products. 2. In vitro, BaP hydroxylase (AHH) activity was found in hepatic microsomes and postmitochondrial fractions from both larvae and adults of the pleurodele. 3. The clastogenic effect of BaP formation of micronuclei in the erythrocytes was shown to be related to the presence of BaP quinones in the tissues of the newt.

Effects of chemical pollution on the activities of hepatic xenobiotic metabolizing enzymes in fish from the River Rhône.

Polychlorobiphenyl (PCBs) levels and hepatic xenobiotic metabolizing enzyme activities were measured in fish from three locations of the River Rhône to study the consequences of a constant loading of PCBs from a PCB incineration plant. Our results show that levels of PCBs and enzyme activities were higher in fish living downstream from the plant than in fish from two locations upstream, suggesting enzyme induction by PCBs (known to be potent inducers in laboratory conditions). Enzyme activities were studied in spring and autumn in three species: nase (Chondrostoma nasus), roach (Rutilus rutilus) and grayling (Thymallus thymallus). Induction was observed for three cytochrome P-450-dependent monooxygenase activities (MO), i.e. 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD). There was a close correlation between EROD and AHH activities (for all species). Glutathione S-transferase activities were also shown to be related to the PCB levels. Conversely, cytochrome P-450 content and benzphetamine N-demethylase activity were not "PCB level-dependent". This study clearly demonstrates a close relationship between PCB contamination and MO activities in fish from the field and thus clearly emphasizes the interest in MO as a monitoring tool for estimating water quality.

Animal and plant cytochrome P-450 systems.

Cytochromes P-450 are a family of hemeproteins catalyzing the metabolism of both endogenous and exogenous (xenobiotic) compounds. This review describes the general features of these enzymes and some aspects of comparative studies, mainly in fish, birds, insects and plants.