Acute effects of benzo[a]pyrene on liver phase I and II enzymes, and DNA damage on sea bream Sparus aurata.

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg(-1)) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 +/- 0.67 microg g(-1) dry weight after only 6 h exposure and 4.67 +/- 0.68 microg g(-1) dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 % DNA in the tail.

Acute effects of cadmium on liver phase I and phase II enzymes and metallothionein accumulation on sea bream Sparus aurata.

This research was designed to study Sparus aurata (sea bream) biotransformation and detoxification responses to acute exposure to cadmium (Cd). Sexually immature gilthead sea bream were treated by intraperitoneal injection of Cd chloride (200 microg kg(-1)) for 6, 12, 24 and 48 h. Cd accumulation was quantified in sea bream liver by graphite furnace atomic absorption spectroscopy after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity as phase I biotransformation parameter, (2) liver glutathione-S-transferase (GST) activity as a phase II conjugation enzyme and metallothionein (MT) content as specific response to Cd contamination. Cd bioaccumulation in the liver resulted in an increasing uptake up to 10.3 microg g(-1) wet weight after 48 h of exposure. EROD showed a significant activation only after 6 h exposure and a return to control levels after 12 h. GST revealed significant activation starting from 12 h exposure. MT accumulation in liver showed the same behavior as GST activation.

Preliminary study of responses in mussel (Mytilus edilus) exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether.

Environmental pollutants with hormonal activity including bisphenol, diallyl phtalate and tetrabromodiphenyl ether, have the potential to alter gonadal development and reproduction in aquatic wildlife. Little is known about the biological impact of environmentally relevant concentrations in mussels. To investigate some aspects of their potential estrogenic action, mussels were continuously exposed during 3 weeks. Gonadal development and vitellogenin like protein levels were examined. Bisphenol (50 microg/l) induced the expression of phospho-proteins in females and spawning in both sexes. Diallyl phthalate and tetrabromodiphenyl ether decreased phospho-protein levels in both sexes and induced spawning in males. Moreover, severe damaging effects on ovarian follicles and ovocytes were observed in both bisphenol A- and tetrabromodiphenyl ether-exposed female mussels.

Effect of unbalanced diets on incorporation of delta-aminolevulinic acid into cytochrome P-450.

The in vivo syntheses of two liver microsomal cytochromes P-450 PB3a, P-450 UT50 [(1987) Eur. J. Biochem., submitted] (Mr 50,000, 52,000) have been estimated by measuring the specific activity 2 h after i.p. administration of delta-[3H]aminolevulinic acid to male Sprague Dawley rats. The animals were fed either a standard rat chow (5% lard, 22% casein) or unbalanced diets (high lipid, 30% lard or low protein, 6% casein) with or without 50 ppm Phenoclor DP6. The high-lipid diet supported a more rapid body weight gain but had little impact on cytochrome P-450 content, expressed either per whole liver or per mg microsomal protein, and on the incorporation of the precursor into cytochrome P-450. The latter was determined by measuring the radioactivity incorporated into the cytochrome P-450 fraction, partially purified by affinity chromatography, as well as into two cytochrome P-450 isozymes (Mr 50,000 or 52,000) purified by DEAE-52 cellulose ion-exchange chromatography. The low-protein diet, on the other hand, severely depressed body weight gain and cytochrome P-450 content as well as incorporation of radioactivity, the lower-Mr cytochrome (Mr 50,000) being particularly affected. However, when a potent inducer, Phenoclor DP6, was added to the low-protein diet, cytochrome synthesis was restored indicating that the effect was reversible.

Effect of nutritional imbalances on cytochrome P-450 isozymes in rat liver.

Male Sprague-Dawley rats were fed for six weeks either a control diet containing 22% casein (C) and 5% fat (F) or a low-protein diet (6% C, 5% F) or high-lipid diet (30% C, 30% F). A group of rats received a control diet containing 50 ppm of Phenoclor DP6. Three major forms of cytochrome P-450, UT 50, BP 3a and MC 2 were purified from livers of DP6-fed rats and only two forms, UT 50 and PB 3a, were purified from control and dietary groups. The amino acid composition and the catalytic activities towards all substrates tested were only significantly modified in the purified UT 50 P-450 isozyme from rats fed the low-protein diet. The N-terminal sequence analysis shows that cytochrome P-450 UT 50 (from control group) and UT 501 (from low-protein group) are two distinct proteins.

Incorporation of labeled 2,4,5,2',4',5'-hexachlorobiphenyl into the nuclear fraction of rat hepatocytes in vivo.

2,4,5,2',4',5'-[14C] Hexachlorobiphenyl (2,4,5-HCB) a slowly metabolized PCB was given to rats by gastric incubation. The hepatocyte nuclei (HCN) were then isolated and treated with specific hydrolytic enzymes in order to separate the nucleic macromolecules, protein RNA and DNA. The results show that 2,4,5-HCB binds in vivo to hepatocyte nuclei. Liver nuclear proteins bind 70% of 2,4,5-HCB and 30% is found in the DNA fraction. No radioactivity was found in the nuclear RNA fraction at this experimental time (16 h).

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.

A time course investigation of vitamin A level and lipid composition of the liver endoplasmic reticulum in rats following treatment with congeneric polychlorobiphenyls.

The drug metabolizing enzyme activities, the vitamin A content and the fatty acid composition in the endoplasmic reticulum membrane were studied in rat liver after a single injection of the polychlorobiphenyls (PCBs) 3,3',4,4'-tetrachlorobiphenyl [(3,4)2Cl] or 2,2',4,4',5,5'-hexachloro-biphenyl [(2,4,5)2Cl], 300 mumol/kg each. The microsomal vitamin A level was markedly lowered 3 days after treatment with (3,4)2Cl, a coplanar type inducer of cytochrome P-450. A marked increase in microsomal AHH and UDPGT activities occurred within 3 days after injection of (3,4)2Cl whereas (2,4,5,)2Cl treatment enhanced APDM activity only. Arachidonic, stearic and linoleic acid microsomal contents were enhanced by the two congeners. (3,4)2Cl caused the proportion of docosahexaenoic acid to decrease. No highly significant correlation was found between the vitamin A content and lipid components in the microsomal membrane. However, the vitamin A level was inversely related to the activities of drug metabolizing enzymes induced by coplanar compounds (cytochrome P-450 towards benzo[a]pyrene and UDP glucuronosyl transferase towards 4-nitrophenol).

A time-course investigation of vitamin A levels and drug metabolizing enzyme activities in rats following a single treatment with prototypic polychlorinated biphenyls and DDT.

Xenobiotics previously characterized as selective inducers of drug-metabolizing enzymes were chosen to probe possible relationships between enzyme induction and vitamin A metabolism. Liver, kidney and serum retinol and retinyl palmitate levels were investigated in male Sprague--Dawley rats receiving a single i.p. injection of the polychlorinated biphenyls (PCBs), 2,2',5,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl or 2,2',4,4',5,5'-hexachlorobiphenyl (300 mumol/kg) or 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane (DDT) (150 mumol/kg). While 2,2',5,5'-tetrachlorobiphenyl, a weak or non-inducer, and 2,2',4,4',5,5'-hexaclorobiphenyl and DDT, phenobarbital-type inducers of cytochrome P-450, led to no reduction in total vitamin A content of liver or kidney during the 7 day time-course, administration of 3,3',4,4'-tetrachlorobiphenyl, a toxic PCB and a potent 3-methylcholanthrene-type inducer of cytochrome P-450, resulted in progressively lowered liver vitamin A levels (to 40% of control values by day 7). During this time, kidney total vitamin A content increased 3-fold. The increase in kidney vitamin A (due primarily to increased retinol content) was only equal to 1/40 of total vitamin A which had disappeared from the liver. Although 3,3',4,4'-tetrachlorobiphenyl specifically induced certain drug-metabolizing enzyme activities, e.g. aryl hydrocarbon hydroxylase and UDP-glucuronosyltransferase (toward 4-nitrophenol), no highly significant correlations were found among the vitamin A levels and drug-metabolizing enzyme activities in the liver (aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aldrin epoxidase, microsomal epoxide hydrolase, UDP-glucuronosyltransferase toward 4-nitrophenol, glutathione transferase toward 1-chloro-2,4-dinitrobenzene and cytochrome P-450 content) as determined by multiple linear regression analysis.

Effects of carbaryl and naphthalene on rat hepatic CYP1A1/2: potential binding to Ah receptor and 4S benzo(a)pyrene-binding protein.

Activation of the CYP1A1 gene has been described to be mediated by the cytosolic Ah receptor (AhR) and a possible cooperative role of the 4S benzo(a)pyrene-binding protein (4S protein). Carbaryl (CAR) has been shown to induce human CYP1A1 gene expression without binding to the human AhR. In this study, Sprague-Dawley rats received a single i.p. dose of 20, 80, 150 micromol/kg CAR or NAPn (naphthalene, the aromatic part of CAR) and were sacrificed after 24 h. CAR increased ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase activities, the level of CYP1A1, 1A2 proteins, and CYP1A1 mRNA at the highest dose, whereas NAPn showed no effects. Moreover, CAR, naphthol (its major metabolite) and NAPn were not ligands in vitro of the TCDD binding site of AhR or the benzo(a)-pyrene binding site of 4S protein in rat, neither was CAR a ligand of these two binding sites in mice, dog, monkey or human. Molecular properties of CAR were evaluated and showed that this molecule is far from the structural characteristics of CYP 1A1 specific inducers although a planar conformation can be achieved with an energy < 5 kJ x mol(-1). The data demonstrated that CAR could also modulate the AhR-mediated responses, even though it did not meet the structural requirements to be ligand of AhR.

Uranium in the environment: occurrence, transfer, and biological effects.

The assessment of uranium chemical and radiological consequences depends on the physicochemical properties of these radioelements and the knowledge of their environmental fate. Although uranium is the source of all these fissionable isotopes, its fate in ecosystems has been poorly investigated. In this review, we have updated information concerning the fate of uranium in the different compartments of the environment, the possibility of transfer to man through the food chain, and the biological and toxicological effects of this metal at cellular, tissular, or organism levels. The physicochemical characteristics of uranium, as well as its regulatory statutes, were reviewed. The fate of uranium in the environment was presented by indicating sources of uranium emission and the possible routes of transfer to man. The biological alterations caused by uranium exposure were discussed, and finally, we presented results collected during our recent study. Some propositions on research to be done to advance the understanding of uranium occurrence in the environment were also given.

Hepatic Ah receptor binding affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin: similarity between beagle dog and cynomolgus monkey.

Hepatic AhR binding affinity for [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) was compared between two species widely used as laboratory animals: beagle dog and cynomolgus monkey (Macaca fascicularis). The enriched 9S fractions from both species were obtained by sucrose gradient sedimentation. After incubation with [3H]TCDD, dextran-coat charcoal treatment (10 mg/ml) revealed that dog and monkey possess an AhR with a low binding affinity for [3H]TCDD. Saturation experiments were then achieved according to the method developed in experiments on human samples. The binding characteristics were determined after analysis of the data by Scatchard and Woolf plots. Receptor concentrations were quite similar in dog and monkey liver (26.6 and 14.4 pmol/mg, respectively) as well as the affinity (Kd) for [3H]TCDD (17.1 and 16.5 nM, respectively). The low binding affinity of dog and monkey AhRs appeared to be similar to those observed in human.

Prochloraz as potent inhibitor of benzo[a]pyrene metabolism and mutagenic activity in rat liver fractions.

We have determined how prochloraz, an imidazole antifungal agent, affects the metabolism of benzo[a]-pyrene by hepatic microsomes from 3-methylcholanthrene treated male rats. The prochloraz-like 7,8-benzoflavone was a potent inhibitor of total benzo[a]pyrene metabolism while miconazole was a weak inhibitor. The proportion of benzo[a]pyrene dihydrodiols formed was decreased whereas phenols were increased by the in vitro addition of prochloraz. Furthermore, a good correlation was obtained between the effects of prochloraz on the microsomal formation of benzo[a]pyrene metabolites and on the mutagenic activity of benzo[a]pyrene in the Salmonella typhimurium test.

Lipid composition and monooxygenase relationship in liver microsomal membranes from rats fed unbalanced diets.

The cholesterol and phospholipid content of microsomal membrane from rats fed either a high lipid (H1) (30% lard) or a low protein (Lp) diet (6% casein) have been compared with those from rats fed a standard (St) diet (22% casein, 5% lard). For each diet, half of the group was treated with Phenochlor DP6. A significant increase in the phospholipid cholesterol ratio was observed in rats fed a high lipid diet or treated with DP6. These effects tend to increase the microsomal membrane fluidity. The protein deficiency decreased the phospholipid/cholesterol ratio and then the fluidity of the endoplasmic reticulum membrane. The specific activity of cytochrome P-450 to hydroxylate aniline which is independent to the enzymic form of cytochrome P-450 was closely correlated with the viscosity status of the microsomal membrane.

Activation of benzo[a]pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction.

The ability of the mussel postmitochondrial fraction (S9) to activate benzo[a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4',5'-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by alpha-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytochrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.

Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1.

The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of alpha-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IIIA, were decreased in the deficient group to -35% and -16%, respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 IIB and IIIA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+20%) and strongly decreased in the supplemented group (-99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.

Effect of vitamins A, C and glutathione on the mutagenicity of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats.

Vitamin A deficiency has been shown to enhance the mutagenicity of benzo[a]pyrene (Narbonne et al., 1985). Here we report that this is not a result of increased benzo[a]pyrene metabolism but might be a consequence of either a lack of vitamin A or a decreased level of scavengers (ascorbic acid and glutathione) in the liver. However, the addition of vitamin A in vitro in the form of retinyl palmitate strongly inhibits the benzo[a]pyrene mutagenicity. An enhancing effect on the mutagenicity of benzo[a]pyrene is observed with addition of ascorbic acid when incubated with high amounts of the precarcinogen. In vivo addition of high levels of glutathione also reduces the mutagenicity of benzo[a]pyrene.

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.

Effects of vitamins A and E on methylazoxymethanol-induced mutagenesis in Salmonella typhimurium strain TA100.

The aim of this study is to report the antimutagenic effect of vitamin A and vitamin E towards methylazoxymethanol (MAM)-induced mutagenesis in Salmonella typhimurium strain TA100 sensitive to alkylating agents. In order to characterize different levels of action of these two fat-soluble vitamins towards the mutagenicity of MAM, several assays have been considered to show the antimutagenic effect and the possible interactions of vitamins with MAM or with the bacteria. Thus, for each vitamin, three different assays with three different incubations have been conducted: (i) MAM, bacteria and vitamins together, (ii) MAM and vitamins, (iii) bacteria and vitamins. The results showed that both vitamins A and E present an antimutagenic effect towards MAM induced mutagenesis. alpha-Tocopherol seems to have an action directly on to the mutagenic agent, whereas the action of retinol is likely due to a protection of the bacterial genoma against MAM. These in vitro results could help to interpret results of colon carcinogenesis studies using animals induced by 1,2-dimethylhydrazine and fed vitamins supplemented diet.

Structure-activity relationships of the N-methylcarbamate series in Salmonella typhimurium.

Aromatic hydrocarbons of low molecular weight, hydroxy and N-methylcarbamate derivatives were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98 and TA1535 in the presence of a rat-liver 9000 X g supernatant fraction. The presence of 2 or 3 aromatic rings resulted in a weak increase in revertants. Hydroxylation and carbamylation of aromatic rings increased the mutagenic activity of these aromatic compounds. In order to evaluate the structure-activity relationship, the specific molecular connectivity indices were calculated. A significant inverse relationship exists between mutagenicity and zero- and second-order specific molecular connectivity indices. Only compounds with second-order specific molecular connectivity indices lower than 0.300 increased mutagenic activity.