The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes.

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.

Diflubenzuron, a benzoyl-urea insecticide, is a potent inhibitor of TCDD-induced CYP1A1 expression in HepG2 cells.

Diflubenzuron (DFB) belongs to a group of compounds called benzoyphenyl ureas acting as chitin synthesis inhibitors, which also inhibit growth of B16 murine melanomas. The present study was designed to investigate the effect of this insecticide, on CYP1A1 expression and induction in human hepatoma cells HepG2. Treatment of HepG2 cells over 72 h with noncytotoxic concentrations of DFB resulted in a strong dose-dependent decrease in constitutive ethoxyresorufin-O-deethylase activity. Moreover, DFB significantly decreased CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after 24 h exposure, as demonstrated by ethoxyresorufin-O-deethylase (EROD) activity and Northern blot analysis. Additional studies were performed both on parental HepG2 cells and HepG2-241c.1, which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene, cloned under the control of the human CYP1A1 promoter (-1140 to +59). Ribonuclease protection assays (RPA) analysis clearly demonstrated an inhibition of CYP1A1 transcription in both cell lines. Surprisingly, in corresponding experiments using 3-methylcholanthrene (3-MC) as a CYP1A1 inducer, DFB was less effective. Finally, in competitive binding studies using a 9S-enriched fraction of HepG2 cytosol, DFB was capable of displacing [(3)H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from its Ah receptor binding site. Taken together, these results support the involvement of a transcriptional mechanism in the inhibition of CYP1A1 expression in HepG2 cells by DFB, possibly via an Ah receptor antagonism.

Molecular characteristics of carbaryl, a CYP1A1 gene inducer.

Carbaryl belongs to a series of compounds that activate the CYP1A1 gene. This study demonstrates the inability of carbaryl to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the rat aryl hydrocarbon (dioxin) receptor. Structural and physicochemical properties of this insecticide, in relation to the requirements for binding to the aryl hydrocarbon receptor, are described. The crystal structure was determined experimentally using X-ray diffraction. A conformational search using molecular mechanics was performed by means of a Monte-Carlo-type method and a stochastic dynamics simulation. Lipophilicity calculations, log P, and molecular lipophilicity potential are also presented. Common and discriminating properties of carbaryl and aryl hydrocarbon receptor ligands are discussed.

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.

Cytochrome 1A1 induction by primaquine in human hepatocytes and HepG2 cells: absence of binding to the aryl hydrocarbon receptor.

Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the world. It represents a crucial problem in public health care, affecting 750 million people annually, of whom at least two million die. Various antimalarials currently used were studied for their capability to induce expression of the cytochrome P450 1A1 (CYP1A1) gene, an enzyme that plays an important role in the activation of xenobiotics to genotoxic derivatives. Studies on human hepatocytes and HepG2 cell lines showed that primaquine was capable of dose dependently increasing both the ethoxyresorufin-O-deethylase activity and CYP1A1 mRNAs, suggesting a transcriptional activation of this gene. Moreover, alpha-naphthoflavone, a partial aryl hydrocarbon receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element, were shown to suppress CYP1A1 induction when added to the cultures. However, neither primaquine nor its metabolites were able to displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin from AhR in competitive binding studies using 9S-enriched fractions of human cytosol. These data, together with the induction of CYP1A1 promoter-directed chloramphenicol acetyl transferase gene expression, suggest that CYP1A1 induction involves the participation of the AhR but not a direct primaquine-receptor interaction. This supports the notion that an alternative ligand-independent mechanism has to be considered. Given the pharmaco-toxicological significance of CYP1A1 induction, these findings may have important implications in the treatment of malaria with primaquine and new analogs.

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.

Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor.

Insecticides deserve particular attention since the general population is potentially exposed to such chemicals through many routes. We therefore tested the comparative acute and chronic toxicity of chemicals belonging to the major insecticides families (DDT, malathion and tetrachlorvinphos, carbaryl, cypermethrin, diflubenzuron), in hepatocytes, HepG2 and HaCaT cell lines. Two kinds of end-points were used: cytotoxicity parameters and CYP1A1 induction. Except for cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect in hepatocytes and HaCaT, but not in HepG2 cells. However, the induction of the EROD activity appeared more sensitive since a response was detected at lower concentrations. Significant differences were observed between the cell types and the insecticides. Furthermore, these chemicals were unable to displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the Ah receptor. The experimental approach used herein may be a good means for predicting the acute and chronic toxicity of pesticides.

The 4S benzo(a)pyrene-binding protein is not a transcriptional activator of Cyp1a1 gene in Ah receptor-deficient (AHR -/-) transgenic mice.

In an effort to better understand the role of the 4S benzo(a)pyrene-binding protein in the induction of CYP1A1 by PAHs, we used a genetically engineered mouse line deficient in Ah receptor (AHR -/-). First, we demonstrated through binding experiments analyzed by sucrose gradient sedimentation and gel permeation chromatography that AHR -/- mice have no detectable AHR protein. In contrast, this AHR-deficient line expressed a 4S protein which efficiently binds BP as it does in hepatic cytosol from C57BL/6 mice. In vivo BP exposure in AHR-deficient mice proved the inability to sustain any CYP1A1 mRNA or CYP1A1 protein induction. These findings demonstrate the requirement of an active AHR to sustain the transactivation pathway leading to CYP1A1 induction. Surprisingly, the 4S BP-binding protein, which was previously characterized as the glycine N-methyltransferase, was completely devoid of such an enzymatic activity after purification by Sephacryl gel permeation chromatography. Moreover, sedimentation and chromatographic experiments, under nondenaturing conditions, do not support the assumption of 4S protein as a subunit of a multimeric protein (GNMT) displaying a molecular mass of 150 kDa.

The 8S benzo(a)pyrene-binding protein is an aldehyde dehydrogenase regulated by the Ah receptor.

8S Benzo(a)pyrene-binding proteins from liver cytosol of mouse and rabbit have been partially purified by gel permeation chromatography and affinity chromatography on 1-aminopyrene-Sepharose columns. These proteins, which bind polycyclic aromatic hydrocarbons and daunorubicine, have been identified, by microsequencing, as aldehyde dehydrogenases composed of polypeptides of 54 kDa. Using Ah receptor-deficient (AHR-/-) transgenic mice it has been shown that the amount as well as the binding capabilities of 8S protein was strongly altered in these mice, suggesting that its expression was partially under the control of the Ah receptor. The function of these proteins is currently unknown.

Knockout animals in toxicology: assessment of toxin bioactivation pathways using mice deficient in xenobiotic metabolizing enzymes.

Genetically engineered animal models represent a substantial improvement in in vivo assessment of toxic pathways. Several transgenic mouse lines have been designed to detect specific toxic markers in response to xenobiotic exposure. They are suitable for in vivo large scale screening of potentially toxic effects of drugs and other xenobiotics, and are used as bioassay models for carcinogenicity testing. This contribution will focus on a different strategy, using transgenic knockout mouse lines, to investigate with more accuracy some metabolic pathways leading to the bioactivation or the bioinactivation of xenobiotics. Through direct knockout of cytochrome P450 (CYP) genes or the knockout of the transcriptional activator of CYP genes AHR (aryl hydrocarbon receptor), the involvement of hepatic metabolic enzymes in xenobiotic bioactivation will be exemplified.

Ah receptor-dependent CYP1A induction by two carotenoids, canthaxanthin and beta-apo-8'-carotenal, with no affinity for the TCDD binding site.

The assays of several phase I and phase II xenobiotic-metabolizing enzyme activities, as well as CYP1A immunoblot analysis, were performed in liver microsomes and cytosol of male C57BL/6 mice (Ah receptor-responsive), of male DBA/2 mice (Ah receptor-low responsive) and of female Ah receptor gene knockout mice that were fed diets containing 300 mg/kg of a nonprovitamin A carotenoid, canthaxanthin, or a provitamin A carotenoid, beta-apo-8'-carotenal for 14 days, or which were injected i.p. with 3-methylcholanthrene. Previous studies have shown that some carotenoids, such as canthaxanthin and beta-apo-8'-carotenal, are strong inducers of liver CYP1A1 and 1A2 when given to rats. In this work, only canthaxanthin induced both CYP1A1 and 1A2 in C57BL/6 mice, whereas beta-apo-8'-carotenal induced only CYP1A2 in this strain. Neither of the two carotenoids modified CYP1A1/2 protein contents or enzyme activities in Ah receptor-low responsive DBA/2 or in Ah receptor gene knockout mice. Cytosol prepared from C57BL/6 mice liver tissue was incubated with [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence of canthaxanthin or beta-apo-8'-carotenal and analyzed by sucrose density gradient sedimentation: neither of the carotenoids, even when present in large excess, competed with TCDD for the TCDD binding site of the cytosolic Ah receptor of C57BL/6 mice. In brief, the carotenoids canthaxanthin or beta-apo-8'-carotenal induced Cyp1a genes in mice through an Ah receptor-dependent pathway, but did not bind to the Ah receptor.

Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor.

In spite of increasing numbers of insecticides used in agriculture, there are serious concerns regarding the potential risks of exposure to these agents. Carbaryl is one of the most important carbamate insecticides and has been used for about 30 years to control a wide range of pests. The study was designed to investigate if, among various insecticides currently used in world agriculture, this compound could induce human CYP1A1, an enzyme known to play an important role in the chemical activation of xenobiotics to genotoxic derivatives. Studies on HepG2 and HaCaT cell lines showed that carbaryl is capable of increasing, in a dose-dependent manner, both the ethoxyresorufin rufin-O-dec, O-deethylase activity and the steady-state concentrations of CYP1A1 mRNA, suggesting a transcriptional activation of this gene. When alpha-naphthoflavone, a partial Ah receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element (XRE), were added to the cultures, CYP1A1 induction was suppressed. However, competitive binding studies using the 9S enriched fraction of human cytosol indicated that carbaryl did not displace [3H]TCDD from AhR. These data, together with the activation of a XRE-directed CAT reporter gene by carbaryl, suggest that induction of CYP1A1 involves the participation of the AhR and the XRE, but is not mediated by a direct carbaryl-receptor interaction. An alternative ligand-independent mechanism should be considered. Therefore, although carbaryl concentration in food is very low, care should be taken because of its possible adverse effects in human health through liver and skin, given the well established toxicological importance of CYP1A1 induction.

Induction of CYP1A1 gene by benzimidazole derivatives during Caco-2 cell differentiation. Evidence for an aryl-hydrocarbon receptor-mediated mechanism.

The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer beta-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either beta-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer beta-naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to beta-naphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.

Effect of exposure of rabbit hepatocytes to sulfur-containing anthelmintics (oxfendazole and fenbendazole) on cytochrome P4501A1 expression.

The expression of cytochrome P4501A1 and 1A2 was investigated in rabbit hepatocytes maintained in primary cultures for 96 hr in the absence or presence of 100 mum of the benzimidazole anthelmintics oxfendazole or fenbendazole. Total cytochrome P-450, ethoxyresorufin O-deethylase and acetanilide hydroxylase activities were significantly increased in cell cultures receiving benzimidazoles. These increases were more marked after exposure of cultured hepatocytes to oxfendazole (OFZ) than to fenbendazole (FBZ). Western and Northern blot analysis of microsomes and RNA prepared from these cultures revealed increased levels of both protein and specific mRNA for P4501A1. The inhibition of these inductions in the presence of actinomycin D suggests a transcriptional way of activation of this gene. The ability of OFZ to bind to the Ah receptor has been examined. Data obtained from competition experiments with dioxin demonstrated that OFZ and other compounds in the benzimidazole series are not ligand of the Ah receptor. From saturation experiments and Scatchard plot analysis, rabbit hepatocyte Ah receptor (K(d) = 10.6 nm) seems to belong, as does the human Ah receptor, to a low-affinity category. Different induction rates obtained with several benzimidazole drugs suggest that the sulfur atom within the molecule is critical for CYP1A1 induction. The widely used benzimidazole anthelmintics OFZ and FBZ may exert an inducing effect through an original pathway that does not require a specific binding step to the Ah receptor.

Evidence for the ligand-independent activation of the AH receptor.

Benzimidazole derivatives are potent inducers of CYP1A1 in rabbit and human hepatocytes, but apparently do not bind the AH receptor. To resolve this paradoxical behaviour, studies have been concerned with the question of whether an alternative ligand-independent mechanism could explain the activation of the AH receptor. From experiments in cultured rabbit hepatocytes we show that benzimidazoles bind early and transiently to an unknown protein. Moreover, they are able to deplete the AHR in a time- and dose-dependent manner. In contrast, benzimidazoles are unable to induce CYP1A1 mRNA in mouse hepa-1 cells and to deplete the high-affinity AHR form from these cells. Taken together these data suggest that a signal transduction pathway, similar to that involved in the ligand-independent activation of steroid receptors, could only activate the low-affinity forms of AHR as those existing in rabbit and human cells.

Thiabendazole is an inducer of cytochrome P4501A1 in cultured rabbit hepatocytes.

The effect of TBZ (30-100 microM) was investigated on cytochromes P450 of cultured rabbit hepatocytes considered 72 h after plating. At the highest concentrations and without apparent cellular toxicity, the drug provokes a dose-dependent increase in total microsomal cytochrome P450 and a rise in EROD activity which was correlated to a specific increase in P4501A1 level. Northern blot analysis of RNA reveals an increased level of mRNA specific to P4501A1. The transcriptional activation of this isoenzyme is proposed because of the significant inhibition of the above-mentioned increases when actinomycin was added to the culture medium. Data obtained from competition experiments demonstrate that TBZ is not a ligand of Ah receptor. A down-regulation process could explain the slight decrease in both ANOH and P4502E1 level observed in hepatocytes treated with the highest dose of TBZ.

The pig as a model for studying AH receptor and other PAH-binding proteins in man.

In this report we compare the three major binding proteins existing in hepatic cytosols of pig and human. Many data suggest that for the AH receptor, which mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a structural as well as a functional variability exists across species. Similar conclusions can be drawn from the interspecies characterization of benzo(a)pyrene binding proteins, namely the 4S protein and the 8S protein. Using fractionation procedures such as sucrose gradient sedimentation and gel permeation chromatography we obtained enriched fractions containing each of these binding proteins. By saturation experiments and analysis of data by Scatchard and Woolf plots we determined binding characteristics and we conclude that pig and human AH receptors were closely related proteins since their Kd (18 +/- 0.4nM) were found quite similar. On the other hand, 4S proteins from pig (Kd = 16.7 nM; Bmax = 5.5 pmol/mg) and from human (Kd = 14 nM; Bmax = 4.5 pmol/mg) as well as 8S proteins (Kd approximately 300 nM) also exhibit remarkable similarities.

Omeprazole and lansoprazole are mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture.

The ability of several gastric antiulcer drugs including lansoprazole, cimetidine and ranitidine to affect the expression of human liver microsomal cytochromes P450 comparatively to omeprazole, reported previously to be a CYP1A inducer, was evaluated in primary cultures of human hepatocytes. Poly (A)+ RNA and microsomes extracted from the cells were analyzed in Northern and Western blots with specific cDNA probes and antibodies, and assayed for form-specific monoxygenase activities. Lansoprazole induced both CYP1A1 and CYP1A2 as omeprazole and did not apparently bind to the aryl hydrocarbon receptor with high affinity. Omeprazole sulfone was not an inducer of CYP1A. Omeprazole, omeprazole sulfone and lansoprazole induced CYP3A in approximately 50% of tested cultures, whereas 100% of tested cultures responded to omeprazole and to rifampicin in terms of CYP1A and CYP3A induction, respectively. Finally, cimetidine and ranitidine were not inducers. We conclude that omeprazole and lansoprazole constitute a new class of mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture and that the induction of CYP3A in response to these molecules could be polymorphic in humans.

Modulating effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on skin carcinogenesis initiated by the weak inducer 7,12-dimethylbenz(a)anthracene.

The effects of topical pretreatment of CF1-Swiss mice with TCDD on the carcinogenesis induced by DMBA were studied. We also determined the intrinsic features of DMBA as an aryl hydrocarbon hydroxylase (AHH) inducer through either its binding ability to Ah receptor or its inducing effects on benzo(a)pyrene (BP) hydroxylase or DMBA hydroxylase. DMBA is a poor ligand of the Ah receptor (26-fold and 4.3-fold weaker than 3-methylcholanthrene and BP respectively) and a very weak AHH inducer (ten million-fold weaker than TCDD). Nevertheless, DMBA induces a specific isozyme of cytochrome P-450 1A1 since, for an equal dose administered to C57BL/6 mice (200 mg/kg body weight), the DMBA-hydroxylase activity was 1.72-fold increased by DMBA while it remained unchanged after BP treatment. In contrast, the BP-hydroxylase activity was 1.91-fold increased by BP and only 1.47-fold by DMBA. A dose-dependent relationship exists between the increasing dose of TCDD (from 0.001 to 1 microg per mouse) applied to mouse skin and the induction of AHH activity of skin microsomes (from 1 to 60-fold increase). For carcinogenesis experiments, mice were either untreated or pretreated with single different doses of TCDD and, after 24h, DMBA (10 or 25 microg per mouse) was applied to the skin. The average number of papillomas per mouse was dependent on 1) the dose of DMBA and 2) the metabolic capacity of the skin. For 10 microg DMBA, the TCDD only exerts an anticarcinogenic effect (from 5.5 to 0.6 tumor per mouse) whereas for 25 microg DMBA, TCDD exerts a dual effect: first, a cocarcinogenic effect (from 6.2 to 9 and 11.5 tumors per mouse for 0.001 and 0.01 microg TCDD respectively) then an anticarcinogenic effect (2.3 and 1.5 tumors per mouse for 0.1 and 1 microg TCDD respectively). The discussion underlines the decisive importance of two factors: 1) the effective dose of the ultimate carcinogen in contact with cellular targets during a sensitive step of the cell cycle and 2) the time-persistence of a high steady state level of the carcinogen.

Detection and characterization of a novel hepatic 8 S binding protein for benzo[a]pyrene distinct from the Ah receptor.

Using fractionation procedures such as sucrose gradient sedimentation and gel permeation chromatography, a novel cytosolic binding protein for benzo[a]pyrene has been detected in liver of nonmammalian and mammalian species including human. This protein, called 8 S protein, cosediments with the Ah receptor after centrifugation in sucrose density gradient but can be separated from the Ah receptor and 4 S protein by gel permeation chromatography. Owing to its binding characteristics, the 8 S protein is clearly distinct from the Ah receptor. The [3H]BP binding parameters have been determined by saturation experiments. According to Scatchard and Woolf plots the Kd are 268 nM and 138 nM for DBA/2 mouse and guinea pig 8 S proteins, respectively. Bmax are 248 and 840 pmol/mg for DBA/2 mouse and guinea pig 8 S proteins, respectively. Apparent molecular mass of 8 S protein, according to Stokes radius (5 +/- 0.2 nm) and sedimentation coefficient, was estimated approximately 170,000 +/- 6000. After in vitro incubation of 8 S proteins with [3H]BP the charcoal, as well as the 4 S protein, exerts potent stripping effects on the [3H]BP binding. In contrast, after administration of [3H]BP to DBA/2 mice the 8 S protein-[3H]BP complex formed in vivo is more resistant to the stripping effects of charcoal and 4 S protein. Competition studies demonstrate that polycyclic aromatic hydrocarbons (PAHs) (BP > 1-aminopyrene > pyrene > 7,12-dimethylbenz[a]anthracene > 3-methylcholanthrene > benzo[e]pyrene > benzo[g, h, i,]perylene) are the best ligands of the 8 S protein. In contrast, the 2,3,7,8-tetrachlorodibenzo-p-dioxin is a poor ligand of this protein. Phenobarbital, steroid hormones, and omeprazole don't bind the 8 S protein. The at present unknown function of the 8 S protein and its role in the toxicology of PAHs are currently under investigation.