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.

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.

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.

Characterization of the 4 S polycyclic aromatic hydrocarbon-binding protein in human liver and cells.

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a soluble protein that binds PAHs with high affinity in mouse, rat, and rabbit. Until now, this protein had not been detected in human placenta or human cells in culture by cytosol labeling and gradient centrifugation assay. Thanks to a preliminary fractionation of cytosol by sedimentation on sucrose gradient or/and gel permeation chromatography, we found that PBP was present in liver, MCF-7 cell line, and hepatocytes of human. To accurately quantitate PBP binding and determine specific binding parameters, a reduction in the amount of charcoal used to adsorb nonspecifically bound benzo[a]pyrene was required. By saturation analysis, the concentration of specific binding sites for [3H]BP in PBP fraction from human liver was 4.6 pmol/mg of protein compared with 14.7 +/- 1.4 pmol/mg in the same fraction from DBA/2J mouse liver. Kinetic studies analyzed by Scatchard and Woolf plots indicate that human liver and MCF-7 cells contain a low-affinity PBP form: the Kd derived from Woolf plot analysis were 14.2 +/- 1.4 and 26.2 +/- 1.8 nM, respectively. DBA/2J mouse possesses a higher-affinity PBP form, the same analysis indicating a Kd of 6.1 +/- 0.3 nM. These data demonstrate that, by comparison to the mouse liver, a lower-affinity form of PBP is present in reduced concentration in human liver, explaining the impossibility of detecting this protein by sedimentation of human cytosol in sucrose gradient.

Omeprazole, an inducer of human CYP1A1 and 1A2, is not a ligand for the Ah receptor.

Omeprazole is a benzimidazole derivative which induces both P450 1A1 and 1A2 in human liver in vitro and in vivo. Northern blot analysis of polyA RNA prepared from primary cultures of human hepatocytes indicates that both 1A1 and 1A2 messages are induced by beta-naphthoflavone and omeprazole. Co-treatment of cells with these inducers and with actinomycin D or cycloheximide results in no accumulation of both mRNA or superinduction of 1A1 mRNA, respectively. 9S enriched fraction of cytosol was prepared either from human hepatocytes in culture or from human liver tissue and analyzed by sucrose density gradient sedimentation for its capacity to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole or omeprazole sulfone (a metabolite of omeprazole in man). Whereas 2 microM TCDD displaced almost totally [3H]TCDD from the Ah receptor, both omeprazole and omeprazole sulfone did not, even at 5000-fold molar excess. In addition, when [14C] omeprazole was incubated with 9S enriched fraction of human liver or hepatocyte cytosol, no interaction could be detected in sucrose density gradient. These experiments suggest that omeprazole is not a ligand for the human liver Ah receptor.

Mechanism of antimutagenicity of wheat sprout extracts.

In this paper we have demonstrated that wheat sprout extract, which has been shown to be antimutagenic towards benzo[a]pyrene (BP), reduced formation of BP metabolites by hepatic microsomes of either benzo[a]pyrene- or phenobarbital-treated rats as analyzed in high-pressure liquid chromatography (HPLC). Comparing the time dependence of profiles and values of BP metabolites, formed in experiments in which the same dose of wheat sprout extract was added to the incubation medium, it has been observed that the later this extract was added the higher the percent of BP that was metabolized. In a bacterial test (cytochrome P450 induction assay) high inhibition of mutagenic activity of cyclophosphamide and ethidium bromide, in the presence of wheat sprout extract, reflected decreased levels of cytochromes P4502B1 and P4501A1 respectively. Decreased levels of both cytochromes P4501A1 and P4502B1 were also observed in either wheat sprout extract- or wheat sprout extract plus benzo[a]pyrene-treated rats. In all of these studies it has been observed that wheat sprout extract displays much more affinity for cytochrome P4501A1 than for the P4502B1 form. On the other hand the wheat sprout extract had higher affinity for carcinogen binding protein (4S protein) than for the aryl hydrocarbon receptor. The strong inhibition of BP mutagenicity and BP metabolism with non-chlorophyllic wheat sprout extract suggests that chlorophyll is not the main compound responsible for the antimutagenic activity of wheat sprout extract. The similar chromatographic behavior of both the main inhibitory fraction, obtained from wheat sprout extract, and two pure glycosides of apigenin--shaftoside, purified from wheat sprout extract and synthetic swertisine--suggests that antimutagenic compound(s) contained in the wheat sprout extract belong(s) to this family of flavonoids.

Antimutagenic effects of several subfractions of extract from wheat sprout toward benzo[a]pyrene-induced mutagenicity in strain TA98 of Salmonella typhimurium.

The aqueous extract from wheat sprouts contains some antimutagenic factor(s). The factor(s) abolish(es) the activity of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in the S9 fraction from Aroclor-treated rat livers and also inhibit(s) the mutagenic activity of benzo[a]pyrene (B(a)P) in the Ames test. The extract (fraction S30) was subjected to initial fractionation by thermal treatment, 3 24-h cycles of dialysis and ultrafiltration. The antigenotoxic activity of fraction S30 amounted to 98% and was unchanged by thermal treatment (100 degrees C, 10 min). Both the dialysate and the dialysis fluid inhibited the mutagenic effect of B(a)P by 48.4 and 48% respectively. The microsomal subfraction inhibited the mutagenicity only in 10%, and the postmicrosomal subfraction in 68%. It is concluded that the extract from wheat sprouts contains at least 2 heat-resistant compounds (or groups of compounds) located within the cell cytosol and showing antimutagenic activity: one group is of low molecular weight and another of high MW. Alternatively, low-molecular compounds could either be free or bound to high-molecular compound(s).

The effect of wheat sprout extract on benzo(a)pyrene and 7,2-dimethylbenz(a)anthracene activity.

Subcutaneous application of aqueous wheat sprout extract to mice resulted in a slight decrease of the ability of fraction S-9 from their skin to activate DMBA to metabolites mutagenic for S. typhimurium TA 98. Induction by benzo(a)pyrene of sperm abnormalities in mice was diminished after oral administration of the wheat sprout extract; however, even high doses of the extract did not completely abolish the effect of benzo(a)pyrene on spermatozoa. In the carcinogenicity studies, the wheat sprout extract, when applied to mouse skin during the initiation phase, enhanced fourfold the induction of papillomas by DMBA and shortened the period of latency from 9 to 5 weeks.