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We used the marine bivalve (Mytilus galloprovincialis) to assess a range of biological or biomarker responses following exposure to a model-engineered nanoparticle, C 60 fullerene, either alone or in combination with a model polycyclic aromatic hydrocarbon, benzo(α)pyrene [B(α)P]. An integrated biomarker approach was used that included: (i) determination of 'clearance rates' (a physiological indicator at individual level), (ii) histopathological alterations (at tissue level), (iii) DNA strand breaks using the comet assay (at cellular level) and (iv) transcriptional alterations of p53 (anti-oncogene) and ras (oncogene) determined by real-time quantitative polymerase chain reaction (at the molecular/genetic level). In addition, total glutathione in the digestive gland was measured as a proxy for oxidative stress. Here, we report that mussels showed no significant changes in 'clearance rates' after 1 day exposure, however significant increases in 'clearance rates' were found following exposure for 3 days. Histopathology on selected organs (i.e. gills, digestive glands, adductor muscles and mantles) showed increased occurrence of abnormalities in all tissues types, although not all the exposed organisms showed these abnormalities. Significantly, increased levels of DNA strand breaks were found after exposure for 3-days in most individuals tested. In addition, a significant induction for p53 and ras expression was observed in a tissue and chemical-specific pattern, although large amounts of inter-individual variability, compared with other biomarkers, were clearly apparent. Overall, biological responses at different levels showed variable sensitivity, with DNA strand breaks and gene expression alterations exhibiting higher sensitivities. Furthermore, the observed genotoxic responses were reversible after a recovery period, suggesting the ability of mussels to cope with the toxicants C 60 and/or B(α)P under our experimental conditions. Overall, in this comprehensive study, we have demonstrated mussels as a suitable model marine invertebrate species to study the potential detrimental effects induced by possible genotoxicants and toxicants, either alone or in combinations at different levels of biological organisation (i.e. molecular to individual levels)., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Motivation: Comparing time courses of gene expression with time courses of phenotypic data may provide new insights in cellular mechanisms. In this study, we compared the performance of five pattern recognition methods with respect to their ability to relate genes and phenotypic data: one classical method (k-means) and four methods especially developed for time series [Short Time-series Expression Miner (STEM), Linear Mixed Model mixtures, Dynamic Time Warping for -Omics and linear modeling with R/Bioconductor limma package]. The methods were evaluated using data available from toxicological studies that had the aim to relate gene expression with phenotypic endpoints (i.e. to develop biomarkers for adverse outcomes). Additionally, technical aspects (influence of noise, number of time points and number of replicates) were evaluated on simulated data., Results: None of the methods outperforms the others in terms of biology. Linear modeling with limma is mostly influenced by noise. STEM is mostly influenced by the number of biological replicates in the dataset, whereas k-means and linear modeling with limma are mostly influenced by the number of time points. In most cases, the results of the methods complement each other. We therefore provide recommendations to integrate the five methods., Availability: The Matlab code for the simulations performed in this research is available in the Supplementary Data (Word file). The microarray data analysed in this paper are available at ArrayExpress (E-TOXM-22 and E-TOXM-23) and Gene Expression Omnibus (GSE39291). The phenotypic data are available in the Supplementary Data (Excel file). Links to the pattern recognition tools compared in this paper are provided in the main text., Contact: d.hendrickx@maastrichtuniversity.nl, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The chain of events leading from a toxic compound exposure to carcinogenicity is still barely understood. With the emergence of high-throughput sequencing, it is now possible to discover many different biological components simultaneously. Using two different RNA libraries, we sequenced the complete transcriptome of human HepG2 liver cells exposed to benzo[a]pyrene, a potent human carcinogen, across six time points. Data were integrated in order to reveal novel complex chemical-gene interactions. Notably, we hypothesized that the inhibition of MGMT, a DNA damage response enzyme, by the over-expressed miR-181a-1_3p induced by BaP, may lead to liver cancer over time., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) induces cytochrome P450 (CYP) 1A1 and 1B1 enzymes, which biotransform PAHs resulting in the formation of DNA adducts. We hypothesised that 2,3',4,5'-tetramethoxystilbene (TMS), an analogue of resveratrol and a potent CYP1B1 inhibitor, may inhibit r7, t8, t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adduct formation in cells exposed to benzo[a]pyrene (BP). To address this, MCF-7 cells were cultured for 96 h in the presence of 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS. Cells were assayed at 2-12 h intervals for: BPdG adducts by r7, t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay; CYP1A1 and 1B1 gene expression changes by relative real-time polymerase chain reaction; and CYP1A1/1B1 enzyme activity by ethoxyresorufin-O-deethylase (EROD) assay. Whereas maximal BPdG levels were similar for all exposure groups, the times at which the maxima were reached increased by 16 and 24 h with the addition of 1 and 4 μM TMS, respectively. The maximal expression of CYP1A1 and CYP1B1 occurred at 16, 24 and 48 h, but the maximal level for EROD-specific activity was reached at 24, 48 and 60 h, in cells exposed to 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS, respectively. The area under the curve from 4 to 96 h of exposure (AUC(4-)(96 h)) for BPdG adduct formation was not increased in the presence of TMS, but for CYP1A1 and CYP1B1 expression fold increase AUC(4-)(96 h) and EROD-specific activity AUC(4-)(96 h), there were significant (P < 0.05) increases in the presence of 4 μM TMS. Therefore, during 96 h of exposure in MCF-7 cells, the combination of BP plus TMS caused a slowing of BP biotransformation, with an increase in CYP1A1 and CYP1B1 expression and EROD activity, and a slowing, but no change in magnitude of BPdG formation.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon produced by cigarette combustion, is implicated as a causative agent in smoking-related cancer and atherosclerosis. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], a potent ligand for the nuclear receptor vitamin D receptor (VDR), has been shown to decrease the risk of osteoporosis, some types of cancer and cardiovascular disease, suggesting an opposing effect of vitamin D3 to cigarette smoking. In this study, we investigated the effects of BaP on the vitamin D3 signaling pathway. BaP effectively enhanced the 1,25(OH)2D3-dependent induction of cytochrome P450 24A1 (CYP24A1) in human monocyte/macrophage-derived THP-1 cells and breast cancer MCF-7 cells. BaP combination was less or not effective on mRNA expression of CD14, arachidonate 5-lipoxygenase, and cathelicidin antimicrobial peptide in THP-1 cells. BaP also increased the expression of CYP24A1 induced by a non-vitamin D VDR ligand, lithocholic acid acetate. Another aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, enhanced CYP24A1 expression by 1,25(OH)2D3 in THP-1 cells. Treatment of cells with an AhR antagonist and a protein synthesis inhibitor inhibited the enhancing effect of BaP on CYP24A1 induction, indicating that the effects of BaP are mediated by AhR activation and de novo protein synthesis. BaP pretreatment increased 1,25(OH)2D3-dependent recruitment of VDR and retinoid X receptor to the CYP24A1 promoter. Analysis of 1,25(OH)2D3 metabolism showed that BaP enhanced the hydroxylation of 1,25(OH)2D3 by CYP24A1 in THP-1 cells. Thus, AhR activation by BaP stimulates vitamin D3 catabolism. Modulation of vitamin D signaling by AhR may represent a mechanism underlying cigarette smoking-related diseases.

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

Benzo[a]pyrene (B[a]P) has been shown to be an inducer of apoptosis in some cell types. To date, due to the lack of an appropriate model system, studies of the cellular and biochemical mechanism(s) by which B[a]P induces apoptosis have been focused on Hepa1c1c7 cells. Moreover, the precise relationship between the bioactivation of B[a]P by CYP1A1 or CYP1B1 and the occurrence of cytotoxicity-mediated apoptosis requires further elucidation. In the present study, we showed that B[a]P-induced apoptosis in RL95-2 cells is accompanied by the activation of caspases. In addition, the mitochondrial changes, including the decrease of mitochondrial potential and the release of mitochondrial cytochrome c and second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low PI (Smac/DIABLO) into the cytosol, support the suggestion that the mitochondrial pathway is robustly associated with B[a]P-evoked apoptosis. This study showed the involvement of the nuclear translocation of mitochondrial apoptosis-inducing factor in B[a]P-induced apoptosis of RL95-2 cells. Exposure to B[a]P up-regulates aryl hydrocarbon receptor, heat-shock protein 90, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and epoxide hydrolase significantly, which might be prerequisites for the conversion of B[a]P to B[a]P-7,8-dihydroxy-9,10-epoxide. Although both CYP1A1 and CYP1B1 proteins were up-regulated significantly by B[a]P, only CYP1A1 exhibited activity. Thus, CYP1A1 is believed to be a central oxidative enzyme that is ultimately required for formation of B[a]P-7,8-dihydroxy-9,10-epoxide from B[a]P in RL95-2 cells. Altogether, our data showed that RL95-2 cells are susceptible to apoptosis by exposure to B[a]P and that B[a]P-evoked apoptosis is mediated predominantly by the activation of CYP1A1. Here we suggest that RL95-2 cells are an excellent model for the investigation of xenobiotic mechanisms associated with CYP1A1 as well as CYP1B1.

Dietary polyphenols, including flavonoids, have been implied to have cancer preventive properties. Suggested mechanisms include inhibition of carcinogen-activating cytochrome P450 (CYP) transcription and activities. These studies have focused mainly on CYP1A1. However, CYP1B1 has recently been shown to be of particular importance in smoking-induced oral and oesophageal cancer. Previous observations in our laboratory demonstrated that methoxylated flavonoids may be effective inhibitors of CYP1A1 transcription and activity as well as being orally bioavailable. In this study, an initial screening of 19 methoxylated flavones, using the ethoxyresorufin de-ethylation assay in human oral squamous cell carcinoma SCC-9 cells pretreated with 1 microM benzo[a]pyrene, identified six strongly inhibitory compounds for further studies. The effect of these flavones on CYP1B1 mRNA expression was measured with quantitative branched DNA methodology. Four of the compounds--3',4'-dimethoxyflavone and 5,7,4'-trimethoxyflavone and, in particular, 7,3'-dimethoxyflavone and 7,4'-dimethoxyflavone--were potent inhibitors of CYP1B1 mRNA expression. Two of the more common unmethylated polyphenols--curcumin and quercetin--were also potent inhibitors. Whereas most unmethylated polyphenols, such as curcumin and quercetin, have very poor bioavailability, the high metabolic stability of the methoxylated flavones studied here suggests that these CYP1B1 inhibitors may also be effective in-vivo.

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17beta-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERalpha, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligands.

Pro-carcinogens, such as benzo[a]pyrene (B[a]P), that are exogenous ligands of the aromatic hydrocarbon receptor may influence the susceptibility of target-cell populations through the up-regulation of cytochrome P450 (CYP) mixed function oxidases. We examined whether the growth kinetics of MCF-7 cells might determine the level of up-regulation of CYP1A1, CYP1A2 or CYP1B1 by B[a]P, and whether this could then influence subsequent levels of DNA damage. Cell cultures manipulated to be G(0)/G(1)-phase concentrated, S-phase concentrated or G(2)/M-phase concentrated were treated with B[a]P and the expression levels of CYP1A1, CYP1A2, CYP1B1, cyclin-dependent kinase inhibitor 1A [CDKN1A (P21(WAF1/CIP1))], B-cell leukaemia/lymphoma-2 (BCL-2), and Bcl-2-associated X levels were determined. Levels of DNA damage were measured as DNA single-strand breaks (SSBs) by the alkaline single-cell gel electrophoresis (comet) assay or as DNA adducts by (32)P-postlabelling analysis. B[a]P-induced up-regulation of CYP1A1 was >100-fold in S-phase-concentrated cells, but in G(0)/G(1)-phase- or G(2)/M-phase-concentrated cultures up-regulation occurred to a significantly lower extent. Consistent with this, B[a]P-treated S-phase-concentrated cultures exhibited markedly up-regulated P21(WAF1/CIP1), higher levels of dose-related increases in DNA SSBs, and increased DNA adduct levels presumably as a result of CYP1A1-mediated activation of B[a]P to B[a]P-diol-epoxide compared with the cultures enriched for the other cell cycle phases. Growth kinetics in vitro may be an important predeterminant of susceptibility to an exogenous pro-carcinogen in short-term test systems and these findings have important implications when extrapolating such results to a particular target-cell population in vivo.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.

We studied the replacement of hepatic S9 with in vivo and in vitro induced hepatocytes as a metabolic activation system with the aim of broadening the possibilities of mutagenic assays. Rats were pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), 3-methylcholanthrene (MC) and a combination of BNF and PB (BNF + PB). Mutagenic activation of benzo[a]pyrene (BP) and 2-aminoanthracene (2AA) by hepatic S9 and hepatocytes was determined in the Ames test. Primary rat hepatocytes were used for in vitro induction and were used as the activating system in the Ames test. In vivo BNF treatment greatly increased the metabolic activation capacity of hepatic S9 and hepatocytes towards BP. With regard to 2AA activation, S9 and hepatocytes showed different BNF induction profiles. PB treatment reduced the mutagenicity of both compounds. Although ethoxyresorufin O-dealkylase (EROD) activity of S9 from BNF + PB-treated animals was almost 30-fold greater than the control, its effectiveness in activation of 2AA was below the control level. A large part of the EROD activity of control cells was lost during culture, together with the ability to activate 2AA, however, 72 h of MC induction increased EROD activity to 200-fold of the control, which corresponds to 28% of that of in vivo induced hepatocytes. The mutagenic potential of BP activated by in vitro induced hepatocytes was 10-fold above the control, which is 47% of the mutagenicity detected following in vivo induction. In vitro induced hepatocytes increased 2AA mutagenicity to 14.6-fold over the control, which corresponds to 68% of in vivo induction. Our results suggest that primary culture of hepatocytes provides a useful model for the study of the role of metabolic activation processes concerning enzyme activity of cytochromes P450 and other metabolic enzymes and induction profiles of different inducers.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.

The effects of lead nitrate on the induction of hepatic cytochrome P450IA (CYP1A) isoforms, mainly CYP1A2, by aromatic amines (2-methoxy-4-aminoazobenzene, 2-amino-3-methyl-9H-pyrido [2,3-b]indole and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and aryl hydrocarbons (3-methylcholanthrene, benzo[a]pyrene and beta-naphthoflavone) in male F344 rats were examined at the levels of mRNA, protein and activity of the enzymes. Pretreatment of rats with lead nitrate suppressed the expression of hepatic CYP1A enzyme(s), especially CYP1A2, at both levels of protein and activity of the enzyme(s) by treatment with an aromatic amine or an aryl hydrocarbon. On the other hand, the lead nitrate pretreatment suppressed the induction of CYP1A mRNA(s) by an aromatic amine but not by an aryl hydrocarbon. These findings indicate that lead nitrate suppresses the expression of CYP1A enzymes at both stages of post-translation of mRNAs and transcriptional activation of the genes, and further suggest that the pathway for the transcriptional activation of the CYP1A genes by the aromatic amines is different from that by the aryl hydrocarbons.

Methyl methacrylate (MMA) is widely used as a cement in dentistry, orthopaedic surgery and ophthalmology. Studies based on short-term genotoxicity tests have produced conflicting results in the last two decades. In the present study, the effects of MMA on the mutagenicity of 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P) were evaluated with the Salmonella typhimurium TA98 strain in the absence and presence of S9 mix. The direct-acting mutagenicity of 1-NP was markedly decreased by MMA in a dose-dependent manner. However, a low inhibitory effect of MMA on the metabolic-acting mutagenicity of B[a]P was observed. MMA did not show mutagenicity within the concentrations of 4.7-37.6 microM either with or without S9 mix. The inhibitory effect of MMA was not due to its cytotoxicity because very low and/or no cytotoxicity of MMA to S. typhimurium TA98 was observed. To confirm the antimutagenicity of MMA against 1-NP and B[a]P, a 32P-postlabelling method was used to determine whether MMA modified DNA adduct formation produced by both compounds in calf thymus DNA. MMA inhibits the formation of 1-NP- and B[a]P-DNA adducts in a dose-dependent manner. The DNA adduct of 1-NP reduced by MMA was greater than that of B[a]P. Thus, we suggested that MMA was possibly acting as an inhibitor of chemical carcinogenesis.

The polycyclic aromatic hydrocarbons are major environmental pollutants. Benzo[a]pyrene and 3-methylcholanthrene are prominent members of this group of compounds. In the present study, we have examined the effect of these carcinogens/mutagens upon human chorionic gonadotrophin (HCG) secretion in the first trimester placenta in vitro. At 7-9 weeks in static cultures, exposure to 50 microM benzo[a]pyrene for 24 h increased beta-HCG secretion by placental explants. The effect after 6 h of incubation was less apparent. The effect of 5 microM benzo[a]pyrene at the two time points also was less significant than 50 microM benzo[a]pyrene. In explants exposed to 50 microM 3-methylcholanthrene, the effect on HCG secretion was also stimulatory. In superfusion of placental explants pretreated overnight with benzo[a]pyrene, there was a similar increase in the pattern of pulsatile beta-HCG secretion, as analysed by the area under the curve and the pulse amplitude, which was most evident at 24 h with the 50 microM dose. No significant effect on pulse frequency was noted. The effect of 50 microM 3-methylcholanthrene was also stimulatory. In order to determine the functional integrity of the explants treated with benzo[a]pyrene, the effect of 1 min pulses of 10(-10) gonadotrophin-releasing hormone (GnRH) analogue (a known HCG stimulator in superfusion) was tested, demonstrating that it increased beta-HCG secretion compared to controls. In addition, there was also an increase in whole HCG, as measured by the Tandem-R assay following exposure to benzo[a]pyrene. In conclusion, short-term exposure to carcinogens increases HCG secretion of placental explants in early pregnancy and this effect is maintained after removal of the xenobiotic.

Freshly isolated rat hepatocytes cultivated in Williams Medium E (WME) quickly lose their cytochrome P-450-dependent metabolic activities. A significantly better maintenance of cytochrome P-450 was achieved by supplementing WME with hormones and nutrients and 2% DMSO and 10% fetal calf serum. After 72-h cultivation in this medium the cytochrome P-450 content was still 72% of the amount measured in freshly isolated cells. Over the same period of time the activity of 7-ethoxycoumarin-O-deethylase dropped to approximately 55% of its initial value. DNA repair induction by UV irradiation, hydrogen peroxide, benzo[a]pyrene and 2-acetylaminofluorene was measured. A clear dose-dependent increase of the incorporation of [3H]thymidine was found with all agents tested, regardless of whether the treatment of the hepatocytes began 4 or 52 h after cell isolation. In the case of UV light and hydrogen peroxide the maximum effects were slightly lower after prolonged cultivation, whereas little change or even a slight increase was found with benzo[a]pyrene and 2-acetylaminofluorene. These results indicated that cell cultivation led to a slight loss of DNA repair activity but did not, despite the loss of cytochrome P-450, cause a decrease in DNA-damaging intermediates produced from the two indirect mutagens. The absolute amounts of [3H]thymidine incorporated by the solvent control cells of the 52-h groups was consistently found to be twice the level measured in the corresponding 4-h control groups. The incorporation of deoxy[3H]cytidine was not changed when tested in parallel. The increase in [3H]thymidine incorporation can therefore be explained by the assumption of a diminution of the thymidine pool during cell cultivation.

The effect of modifying the T7 promoter-containing plasmid pDR100 with aminofluorene (AF), acetylaminofluorene (AAF), and benzo[a]pyrene (B[a]P) adducts on RNA synthesis by the T7 RNA polymerase was determined. We found that increasing levels of each of the three adducts caused a progressive decline in RNA synthesis, but that the inhibition produced by benzo[a]pyrene adducts was substantially greater than that caused by either AF or AAF adducts. Polyacrylamide gel electrophoresis analysis confirmed that the B[a]P adducts more strongly inhibited chain elongation since their presence resulted in the production of RNA fragments having average chain lengths very close to that predicted if each adduct permanently blocked the polymerase during transcription. We have also determined the effect of each type of adduct on the initiation of transcription and show that the T7 RNA polymerase initiates as efficiently on both AF or AAF-containing templates as it does on unmodified DNA, while B[a]P adducts were found to increase in the number of chain starts.

Previous work from this laboratory demonstrated the presence of micronuclei in erythrocytes from larvae of the urodele amphibian Pleurodeles waltl reared in water containing clastogenic substances. In order to investigate the generality of this finding, larvae from another urodele Ambystoma mexicanum (axolotl) were reared in water containing one of the two following compounds: benzo[a]pyrene (BaP) or ethylmethane sulphonate (EMS). The level of micronucleated erythrocytes on blood smears was compared with control samples from larvae reared in fresh water. The optimum larval stage for this test system was determined. The effects of the indirect mutagen (BaP), and the direct mutagen (EMS) were found to depend on both dose and exposure to the clastogen. Positive results were obtained for BaP after 8 days of treatment at a concentration of 0.025 p.p.m. After 10 days of treatment at a concentration of 0.1 p.p.m. numerous micronuclei were seen (greater than 250%). Positive results were also obtained with EMS after 8 days of treatment at a concentration of 24 p.p.m. At 62 p.p.m., positive results were found after 6 days of treatment, while at 124 p.p.m. positive results were found after only 4 days. The results with both these agents show that the axolotl holds promise as an in vivo test system for the detection of low concentrations of clastogens in an aquatic environment.