Relative effective potencies of dioxin-like compounds in rodent and human lung cell models.

Toxicity of dioxin-like compounds (DLCs), such as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls, is largely mediated via aryl hydrocarbon receptor (AhR) activation. AhR-mediated gene expression can be tissue-specific; however, the inducibility of AhR in the lungs, a major target of DLCs, remains poorly characterized. In this study, we developed relative effective potencies (REPs) for a series of DLCs in both rodent (MLE-12, RLE-6TN) and human (A549, BEAS-2B) lung and bronchial epithelial cell models, using expression of both canonical (CYP1A1, CYP1B1) and less well characterized (TIPARP, AHRR, ALDH3A1) AhR target genes. The use of rat, murine and human cell lines allowed us to determine both species-specific differences in sensitivity of responses to DLCs in lung cellular models and deviations from established WHO toxic equivalency factor values (TEF) values. Finally, expression of selected AhR target genes was determined in vivo, using lung tissues of female rats exposed to a single oral dose of DLCs and compared with the obtained in vitro data. All cell models were highly sensitive to DLCs, with murine MLE-12 cells being the most sensitive and human A549 cells being the least sensitive. Interestingly, we observed that four AhR target genes were more sensitive than CYP1A1 in lung cell models (CYP1B1, AHRR, TIPARP and/or ALDH3A1). We found some deviations, with strikingly low REPs for polychlorinated biphenyls PCBs 105, 167, 169 and 189 in rat RLE-6TN cells-derived REPs for a series of 20 DLCs evaluated in this study, as compared with WHO TEF values. For other DLCs, including PCBs 126, 118 and 156, REPs were generally in good accordance with WHO TEF values. This conclusion was supported by in vivo data obtained in rat lung tissue. However, we found that human lung REPs for 2,3,4,7,8-pentachlorodibenzofuran and PCB 126 were much lower than the respective rat lung REPs. Furthermore, PCBs 118 and 156 were almost inactive in these human cells. Our observations may have consequences for risk assessment. Given the differences observed between rat and human data sets, development of human-specific REP/TEFs, and the use of CYP1B1, AHRR, TIPARP and/or ALDH3A1 mRNA inducibility as sensitive endpoints, are recommended for assessment of relative effective potencies of DLCs.

Butyrate and docosahexaenoic acid interact in alterations of specific lipid classes in differentiating colon cancer cells.

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.

AhR-mediated changes in global gene expression in rat liver progenitor cells.

Although the tumor-promoting effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar polychlorinated biphenyls (PCBs), and related compounds in liver tissue are primarily attributed to the activation of the aryl hydrocarbon receptor (AhR), the underlying molecular mechanisms are still unclear. Liver progenitor (oval) cells have been suggested to constitute a potential target for hepatocarcinogenic chemicals. To better understand AhR-driven pathways, we analyzed the transcriptional program in response to coplanar PCB 126 in contact-inhibited rat liver progenitor WB-F344 cells using high-density microarrays. After 6-h treatment, we identified 145 significantly deregulated genes considered to be direct AhR-dependent target genes. The number of differentially regulated genes increased to 658 and 968 genes after 24 and 72 h, respectively. Gene ontology analysis revealed that these genes were primarily involved in drug and lipid metabolism, cell cycle and growth control, cancer developmental processes, cell-cell communication, and adhesion. Interestingly, the Wnt and TGF-ß signaling pathways, both being involved in developmental and tumorigenic processes, belonged to the most affected pathways. AhR- and ARNT-dependent regulation of selected target genes of interest was then confirmed using TCDD as a model AhR agonist, together with pharmacological inhibition of the AhR and by RNA-interference techniques. We demonstrated AhR-dependent regulation of emerging and novel AhR target genes, such as Fst, Areg, Hbegf, Ctgf, Btg2, and Foxq1. Among them, the transcription factor Foxq1, recently suggested to contribute to tumor promotion and/or progression, was found to be regulated at both mRNA and protein levels by AhR/ARNT activation.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its methyl derivatives in human keratinocytes.

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression.

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.

Benzo[a]pyrene and tumor necrosis factor-α coordinately increase genotoxic damage and the production of proinflammatory mediators in alveolar epithelial type II cells.

Alveolar type II epithelial (AEII) cells regulate lung inflammatory response and, simultaneously, they are a target of environmental carcinogenic factors. We employed an in vitro model of rat AEII cells, the RLE-6TN cell line, in order to analyze the interactive effects of tumor necrosis factor-α (TNF-α), a cytokine which plays a key role in the initiation of inflammatory responses in the lung, and benzo[a]pyrene (BaP), a highly carcinogenic polycyclic aromatic hydrocarbon. TNF-α strongly augmented the formation of stable BaP diol epoxide-DNA adducts in AEII cells, which was associated with enhanced p53-Ser15 phosphorylation and decreased cell survival. The increased genotoxicity of BaP was associated with altered expression of cytochrome P450 (CYP) enzymes involved in its bioactivation, a simultaneous suppression of CYP1A1 and enhancement of CYP1B1 expression. Importantly, BaP and TNF-α acted synergistically to upregulate key inflammatory regulators in AEII cells, including the expression of inducible NO synthase and cyclooxygenase-2 (COX-2), and enhanced prostaglandin E2 production and expression of proinflammatory cytokines, such as TNF-α, interleukin-1ß and interleukin-6. We observed that BaP and TNF-α together strongly activated p38 kinase, a principal regulator of inflammatory response. SB202190, a specific p38 inhibitor, prevented induction of both COX-2 and proinflammatory cytokines, thus confirming that p38 activity was crucial for the observed inflammatory reaction. Taken together, our data demonstrated, for the first time, that a proinflammatory cytokine and an environmental PAH may interact to potentiate both DNA damage and the inflammatory response in AEII cells, which may occur through coordinated upregulation of p38 activity.

Toxic effects of methylated benzo[a]pyrenes in rat liver stem-like cells.

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Genotoxic polycyclic aromatic hydrocarbons fail to induce the p53-dependent DNA damage response, apoptosis or cell-cycle arrest in human prostate carcinoma LNCaP cells.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been positively associated with prostate cancer, but knowledge of the formation of PAH-DNA adducts and related genotoxic events in prostatic cells is limited. In the present study, benzo[a]pyrene (BaP), a potent mutagenic PAH, formed significant levels of DNA adducts in cell lines derived from human prostate carcinoma. When analyzing the effect of BaP on the induction of CYP1 enzymes participating in the metabolic activation of PAHs in LNCaP cells, we found that BaP induced expression of CYP1A1 and CYP1A2, but not CYP1B1 enzyme. Despite a significant amount of DNA adducts being formed by BaP and, to a lesser extent also by another strong genotoxin, dibenzo[a,l]pyrene, neither apoptosis nor cell-cycle arrest were induced in LNCaP cells. LNCaP cells were not sensitized to the induction of apoptosis by PAHs even through inhibition of the phosphoinositide-3-kinase/Akt pro-survival pathway. The lack of apoptosis was not due a disruption of expression of pro-apoptotic and pro-survival members of the Bcl-2 family of apoptosis regulators. In contrast to other genotoxic stimuli, genotoxic PAHs failed to induce DNA double-strand breaks, as illustrated by the lack of phosphorylation of histone H2AX or checkpoint kinase-2. BaP did not activate p53, as evidenced by the lack of p53 accumulation, phosphorylation at Ser15, or induction of p53 transcriptional targets. Taken together, although genotoxic PAHs produced significant levels of DNA adducts in a model of human prostate carcinoma cells, they did not activate the mechanisms leading to elimination of cells with significant damage to DNA, presumably due to their failure to activate the p53-dependent DNA damage response.

The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells.

In contrast to hepatocytes, there is only limited information about the expression and activities of enzymes participating in metabolic activation of environmental mutagens, including polycyclic aromatic hydrocarbons (PAHs), in liver progenitor cells. In rat liver "stem-like" WB-F344 cell line, sharing many characteristics with rat liver progenitor cells, PAHs are efficiently activated to their ultimate genotoxic metabolites forming DNA adducts. The present study aimed to characterize expression/activities of enzymes of two major pathways involved in the metabolism of benzo[a]pyrene (BaP): cytochrome P450 (CYP) family 1 enzymes and cytosolic aldo-keto reductases (AKRs). We report here that, apart from induction of CYP1A1 and CYP1B1 expression and the corresponding enzymatic activity, both BaP and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) expression and activity. In contrast, the aldehyde reductase AKR1A1 was not induced by either treatment. Thus, both CYP1 and AKR metabolic pathways were inducible in the model of liver progenitor cells. BaP and TCDD were efficient inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in WB-F344 cells, a principal enzyme of cellular antioxidant defense. Both compounds also induced expression of transcription factor NRF2, involved in control of enzymes protecting cells from oxidative stress. However, although BaP induced a significant formation of reactive oxygen species, it did not induce expression of heme oxygenase-1, suggesting that induction of oxidative stress by BaP was limited. Using shRNA against the aryl hydrocarbon receptor (AhR), we found that similar to CYP1A1 and CYP1B1, the AKR1C9 induction was AhR-dependent. Moreover, constitutive AKR1C9 levels in AhR-deficient rat BP8 hepatoma cells were significantly lower than in their AhR-positive 5L variant, thus supporting possible role of AhR in regulation of AKR1C9 expression. Taken together, both CYP1 and AKR1C9 appear to be AhR-regulated metabolic pathways, which may contribute to formation of pro-carcinogenic PAH metabolites in liver progenitor cells.

Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs.

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID-indomethacin. RNAi provided evidence for the involvement of p21(Cip1/Waf1), but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21(Cip1/Waf1) and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21(Cip1/Waf1) level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.

The 2,2',4,4',5,5'-hexachlorobiphenyl-enhanced degradation of connexin 43 involves both proteasomal and lysosomal activities.

One of the toxic effects of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) is the acute inhibition of gap junctional intercellular communication (GJIC), an event possibly associated with tumor promotion. The model NDL-PCB-2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-induces a sustained GJIC inhibition in rat liver epithelial WB-F344 cells. As this effect might be related to deregulation of connexin 43 (Cx43) synthesis, trafficking, or degradation, we investigated the impact of PCB 153 on these events. Although PCB 153 had no effect on Cx43 mRNA levels, it induced a gradual loss of Cx43 protein and significantly decreased the amount of gap junction plaques in plasma membrane. PCB 153 contributed to extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent accumulation of hyperphosphorylated Cx43-P3 form, thus indicating that ERK1/2 activation by PCB 153 might contribute to its effects on Cx43 internalization or degradation. Inhibition of either proteasomes or lysosomes with their specific inhibitors largely restored total Cx43 protein levels, thus suggesting that both proteasomes and lysosomes may participate in the PCB 153-enhanced Cx43 internalization and degradation. However, neither the proteasomal nor the lysosomal inhibitors restored normal GJIC or number/size of gap junction plaques. Finally, PCB 153 also interfered with restoration of gap junction plaques following the inhibition of Cx43 transport to plasma membrane. Taken together, multiple modes of action seem to contribute to downregulation of Cx43 in PCB 153-treated rat liver epithelial cells. The enhanced degradation of Cx43, together with persistent inhibition of GJIC, might contribute to tumor-promoting effects of NDL-PCBs.

Reduction of doxorubicin and oracin and induction of carbonyl reductase in human breast carcinoma MCF-7 cells.

In cancer cells, the drug-metabolizing enzymes may deactivate cytostatics, thus contributing to their survival. Moreover, the induction of these enzymes may also contribute to development of drug-resistance through acceleration of cytostatics deactivation. However, the principal metabolic pathways contributing to deactivation of many cytostatics still remain poorly defined. The main aims of the present study were: (i) to compare the reductive deactivation of cytostatic drugs doxorubicin (DOX) and oracin (ORC) in human breast cancer MCF-7 cells; (ii) to identify major enzyme(s) involved in the carbonyl reduction; and iii) to evaluate the activities and expression of selected carbonyl reducing enzymes in MCF-7 cells upon a short-term (48 h) exposure to either DOX or ORC. We found that MCF-7 cells were able to effectively metabolize both DOX and ORC through reduction of their carbonyl groups. The reduction of ORC was stereospecific, with a preferential formation of + enantiomer of dihydrooracin (DHO). The cytosolic carbonyl reductase CBR1 seemed to be a principal enzyme reducing both drugs, while cytosolic aldo-keto reductase AKR1C3 or microsomal reductases probably did not play important role in metabolism of either DOX or ORC. The exposure of MCF-7 cells to low (nanomolar) concentrations of DOX or ORC caused a significant elevation of reduction rates of both cytostatics, accompanied with an increase of CBR1 protein levels. Taken together, the present results seem to suggest that the accelerated metabolic deactivation of ORC or DOX might contribute to the survival of breast cancer cells during exposure to these cytostatics.

DNA adducts formation and induction of apoptosis in rat liver epithelial 'stem-like' cells exposed to carcinogenic polycyclic aromatic hydrocarbons.

The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[a,l]pyrene, and partly also benzo[g]chrysene and benz[a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[a,l]pyrene, and to a lesser extent also benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[a]anthracene, chrysene, benzo[b]fluoranthene and dibenzo[a,h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3',5'-tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[a,l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[a,l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells.

Concentrations of methylated naphthalenes, anthracenes, and phenanthrenes occurring in Czech river sediments and their effects on toxic events associated with carcinogenesis in rat liver cell lines.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. In the present study, we determined levels of monomethylated naphthalenes (MeNap), phenanthrenes (MePhe), and anthracenes (MeAnt) in Czech river sediments. The levels of MePhe generally were lower than the concentrations of phenanthrene. In contrast, both MeNap and MeAnt were found at levels higher than their respective parent compounds in the majority of sampling sites. We then investigated their aryl hydrocarbon receptor (AhR)-mediated activity, accumulation of phosphorylated p53 protein, induction of expression of cytochrome P450 1A1 (CYP1A1), inhibition of gap junctional intercellular communication (GJIC), and effects on cell proliferation in rat liver cell models to evaluate the relative importance of these toxicity mechanisms of low-molecular-weight methylated PAHs. Methylated phenanthrene and anthracene compounds were weak inducers of AhR-mediated activity as determined both in a reporter gene assay system and by detection of the endogenous gene (Cyp1a1) induction. 2-Methylphenanthrene was the most potent AhR ligand. Contribution of MeAnt and MePhe to overall AhR-inducing potencies should be taken into account in PAH-contaminated environments. Nevertheless, their effects on AhR were not sufficient to modulate cell proliferation in a normal rat liver progenitor cell model system. These PAHs only had a marginal effect on p53 phosphorylation at high doses of 1-, 3-, and 9-MePhe as well as 1 MeAnt. On the other hand, both 2- and 9-MeAnt as well as all the MePhe under study were efficient inhibitors of GJIC, suggesting that these compounds might act as tumor promoters. In summary, inhibition of GJIC and partial activation of AhR seem to be the most prominent toxic effects of the methylated PAHs in the present study.

Tumor necrosis factor-alpha modulates effects of aryl hydrocarbon receptor ligands on cell proliferation and expression of cytochrome P450 enzymes in rat liver "stem-like" cells.

Various liver diseases lead to an extensive inflammatory response and release of a number of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). This cytokine is known to play a major role in liver regeneration as well as in carcinogenesis. We investigated possible interactions of TNF-alpha with ligands of the aryl hydrocarbon receptor (AhR) and known liver carcinogens, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and coplanar 3,3',4,4',5-pentachlorobiphenyl (PCB 126). These compounds have been previously found to disrupt cell cycle control in contact-inhibited rat liver WB-F344 cells, an in vitro model of adult liver progenitor cells. TNF-alpha itself had no significant effect on the proliferation/apoptosis ratio in the WB-F344 cell line. However, it significantly potentiated proliferative effects of low picomolar range doses of both TCDD and PCB 126, leading to an increase in cell numbers, as well as an increased percentage of cells entering the S-phase of the cell cycle. The combination of TNF-alpha with low concentrations of AhR ligands increased both messenger RNA (mRNA) and protein levels of cyclin A, a principle cyclin involved in disruption of contact inhibition. TNF-alpha temporarily inhibited AhR-dependent induction of cytochrome P450 1A1 (CYP1A1). In contrast, TNF-alpha significantly enhanced induction of CYP1B1 at both mRNA and protein levels, by a mechanism, which was independent of nuclear factor-kappaB activation. These results suggest that TNF-alpha can significantly amplify effects of AhR ligands on deregulation of cell proliferation control, as well as on expression of CYP1B1, which is involved in metabolic activation of a number of mutagenic compounds.

beta-Naphthoflavone and 3'-methoxy-4'-nitroflavone exert ambiguous effects on Ah receptor-dependent cell proliferation and gene expression in rat liver 'stem-like' cells.

Both natural and synthetic flavonoids are known to interact with the aryl hydrocarbon receptor (AhR); however, their agonist/antagonist properties in vitro have been so far studied mostly in the context of cytochrome P450 1A1 gene (Cyp1a1) regulation. We investigated effects of two synthetic flavones known either as AhR agonist (beta-naphthoflavone; BNF) or antagonist (3'-methoxy-4'-nitroflavone; 3M4NF), using an in vitro model of liver 'stem-like' cells, on expression of various AhR target genes and AhR-dependent cell proliferation. We found that the presumed antagonist 3M4NF induces a partial nuclear translocation and activation of AhR. Although inhibiting the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced Cyp1a1 expression, 3M4NF alone induced a minor increase of CYP1A1 mRNA and protein. However, 3M4NF did not induce AhR binding to synthetic dioxin response elements (DRE). In contrast to Cyp1a1, 3M4NF induced a marked expression of other AhR-regulated genes, such as Cyp1b1 and Nqo1, as well as transcriptional repression of Cdh13 gene, confirming that its effects may be promoter-context specific. Like BNF, 3M4NF induced AhR-dependent cell proliferation of contact-inhibited rat liver 'stem-like' WB-F344 cells, associated with a marked upregulation of Cyclin A, as well as the downregulation of proteins involved in formation of cell-cell contacts. Based on these experimental findings, we conclude that partial agonists/antagonists of AhR can increase cell proliferation rate and AhR-dependent genes expression in both cell type- and gene-specific manner. The specificity of effects of flavones on diverse AhR targets should be taken into account, when studying AhR signaling using presumed AhR antagonists.

The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells.

Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. We investigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 'stem-like' cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenic benzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxic BaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression of Cyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhR mutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27(Kip1), or pRb phosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentage of cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulation of p27(Kip1) and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, although S-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated in dnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant role both in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhanced cell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations.

7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial 'stem-like' cells.

Immature liver progenitor cells have been suggested to be an important target of hepatotoxins and hepatocarcinogens. The goal of the present study was to assess the impact of 7H-dibenzo[c,g]carbazole (DBC) and its tissue-specific carcinogenic N-methyl (N-MeDBC) and 5,9-dimethyl (DiMeDBC) derivatives on rat liver epithelial WB-F344 cells, in vitro model of liver progenitor cells. We investigated the cellular events associated with both tumor initiation and promotion, such as activation of aryl hydrocarbon receptor (AhR), changes in expression of enzymes involved in metabolic activation of DBC and its derivatives, effects on cell cycle, cell proliferation/apoptosis and inhibition of gap junctional intercellular communication (GJIC). N-MeDBC, a tissue-specific sarcomagen, was only a weak inhibitor of GJIC or inducer of AhR-mediated activity, and it did not affect either cell proliferation or apoptosis. DBC was efficient GJIC inhibitor, while DiMeDBC manifested the strongest AhR inducing activity. Accordingly, DiMeDBC was also the most potent inducer of cytochrome P450 1A1 (CYP1A1) and CYP1A2 expression among the three compounds tested. Both DBC and DiMeDBC induced expression of CYP1B1 and aldo-keto reductase 1C9 (AKR1C9). N-MeDBC failed to significantly upregulate CYP1A1/2 and it only moderately increased CYP1B1 or AKR1C9. Only the potent liver carcinogens, DBC and DiMeDBC, caused a significant increase of p53 phosphorylation at Ser15, an increased accumulation of cells in S-phase and apoptosis at micromolar concentrations. In addition, DiMeDBC was found to stimulate cell proliferation of contact-inhibited WB-F344 cells at 1 microM concentration, which is a mode of action that might further contribute to its hepatocarcinogenicity. The present data seem to suggest that the AhR activation, induction of enzymes involved in metabolic activation, inhibition of GJIC or stimulation of cell proliferation might all contribute to the hepatocarcinogenic effects of DBC and DiMeDBC.

Quantitative detection of species-specific DNA in feedstuffs and fish meals.

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.

Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.

Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The 'dioxin-like' PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the 'non-dioxin-like' compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 muM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.