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Cytochrome P450 (P450)1A1 plays a critical role in the metabolic activation and detoxification of polycyclic aromatic hydrocarbons (PAHs), many of which are potent human carcinogens. In this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1A1 expression in human hepatoma cells (HepG2) and that this phenomenon is mediated by sustained transcriptional activation of the CYP1A1 promoter. Treatment of HepG2 cells with MC resulted in marked induction (8-20-fold) of ethoxyresorufin O-de-ethylase activities, CYP1A1 apoprotein contents, and mRNA levels, which persisted for up to 96 h. MC also caused sustained transcriptional activation of the human CYP1A1 promoter for up to 96 h, as inferred from transient transfection experiments. Experiments with deletion constructs indicated that Ah response elements located at -886, -974, and -1047, but not -491, nucleotides from the start site, contributed to the sustained transcriptional activation of the CYP1A1 promoter. Electrophoretic mobility-shift and chromatin immunoprecipitation assays suggested that prolonged CYP1A1 induction was mediated by Ah receptor (AHR)-independent mechanisms. Experiments with [3H]MC and liquid chromatography-tandem mass spectrometry demonstrated rapid elimination of MC and its metabolites from the cells by 12 to 24 h, suggesting that these compounds did not elicit sustained CYP1A1 induction via the classical AHR-mediated pathway. In conclusion, the results of this study support the hypothesis that MC causes persistent induction of CYP1A1 in human hepatoma cells by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter via AHR-independent mechanisms. These observations have important implications for human carcinogenesis mediated by PAHs.

Recent epidemiological studies have revealed a possible correlation between exposure to high levels of dioxins or dioxin-like compounds and diabetes. Yet the interaction between insulin and dioxin actions remains elusive. We studied the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), a protein involved in glucose homeostasis and whose expression is down-regulated by insulin. We showed that 2,3,7,8-tetrachorodibenzo-p-dioxin (TCDD) specifically induced IGFBP-1 mRNA in human hepatocytes and HepG2 human hepatoma cells (2.5- and 8-fold, respectively). Cellular and secreted IGFBP-1 protein levels were also up-regulated. Transfection and reporter assays showed that the IGFBP-1 promoter was activated by TCDD and that this activation was dependent on the integrity of a proximal xenobiotic-responsive element (XRE). This XRE, located near the insulin-glucocorticoid regulatory region, binds the aryl-hydrocarbon receptor. In agreement with previous studies, the IGFBP-1 promoter was down-regulated by insulin (50%); we show here that although TCDD activated the IGFBP-1 promoter 5- to 6-fold, the combination of TCDD and insulin led to an expression level of IGFBP-1 that was higher than basal level (2- to 3-fold activation). Similar regulations were observed for the endogenous IGFBP-1 mRNA. These data suggest that the xenobiotic-hormonal regulatory region of the IGFBP-1 promoter mediates an up-regulation of IGFBP-1 expression by TCDD even in the presence of insulin. Because IGFBP-1 modulates blood glucose levels, the up-regulation of IGFBP-1 by dioxins might account for the disruptive effects of these pollutants on glucose metabolism.

The dioxin 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) induces phase I and II xenobiotic metabolizing enzymes (XME) which act sequentially to eliminate different classes of xenobiotics. The transcriptional effects of TCDD are generally mediated by the arylhydrocarbon receptor (AhR). We hypothesized that TCDD could also act indirectly, by increasing the activity of cytochrome P450 1A1 (CYP1A1), a phase I gene, which could then mediate the induction of other XME genes, such as the NAD(P)H:quinone oxidoreductase 1 (NQO1). To test this hypothesis, NQO1 gene expression was monitored after either overexpression of CYP1A1 or siRNA-mediated knock-down of CYP1A1 activity in the hepatoma cell line HepG2. Overexpression of CYP1A1 in the absence of TCDD was carried out using either adenoviral infection or the "Tet-off" system. Recombinant adenoviruses were produced encoding no protein, CYP1A1 (Ad1A1), or a mutated inactive CYP1A1 (Ad1A1mut). In the HepG2 Tet-off cell line, CYP1A1 expression was induced by the removal of doxycycline (dox) from the cell medium. Ad1A1 infection or dox removal induced CYP1A1 activity and H(2)O(2) production similarly to TCDD treatment. Moreover, in both systems, the amount of NQO1 mRNA increased to the same level as after TCDD treatment (approximately 2-fold). The UDP-glucuronosyl transferase 1A6 (UGT1A6) gene is also similarly regulated. NQO1 gene expression was not induced when mutant, inactive CYP1A1 was overexpressed or when the antioxidant N-acetyl cysteine (NAC) was added to Ad1A1. Finally, either NAC or siRNA directed against CYP1A1 mRNA decreased the induction of NQO1 gene expression by TCDD. We conclude that, after exposure to TCDD, the NQO1 gene expression can be controlled by CYP1A1 activity through an oxidative stress mediated pathway.

The human paraoxonase-1 (PON-1) is a serum high-density lipoprotein-associated phosphotriesterase secreted mainly by the liver. This enzyme is able to hydrolyze toxic organophosphate xenobiotics, endogenous oxidized phospholipids, and homocysteine thiolactone. Physiologically, it is thought to protect against cardiovascular diseases. The level of PON-1 gene expression is a major determinant of paraoxonase-1 status but little is known regarding the regulation of this gene. We identified several transcription start sites and characterized the regulation of its promoter by fibrates and statins. In HuH7 human hepatoma cells, the PON-1 secreted enzymatic activity and mRNA levels were increased by fenofibric acid (approximately 70%) and decreased by several statins (approximately 50%). Transient and stable transfection assays in HuH7 cells indicated that the modulation of the mRNA and enzymatic activity levels could be accounted for by the regulation of the PON-1 gene promoter activity by these drugs. These effects are probably not mediated by the PPAR alpha because over-expression of this receptor decreased the fibrate effect and did not modify statins activity. The repressive effect of statins is reversed by mevalonate and 22(R)-hydroxycholesterol, suggesting the involvement of the liver X receptor in the mechanism. The opposite effects of fenofibrate and statins could be consistent with clinical data on homocysteine levels after hypolipidemic drug treatment. Regarding the toxicological aspects, the induction achieved with fenofibric acid, although limited, could increase organophosphate metabolism and may be relevant in certain conditions for protective treatments.

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.

The adaptive response to cellular stress requires the reprogramming of gene expression. So far, research has focused on induction mechanisms; several transcription factors activated by cellular stress have been shown to trigger the induction of repair and detoxification enzymes. Using the hepatoma cell line HepG2, we report that the trans-activating function of the nuclear factor I/CCAAT box transcription factor (NFI/CTF-1) is, on the contrary, repressed by various stress conditions, including inflammatory cytokine treatment, glutathione depletion, heat and osmotic shocks, and chemical stress. Under the same conditions, other transcription factors were not affected. We show that when Cys-427 within the trans-activating domain of NFI/CTF-1 is mutated into a serine, the repressive effect triggered by cellular stresses is no longer observed. In addition, this effect is abolished in cells transfected with a thioredoxin expression vector. Using the dichlorofluorescein fluorescent probe, we provide direct evidence that the stress conditions elicit an intracellular reactive oxygen species generation, which can, in turn, negatively regulate NFI/CTF-1. In agreement with these observations, we show that the CYP1A1 mRNA and the CYP1A1 gene promoter, which is a target of NFI/CTF-1, are repressed by stress conditions. Thus, through the redox regulation of its trans-activating function, NFI/CTF-1 constitutes a novel biologically relevant negative sensor of several stress stimuli.

Cytochrome P450 enzymes catalyze the first step of the metabolism and subsequent elimination of hydrophobic xenobiotics. However, the activity of some isoforms, among them CYP1A1 and CYP2E1, may result in cellular insults such as oxidative stress and activation of procarcinogen compounds into reactive metabolites. The regulation of the expression of these enzymes is therefore important. We have previously shown that the CYP1A1 gene promoter was repressed by oxidative stress. We show here that the CYP2E1 gene promoter is down-regulated by exogenous H(2)O(2) addition and glutathione depletion. It is also repressed by the transfection of a CYP2E1 expression vector, which elicits an intracellular H(2)O(2) generation. This autoregulation is limited by catalase (which catalyzes the catabolism of H(2)O(2)), thus implying H(2)O(2) as a mediator of the negative feedback mechanism. Furthermore, we observed that the activity of CYP1A1 resulting either from the stimulation of the endogenous gene by benzo[a]pyrene treatment or from the transfection of an expression vector, repressed the activity of the CYP2E1 gene promoter. Conversely, CYP2E1 overexpression repressed the activity of the CYP1A1 gene promoter. In both cases, catalase and a specific inhibitor of one enzyme prevented the repression of the other. This suggests that the generation of H(2)O(2) during the catalytic cycle of these enzymes is a mediator of the cross-regulatory mechanisms. These novel repressive mechanisms of autoregulation and cross-regulation using H(2)O(2) as a common mediator may limit the potential toxicity resulting from high cytochrome P450 activity within the cell.

The agonist activity of the antimineralocorticoid spironolactone was evaluated in various cell lines through the use of transfection experiments. The target promoters were derived from the deltaMTV promoter in which one or several glucocorticoid-responsive elements (GRE) were inserted in tandem. Spironolactone at 100 nM activated by 6-fold the GRE/deltaMTV promoter in the human hepatoma HepG2 cell line and only partially prevented the 10-fold activation of this promoter by 0.1 nM aldosterone. Both effects were completely dependent on the cotransfection of an expression vector for the mineralocorticoid receptor. The half-maximal agonist effect of spironolactone was similar to its half-maximal antagonist effect (approximately 10 nM). For the GRE-2/deltaMTV, GRE-4/deltaMTV, and wild-type MMTV promoters, the activation by aldosterone was much more potent (70-, 100-, and 110-fold, respectively), whereas spironolactone elicited a 10-, 24-, and 25-fold activation, respectively. Thus, the effect of both compounds and the relative efficiency of spironolactone, compared with that of aldosterone, were dependent on the number of GREs present in the regulatory region of the promoter. The agonist effect of spironolactone was cell specific. Indeed, although spironolactone agonist activity was observed in H5 kidney tubule cells, none could be detected at concentrations of < or = 1 microM in the CV1 monkey fibroblast cells. In contrast, the antagonist effect was observed in all cells. Furthermore, other antimineralocorticoids, such as RU 26752 and progesterone, also displayed mineralocorticoid receptor-dependent agonist activity in the HepG2 cells. The antiprogesterone RU 486 and the antiandrogen cyproterone acetate were ineffective at < or = 1 microM. In conclusion, we show that under certain experimental conditions, several antimineralocorticoids display significant agonist activity in a cell-specific and promoter-dependent manner.

Cytochromes P450 (P450s) are inducible drug-metabolizing enzymes involved in the metabolism of numerous endogenous and exogenous substrates. The regulation of some of these enzymes during experimental diabetes has been reported, but the direct involvement of insulin and the mechanism of its action remain unclear. The aim of our work was to study the effects of insulin on P450 2B and 2E expression in differentiated Fao hepatoma cells. Exposure of the cells to 0.1 microM insulin caused 60% and 80% decreases in the steady state levels of P450 2B and 2E proteins, respectively, within 24 hr. Before this, a rapid decrease in the corresponding messages was observed. Indeed, 5-6 hr of insulin treatment produced 80 and 50% decreases in P450 2B and 2E mRNA levels, respectively. Nuclear run-on transcription and mRNA turnover studies were performed to determine the mechanism (transcriptional and/or post-transcriptional) by which insulin modulated these mRNA levels. From our results, it can be concluded that insulin down-regulates the expression of P450 2B by shortening the half-life of its mRNA (half-lives of 6.9 hr without insulin and 3.6 hr with insulin), whereas it down-regulates the expression of P450 2E both by weak repression of the transcription rate (-30%) and, in particular, by acceleration of its mRNA turnover (half-lives of 8.5 hr without insulin and 3.3 hr with insulin).