The gene expression profile of a drug metabolism system and signal transduction pathways in the liver of mice treated with tert-butylhydroquinone or 3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate of sodium.

Tert-butylhydroquinone (tBHQ) is a highly effective phenolic antioxidant used in edible oils and fats in foods as well as in medicines and cosmetics. TBHQ has been shown to have both chemoprotective and carcinogenic effects. Furthermore, it has potential anti-inflammatory, antiatherogenic, and neuroprotective activities. TBHQ induces phase II detoxification enzymes via the Keap1/Nrf2/ARE mechanism, which contributes to its chemopreventive functions. Nonetheless, there is growing evidence that biological effects of tBHQ may be mediated by Nrf2-independent mechanisms related to various signaling cascades. Here, we studied changes in gene expression of phase I, II, and III drug metabolizing enzymes/transporters as well as protein levels and activities of cytochromes P450 (CYPs) elicited by tBHQ and its structural homolog TS-13 in the mouse liver. Next, we carried out gene expression analysis to identify signal transduction pathways modulated by the antioxidants. Mice received 100 mg/kg tBHQ or TS-13 per day or only vehicle. The liver was collected at 12 hours and after 7 days of the treatment. Protein and total RNA were extracted. Gene expression was analyzed using Mouse Drug Metabolism and Signal Transduction PathwayFinder RT2Profiler™PCR Arrays. A western blot analysis was used to measure protein levels and a fluorometric assay was employed to study activities of CYPs. Genes that were affected more than 1.5-fold by tBHQ or TS-13 treatment compared with vehicle were identified. Analysis of the gene expression data revealed changes in various genes that are important for drug metabolism, cellular defense mechanisms, inflammation, apoptosis, and cell cycle regulation. Novel target genes were identified, including xenobiotic metabolism genes encoding CYPs, phase II/III drug metabolizing enzymes/transporters. For Cyp1a2 and Cyp2b, we observed an increase in protein levels and activities during tBHQ or TS-13 treatment. Changes were found in the gene expression regulated by NFκB, androgen, retinoic acid, PI3K/AKT, Wnt, Hedgehog and other pathways.

Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism.

Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A-pAhR repressor (AhRR)-was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.

Association of cytochrome P450 genetic polymorphisms with neoadjuvant chemotherapy efficacy in breast cancer patients.

BACKGROUND: The enzymes of the cytochrome P450 family (CYPs) play an important role in the metabolism of a great variety of anticancer agents; therefore, polymorphisms in genes encoding for metabolizing enzymes and drugs transporters can affect drug efficacy and toxicity. METHODS: The genetic polymorphisms of cytochrome P450 were studied in 395 patients with breast cancer by RLFP analysis. RESULTS: Here, we studied the association of functionally significant variant alleles of CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 with the clinical response to neoadjuvant chemotherapy in breast cancer patients. A significant correlation was observed between the CYP2C9*2 polymorphism and chemotherapy resistance (OR = 4.64; CI 95% = 1.01 - 20.91), as well as between CYP2C9*2 heterozygotes and chemotherapy resistance in women with nodal forms of breast cancer and a cancer hereditary load (OR = 15.50; CI 95% = 1.08 - 826.12) when the potential combined effects were examined. No significant association between chemotherapy resistance and the other examined genotypes and the potential combined clinical and tumour-related parameters were discovered. CONCLUSION: In conclusion, CYP2C9*2 was associated with neoadjuvant chemotherapy resistance (OR = 4.64; CI 95% = 1.01 - 20.91) in the population of interest.

Comparative study of CYP2B induction in the liver of rats and mice by different compounds.

Male Wistar rats and C57BL mice were treated by phenobarbital (PB), 2,4,6-triphenyldioxane-1,3 (TPD) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). The CYP2B specific activities (PROD and BROD) were determined in the animal livers. PB administration significantly increased levels of PROD- and BROD-activity in the rat and mouse livers, whereas TPD induced CYP2B activities only in rat liver and TCPOBOP--only in mouse liver. The result of Western-blot analysis showed that PB and TPD increased CYP2B protein content in rat liver, PB and TCPOBOP--in mouse liver. Results of multiplex RT-PCR showed that the increase in CYP2B enzymatic activities reflected at least in part an increased mRNA levels. Thus, our results provide evidence to support the conclusion that the species-dependent differences of CYP2B induction occur because of differences of transcriptional activation of CYP2B genes.

CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers.

The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca(2+)/calmodulin-dependent kinase II (W7) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes.

In vivo effects of protein kinase and phosphatase inhibitors on CYP2B induction in rat liver.

Effects of inhibiting protein kinases and phosphatases on induction of CYP2B by triphenyldioxane (TPD) and phenobarbital (PB) were investigated. Male Wistar rats were treated with test inhibitors before TPD or PB administration. Inhibitors of phosphatidylinositol-3-kinase (Wortmannin) and protein kinase C (bisindolylmaleimide I) did not have appreciable effects on TPD- or PB-induced pentoxyresorufin O-dealkylase (PROD) activity specific for CYP2B, although bisindolylmaleimide I did give substantial induction alone. W-7, an inhibitor of Ca2+/calmodulin-dependent kinase II, produced a 6-fold increase in the TPD-induced PROD activity and did not lead to a significant increase in basal PROD activity. Treatment of rats with okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, caused considerable decreases in PROD activity during the induction by TPD and PB (8- and 2.5-fold, respectively). Results of multiplex RT-PCR showed that the increase in enzymatic activity from W7 and OA treatment reflected at least in part increased mRNA levels. CYP2B mRNA level in the liver of rats treated with W-7 and TPD was 1.5 times higher than in the liver of TPD-treated rats. This effect was not observed for PB-induction. OA treatment caused a decrease of the CYP2B mRNA levels of 44% and 33% respectively, for TPD- and PB-induction. Thus, our results are consistent with the hypothesis that phosphorylation/dephosphorylation signaling pathways are involved in regulation of CYP2B induction in rat liver.

Cytochrome P4501A1 and 1A2 gene expression in the liver of 3-methylcholanthrene- and o-aminoazotoluene-treated mice: a comparison between PAH-responsive and PAH-nonresponsive strains.

The objective of this study was to investigate cytochrome P4501A1 and 1A2 mRNA, protein, and enzyme activity in the liver of male mice differing in the aryl hydrocarbon receptor (AhR) genotype during treatment with the carcinogenic compounds 3-methylcholanthrene (MC) and o-aminoazotoluene (OAT). The basal levels of the CYP1A1 and CYP1A2 enzyme activities were comparable among the mouse strains examined. Significant interstrain variations were observed after treatment by the inducers: EROD and MROD activities were considerably increased in C57BL and A/Sn mice, but not in AKR, SWR, and DBA mice. Western blot analysis did not detect CYP1A1 in the liver of untreated mice. Treatment of mice with MC or OAT caused CYP1A1 accumulation in the liver of C57BL and A/Sn mice, but not in AKR, SWR, and DBA mice. CYP1A2 was detected in all studied mouse strains in both untreated and inducer-treated livers. The results of multiplex RT-PCR showed that the CYP1A1 mRNA in the liver of untreated mice was hardly detectable while constitutive expression of the CYP1A2 gene was rather high. After treatment with MC and OAT the CYP1A1 mRNA level dramatically increased in all strains examined while the increase in the CYP1A2 mRNA level was not striking. This finding did not correlate with the data on the enzyme activity. Our results demonstrated a discrepancy between the transcription of CYP1A1 and CYP1A2 genes and the inducibility of these enzymes in the liver of mice, suggesting a posttranscriptional mechanism of cytochrome P4501A regulation. This comparison between aromatic hydrocarbon-responsive and -nonresponsive strains could contribute to understanding of cytochrome P4501A gene regulation in the liver under the influence of environmental factors.