Cytochrome P450 1A1 Gene Expression Research Articles

Effect of interleukin (IL)-8 on benzo[a]pyrene metabolism and DNA damage in human lung epithelial cells.

It has been well established that inflammation and concurrent mutagenic exposures drive the carcinogenic process in a synergistic way. To elucidate the role of the inflammatory cytokine IL-8 in this process, we studied its effect on the activation and deactivation of the chemical mutagen benzo[a]pyrene B[a]P in the immortalized pulmonary BEAS-2B cell line. After 24h incubation with B[a]P in the presence or absence of IL-8, the B[a]P induced cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) gene expression and CYP1A1 enzyme activity was significantly higher in the presence of the cytokine. Consistent with these findings, we observed higher concentration of the metabolite B[a]P-7,8-diol under concurrent IL-8 treatment conditions. Interestingly, we also found higher concentrations of unmetabolized B[a]P. To explain this, we examined the downstream effects of IL-8 on NADPH oxidases (NOXes). IL-8 lowered the intracellular NADPH level, but this effect could not explain the changes in B[a]P metabolism. IL-8 also significantly depleted intracellular glutathione (GSH), which also resulted in enhanced levels of unmetabolized B[a]P, but increased concentrations of the metabolite B[a]P-7,8-diol. No differences in B[a]P-DNA adducts level were found between B[a]P and B[a]P combined with IL-8, and this might be due to a 3-fold increase in nucleotide excision repair (NER) after IL-8 treatment. These findings suggest that IL-8 increased the formation of B[a]P-7,8-diol despite an overall delayed B[a]P metabolism via depletion of GSH, but DNA damage levels were unaffected due to an increase in NER capacity.

Relative effect potency estimates of dioxin-like activity for dioxins, furans, and dioxin-like PCBs in adults based on cytochrome P450 1A1 and 1B1 gene expression in blood

BackgroundIn the risk assessment of PCDDs, PCDFs, and dioxin-like (DL) PCBs, regulatory authorities support the use of the toxic equivalency factor (TEF)-scheme derived from a heterogeneous data set of the relative effect potency (REPs) estimates. ObjectivesWe sought to determine REPs for dioxin-like compounds (DLCs) using expression of cytochrome P450 (CYP) 1A1 and 1B1 mRNA in human peripheral blood mononuclear cells representing two different pathways. MethodsWe used a sex and age adjusted regression-based approach comparing the strength of association between each DLC and the cytochrome P450 (CYP) 1A1 and 1B1 mRNA expression in 320 adults residing in an organochlorine-polluted area of eastern Slovakia. ResultsWe calculated REPs based on CYP1A1 expression for 4 PCDDs, 8 PCDFs, and 1 PCB congener, and based on CYP1B1 expression for 5 PCDFs and 11 PCB congeners. REPs from CYP1A1 correlated with REPs previously derived from thyroid volume (ρ=0.85; p<0.001) and serum FT4 (ρ=0.77; p=0.009). The 13 log REPs from CYP1A1 correlated with log WHO-TEFs (r=0.63; p=0.015) and 11 log PCB REPs with PCB consensus toxicity factors (CTFs) for compounds with WHO-TEFs (r=0.80; p=0.003). The complete set of derived 56 log REPs correlated with the log CTFs (r=0.77; p=0.001) and log WHO-TEFs (r=0.81; p<0.001). ConclusionsREPs calculated from thyroid and cytochrome P450 endpoints realistically reflect human exposure scenarios because they are based on human chronic and low-dose exposures. While the CYP 1A1 seems more suitable for toxicity evaluation of PCDD/Fs, the CYP 1B1 is more apt for PCDFs and PCBs and reflects different pathways.

In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus)

Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERβ1, ERβ2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERβ, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish.

CYP1A1 mediates the suppression of major inflammatory cytokines in pulmonary alveolar macrophage (PAM) cell lines caused by Mycoplasma hyponeumoniae

Mycoplasmal pneumonia is a lung infection disease caused by Mycoplasma hyopneumoniae in swine. We previously reported that Cytochrome P450 1A1 (CYP1A1) expression was significantly downregulated in pigs infected with M. hyopneumoniae compared to the healthy controls. In this study, pulmonary alveolar macrophage (PAM) cell lines with CYP1A1 overexpression or siRNA-mediated CYP1A1 silencing were used to explore the biological function and regulatory mechanism of CYP1A1 gene expression changed on the inflammatory response of pigs infected with M. hyopneumoniae. The results showed that the cells overexpressing CYP1A1 infected with M. hyopneumoniae led to a rapid increase in PPAR-γ expression, which resulted in decreasing the levels of several inflammatory cytokines including IL-1β, IL-6, IL-8 and TNF-α. On the contrary, this effect was just opposite in CYP1A1-RNAi cells infected with M. hyopneumoniae. We suggest that CYP1A1 suppress the inflammatory response caused by M. hyopneumoniae infection, via PPAR-γ signaling pathway in pigs.

Deleterious effects of incense smoke exposure on kidney function and architecture in male albino rats

Context: Previous studies, including ours, have shown adverse effects of incense smoke on human health. However, the effect of incense smoke on kidney function and structure remains unknown.Objective: To evaluate possible adverse effects of incense smoke on kidney function and architecture in albino rats after chronic exposure to Arabian incense.Materials and methods: Emission characteristics including particle size distribution, volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) were determined by gravimetric and GCMS analyses. Kidney functional markers, oxidative stress and inflammatory markers were measured by standard or ELISA based procedures. Ultrastructural changes in kidney were examined by transmission electron microscope (TEM) and the gene expression of xenobiotic metabolizing enzymes including cytochrome P-450-1A1 (CYP1A1) and CYP1A2 were studied by real time PCR.Results: Rats exposed to incense smoke demonstrated a significant increase in serum creatinine, uric acid, blood urea nitrogen (BUN), tissue malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α) and interleukin-4 (IL-4) levels and a significant decline in tissue reduced glutathione (GSH) and catalase activity. Incense smoke exposed rats also displayed marked ultrastructural changes in kidney tissue. Further, a significant increase in tissue gene expression of both CYP1A1 and CYP1A2 was noted in exposed rats.Discussion: Changes to kidney functional markers and architecture appear to be mediated through augmented oxidative stress and inflammation.Conclusion: Long-term exposure to incense smoke may have deleterious effects on kidney function and architecture. Though, inhalation is the rout of exposure, findings of this study underscore that incense smoke may also have an effect on non-pulmonary tissues.

Vinorelbine Inhibits Pregnane X Receptor‐Mediated Induction of Cytochrome P450 3A4 Gene Expression in Human Hepatocytes

Pregnane X receptor (PXR), a ligand‐dependent nuclear receptor, is involved in regulating the gene expression of enzymes and proteins involved in endobiotic and xenobiotic metabolism. In particular, PXR plays a crucial role in regulating the gene expression of drug‐metabolizing enzymes, including cytochrome p450 3A4 (CYP3A4). In cancer patients, upregulation of the PXR target genes, including CYP3A4, can lead to not only adverse drug interactions but also chemoresistance. It is possible to inhibit the activity of PXR in order to overcome PXR‐mediated adverse drug interactions and chemoresistance. In this regard, a few PXR inhibitors or antagonists have been identified and characterized. However, these compounds fall short of reaching their clinical utility as they are non‐selective and/or highly‐toxic. Therefore, there is a practical need to develop a selective and less‐toxic PXR antagonist to overcome PXR‐mediated adverse drug interactions and chemoresistance. We sought to determine whether vinorelbine (VNB), a clinically‐used antimicrotubule agent, inhibits the transcriptional activity of PXR in human hepatocytes. PXR transactivation and cell viability assays were performed to determine CYP3A4 promoter activity and cytotoxicity, respectively, in HepG2 human liver carcinoma cells. Quantitative RT‐PCR assays were conducted to study gene expression of CYP3A4 in human primary hepatocytes. VNB, at its non‐cytotoxic and therapeutically‐relevant concentrations, inhibited PXR transactivation of CYP3A4 promoter activity. Additionally, VNB attenuated the PXR agonist‐induced gene expression of CYP3A4, suggesting that VNB downregulates CYP3A4 gene expression by inhibiting the activity of PXR. These results support our conclusion that VNB inhibits the transcriptional activity of PXR at its non‐cytotoxic and therapeutically relevant concentrations. Future studies will be directed to study the selectivity of VNB to PXR as well as the mechanisms of VNB inhibition of PXR‐mediated CYP3A4 induction.Support or Funding InformationThis work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR. The authors would like to thank Drs. Coleman, Tao, and Schwartz for sharing their research facilities.

Differential effects of metal ions on TCDD-induced cytotoxicity and cytochrome P4501A1 gene expression in a zebrafish liver (ZFL) cell-line.

Trace metal ions and trace organic compounds are common co-contaminants in the environment that pose risks to human health. We evaluated the effects of four metal ions (As(3+), Cu(2+), Hg(2+), and Zn(2+)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced cytotoxicity and the expression of the cytochrome P4501A1 gene (cyp1a1) in the zebrafish liver (ZFL) cell line. A metal accumulation study showed that Cu and Zn did not accumulate in ZFL cells. However, As and Hg did accumulate, which resulted in the inhibition of TCDD-mediated induction of cyp1a1 mRNA and protein expression, and 7-ethoxyresorufin O-deethylase activity. A luciferase assay showed that both As(3+) and Hg(2+) inhibited the TCDD-induced activity of gene constructs containing either synthetic 3XRE or a distal cyp1a1 promoter region, implying that the decreased levels of TCDD-induced cyp1a1 were due to transcriptional effects. A proteomic study showed that the toxic effects of As(3+) might be due to changes in cellular metabolic processes, the cellular stimulation response and the cellular redox state in ZFL cells.

Gene expression of cytochrome P4501A1 was involved in the alteration of cell function by dioxinvia estrogen receptor in cellular and xenografted ovarian cancer models

Gene expression of cytochrome P4501A1 was involved in the alteration of cell function by dioxinvia estrogen receptor in cellular and xenografted ovarian cancer models

Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells.

A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

Galactomyces fermentation filtrate prevents T helper 2-mediated reduction of filaggrin in an aryl hydrocarbon receptor-dependent manner.

The aryl hydrocarbon receptor (AhR) recognizes diverse small molecules such as dioxins, tryptophan photoproducts and phytochemicals. It also plays crucial roles in epidermal homeostasis by upregulating epidermal barrier proteins. In preliminary screening, we found that Galactomyces fermentation filtrate (GFF), a cosmetic compound, was capable of activating AhR. To examine whether GFF upregulates the expression of the filaggrin and loricrin genes, FLG and LOR, in an AhR-dependent manner. The activation (cytoplasmic to nuclear translocation) of AhR was confirmed by immunofluorescence study and by upregulation of an AhR-specific marker, cytochrome P450-1A1 (CYP1A1). Gene expression levels were compared by quantitative reverse transcription PCR with or without GFF, interleukin (IL)-4 or IL-13 in normal human keratinocytes. AhR or control knockdown was carried out by transfection with AhR or control small interfering RNA. The protein expression of FLG and LOR was examined by immunohistochemistry using a three-dimensional epidermal equivalent treated with or without GFF or T helper (Th)2 cytokines. GFF induced the nuclear translocation of AhR with significant and dose-dependent upregulation of CYP1A1, FLG and LOR gene expression. The enhancing effects of GFF were abolished in AhR-knockdown keratinocytes. Th2 cytokines decreased expression of genes for FLG and LOR, and this expression was completely restored in the presence of GFF. The downregulated expression of the FLG gene with its restoration by GFF was also evident in the epidermal equivalent. GFF also upregulated the gene expression of genes encoding occludin, claudin-1 and 4, and kallikrein 5 and 7. Use of GFF is feasible to prevent the Th2-mediated reduction of FLG in an AhR-dependent fashion.

Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism.

Mg2+/Mn2+-dependent phosphatase 1A is involved in regulating pregnane X receptor-mediated cytochrome p450 3A4 gene expression.

Variations in the expression of human pregnane X receptor (hPXR)-mediated cytochrome p450 3A4 (CYP3A4) in liver can alter therapeutic response to a variety of drugs and may lead to potential adverse drug interactions. We sought to determine whether Mg(2+)/Mn(2+)-dependent phosphatase 1A (PPM1A) regulates hPXR-mediated CYP3A4 expression. PPM1A was found to be coimmunoprecipitated with hPXR. Genetic or pharmacologic activation of PPM1A led to a significant increase in hPXR transactivation of CYP3A4 promoter activity. In contrast, knockdown of endogenous PPM1A not only attenuated hPXR transactivation, but also increased proliferation of HepG2 human liver carcinoma cells, suggesting that PPM1A expression levels regulate hPXR, and that PPM1A expression is regulated in a proliferation-dependent manner. Indeed, PPM1A expression and hPXR transactivation were found to be significantly reduced in subconfluent HepG2 cells compared with confluent HepG2 cells, suggesting that both PPM1A expression and hPXR-mediated CYP3A4 expression may be downregulated in proliferating livers. Elevated PPM1A levels led to attenuation of hPXR inhibition by tumor necrosis factor-α and cyclin-dependent kinase-2, which are known to be upregulated and essential during liver regeneration. In mouse regenerating livers, similar to subconfluent HepG2 cells, expression of both PPM1A and the mouse PXR target gene cyp3a11 was found to be downregulated. Our results show that PPM1A can positively regulate PXR activity by counteracting PXR inhibitory signaling pathways that play a major role in liver regeneration. These results implicate a novel role for PPM1A in regulating hPXR-mediated CYP3A4 expression in hepatocytes and may explain a mechanism for CYP3A repression in regenerating livers.

Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively.

Sex hormone modulation of both induction and inhibition of CYP1A by genistein in HepG2/C3A cells.

Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.

Differential relative effect potencies of some dioxin-like compounds in human peripheral blood lymphocytes and murine splenic cells

Human risk assessment for dioxin-like compounds is typically based on the concentration measured in blood serum multiplied by their assigned toxic equivalency factor (TEF). Consequently, the actual value of the TEF is very important for accurate human risk assessment. In this study we investigated the effect potencies of three polychlorinated dibenzo-p-dioxins (PCDDs), six polychlorinated dibenzofurans (PCDFs) and 10 polychlorinated biphenyls (PCBs) relative to the reference congener 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) in in vitro exposed primary human peripheral blood lymphocytes (PBLs) and mouse splenic cells. REPs were determined based on cytochrome P450 (CYP) 1A1, 1B1 and aryl hydrocarbon receptor repressor (AhRR) gene expression as well as CYP1A1 activity in human PBLs and Cyp1a1 gene expression in murine splenic cells. Estimated median human REPs for 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1234678-HpCDD), 2,3,4,7,8,-pentachlorodibenzofuran (23478-PeCDF), 1,2,3,4,7,8-hexachlorodibenzofuran (123478-HxCDF) and 1,2,3,4,7,8,9-heptachlorodibenzofuran (1234789-HpCDF) were with 0.1, 1.1, 1 and 0.09, respectively, significantly higher compared to those estimated for mouse with REPs of 0.05, 0.45, 0.09 and 0.04, respectively. Opposite to these results, the estimated median human REP of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), was with 0.001 30-fold lower compared to the mouse REP of 0.03. Furthermore, human REPs for 1234678-HpCDD, 23478-PeCDF, 123478-HxCDF, 1234789-HpCDF and PCB 126 were all outside the ± half log uncertainty range that is taken into account in the WHO-assigned TEFs. Together, these data show congener- and species-specific differences in REPs for some, but not all dioxin-like congeners tested. This suggests that, more emphasis should be placed on human-tissue derived REPs in the establishment of a TEF for human risk assessment.

Regulation of Human Pregnane X Receptor and its Target Gene Cytochrome P450 3A by Praeruptorin A Isolated from the Herbal Medicine Peucedanum praeruptorum

Qianhu, the dried roots of Peucedanum praeruptorum, is a well-known traditional Chinese medicinal herb which was officially listed in the Chinese Pharmacopoeia. Praeruptorin A is the major active constituent of Qianhu. Our previous studies show that praeruptorin effectively transactivated the protein expression and catalytic activity of cytochrome P450 3A4 via the constitutive androstane receptor-mediated pathway. However, the effect of praeruptorin on the transactivation of cytochrome P450 3A4 through pregnane X receptor pathway is still unclear. To further elucidate the role of the pregnane X receptor pathway in the up-regulation of cytochrome P450 3A4 by praeruptorin, in this study, the effect of praeruptorin on the cytochrome P450 3A4 gene expression was investigated in mouse primary hepatocytes after knockdown of the pregnane X receptor by transient transfection of its siRNA; and the gene expression, protein expression, and catalytic activity of cytochrome P450 3A4 in the LS174T cells with pregnane X receptor overexpression were determined by real-time PCR, Western blot analysis, and LC-MS/MS-based cytochrome P450 3A4 substrate assay, respectively. We found that the level of cytochrome P450 3a11 gene expression in mouse primary hepatocytes was significantly increased by praeruptorin, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. Praeruptorin significantly induced cytochrome P450 3A4 mRNA, protein expression, and functional activity through pregnane X receptor-mediated pathway in pregnane X receptor-overexpression LS174T cells; conversely, induction was not found in LS174T cells untransfected with pregnane X receptor plasmids. These findings suggest that praeruptorin can significantly up-regulate cytochrome P450 3A4 gene via the pregnane X receptor-mediated pathway, and this should be taken into consideration in potential herb-drug interactions.

CYP1A expression in liver and gills of roach (Rutilus rutilus) after waterborne exposure to two phenanthrene derivatives, 1‐methylphenanthrene and 4‐methylphenanthrene

Phenanthrenes (Phs) substituted with alkyl groups are a class of compound present in the environment, and they appear to be toxic to developing fish. The present study aimed to investigate the effect of waterborne exposure to two monomethyl derivatives of phenanthrene, 1-methylphenanthrene (1M-Ph) and 4-methylphenanthrene (4M-Ph), on cytochrome P450 1A (CYP1A) gene expression in fish gills and liver. Juvenile common roaches (Rutilus rutilus) were exposed to water with dimethyl sulfoxide (DMSO) solutions of 1M-Ph, 4M-Ph, benzo[a]pyrene (BaP; positive control), each at a dose of 100 µg/L, or to water with DMSO alone (negative control group) for 2 d and 7 d. Significant CYP1A responses with regard to treatment and exposure duration were noted (2-way analysis of variance [ANOVA]) in gills (p = 0.013 and p = 0.003, respectively) and liver (p < 0.001). The 2 monomethyl Phs did not induce consistent gene expression changes, except for 4-MPh, which elevated the CYP1A messenger ribonucleic acid (mRNA) level in the liver at the end of the treatment (almost 4-fold; p < 0.05; 7 d). As was expected, exposure to BaP resulted in elevation of CYP1A mRNA expression in treated fish compared with the control group. Expressions after 2 d and 7 d were approximately 220- and 180-fold higher in liver and 8- and 6-fold higher in gills respectively. The CYP1A protein levels remained stable in both tissues, with one notable exception in roach liver treated for 2 d with BaP (∼ 6-fold increase; p < 0.05). The different effects of the 1- and 4-methylphenanthrenes on CYP1A gene expression in roach liver suggest a relationship between chemical or 3-D structure of the differentially substituted monomethyl Phs and their biological activity.

Up-regulatation of CYP3A expression through pregnent X receptor by praeruptorin D isolated from Peucedanum praeruptorum Dunn

Ethnopharmacological relevanceQianhu, the dried roots of Peucedanum praeruptorum DUNN (Umbelliferae), is a well-known traditional Chinese medicinal herb which was officially listed in the Chinese Pharmacopoeia. Praeruptorin D (PD) is one of the major active constituents of Peucedanum praeruptorum Dunn (Qianhu). The Pregnane X receptor (PXR) is an orphan nuclear receptor and plays a pivotal role in the activation of human cytochrome P450 3A4 (CYP3A4) gene. Aim of the studyThe purpose of this study was to investigate the effect of PD on the PXR-mediated transactivation of CYP3A4, and thus to predict potential herb–drug interactions between PD, Qianhu, and the other co-administered drugs that metabolized by CYP3A4. Materials and methodsThe effect of PD on the Cyp3a11, mPXR mRNA expression in mice primary hepatocytes was measured using real-time PCR. The gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells after transfected with PXR expression plasmids were determined by real-time PCR, Western blot analysis, and LC–MS/MS based CYP3A4 substrate assay. ResultsThe results revealed that the level of Cyp3a11 gene expression in mice primary hepatocytes was significantly increased by PD, but PD cannot induce the mPXR gene expression. On the other hand, CYP3A4 mRNA, protein expression and functional activity in PXR-over-expression LS174T cells were significantly increased by PD through PXR-mediated pathway; conversely, no significant change was found in the untransfected cells. ConclusionsThese findings suggest that PD can significantly up-regulate CYP3A4 expression and activity via the PXR-mediated pathway and this should be taken into consideration to predict any potential herb–drug interactions when PD and Peucedanum praeruptorum Dunn are co-administered with other drugs.

Effects of steroid hormones on reproduction- and detoxification-related gene expression in adult male mosquitofish, Gambusia affinis

The molecular mechanisms that mediate fish reproduction and detoxification in response to steroid hormones were studied by using adult male western mosquitofish (Gambusia affinis) as sentinel species. The expression patterns of three vitellogenins (VtgA, VtgB and VtgC), two estrogen receptors (ERα and ERβ), two androgen receptors (ARα and ARβ), metallothionein (MT) and cytochrome P450 1A (CYP1A) in the liver and testis of adult male mosquitofish were assessed through exposure treatments with progesterone (P), testosterone (T) and 17β-estradiol (E2), alone and in combination for eight days. The results showed that expression patterns of Vtg subtype, ER subtype, AR subtype, MT and CYP1A genes in male mosquitofish varied according to tissue and specific hormone stress. Vtg subtype mRNA expression was induced in the liver in E2-added treatments, and an up-regulation of ERα mRNA expression was also observed. In addition, hormone treatments increased three Vtg subtype mRNA expression levels in the testis, at least to some extent. All hormone treatments significantly inhibited ERα, ERβ and ARβ mRNA expression in the testis. Some of hormone treatments could affect MT and CYP1A gene expression in mosquitofish. In general, multiple hormone treatments showed different effects on target gene expression compared with corresponding hormone alone. The results from the present study provided valuable information on the toxicological effects of steroid hormones in mosquitofish.

Chemopreventive Role of Metformin during DMBA–induced Breast Carcinogenesis in MCF10A Cells through CYP1A1‐AhR Signaling Pathway

Recent studies have established that metformin (MET), an oral hypoglycemic drug, possesses antioxidant activity and effective against different types of cancer. Although several postulated mechanisms were reported, the role of the cytochrome P450 1A1 (CYP1A1)‐aryl hydrocarbon receptor (AhR) signaling pathway in the protective effect of MET against breast cancer has not been reported before. Therefore, we examined the capacity of MET to inhibit CYP1A1 gene expression and DNA adducts formation in human breast cells MCF10A by dimethylbenz[a]anthracene (DMBA), a well‐known breast chemical carcinogen. Our results showed that DMBA‐induced DNA adduct was completely prevented by MET as evidenced by decrease in DNA repair genes and 8‐hydroxy‐2′‐deoxyguanosine level. Importantly, MET inhibited the induction of CYP1A1 mRNA, protein and activity levels by DMBA, suggesting a direct role of CYP1A1. Mechanistically, this was strongly correlated with MET ability to inhibit AhR‐dependent luciferase activity, but there was no effect on AhR activation, as evidenced by binding assay, suggesting that MET is not an AhR ligand. In conclusion, the present work provides the first evidence that MET inhibits the DMBA‐mediated carcinogenicity by inhibiting the expression of CYP1A1 through an AhR ligand‐independent mechanism. This work was supported by the Deanship of Scientific Research grant no. RGP‐VPP‐141.