Mercury modulates the cytochrome P450 1a1, 1a2 and 1b1 in C57BL/6J mice: in vivo and in vitro studies.

In the current study C57BL/6J mice were injected intraperitoneally with Hg(2+) in the absence and presence of TCDD. After 6 and 24h the liver was harvested and the expression of Cyps was determined. In vitro, isolated hepatocytes were incubated with TCDD in the presence and absence of Hg(2+). At the in vivo level, Hg(2+) significantly decreased the TCDD-mediated induction of Cyps at 6h while potentiating their levels at 24h. In vitro, Hg(2+) significantly inhibited the TCDD-mediated induction of Cyp1a1 in a concentration- and time-dependent manner. Interestingly, Hg(2+) increased the serum hemoglobin (Hb) levels in mice treated for 24h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the AhR-dependent luciferase activity with a subsequent increase in Cyp1a1 protein and catalytic activity levels. Importantly, when hepatocytes were treated for 2h with Hg(2+) in the presence of TCDD, then the medium was replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. In addition, Hg(2+) increased heme oxygenase-1 (HO-1) mRNA, which coincided with a decrease in the Cyp1a1 activity level. When the competitive HO-1 inhibitor, tin mesoporphyrin was applied to the hepatocytes there was a partial restoration of Hg(2+)-mediated inhibition of Cyp1a1 activity. In conclusion, we demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by Hg(2+) in C57BL/6J mice livers and isolated hepatocytes. Moreover, this study implicates Hb as an in vivo specific modulator of Cyp1 family.

Modulation of cytochrome P450 1 (Cyp1) by vanadium in hepatic tissue and isolated hepatocyte of C57BL/6 mice.

The objective of the current study was to investigate the effect of vanadium (V(5+)) on Cyp1 expression and activity in C57BL/6 mice liver and isolated hepatocytes. For this purpose, C57BL6 mice were injected intraperitoneally with V(5+) (5 mg/kg) in the absence and presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (15 µg/kg) for 6 and 24 h. Furthermore, isolated hepatocytes from C57BL6 mice were treated with V(5+) (5, 10, and 20 µM) in the absence and presence of TCDD (1 nM) for 3, 6, 12, and 24 h. In vivo, V(5+) alone did not significantly alter Cyp1a1, Cyp1a2, or Cyp1b1 mRNA, protein, or catalytic activity levels. Upon co-exposure to V(5+) and TCDD, V(5+) significantly potentiated the TCDD-mediated induction of the Cyp1a1, Cyp1a2, and Cyp1b1 mRNA, protein, and catalytic activity levels at 24 h. In vitro, V(5+) decreased the TCDD-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels. Furthermore, V(5+) significantly inhibited the TCDD-induced AhR-dependent luciferase activity. V(5+) also increased serum hemoglobin (Hb) levels in animals treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone or in the presence of TCDD, there was an increase in the AhR-dependent luciferase activity. When isolated hepatocytes were treated for 2 h with V(5+) in the presence of TCDD, followed by replacement of the medium with new medium containing Hb, there was further potentiation to the TCDD-mediated effect. The present study demonstrates that there is a differential modulation of Cyp1a1 by V(5+) in C57BL/6 mice livers and isolated hepatocytes and demonstrates Hb as an in vivo specific modulator.

Modulation of aryl hydrocarbon receptor-regulated genes by acute administration of ammonium metavanadate in kidney, lung and heart of C57BL/6 mice.

We recently reported that vanadium (V(5+) ) was able to decrease the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 and Nqo1 at mRNA, protein and catalytic activity levels in mouse hepatoma Hepa 1c1c7 and human hepatoma HepG2 cells. However, little is known regarding the in vivo effects. Thus, the objective of this study was to investigate whether similar effects would occur at the in vivo level. Therefore, we examined the effect of exposure to V(5+) (5 mg kg(-1) ) with or without TCDD (15 µg kg(-1) ) on the AhR-regulated genes in kidney, lung and heart of C57BL/6 J mice. Our results demonstrated that V(5+) alone significantly decreased Cyp1b1 protein and catalytic activity levels in kidney at 24 h. Moreover, it significantly potentiated Nqo1 and Gsta1 gene expression in the heart, and only Gsta1 gene expression in the lung. Upon co-exposure, we found that V(5+) significantly inhibited the TCDD-mediated induction of Cyp1a1, Cyp1a2 and Cyp1b1 mRNA, protein and catalytic activity levels in the kidney at 24 h. On the other hand, V(5+) significantly potentiated the TCDD-mediated induction of Nqo1 and Gsta1 protein and activity levels in the kidney. Cyp1a1, Cyp1b1, Nqo1 mRNA, protein and catalytic activity levels in the lung were significantly potentiated at 6 h. Interestingly, all tested genes in the heart were significantly decreased at 6 h with the exception of Gsta1 mRNA. The present study demonstrates that V(5+) modulates TCDD-induced AhR-regulated genes. Furthermore, the effect on one of these enzymes could not be generalized to other enzymes even if it was in the same organ.

Differential modulation of aryl hydrocarbon receptor regulated enzymes by arsenite in the kidney, lung, and heart of C57BL/6 mice.

During the last couple of decades, efforts have been made to study the toxic effects of individual aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by arsenic As(III). However, little is known about the combined toxic effects of TCDD and As(III) in vivo. Previous reports from our laboratory and others have demonstrated that As(III), by itself or in the presence of AhR ligands, such as TCDD, is capable of differentially altering the expression of various phase I and phase II AhR-regulated genes in in vitro systems. Thus, the objective of the current study was to investigate whether or not similar effects would occur at the in vivo level. Therefore, we examined the effect of exposure to As(III) (12.5 mg/kg) in the absence and presence of TCDD (15 µg/kg) on the AhR-regulated genes using C57Bl/6 mice. Our results demonstrated that As(III) alone inhibited Cyp1a1 and Cyp1a2 in the kidney, while it induced their levels in the lung and did not affect their mRNA levels in the heart. As(III) also induced Nqo1 and Gsta1 in all tested tissues. Upon co-exposure to As(III) and TCDD, As(III) inhibited the TCDD-mediated induction of Cyp1a1 in the kidney and heart, Cyp1a2 in the kidney and heart, while it potentiated TCDD-mediated induction of Cyp1a1 in the lung, and Nqo1 and Gsta1 in the kidney and lung. In conclusion, the present study demonstrates for the first time that As(III) modulates constitutive and TCDD-induced AhR-regulated genes in a time-, tissue-, and AhR-regulated enzyme-specific manner.

Acute mercury toxicity modulates cytochrome P450, soluble epoxide hydrolase and their associated arachidonic acid metabolites in C57Bl/6 mouse heart.

Mercury exposure is associated with increased risk of cardiovascular disease and profound cardiotoxicity. However, the correlation between Hg(2+)-mediated toxicity and alteration in cardiac cytochrome P450s (Cyp) and their dependent arachidonic acid metabolites has never been investigated. Therefore, we investigated the effect of acute mercury toxicity on the expression of Cyp-epoxygenases and Cyp-ω-hydroxylases and their associated arachidonic acid metabolites in mice hearts. In addition, we examined the expression and activity of soluble epoxide hydrolase (sEH) as a key player in arachidonic acid metabolism pathway. Mercury toxicity was induced by a single intraperitoneal injection (IP) of 2.5 mg/kg of mercuric chloride (HgCl2). Our results showed that mercury treatment caused a significant induction of the cardiac hypertrophy markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); in addition to Cyp1a1, Cyp1b1, Cyp2b9, Cyp2b10, Cyp2b19, Cyp2c29, Cyp2c38, Cyp4a10, Cyp4a12, Cyp4a14, Cyp4f13, Cyp4f15, Cyp4f16 and Cyp4f18 gene expression. Moreover, Hg(2+) significantly increased sEH protein expression and activity levels in hearts of mercury-treated mice, with a consequent decrease in 14,15-, and 11,12-epoxyeicosatrienoic acids (EETs) levels. Whereas the formation of 14,15-, 11,12-, 8,9-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. In conclusion, acute Hg(2+) toxicity modulates the expression of several Cyp and sEH enzymes with a consequent decrease in the cardioprotective EETs which could represent a novel mechanism by which mercury causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent Hg(2+)-induced hypertrophy.

Posttranslational mechanisms modulating the expression of the cytochrome P450 1A1 gene by methylmercury in HepG2 cells: a role of heme oxygenase-1.

Recently we demonstrated the ability of mercuric chloride (Hg(2+)) in human hepatoma HepG2 cells to significantly decrease the TCDD-mediated induction of Cytochrome P450 1A1 (CYP1A1) mRNA, protein, and catalytic activity levels. In this study we investigated the effect of methylmercury (MeHg) on CYP1A1 in HepG2 cells. For this purpose, cells were co-exposed to MeHg and TCDD and the expression of CYP1A1 mRNA, protein, and catalytic activity levels were determined. Our results showed that MeHg did not alter the TCDD-mediated induction of CYP1A1 mRNA, or protein levels; however it was able to significantly decrease CYP1A1 catalytic activity levels in a concentration-dependent manner. Importantly, this inhibition was specific to CYP1A1and was not radiated to other aryl hydrocarbon receptor (AhR)-regulated genes, as MeHg induced NAD(P)H:quinone oxidoreductase 1 mRNA and protein levels. Mechanistically, the inhibitory effect of MeHg on the induction of CYP1A1 coincided with an increase in heme oxygenase-1 (HO-1) mRNA levels. Furthermore, the inhibition of HO-1 activity, by tin mesoporphyrin, caused a complete restoration of MeHg-mediated inhibition of CYP1A1 activity, induced by TCDD. In addition, transfection of HepG2 cells with siRNA targeting the human HO-1 gene reversed the MeHg-mediated inhibition of TCDD-induced CYP1A1. In conclusion, this study demonstrated that MeHg inhibited the TCDD-mediated induction of CYP1A1 through a posttranslational mechanism and confirms the role of HO-1 in a MeHg-mediated effect.

Differential modulation of cytochrome P450 1a1 by arsenite in vivo and in vitro in C57BL/6 mice.

Heavy metals, typified by arsenite (As(III)), have been implicated in altering the carcinogenicity of aryl hydrocarbon receptor (AhR) ligands, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), by modulating the induction of the Cyp1a1 enzyme, but the mechanism remains unresolved. In this study, the effects of As(III) on Cyp1a1 expression and activity were investigated in C57BL/6 mouse livers and isolated hepatocytes. For this purpose, C57BL/6 mice were injected intraperitoneally with As(III) (12.5 mg/kg) in the absence and presence of TCDD (15 µg/kg) for 6 and 24 h. Furthermore, isolated hepatocytes from C57BL/6 mice were treated with As(III) (1, 5, and 10 µM) in the absence and presence of TCDD (1 nM) for 3, 6, 12, and 24 h. At the in vivo level, As(III) decreased the TCDD-mediated induction of Cyp1a1 mRNA at 6h while potentiating its mRNA, protein, and catalytic activity levels at 24 h. At the in vitro level, As(III) decreased the TCDD-mediated induction of Cyp1a1 mRNA in a concentration- and time-dependent manner. Moreover, As(III) decreased the TCDD-mediated induction of Cyp1a1 protein and catalytic activity levels at 24 h. Interestingly, As(III) increased the serum hemoglobin (Hb) levels in animals treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the nuclear accumulation of AhR and AhR-dependent luciferase activity. Furthermore, Hb potentiated the TCDD-induced AhR-dependent luciferase activity. Importantly, when isolated hepatocytes were treated for 5h with As(III) in the presence of TCDD and the medium was then replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. Taken together, these results demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by As(III) in C57BL/6 mouse livers and isolated hepatocytes. Thus, this study implicates Hb as an in vivo-specific modulator.

Effect of mercury on aryl hydrocarbon receptor-regulated genes in the extrahepatic tissues of C57BL/6 mice.

The individual toxic effects of aryl hydrocarbon receptors (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or heavy metals typified by mercury (Hg(2+)) has been previously demonstrated. However, little is known about the combined toxic effects of TCDD and Hg(2+)in vivo. Therefore, we examined the effect of exposure to Hg(2+) (2.5mg/kg) in the absence and presence of TCDD (15 µg/kg) on the AhR-regulated genes using C57Bl/6 mice. Hg(2+) alone did not affect kidney, lung, or heart Cyp1a1/1a2/1b1 mRNA levels. On the contrary, Hg(2+) alone significantly induced kidney Cyp1a1/1a2/1b1 and lung Cyp1b1 protein and catalytic activities. Hg(2+) also induced Nqo1, Gsta1, and HO-1 at the mRNA, protein, and activity levels in the kidney and heart but not in the lung. Upon co-exposure to Hg(2+) and TCDD, Hg(2+) significantly potentiated the TCDD-mediated induction of kidney and lung Cyp1a1/1a2/1b1 mRNA levels, while it decreased their kidney protein and catalytic activity and it increased their lung protein. In addition, Hg(2+) potentiated the TCDD-mediated induction of Nqo1, Gsta1, and HO-1 at mRNA, protein and activity levels in all tissues. The present study demonstrates that Hg(2+) modulates the constitutive and TCDD-induced AhR-regulated genes in a time-, tissue- and, AhR-regulated enzyme genes manner.

Transcriptional modulation of the NAD(P)H:quinone oxidoreductase 1 by mercury in human hepatoma HepG2 cells.

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones plays a critical role in cancer prevention. Heavy metals such as mercury (Hg(2+)) alter the carcinogenicity of aryl hydrocarbon receptor ligands, mainly by modifying various xenobiotic-metabolizing enzymes such as NQO1. Therefore, we examined the effect of Hg(2+) on the expression of NQO1 in human hepatoma HepG2 cells. For this purpose HepG2 cells were incubated with various concentrations of Hg(2+) (2.5, 5, and 10µM) in the presence and absence of two NQO1 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL), as bifunctional and monofunctional inducers, respectively. Analysis of the time-dependent effect of Hg(2+) revealed that Hg(2+) increased the expression of NQO1 mRNA in a time-dependent manner. In addition, Hg(2+) increased NQO1 at the mRNA, protein, and activity levels in the presence and absence of both NQO1 inducers, TCDD and SUL, which coincided with increased nuclear accumulation of Nrf2 protein. Investigating the effect of Hg(2+) at the transcriptional level revealed that Hg(2+) significantly induced the antioxidant-responsive element-dependent luciferase reporter gene expression in the absence and the presence of both NQO1 inducers. NQO1 mRNA and protein decay experiments revealed a lack of posttranscriptional and posttranslational mechanisms. Transfecting HepG2 cells with siRNA for Nrf2 significantly decreased the Hg(2+)-mediated induction of NQO1 mRNA and catalytic activity by approximately 90%. In conclusion, we demonstrated that Hg(2+) regulates the expression of the NQO1 gene through a transcriptional mechanism in human hepatoma HepG2 cells. In addition, Nrf2 is involved in the modulation of NQO1 by Hg(2+).

Mercury modulates the CYP1A1 at transcriptional and posttranslational levels in human hepatoma HepG2 cells.

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and metals, such as mercury (Hg(2+)), are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of the carcinogen-activating enzyme cytochrome P450 1A1 (CYP1A1). Therefore, we examined the effect of co-exposure to Hg(2+) and TCDD on the expression of the CYP1A1 in HepG2 cells. Our results showed that Hg(2+) significantly inhibited the TCDD-mediated induction of CYP1A1 at the mRNA, protein, and catalytic activity levels. At the transcriptional level, co-exposure to Hg(2+) and TCDD significantly decreased the TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Moreover, Hg(2+) did not affect CYP1A1 mRNA stability, while decreasing its protein half-life, suggesting the involvement of a posttranslational mechanism. Importantly, Hg(2+) increased the expression of heme oxygenase-1 (HO-1), a rate limiting enzyme in heme degradation, which coincided with further decrease in the CYP1A1 catalytic activity levels. Upon using a competitive HO-1 inhibitor, tin mesoporphyrin, heme precursor, hemin, or transfecting the HepG2 cells with siRNA for HO-1 there was a partial restoration of the inhibition of TCDD-mediated induction of CYP1A1 catalytic activity. In conclusion, we demonstrate that Hg(2+) down-regulates the expression of CYP1A1 at the transcriptional and posttranslational levels in HepG2 cells. In addition, HO-1 is involved in the modulation of CYP1A1 at the posttranslational level.