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It has been shown by previous studies that mandibular growth can be enhanced by the systemic administration of recombinant growth hormone (rGH) and/or local application of therapeutic low intensity pulsed ultrasound (LIPUS). The purpose of this study was to determine if local injection of rGH and application of LIPUS to the temporomandibular joint (TMJ) would synergistically enhance mandibular growth. In an animal study, the effect of rGH, LIPUS, and combination of rGH and LIPUS on male Sprague-Dawley rats was observed. Mandibular growth was evaluated by measuring total hemimandibular and condylar bone volume and bone surface area as well as condylar bone mineral density (BMD) after 21 days on dissected rats' mandibles using micro-computed tomography (MicroCT). The expression of c-jun mRNA extracted from the liver of each of these rats was also quantified by real-time polymerase chain reaction to evaluate possible systemic effect of local rGH administration. Significant growth stimulation was observed in the mandibular and condylar bone of the animals treated with rGH, LIPUS, and rGH/LIPUS combined when compared with the control group. Bone volume, surface area, condylar bone mineral density, and c-jun expression were also compared between the treatment groups and the control in the liver. The results suggest that mandibular growth may be enhanced by injection of rGH or LIPUS application. The current study although showed synergetic effect of rGH and LIPUS application in increasing mandibular condylar head length, there was no significant changes in mandibular bone volume using both treatments together when compared to the two individual treatments. Moreover, combined rGH and LIPUS decreased condylar bone mineral density than each treatment separately. Future research could be directed to investigate the effects of different rGH doses and/or different LIPUS exposures parameters on lower jaw growth., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previous reports have proposed a cross-talk between the nuclear factor erythroid-2 p45-related factor-2 (Nrf2)/antioxidant response element (ARE) and the aryl hydrocarbon receptor (AhR)/xenobiotic response element (XRE) signaling pathways. Therefore, the aim of the current study was to examine the level of phase I, phase II drug metabolizing enzymes (DMEs), and phase III transporters and their related transcription factors in the Nrf2 knockout model. Our results showed that phase II DMEs that are under the control of Nrf2 typified by NAD(P)H: quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase (Gst) were significantly lower at the mRNA, protein, and catalytic activity levels in the livers of Nrf2 knockout mice compared to wild type. Furthermore, phase I cytochrome P450s (CYPs), Cyp1, and Cyp2b10 at mRNA, protein, and catalytic activity levels were significantly lower in the livers of Nrf2 knockout mice. Interestingly, our results showed that the transcription factors AhR, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) at mRNA, and protein expression levels were significantly lower in the livers of Nrf2 knockout mice compared to wild type. Importantly, phase III drug transporters mRNA levels of the multiple drug resistance associated proteins (Mrp2 and Mrp3), and solute carrier organic anion transporters (Slco1a6 and Slco2b1) were significantly lower in the liver of Nrf2 knockout mice. Co-activators, Ncoa1, Ncoa2, and Ncoa3 mRNA levels were not altered while co-repressors, Ncor1 and Ncor2 were significantly lower in the livers of Nrf2 knockout mice. In conclusion, knockout of Nrf2 causes disruption to the coordination of phase I, phase II drug DMEs, and phase III drug transporters through altering the transcription factors controlling them., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Recent studies demonstrated the carcinogenicity and the mutagenicity of vanadium compounds. In addition, vanadium (V(5+)) was found to enhance the effects of other genotoxic agents. However, the mechanism by which V(5+) induce toxicity remain unknown. In the current study we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in human hepatoma HepG2 cells. Therefore, HepG2 cells were treated with increasing concentrations of V(5+) in the presence of two NQO1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V(5+) inhibited the TCDD- and SUL-mediated induction of NQO1 at mRNA, protein and activity levels. Investigating the effect of V(5+) at transcriptional levels revealed that V(5+) significantly inhibited the TCDD- and SUL-mediated induction of antioxidant responsive element (ARE)-dependent luciferase reporter gene expression. In addition, V(5+) was able to decrease the TCDD- and SUL-induced nuclear accumulation of nuclear factor erythroid 2-related factor-2 (Nrf2) without affecting Nrf2 mRNA or protein levels. Looking at the post-transcriptional level, V(5+) did not affect NQO1 mRNA stability, thus eliminating the possible role of V(5+) in decreasing NQO1 mRNA levels through this mechanism. In contrast, at post-translational level, V(5+) was able to significantly decrease NQO1 protein half-life. The present study demonstrates for the first time that V(5+) down-regulates NQO1 at the transcriptional and post-translational levels in the human hepatoma HepG2 cells via AhR- and Nrf2-dependent mechanisms., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and beta-naphthoflavone (BNF), for 24 and 48h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Cardiovascular diseases (CVDs) remain the leading cause of death in the developed countries. Taking into account the mounting evidence about the role of cytochrome P450 (CYP) enzymes in cardiovascular physiology, CYP polymorphisms can be considered one of the major determinants of individual susceptibility to CVDs. One of the important physiological roles of CYP enzymes is the metabolism of arachidonic acid. CYP epoxygenases such as CYP1A2, CYP2C, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which generally possess vasodilating, anti-inflammatory, anti-apoptotic, anti-thrombotic, natriuretic, and cardioprotective effects. Therefore, genetic polymorphisms causing lower activity of these enzymes are generally associated with an increased risk of several CVDs such as hypertension and coronary artery disease. EETs are further metabolized by soluble epoxide hydrolase (sEH) to the less biologically active dihydroxyeicosatrienoic acids (DHETs). Therefore, sEH polymorphism has also been shown to affect arachidonic acid metabolism and to be associated with CVDs. On the other hand, CYP omega-hydroxylases such as CYP4A11 and CYP4F2 metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) which has both vasoconstricting and natriuretic effects. Genetic polymorphisms causing lower activity of these enzymes are generally associated with higher risk of hypertension. Nevertheless, some studies have denied the association between polymorphisms in the arachidonic acid pathway and CVDs. Therefore, more research is needed to confirm this association and to better understand the pathophysiologic mechanisms behind it., (2010 Elsevier Inc. All rights reserved.)

NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 microM), Cd2+ (5 microM), and Cr6+ (25 microM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway.

Co-contamination with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs) is a common environmental problem with multiple biological consequences. In this study, we tested in human hepatoma HepG2 cells the potential effects of three prominent environmental heavy metals, mercury (Hg(2+)), lead (Pb(2+)), and copper (Cu(2+)), on the induction of cytochrome P450 1A1 (CYP1A1) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent PAH. Our results show that TCDD in the absence and presence of heavy metals did not significantly affect HepG2 cell viability using MTT and LDH leakage assays. Exposure of HepG2 cells with either Hg(2+) or Pb(2+) significantly decreased, whereas Cu(2+) potentiated the CYP1A1 induction mediated by TCDD at the activity levels. In a manner similar to CYP1A1 activity, both Hg(2+) and Pb(2+) significantly down-regulated, while Cu(2+) up-regulated, the induction of CYP1A1 protein mediated by TCDD, suggesting that the modulations of CYP1A1 by heavy metals are mediated at least in part at the translational level. Based on these results, exposure to metal/PAH mixtures would differentially modulate PAHs-mediated carcinogenicity.

Cardiomyocytes are a valuable tool for studying the drug metabolizing enzymes in the heart. However, isolated cardiomyocytes are rather fragile and difficult to isolate. Therefore, there is an urgent need for an in vitro cell line model. The H9c2 cells are commonly used as an in vitro model for studying the cellular mechanisms and signaling pathways involved in drug-induced cardiotoxicity. These cells maintain many molecular markers of cardiomyocytes and show morphological characteristics of immature embryonic cardiomyocytes. Therefore, in the present study we examined the expression and inducibility of CYP1A1 in the H9c2 rat cardiomyoblast cells. Our results showed that treatment of H9c2 cells with the CYP1A1 inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 at mRNA, protein, and activity levels in a concentration-dependent manner. The RNA synthesis inhibitor, actinomycin D, completely blocked the CYP1A1 mRNA induction by TCDD, indicating the requirement of de novo RNA synthesis through transcriptional activation. In conclusion, we demonstrated for the first time the constitutive expression and inducibility of CYP1A1 in H9c2 cells. Therefore, this cell line offers a unique in vitro model to study the role of CYP1A1 in the pathogenesis of various cardiovascular diseases.

Maltol is used extensively as a flavor-enhancing agent, food preservative, antioxidant, and also in cosmetic and pharmaceutical formulations. However, a number of studies have shown that maltol may induce carcinogenicity and toxicity but the mechanisms involved remain unknown. Therefore, we examined the ability of maltol to induce the cytochrome P450 1a1 (Cyp1a1), an enzyme known to play an important role in the chemical activation of xenobiotics to carcinogenic derivatives. Our results showed that treatment of Hepa 1c1c7 cells with maltol significantly induced Cyp1a1 at mRNA, protein, and activity levels in a concentration-dependent manner. The RNA synthesis inhibitor, actinomycin D, completely blocked the Cyp1a1 mRNA induction by maltol, indicating a requirement of de novo RNA synthesis through transcriptional activation. In addition, maltol induced aryl hydrocarbon receptor (AhR)-dependent luciferase reporter gene expression in stably transfected H1L1.1c2 cells, suggesting an AhR-dependent mechanism. This is the first demonstration that the food flavoring agent, maltol, can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which maltol promotes carcinogenicity and toxicity.

Cardiovascular disease (CVD) is a human health crisis that remains the leading cause of death worldwide. The cytochrome P450 (CYP) class of enzymes are key metabolizers of both xenobiotics and endobiotics. Many CYP enzyme families have been identified in the heart, endothelium and smooth muscle of blood vessels. Furthermore, mounting evidence points to the role of endogenous CYP metabolites, such as epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), prostacyclin (PGI(2)), aldosterone, and sex hormones, in the maintenance of cardiovascular health. Emerging science and the development of genetic screening have provided us with information on the differences in CYP expression among populations and groups of individuals. With this information, a link between CYP expression and activity and CVD, such as hypertension, coronary artery disease (CAD), myocardial infarction, heart failure, stroke, and cardiomyopathy and arrhythmias, has been established. In fact many currently used therapeutic modalities in CVD owe their therapeutic efficacy to their effect on CYP metabolites. Thus, the evidence for the involvement of CYP in CVD is numerous. Concentrating on treatment modalities that target the CYP pathway makes ethical sense for the affected individuals and decreases the socioeconomic burden of this disease. However, more research is needed to allow the integration of this information into a clinical setting.