Dr.Mohamed welcomes you to his website!

Our main long-term goal is always to striving product world class health research

Anders LC, Lang AL, Anwar-Mohamed A, Douglas AN, Bushau AM, Falkner KC, Hill BG, Warner NL, Arteel GE, Cave M, McClain CJ, Beier JI. Vinyl chloride (VC) is a ubiquitous environmental contaminant for which human risk is incompletely understood. We have previously reported that high occupational exposure to VC directly caused liver damage in humans. However, whether VC may also potentiate liver injury from other causes is not known. C57Bl/6J mice were administered chloroethanol (CE), a major metabolite of VC, and lipopolysaccharide (LPS) 24 h after CE. Samples were harvested for determination of liver damage, inflammation, and changes in carbohydrate and lipid metabolism. In mice, CE exposure alone caused no detectable liver damage. LPS exposure caused inflammatory liver damage, oxidative stress, lipid accumulation, and glycogen depletion; the effect of all of these variables was potentiated by CE pre-exposure. In vitro experiments suggest that VC metabolite chloroacetaldehyde (CAA) directly damages mitochondria, which may explain the sensitization effect observed in vivo Moreover, co-exposure of cells to CAA and TNFα caused increased cell death, supporting the hypothesis of sensitization by VC metabolites. Taken together, these data demonstrate that exposure to VC/metabolites at levels that are not overtly hepatotoxic can potentiate liver injury caused by another hepatotoxicant. This serves as proof-of-concept that VC hepatotoxicity may be modified by an additional metabolic stress such as endotoxemia, which commonly occurs in acute (eg, sepsis) and chronic (eg, NAFLD) diseases. Toxicol Sci. 2016 Jun;151(2):312-23. doi: 10.1093/toxsci/kfw045. Epub 2016 Mar 8. DOI: 10.1093/toxsci/kfw045 PMCID: PMC4880135 PMID: 26962056

source: www.ncbi.nlm.nih.gov | europepmc.org | scholar.google.ca | pubag.nal.usda.gov | onlinelibrary.wiley.com | tandfonline.com

Anwar-Mohamed A, El-Sherbeni AA, Hamdy DA, Korashy HM, Brocks DR, El-Kadi AO. Ketoconazole (KTZ) has 2 chiral centers with the therapeutically active form being a racemic mixture of 2 cis-enantiomers, namely, (2R,4S)-(+)-KTZ and (2S,4R)-(-)-KTZ. The aims of the present study were to examine the effects of (+)-KTZ, (-)-KTZ, and (±)-KTZ on aryl hydrocarbon receptor activation and subsequently CYP1A1 induction in both human HepG2 and murine Hepa1c1c7 hepatoma cells, and to further test their inhibitory effect using recombinant human and mouse CYP1A1 enzymes. Our results demonstrated that (+)-KTZ induced human CYP1A1 more than (-)-KTZ, whereas on the other hand (-)-KTZ induced murine Cyp1a1 more than (+)-KTZ at the mRNA, and activity levels. Human CYP1A1 showed higher affinity to 7ER compared with murine Cyp1a1 (Km values 13.29 nM for human vs. 168.1 nM for murine). The intrinsic clearance values for human and murine CYP1A1 were 194.1 and 87.6 μL/pmol P450/min, respectively, whereas, Vmax values were 2.58 and 14.73 pmol/pmol P450/min, respectively. (+)-KTZ and (-)-KTZ directly inhibited CYP1A1 activity by noncompetitive mechanism. The affinity of (-)-KTZ to interact with human CYP1A1 and murine Cyp1a1 was significantly different from (+)-KTZ, as the Ki values for human CYP1A1 and murine Cyp1a1 were 199.4 and 413.7 nM, respectively, for (+)-KTZ, and 269.3 and 230.8 nM, respectively, for (-)-KTZ. J Pharm Sci. 2016 Mar;105(3):1318-26. doi: 10.1016/j.xphs.2015.12.009. Epub 2016 Feb 6. DOI: 10.1016/j.xphs.2015.12.009 PMID: 26886340 [Indexed for MEDLINE]
Douglas DN, Pu CH, Lewis JT, Bhat R, Anwar-Mohamed A, Logan M, Lund G, Addison WR, Lehner R, Kneteman NM. Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and β-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced β-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced β-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis. J Biol Chem. 2016 Jan 22;291(4):1974-90. doi: 10.1074/jbc.M115.674861. Epub 2015 Dec 1. DOI: 10.1074/jbc.M115.674861 PMCID: PMC4722472 PMID: 26627833 [Indexed for MEDLINE]
Anwar-Mohamed A, Barakat KH, Bhat R, Noskov SY, Tyrrell DL, Tuszynski JA, Houghton M. Acquired cardiac long QT syndrome (LQTS) is a frequent drug-induced toxic event that is often caused through blocking of the human ether-á-go-go-related (hERG) K(+) ion channel. This has led to the removal of several major drugs post-approval and is a frequent cause of termination of clinical trials. We report here a computational atomistic model derived using long molecular dynamics that allows sensitive prediction of hERG blockage. It identified drug-mediated hERG blocking activity of a test panel of 18 compounds with high sensitivity and specificity and was experimentally validated using hERG binding assays and patch clamp electrophysiological assays. The model discriminates between potent, weak, and non-hERG blockers and is superior to previous computational methods. This computational model serves as a powerful new tool to predict hERG blocking thus rendering drug development safer and more efficient. As an example, we show that a drug that was halted recently in clinical development because of severe cardiotoxicity is a potent inhibitor of hERG in two different biological assays which could have been predicted using our new computational model. Toxicol Lett. 2014 Nov 4;230(3):382-92. doi: 10.1016/j.toxlet.2014.08.007. Epub 2014 Aug 13. DOI: 10.1016/j.toxlet.2014.08.007 PMID: 25127758 [Indexed for MEDLINE]
Anwar-Mohamed A, Elshenawy OH, El-Sherbeni AA, Abdelrady M, El-Kadi AO. The toxic effects of arsenic on the whole brain, as well as the discrete regions, has been previously reported for mice. We investigated the effects of acute arsenite (As(III)) on brain levels of arachidonic acid (AA) and its associated metabolites generated through cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) pathways. Our results demonstrated that acute As(III) treatment (12.5 mg·(kg body mass)(-1)) decreases cytosolic phospholipase A2 (cPLA2) with a subsequent decrease in its catalytic activity and brain AA levels. In addition, As(III) differentially altered CYP epoxygenases and CYP ω-hydroxylases, but it did not affect brain Ephx2 mRNA or sEH catalytic activity levels. As(III)-mediated effects on Cyps caused an increase in brain 5,6-epoxyeicosatrienoic acid (5,6-EET) and 16/17-hydroxyeicosatetreinoic acid (16/17-HETE) levels, and a decrease in 18- and 20-HETE levels. Furthermore, As(III) increased cyclooxygenase-2 (COX-2) mRNA while decreasing prostaglandins F2α (PGF2α) and PGJ2. As(III) also increased brain 5-lipoxygenase (5-LOX) and 15-LOX mRNA, but decreased 12-LOX mRNA. These changes in LOX mRNA were associated with a decrease in 8/12-HETE levels only. In conclusion, this is the first demonstration that As(III) decreases AA levels coinciding with alterations to EET, HETE, and PG levels, which affects brain development and neurochemistry. Can J Physiol Pharmacol. 2014 Aug;92(8):693-702. doi: 10.1139/cjpp-2014-0136. DOI: 10.1139/cjpp-2014-0136 PMID: 25065748 [Indexed for MEDLINE]
Barakat KH, Anwar-Mohamed A, Tuszynski JA, Robins MJ, Tyrrell DL, Houghton M. Many direct-acting antiviral agents (DAAs) that selectively block hepatitis C virus (HCV) replication are currently under development. Among these agents is Daclatasvir, a first-in-class inhibitor targeting the NS5A viral protein. Although Daclatasvir is the most potent HCV antiviral molecule yet developed, its binding location and mode of binding remain unknown. The drug exhibits a low barrier to resistance mutations, particularly in genotype 1 viruses, but its efficacy against other genotypes is unclear. Using state-of-the-art modeling techniques combined with the massive computational power of Blue Gene/Q, we identified the atomic interactions of Daclatasvir within NS5A for different HCV genotypes and for several reported resistant mutations. The proposed model is the first to reveal the detailed binding mode of Daclatasvir. It also provides a tool to facilitate design of second generation drugs, which may confer less resistance and/or broader activity against HCV. J Chem Inf Model. 2015 Feb 23;55(2):362-73. doi: 10.1021/ci400631n. Epub 2014 Apr 14. DOI: 10.1021/ci400631n PMID: 24730573 [Indexed for MEDLINE]

Hamann I, Petroll K, Hou X, Anwar-Mohamed A, El-Kadi AO, Klotz LO.

 Epidemiological studies have indicated a relationship between the prevalence of diabetes and exposure to arsenic. Mechanisms by which arsenic may cause this diabetogenic effect are largely unknown. The phosphoinositide 3'-kinase (PI3K)/Akt signaling pathway plays an important role in insulin signaling by controlling glucose metabolism, in part through regulating the activity of FoxO transcription factors. The present study aimed at investigating the effect of short and long-term exposure to arsenite on insulin signaling in HepG2 human hepatoma cells, the role of PI3K/Akt signaling therein and the modulation of target genes controlled by insulin. Exposure of cells to arsenite for 24 h rendered cells less responsive toward stimulation of Akt by insulin. At the same time, short-term exposure to arsenite induced a concentration-dependent increase in phosphorylation of Akt at Ser-473, followed by phosphorylation of FoxO proteins at sites known to be phosphorylated by Akt. Phosphorylation of FoxOs was prevented by wortmannin, pointing to the involvement of PI3K. Arsenite exposure resulted in attenuation of FoxO DNA binding and in nuclear exclusion of FoxO1a-EGFP. A 24-h exposure of HepG2 cells to submicromolar concentrations of arsenite resulted in downregulation of glucose 6-phosphatase (G6Pase) and selenoprotein P (SelP) mRNA levels. Curiously, arsenite had a dual effect on SelP protein levels, inducing a small increase in the nanomolar and a distinct decrease in the micromolar concentration range. Interestingly, arsenite-induced long-term effects on G6Pase and SelP mRNA or SelP protein levels were not blocked by the PI3K inhibitor, wortmannin. In conclusion, arsenite perturbs cellular signaling pathways involved in fuel metabolism: it impairs cellular responsiveness toward insulin, while at the same time stimulating insulin-like signaling to attenuate the expression of genes involved in glucose metabolism and the release of the hepatokine SelP, which is known to modulate peripheral insulin sensitivity.

 

Biometals. 2014 Apr;27(2):317-32. doi: 10.1007/s10534-014-9714-y. Epub 2014 Feb 18.

 

DOI: 10.1007/s10534-014-9714-y

PMID: 24535192 [Indexed for MEDLINE]

 

 

Anwar-Mohamed A, Elshenawy OH, Soshilov AA, Denison MS, Chris Le X, Klotz LO, El-Kadi AO.

 

Activation of the aryl hydrocarbon receptor (AhR) ultimately leads to the induction of the carcinogen-activating enzyme cytochrome P450 1A1 (CYP1A1), and activation of the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in addition to the AhR pathway induces the expression of the NADP(H):quinone oxidoreductase (NQO1). Therefore, the aim of this study was to examine the effect of As(III) pentavalent metabolites, MMA(V), DMA(V), and TMA(V), on AhR and Nrf2 activation and on the expression of their prototypical downstream targets CYP1A1 and NQO1, respectively. Our results showed that treatment of HepG2 cells with MMA(V), DMA(V), or TMA(V) in the absence and presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin or sulforaphane significantly induced both CYP1A1 and NQO1 at the mRNA, protein, and catalytic activity levels. Furthermore, these metabolites increased the AhR-dependent XRE-driven and the Nrf2-dependent ARE-driven luciferase reporter activities, which coincided with increased nuclear accumulation of both transcription factors. However, none of these metabolites were shown to be AhR ligands. The induction of CYP1A1 by these metabolites seems to be ligand-independent, possibly through a decrease in HSP90 protein expression levels. The metabolites also increased ROS production, which was significantly higher than that produced by As(III). Upon knockdown of AhR and Nrf2 the MMA(V)-, DMA(V)-, and TMA(V)-mediated induction of both CYP1A1 and NQO1 proteins was significantly decreased. In conclusion, this study demonstrates for the first time that methylated pentavalent arsenic metabolites are bifunctional inducers, as they increase CYP1A1 by activating the AhR/XRE signaling pathway and they increase NQO1 by activating the Nrf2/ARE signaling pathway in addition to the AhR/XRE pathway.

 

Free Radic Biol Med. 2014 Feb;67:171-87. doi: 10.1016/j.freeradbiomed.2013.10.810. Epub 2013 Oct 24.

 

DOI: 10.1016/j.freeradbiomed.2013.10.810

PMID: 24161444 [Indexed for MEDLINE]

Gordon M(1), El-Kalla M, Zhao Y, Fiteih Y, Law J, Volodko N, Anwar-Mohamed A,

El-Kadi AO, Liu L, Odenbach J, Thiesen A, Onyskiw C, Ghazaleh HA, Park J, Lee SB,

Yu VC, Fernandez-Patron C, Alexander RT, Wine E, Baksh S.

 

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a(+/-) , Rassf1a(-/-) and an intestinal epithelial cell specific knockout mouse (Rassf1a (IEC-KO) ) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a(-/-) mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a(-/-) background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a(-/-) mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.

 

PLoS One. 2013 Oct 16;8(10):e75483. doi: 10.1371/journal.pone.0075483. eCollection 2013.

 

DOI: 10.1371/journal.pone.0075483

PMCID: PMC3797720

PMID: 24146755 [Indexed for MEDLINE]

 

Elshenawy OH, Anwar-Mohamed A, El-Kadi AO.

 

Arachidonic acid (AA) is metabolized by enzymes of the cytochrome P450 (CYP) 4A and CYP4F subfamilies to 20- hydroxyeicosatetraeonic acid (20-HETE), which plays an important role in the cardiovascular system. In the current work, we reviewed the formation of 20-HETE in different species by different CYPs; 20-HETE metabolism by cyclooxygenases (COXs) and different isomerases; and the current available inducers and inhibitors of 20-HETE formation in addition to its agonists and antagonists. Moreover we reviewed the negative role of 20-HETE in cardiac hypertrophy, cardiotoxicity, diabetic cardiomyopathy, and in ischemia/reperfusion (I/R) injury. Lastly, we reviewed the role of 20-HETE in different hypertension models such as the renin/angiotensin II model, Goldblatt model, spontaneously hypertensive rat model, androgen-induced model, slat- and deoxycorticosterone acetate (DOCA)-salt-induced models, and high fat diet model. 20-HETE can affect pro- and anti-hypertensive mechanisms dependent upon where, when, and by which isoform it has been produced. In contrast to hypertension we also reviewed the role of 20-HETE in endotoxin-induced hypotension and the natriuretic effects of 20-HETE. Based on the recent studies, 20-HETE production and/or action might be a therapeutic target to protect against the initiation and progression of cardiovascular diseases.

 

Curr Drug Metab. 2013 Jul;14(6):706-19.

 

PMID: 23701160 [Indexed for MEDLINE]

 

Amara IE, Anwar-Mohamed A, El-Kadi AO.

 

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.

 

Toxicol Lett. 2013 Jun 7;219(3):239-47. doi: 10.1016/j.toxlet.2013.03.018. Epub 2013 Mar 26.

 

DOI: 10.1016/j.toxlet.2013.03.018

PMID: 23541843 [Indexed for MEDLINE]

 

Abdelhamid G, Amara IE, Anwar-Mohamed A, El-Kadi AO.

 

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.

 

Arch Toxicol. 2013 Aug;87(8):1531-43. doi: 10.1007/s00204-013-1023-7. Epub 2013 Feb 20.

 

DOI: 10.1007/s00204-013-1023-7

PMID: 23423713 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Sherbeni A, Kim SH, Elshenawy OH, Althurwi HN, Zordoky BN, El-Kadi AO.

 

Arsenic (As(III)) toxicity has received increasing attention as human exposure to arsenic is associated with pulmonary, hepatic and renal toxicities. Therefore, in the present study, we investigated the effect of acute As(III) treatment on pulmonary, hepatic and renal cytochrome (CYP) P450-mediated arachidonic acid metabolism. 2. Our results demonstrated that acute As(III) treatment (12.5 mg/kg) altered CYP epoxygenases, CYP ω-hydroxylases and EPHX2 mRNA levels that were isozyme and tissue specific. 3. Furthermore, As(III) increased the formation of epoxyeicosatrienoic acids (EETs) in the kidney without affecting their levels in the lung or liver. In addition, acute As(III) treatment increased dihydroxyeicosatrienoic acid (DHETs) formation in the lung, while it did not affect liver DHETs formation and decreased kidney DHETs formation. 4. As(III) also increased total epoxygenases activity in the lung while it decreased its levels in the kidney and had no effect on the liver. Furthermore, As(III) increased 20-hydroxyeicosatetraenoic acid formation in the liver while it decreased its formation in the kidney. 5. Lastly, As(III) increased soluble epoxide hydrolase activity in the lung, while it decreased its levels in the kidney and had no effect on the liver. In conclusion, this is the first demonstration that As(III) alters arachidonic acid metabolism in a tissue specific manner.

 

Xenobiotica. 2013 Aug;43(8):719-29. doi: 10.3109/00498254.2012.754113. Epub 2013 Feb 15.

 

DOI: 10.3109/00498254.2012.754113

PMID: 23409951 [Indexed for MEDLINE]

 

Anwar-Mohamed A, Abdelhamid G, Amara IE, El-Kadi AO.

 

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.

 

Free Radic Biol Med. 2013 May;58:52-63. doi: 10.1016/j.freeradbiomed.2013.01.012. Epub 2013 Jan 29.

 

DOI: 10.1016/j.freeradbiomed.2013.01.012

PMID: 23369756 [Indexed for MEDLINE]

 

Elshenawy OH, Anwar-Mohamed A, Abdelhamid G, El-Kadi AO.

 

HL-1 cells are currently the only cells that spontaneously contract while maintaining a differentiated cardiac phenotype. Thus, our objective was to examine murine HL-1 cells as a new in vitro model to study drug metabolizing enzymes. We examined the expression of cytochrome P450s (Cyps), phase II enzymes, and nuclear receptors and compared their levels to mice hearts. Our results demonstrated that except for Cyp4a12 and Cyp4a14 all Cyps, phase II enzymes: glutathione-S-transferases (Gsts), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase (Nqo1), nuclear receptors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator activated receptor (PPAR-alpha) were all constitutively expressed in HL-1 cells. Cyp2b19, Cyp2c29, Cyp2c38, Cyp2c40, and Cyp4f16 mRNA levels were higher in HL-1 cells compared to mice hearts. Cyp2b9, Cyp2c44, Cyp2j9, Cyp2j11, Cyp2j13, Cyp4f13, Cyp4f15 mRNA levels were expressed to the same extent to that of mice hearts. Cyp1a1, Cyp1a2, Cyp1b1, Cyp2b10, Cyp2d10, Cyp2d22, Cyp2e1, Cyp2j5, Cyp2j6, Cyp3a11, Cyp4a10, and Cyp4f18 mRNA levels were lower in HL-1 cells compared to mice hearts. Moreover, 3-methylcholanthrene induced Cyp1a1 while fenofibrate induced Cyp2j9 and Cyp4f13 mRNA levels in HL-1 cells. Examining the metabolism of arachidonic acid (AA) by HL-1 cells, our results demonstrated that HL-1 cells metabolize AA to epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and 20-hydroxyeicosatetraenoic acids. In conclusion, HL-1 cells provide a valuable in vitro model to study the role of Cyps and their associated AA metabolites in addition to phase II enzymes in cardiovascular disease states.

 

Vascul Pharmacol. 2013 Apr;58(4):326-33. doi: 10.1016/j.vph.2012.12.002. Epub 2012 Dec 23.

 

DOI: 10.1016/j.vph.2012.12.002

PMID: 23268359 [Indexed for MEDLINE]

 

Amara IE, Anwar-Mohamed A, Abdelhamid G, El-Kadi AO.

 

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.

 

Toxicol Appl Pharmacol. 2013 Feb 1;266(3):419-29. doi: 10.1016/j.taap.2012.11.027. Epub 2012 Dec 8.

 

DOI: 10.1016/j.taap.2012.11.027

PMID: 23231920 [Indexed for MEDLINE]

 

El-Sherbeni AA, Aboutabl ME, Zordoky BN, Anwar-Mohamed A, El-Kadi AO.

 

Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography-electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19- and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ω-hydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.

 

AAPS J. 2013 Jan;15(1):112-22. doi: 10.1208/s12248-012-9425-7. Epub 2012 Nov 10.

 

DOI: 10.1208/s12248-012-9425-7

PMCID: PMC3535098

PMID: 23139020 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Sherbeni AA, Kim SH, Althurwi HN, Zordoky BN, El-Kadi AO.

 

Acute arsenic (As(III)) exposure has been reported to cause cardiac toxicity, however this toxicity was never linked to the disturbance in cytochrome P450 (P450)-mediated arachidonic acid metabolism. Therefore, we investigated the effect of acute As(III) toxicity on the expression of P450 and soluble epoxide hydrolase (sEH) and their associated arachidonic acid metabolism in mice hearts. As(III) toxicity was induced by a single intraperitoneal injection of 12.5 mg/kg of As(III). Our results showed that As(III) treatment caused a significant induction of the cardiac hypertrophic markers in addition to Cyp1b1, Cyp2b, Cyp2c, Cyp4f, and sEH gene expression in mice hearts. Furthermore, As(III) increased sEH protein expression and activity in hearts with a consequent decrease in 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) formation. Whereas the formation of 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acids (DHETs) was significantly increased. As(III) also increased sEH mRNA and protein expression levels in addition to the hypertrophic markers which was reversed by knockdown of sEH in H9c2 cells. In conclusion, acute As(III) toxicity alters the expression of several P450s and sEH enzymes with a consequent decrease in the cardioprotective EETs which may represent a novel mechanism by which As(III) causes progressive cardiotoxicity. Furthermore, inhibiting sEH might represent a novel therapeutic approach to prevent As(III)-induced hypertrophy.

 

Xenobiotica. 2012 Dec;42(12):1235-47. doi: 10.3109/00498254.2012.693971. Epub 2012 Jun 9.

 

DOI: 10.3109/00498254.2012.693971

PMID: 22680237 [Indexed for MEDLINE]

 

Abdelhamid G, Amara IE, Anwar-Mohamed A, El-Kadi AO.

 

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.

 

J Appl Toxicol. 2013 Nov;33(11):1230-40. doi: 10.1002/jat.2774. Epub 2012 May 18.

 

DOI: 10.1002/jat.2774

PMID: 22611038 [Indexed for MEDLINE]

 

Amara IE, Anwar-Mohamed A, Abdelhamid G, El-Kadi AO.

 

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.

 

Food Chem Toxicol. 2012 Jul;50(7):2325-34. doi: 10.1016/j.fct.2012.04.028. Epub 2012 May 2.

 

DOI: 10.1016/j.fct.2012.04.028

PMID: 22579925 [Indexed for MEDLINE]

 

Anwar-Mohamed A, Abdelhamid G, Amara IE, El-Kadi AO.

 

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.

 

Arch Toxicol. 2012 Jun;86(6):897-910. doi: 10.1007/s00204-012-0855-x. Epub 2012 Apr 26.

 

DOI: 10.1007/s00204-012-0855-x

PMID: 22535251 [Indexed for MEDLINE]

 

Anwar-Mohamed A, Klotz LO, El-Kadi AO.

 

Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.

 

Drug Metab Dispos. 2012 Mar;40(3):504-14. doi: 10.1124/dmd.111.042564. Epub 2011 Dec 8.

 

DOI: 10.1124/dmd.111.042564

PMID: 22159698 [Indexed for MEDLINE]

Eckers A, Sauerbier E, Anwar-Mohamed A, Hamann I, Esser C, Schroeder P, El-Kadi AO, Klotz LO.

 

FHRE-Luc is a promoter reporter construct that is widely used to assess the activity of FoxO (forkhead box, class O) transcription factors. We here demonstrate that this promoter construct responds to exposure of HepG2 human hepatoma cells to known agonists of the aryl hydrocarbon receptor (AhR), 3-methylcholanthrene, benzo(a)pyrene, and 6-formylindolo[3,2-b]carbazole. However, FHRE-Luc activation did not coincide with FoxO DNA binding or changes in Akt-induced FoxO phosphorylation after treatment with AhR agonists. Testing FHRE-Luc deletion constructs and using AhR-deficient cells, we found that FHRE-Luc activation by AhR agonists is due to a functional xenobiotic-response element (XRE) spanning the backbone/insert border of the reporter plasmid. In conclusion, care must be taken when using FHRE-Luc to assess FoxO activity in response to stimuli that potentially interfere with xenobiotic signaling.

 

Arch Biochem Biophys. 2011 Dec 15;516(2):138-45. doi: 10.1016/j.abb.2011.10.006. Epub 2011 Oct 15.

 

DOI: 10.1016/j.abb.2011.10.006

PMID: 22019820 [Indexed for MEDLINE]

 

 Korashy HM, Anwar-Mohamed A, Soshilov AA, Denison MS, El-Kadi AO.

 

We have previously shown that the p38 MAPK inhibitor SB203580 (SB) significantly induced Cyp1a1 gene expression at the mRNA and activity levels, whereas it dramatically inhibited the induction of Cyp1a1 by TCDD in murine hepatoma Hepa 1c1c7 cells. However, the molecular mechanisms involved were not investigated yet. Therefore, the current study aims to examine the capacity of SB to induce the constitutive CYP1A1 gene expression in Hepa 1c1c7 and HepG2 cells and to explore the mechanisms involved. Our results showed that SB induced the Cyp1a1 mRNA, protein, and activity levels in a concentration-dependent manner in Hepa 1c1c7 cells. The increase in Cyp1a1 mRNA by SB was completely blocked by the transcriptional inhibitor, actinomycin D, implying that SB increased de novo RNA synthesis. In addition, the lack of Cyp1a1 induction by SB in mutant aryl hydrocarbon receptor (AhR)-deficient C12 cells and with cotreatment with the AhR antagonist, α-naphthoflavone, clearly suggests an AhR-dependent induction. This was further supported by the ability of SB to induce Cyp1a1 independent from its effect on MAPKs, and to bind to and activate AhR transformation and its subsequent binding to the xenobiotic responsive element (XRE). This is the first demonstration that the p38 MAPK inhibitor, SB can directly bind to and activate AhR-induced Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SB induces this enzyme.

 

Chem Res Toxicol. 2011 Sep 19;24(9):1540-8. doi: 10.1021/tx200141p. Epub 2011 Jul 27.

 

DOI: 10.1021/tx200141p

PMCID: PMC3263331

PMID: 21732638 [Indexed for MEDLINE]

 

Zordoky BN, Anwar-Mohamed A, Aboutabl ME, El-Kadi AO.

 

The use of doxorubicin (DOX) is limited by significant cardiotoxicity, nephrotoxicity, and hepatotoxicity. We have previously shown that DOX cardiotoxicity induces several cardiac cytochrome P450 (P450) enzymes with subsequent alteration in P450-mediated arachidonic acid metabolism. Therefore, in the current study, we investigated the effect of acute DOX toxicity on P450 expression and arachidonic acid metabolism in the kidney and liver of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection (15 mg/kg) of the drug. After 6 and 24 h, the kidneys and livers were harvested, and several P450 gene and protein expressions were determined by real-time polymerase chain reaction and Western blot analyses, respectively. Kidney and liver microsomal protein from control or DOX-treated rats was incubated with arachidonic acid, and its metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that acute DOX toxicity caused an induction of CYP1B1 and CYP4A enzymes and an inhibition of CYP2B1 and CYP2C11 in both the kidney and liver. CYP2E1 was induced and soluble epoxide hydrolase (sEH) was inhibited in the kidney only. In addition, DOX toxicity caused a significant increase in epoxyeicosatrienoic acids formation in the kidney and a significant increase in 20-hydroxyeicosatetraenoic acid formation in both the kidney and the liver. In conclusion, acute DOX toxicity alters the expression of several P450 and sEH enzymes in an organ-specific manner. These changes can be attributed to DOX-induced inflammation and resulted in altered P450-mediated arachidonic acid metabolism.

 

Drug Metab Dispos. 2011 Aug;39(8):1440-50. doi: 10.1124/dmd.111.039123. Epub 2011 May 13.

 

DOI: 10.1124/dmd.111.039123

PMID: 21571947 [Indexed for MEDLINE]

 

Anwar-Mohamed A, Degenhardt OS, El Gendy MA, Seubert JM, Kleeberger SR,

El-Kadi AO.

 

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.

 

Toxicol In Vitro. 2011 Jun;25(4):785-95. doi: 10.1016/j.tiv.2011.01.014. Epub 2011 Jan 31.

 

DOI: 10.1016/j.tiv.2011.01.014

PMID: 21281708 [Indexed for MEDLINE]

 

Amara IE, Anwar-Mohamed A, El-Kadi AO.

 

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.

 

Toxicol Lett. 2010 Dec 15;199(3):225-33. doi: 10.1016/j.toxlet.2010.09.003. Epub 2010 Sep 15.

 

DOI: 10.1016/j.toxlet.2010.09.003

PMID: 20837117 [Indexed for MEDLINE]

 

Abdelhamid G, Anwar-Mohamed A, Elmazar MM, El-Kadi AO.

 

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.

 

Toxicol In Vitro. 2010 Sep;24(6):1554-61. doi: 10.1016/j.tiv.2010.06.017. Epub 2010 Jul 3.

 

DOI: 10.1016/j.tiv.2010.06.017

PMID: 20599494 [Indexed for MEDLINE]

 

 

Al-Hallak KM, Azarmi S, Anwar-Mohamed A, Roa WH, Löbenberg R.

 

Local treatment of lung cancer using inhalable nanoparticles (NPs) is an emerging and promising treatment option. The aim of this study was to investigate the activation of alveolar macrophages by poly (isobutyl cyanoacrylate) (BIPCA) NPs and the consequences of this activation on H460 lung cancer cells. A methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to determine the primary cytotoxicity, that is, the immediate and direct cytotoxicity of doxorubicin (DOX)-loaded NPs on both cell lines. Macrophages were then treated using EC(50) concentrations of different treatments and co-cultured in a two-compartment system with H460 lung cancer cells. These treatments included DOX solution, blank NPs, and DOX-loaded NPs. The results showed that alveolar macrophages exposed to blank or DOX-loaded NPs showed cytotoxicity against cancer cells after 8 and 24h; this behavior was not expressed by naïve macrophages or macrophages treated with DOX solution. Sample analysis indicated that macrophages have the ability to release back fragments of NPs that were previously phagocytized. Further investigations showed that NPs can induce an increase in the excretion of Th1 cytokines namely, monocytes chemoattractant protein-1 (MCP-1), macrophages inflammatory protein (MIP-1), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). The Th1 cytokines released by the alveolar macrophages might explain the significant secondary cytotoxicity effect on H460 cancer cells. Secondary cytotoxicity mediated by macrophages might compliment the direct cytotoxic effect that NPs have on cancer cells.

 

Eur J Pharm Biopharm. 2010 Sep;76(1):112-9. doi: 10.1016/j.ejpb.2010.05.002. Epub 2010 May 7.

 

DOI: 10.1016/j.ejpb.2010.05.002

PMID: 20452423 [Indexed for MEDLINE]

 

Abdelhamid G, Anwar-Mohamed A, Badary OA, Moustafa AA, El-Kadi AO.

 

We recently demonstrated that V(5+) downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels in Hepa 1c1c7 cells through transcriptional mechanism. Therefore, it is important to investigate whether similar changes occur in humans. For this purpose, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of aryl hydrocarbon receptor (AhR)-regulated gene; cytochrome P450 1A1 (CYP1A1) at each step of the AhR signal transduction pathway in human hepatoma HepG2 cells. Our results show a significant reduction in TCDD-mediated induction of CYP1A1 mRNA, protein, and activity levels after V(5+) treatment in a dose-dependent manner. Investigating the effect of co-exposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Looking at the posttranscriptional level, V(5+) did not affect CYP1A1 mRNA stability, thus eliminating the possible role of V(5+) in modifying CYP1A1 gene expression through this mechanism. On the other hand, at the posttranslational level, V(5+) was able to significantly decrease CYP1A1 protein half-life contributing to the inconsistency between catalytic activity and transcriptional level. Importantly, we showed that V(5+) did not significantly alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V(5+) might have decreased CYP1A1 activity through affecting its heme content. This study demonstrates for the first time that V(5+) downregulates the expression of CYP1A1 at the transcriptional, posttranscriptional and posttranslational mechanisms in the human hepatoma HepG2 cells.

 

Cell Biol Toxicol. 2010 Oct;26(5):421-34. doi: 10.1007/s10565-010-9153-7. Epub 2010 Mar 11.

 

DOI: 10.1007/s10565-010-9153-7

PMID: 20221682 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Kadi AO.

 

Aryl hydrocarbon receptor (AhR) ligands, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and metals, typified by arsenite (As(III)), are environmental cocontaminants, and their molecular interaction may disrupt the coordinated regulation of the carcinogen activating enzyme cytochrome P450 1A1 (CYP1A1). Therefore, in this study we examined the effects of coexposure to As(III) and TCDD on the expression of CYP1A1 in HepG2 cells. Our results showed that As(III) caused a dose-dependent decrease in TCDD-mediated induction of CYP1A1 mRNA, protein, and catalytic activity levels. As(III) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression without altering CYP1A1 mRNA stability. In addition, As(III) increased heme oxygenase-1 (HO-1) mRNA, which coincided with a further decrease in the CYP1A1 catalytic activity levels. When a competitive HO-1 inhibitor, tin mesoporphyrin, was applied to HepG2 cells or the cells were transfected with siRNA for HO-1 there was a partial restoration of the inhibition of TCDD-mediated induction of CYP1A1 catalytic activity. Treatment of cells with heme or hemoglobin partially restored the As(III)-mediated inhibition of CYP1A1 catalytic activity. On the other hand, cobalt protoporphyrin increased HO-1 mRNA, with a concomitant decrease in CYP1A1 activity, without affecting CYP1A1 mRNA, which was reversed by HO-1 siRNA transfection. This study demonstrates that As(III) down-regulates CYP1A1 through transcriptional and posttranslational mechanisms. In addition, HO-1 is involved in the As(III)-mediated down-regulation of CYP1A1 at the catalytic activity level.

 

Free Radic Biol Med. 2010 May 15;48(10):1399-409. doi: 10.1016/j.freeradbiomed.2010.02.027. Epub 2010 Feb 25.

 

 

DOI: 10.1016/j.freeradbiomed.2010.02.027

PMID: 20188822 [Indexed for MEDLINE]

 

Anwar-Mohamed A, Zordoky BN, Aboutabl ME, El-Kadi AO.

 

Cytochrome P450 (CYP) generated cardioprotective metabolites, epoxyeicosatrienoic acids (EETs), and cardiotoxic metabolites, hydroxyeicosatetraenoic acids (HETEs) levels are determined by many factors, including the induction or repression of the CYP enzymes, responsible for their formation. Therefore, we examined the effect of acute inflammation on the expression of CYP epoxygenases and CYP omega-hydroxylases in the heart, kidney, and liver and the cardiac CYP-mediated arachidonic acid metabolism. For this purpose, male Sprague-Dawley rats were injected intraperitoneally with LPS (1mg/kg). After 6, 12, or 24h, the tissues were harvested and the expression of CYP genes and protein levels were determined using real time-PCR, and Western blot analyses, respectively. Arachidonic acid metabolites formations were determined by liquid chromatography-electron spray ionization-mass spectrometry LC-ESI-MS. Our results showed that inflammation significantly decreased the CYP epoxygenases expression in the heart, kidney and liver with a concomitant decrease in the EETs produced by these enzymes. In contrast to CYP expoxygenses, inflammation differentially altered CYP omega-hydroxylases expression with a significant increase in 20-HETE formation. The present study demonstrates for the first time that acute inflammation decreases CYP epoxygenases and their associated cardioprotective metabolites, EETs while on the other hand increases CYP omega-hydroxylases and their associated cardiotoxic metabolites, 20-HETE. These changes may be involved in the development and/or progression of cardiovascular diseases by inflammation.

 

Pharmacol Res. 2010 May;61(5):410-8. doi: 10.1016/j.phrs.2009.12.015. Epub 2010 Jan 4.

 

DOI: 10.1016/j.phrs.2009.12.015

PMID: 20045729 [Indexed for MEDLINE]

 

Zordoky BN, Anwar-Mohamed A, Aboutabl ME, El-Kadi AO.

 

Doxorubicin (DOX) is a potent anti-neoplastic antibiotic used to treat a variety of malignancies; however, its use is limited by dose-dependent cardiotoxicity. Moreover, there is a strong correlation between cytochrome P450 (CYP)-mediated arachidonic acid metabolites and the pathogenesis of many cardiovascular diseases. Therefore, in the current study, we have investigated the effect of acute DOX toxicity on the expression of several CYP enzymes and their associated arachidonic acid metabolites in the heart of male Sprague-Dawley rats. Acute DOX toxicity was induced by a single intraperitoneal injection of 15 mg/kg of the drug. Our results showed that DOX treatment for 24 h caused a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A1, CYP4A3, CYP4F1, CYP4F4, and EPHX2 gene expression in the heart of DOX-treated rats as compared to the control. Similarly, there was a significant induction of CYP1A1, CYP1B1, CYP2C11, CYP2J3, CYP4A, and sEH proteins after 24 h of DOX administration. In the heart microsomes, acute DOX toxicity significantly increased the formation of 20-HETE which is consistent with the induction of the major CYP omega-hydroxylases: CYP4A1, CYP4A3, CYP4F1, and CYP4F4. On the other hand, the formation of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) was significantly reduced, whereas the formation of their corresponding dihydroxyeicosatrienoic acids was significantly increased. The decrease in the cardioprotective EETs can be attributed to the increase of sEH activity parallel to the induction of the EPHX2 gene expression in the heart of DOX-treated rats. In conclusion, acute DOX toxicity alters the expression of several CYP and sEH enzymes with a consequent alteration in arachidonic acid metabolism. These results may represent a novel mechanism by which this drug causes progressive cardiotoxicity.

 

Toxicol Appl Pharmacol. 2010 Jan 1;242(1):38-46. doi: 10.1016/j.taap.2009.09.012. Epub 2009 Sep 29.

 

DOI: 10.1016/j.taap.2009.09.012

PMID: 19796650 [Indexed for MEDLINE]

 

 

Anwar-Mohamed A, Elbekai RH, El-Kadi AO.

 

Cytochrome P450 1A1 (CYP1A1) is a hepatic and extrahepatic enzyme that is regulated by the aryl hydrocarbon receptor signaling pathway. With the growing human exposure to heavy metals, emerging evidence suggests that heavy metals exposure alter CYP1A1 enzyme activity. Heavy metals regulate CYP1A1 at different levels of its aryl hydrocarbon receptor signaling pathway in a metal- and species-dependent manner. The importance of CYP1A1 emerges from the fact that it has been always associated with the metabolism of pro-carcinogenic compounds to highly carcinogenic metabolites. However, recently CYP1A1 has gained status along with other cytochrome P450 enzymes in the metabolism of drugs and mediating drug-drug interactions. In addition, CYP1A1 has become a therapeutic tool for the bioactivation of prodrugs, particularly cytotoxic agents. In this review, we shed light on the effect of seven heavy metals, namely arsenic, mercury, lead, cadmium, chromium, copper and vanadium, on CYP1A1 and the consequences on drug metabolism.

 

Expert Opin Drug Metab Toxicol. 2009 May;5(5):501-21. doi: 10.1517/17425250902918302 .

 

DOI: 10.1517/17425250902918302

PMID: 19416086 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Kadi AO.

 

Recent data suggest that vanadium (V5+) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V5+ down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V5+ is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P) H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhRdeficient version, c12, were treated with increasing concentrations of V5+ in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V5+ inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V5+ was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V5+ did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V5+ increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V5+ down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2.

 

Toxicol Appl Pharmacol. 2009 May 1;236(3):261-9.

 

PMID: 19367690 [Indexed for MEDLINE]

 

 

Anwar-Mohamed A, El-Kadi AO.

 

Recent reports have proposed that some naturally occurring phytochemicals can function as anticancer agents mainly through inducing phase II drug detoxification enzymes. Of these phytochemicals, isothiocyanates sulforaphane (SUL), present in broccoli, is by far the most extensively studied. In spite of its positive effect on phase II drug metabolizing enzymes, its effect on the phase I bioactivating enzyme cytochrome P450 1a1 (Cyp1a1) is still a matter of debate. As a first step to investigate this effect, Hepa 1c1c7 and HepG2 cells were treated with various concentration of SUL. Our results showed that SUL-induced CYP1A1 mRNA in a dose- and time-dependent manner. Furthermore, this induction was further reflected on the protein and catalytic activity levels. Investigating the effect of SUL at the transcriptional level revealed that SUL increases the Cyp1a1 mRNA as early as 1h. The RNA polymerase inhibitor actinomycin D (Act-D) completely abolished the SUL-induced Cyp1a1 mRNA. Furthermore, SUL successfully activated AhR transformation and its subsequent binding to the XRE. At the post-transcriptional level, SUL did not affect the levels of existing Cyp1a1 mRNA transcripts. This is the first demonstration that the broccoli-derived SUL can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SUL induces this enzyme.

 

Cancer Lett. 2009 Mar 8;275(1):93-101. doi: 10.1016/j.canlet.2008.10.003. Epub 2008 Nov 13.

 

DOI: 10.1016/j.canlet.2008.10.003

PMID: 19013013 [Indexed for MEDLINE]

 

 

Anwar-Mohamed A, Elbekai RH, El-Kadi AO.

 

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 (Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 microM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content.

 

Toxicol Lett. 2008 Nov 10;182(1-3):121-6. doi: 10.1016/j.toxlet.2008.09.007. Epub 2008 Sep 18.

 

DOI: 10.1016/j.toxlet.2008.09.007

PMID: 18835339 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Kadi AO.

 

Vanadium (V(5+)), a heavy metal contaminant with important toxicological consequences, has received considerable attention as an anticancer agent, although the mechanisms remain unknown. As a first step to investigate these mechanisms, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene: cytochrome P450 1a1 (Cyp1a1) at each step of the AhR signal transduction pathway, using Hepa 1c1c7 cells. Our results showed a significant reduction in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein and activity levels after V(5+) treatments in a dose-dependent manner. Investigation of the effect of coexposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Furthermore, despite not affecting the direct activation of the cytosolic AhR by TCDD and subsequently transforming it to a DNA-binding form, V(5+) inhibited the nuclear accumulation of liganded AhR and subsequent formation of the AhR/aryl hydrocarbon nuclear translocator (Arnt)/xenobiotic responsive element (XRE) complex. Importantly, the V(5+)-mediated inhibition of AhR/Arnt/XRE complex formation coincided with a significant decrease in ecto-ATPase activity. Looking at the post-transcriptional and post-translational effects of V(5+) on existing Cyp1a1 mRNA and protein levels, we showed that V(5+) did not affect Cyp1a1 mRNA or protein stability, thus eliminating possible role of V(5+) in modifying Cyp1a1 gene expression through these mechanisms. This study provides the first evidence that V(5+) down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism.

 

Drug Metab Dispos. 2008 Sep;36(9):1819-27. doi: 10.1124/dmd.108.021154. Epub 2008 Jun 9.

 

DOI: 10.1124/dmd.108.021154

PMID: 18541696 [Indexed for MEDLINE]

 

Anwar-Mohamed A, El-Kadi AO.

 

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.

 

Toxicol In Vitro. 2007 Jun;21(4):685-90. Epub 2007 Jan 15.

 

DOI: 10.1016/j.tiv.2007.01.003

PMID: 17317091 [Indexed for MEDLINE]

 

 anwar anwar 

Abstract Anwar Mohamed 

Abstract: Anwar Mohamed 

Anwar Gamal Mohamed Frankfort We recently demonstrated that V5+ downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-
mediated induction of Cyp1a1 mRNA, protein, and
catalytic activity levels in Hepa 1c1c7 cells through
transcriptional mechanism. Therefore, it is important to
investigate whether similar changes occur in humans.
For this purpose, we examined the effect of V
5+ (as
ammonium metavanadate, NH
4VO3) on the expression
of aryl hydrocarbon receptor (AhR)-regulated gene;
cytochrome P450 1A1 (CYP1A1) at each step of the
AhR signal transduction pathway in human hepatoma
HepG2 cells. Our results show a significant reduction
in TCDD-mediated induction of CYP1A1 mRNA,
protein, and activity levels after V
5+ treatment in a
dose-dependent manner. Investigating the effect of coexposure to V
5+ and TCDD at transcriptional levels
revealed that V
5+ significantly inhibited TCDDmediated induction of AhR-dependent luciferase
reporter gene expression. Looking at the posttranscriptional level, V
5+ did not affect CYP1A1 mRNA
stability, thus eliminating the possible role of V
5+ in
modifying CYP1A1 gene expression through this
mechanism. On the other hand, at the posttranslational
level, V
5+ was able to significantly decrease CYP1A1
protein half-life contributing to the inconsistency
between catalytic activity and transcriptional level.
Importantly, we showed that V
5+ did not significantly
alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V
5+ might have decreased
CYP1A1 activity through affecting its heme content.
This study demonstrates for the first time that V
5+
downregulates the expression of CYP1A1 at the
transcriptional, posttranscriptional and posttranslational
mechanisms in the human hepatoma HepG2 cells.

 

Materials and methods
Materials


Ammonium metavanadate (NH
4VO3), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), cycloheximide (CHX), 7-ethoxyresorufin,
and protease inhibitor cocktail were purchased
from Sigma
Aldrich (St. Louis, MO). 2,3,7,8-
Tetrachlorodibenzo-
p-dioxin, >99% pure, was pur
chased from Cambridge Isotope Laboratories (Woburn,
MA). TRIzol reagent and Lipofectamine 2000 reagents
were purchased from Invitrogen (San Diego, CA). The
High-Capacity cDNA Reverse Transcription Kit and
SYBR Green PCR Master Mix were purchased from
  

Applied Biosystems (Foster City, CA). Actinomycin-D
(Act-D) was purchased from Calbiochem (San Diego,
CA). Chemiluminescence Western blotting detection
reagents were from GE Healthcare Life Sciences
(Piscataway, NJ). Nitrocellulose membrane was pur
chased from Bio-Rad (Hercules, CA). CYP1A1 D-15
mouse anti-rat polyclonal primary antibody was pur
chased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), anti-mouse IgG peroxidase secondary
antibody was purchased from R&D Systems (Minne
apolis, MN, USA). pRL-CMV plasmid and luciferase
assay reagents were obtained from Promega (Madison,
WI). All other chemicals were purchased from Thermo
Fisher Scientific (Toronto, ON, Canada). Primers were
purchased from Integrated DNA Technologies, Inc.
(Coralville, IA) and are listed in Table
1.
Cell culture
Human hepatoma HepG2 cell line, ATCC number
HB-8065 (Manassas, VA), passages 4
15, was main
tained in Dulbecco
s modified Eagles medium
supplemented with 10% heat-inactivated fetal bovine
serum and 1% penicillin
streptomycin. Cells were
grown in 75-cm
2 cell culture flasks at 37°C in a 5%
CO
2 humidified incubator.

Anwar Gamal Mohamed research shows that
Effect of V
5+ on cell viability
The effect of V
5+ on cell viability was determined
using the MTT assay as described previously (Anwar
Mohamed and El-Kadi
2009b). MTT assay measures
the conversion of MTT to formazan in living cells via
mitochondrial enzymes of viable cells. In brief,
HepG2 cells were seeded onto 96-well microtiter cell
culture plates and incubated for 24 h at 37°C in a 5%
CO
2 humidified incubator. Cells were treated with
various concentrations of V
5+ (251,000 μM) in the
absence and presence of 1 nM TCDD. After 24 h
incubation, the medium was removed and replaced
with cell culture medium containing 1.2 mM MTT
dissolved in phosphate-buffered saline (PBS, pH 7.4).
After 2 h of incubation, the formed crystals were
dissolved in isopropanol. The intensity of the color in
each well was measured at a wavelength of 550 nm
using the Bio-Tek EL 312e microplate reader (Bio
Tek Instruments, Winooski, VT).
RNA extraction and cDNA synthesis
Total RNA was isolated using TRIzol reagent
(Invitrogen) according to the manufacturer
s instruc
tions and quantified by measuring the absorbance at
260 nm. Thereafter, first-strand cDNA synthesis was

 

Real-time PCR data analysis
The real-time PCR data were analyzed using the relative
gene expression, i.e., (
ΔΔCT) method as described in
Applied Biosystems User Bulletin No. 2 and explained
further by Livak and Schmittgen (
2001). In brief, the
primers used in the current study were tested to avoid
primer dimers, self-priming formation, or unspecific
amplification. To ensure the quality of the measurements, each plate included, for each gene, a negative
control and a positive control. For each sample, a
threshold cycle (
CT) was calculated based on the time
(measured by the number of PCR cycles) at which the
reporter fluorescent emission increased beyond a
threshold level (based on the background fluorescence
of the system). The triplicate measurements for each
sample were averaged to give an average
CT value for
each group, after removing of outliers (Oscar Aparicio
et al.
2005). The samples were diluted in such a
manner that the
CT value was observed between 15
and 30 cycles. Results were expressed using the
comparative
CT method as described in User Bulletin
2 (Applied Biosystems). Briefly, the
ΔCT values
were calculated in every sample for each gene of
interest as follows:
CT gene of interest-CT reporter gene,
with
β-actin as the reporter gene. Calculation of
relative changes in the expression level of one
specific gene (
ΔΔCT) was performed by subtraction
of
ΔCT of control (untreated cells or 0 h time point)
from the
ΔCT of the corresponding treatment groups.
The values and ranges given in different figures were
determined as follows: 2
ΔΔCT with ΔΔCT+SE and
ΔΔCT-SE, where SE is the standard error of the mean of the ΔΔCT value (User Bulletin 2, Applied

Biosystems)

 

 

© Copyright 2017 Anwar Mohamed