Hesham M. Korashy

The role of aryl hydrocarbon receptor in the pathogenesis of cardiovascular diseases.

Numerous experimental and epidemiological studies have demonstrated that polycyclic aromatic hydrocarbons (PAHs), which are major constituents of cigarette tobacco tar, are strongly involved in the pathogenesis of the cardiovascular diseases (CVDs). Knowing that PAH-induced toxicities are mediated by the activation of a cytosolic receptor, aryl hydrocarbon receptor (AhR), which regulates the expression of a group of xenobiotic metabolizing enzymes (XMEs) such as CYP1A1, CYP1A2, CYP1B1, NQO1, and GSTA1, suggests a direct link between AhR-regulated XMEs and CVDs. Therefore, identifying the localization and expression of the AhR and its regulated XMEs in the cardiovascular system (CVS) is of major importance in understanding their physiological and pathological roles. Generally, it was believed that the levels of AhR-regulated XMEs are lower in the CVS than in the liver; however, it has been shown that similar or even higher levels of expression are demonstrated in the CVS in a tissue- and species-specific manner. Moreover, most, if not all, AhR-regulated XMEs are differentially expressed in most of the CVS, particularly in the endothelium cells, aorta, coronary arteries, and ventricles. Although the exact mechanisms of PAH-mediated cardiotoxicity are not fully understood, several mechanisms are proposed. Generally, induction of CYP1A1, CYP1A2, and CYP1B1 is considered cardiotoxic through generating reactive oxygen species (ROS), DNA adducts, and endogenous arachidonic acid metabolites. However the cardioprotective properties of NQO1 and GSTA1 are mainly attributed to the antioxidant effect by decreasing ROS and increasing the levels of endogenous antioxidants. This review provides a clear understanding of the role of AhR and its regulated XMEs in the pathogenesis of CVDs, in which imbalance in the expression of cardioprotective and cardiotoxic XMEs is the main determinant of PAH-mediated cardiotoxicity.

Metformin attenuates streptozotocin-induced diabetic nephropathy in rats through modulation of oxidative stress genes expression

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion and/or action. One of the most important complications of this metabolic disease is diabetic nephropathy. Hyperglycemia promotes oxidative stress and hence generation of reactive oxygen species (ROS), which is known to play a crucial role in the pathogenesis of diabetic nephropathy. Recent studies have established that metformin, an oral hypoglycemic drug, possesses antioxidant effects. However, whether metformin can protect against diabetic nephropathy has not been reported before. The overall objectives of the present study are to elucidate the potential nephroprotective effect of metformin in a rat diabetic nephropathy model and explore the exact underlying mechanism(s) involved. The effect of metformin on the biochemical changes associated with hyperglycemia induced by streptozotocin was investigated in rat kidney tissues. In addition, energy nucleotides (AMP and ATP), and Acetyl-CoA in the kidney homogenates and mitochondria, and the mRNA expression of oxidative stress and pro-inflammatory mediators were assessed. Our results showed that treatment of normoglycemic rats with metformin caused significant increase in ATP, Acetyl-CoA, and CoA-SH contents in kidney homogenates and mitochondria along with profound decrease in AMP level. On the other hand, treatment of diabetic nephropathy rats with metformin normalized all biochemical changes and the energy status in kidney tissues. At the transcriptional levels, metformin treatment caused significant restoration in diabetic nephropathy-induced oxidative stress mRNA levels, particularly GSTα, NQO1, and CAT genes, whereas inhibited TNF-α and IL-6 pro-inflammatory genes. Our data lend further credence for the contribution of metformin in the nephroprotective effect in addition to its well known hypoglycemic action.

The Effect of Liver Cirrhosis on the Regulation and Expression of Drug Metabolizing Enzymes

Cirrhosis is the end stage of many forms of liver pathologies including hepatitis. The liver is known for its vital role in the processing of xenobiotics, including drugs and toxic compounds. Cirrhosis causes changes in the architecture of the liver leading to changes in blood flow, protein binding, and drug metabolizing enzymes. Drug metabolizing enzymes are primarily decreased due to loss of liver tissue. However, not all enzyme activities are reduced and some are only altered in specific cases. There is a great deal of discrepancy between various reports on cytochrome p450 alterations in liver cirrhosis, likely due to differences in disease severity and other underlying conditions. In general, however, CYP1A and CYP3A levels and related enzyme activities are usually reduced and CYP2C, CYP2A, and CYP2B are mostly unaltered. Both alcohol dehyrogenases and aldehyde dehydrogenases are altered in liver cirrhosis, although the etiology of the disease may determine the expression of alcohol dehydrogenases. Glucuronidation is mainly preserved, but there are a number of factors that determine whether glucuronidation is affected in patients with liver cirrhosis. Low sulphation rates are usually found in patients with liver disease but a decrease in sulfatase activity compensates for the decrease in sulphation rates. In all cases, a reduction in drug metabolizing enzyme activities in liver cirrhosis contributes to decreased clearance of drugs seen in patients with liver abnormalities. The reduction in drug metabolizing enzyme activity must be taken into consideration when adjusting doses, especially in patients with severe liver disease.

TRANSCRIPTIONAL REGULATION OF THE NAD(P)H:QUINONE OXIDOREDUCTASE 1 AND GLUTATHIONE S-TRANSFERASE YA GENES BY MERCURY, LEAD, AND COPPER

Recently, we demonstrated the ability of heavy metals, particularly Hg2+, Pb2+, and Cu2+, to differentially modulate in Hepa 1c1c7 cells the expression of the phase II xenobiotic metabolizing enzymes NAD(P)H:quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase subunit Ya (Gst ya) genes, yet the mechanisms involved remain unknown. To investigate the molecular mechanisms involved in the regulation of Nqo1 and Gst ya genes by heavy metals, Hepa 1c1c7 cells were treated with Hg2+, Pb2+, or Cu2+ in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of Nqo1, Gst ya, and Cyp1a1 genes. Analysis of the time-dependent effect of heavy metals revealed that Hg2+ and Pb2+ increased whereas Cu2+ inhibited the constitutive and inducible expression of Nqo1 and Gst ya mRNAs in a time-dependent manner. The RNA synthesis inhibitor actinomycin D significantly inhibited the Nqo1 and Gst ya mRNA induction in response to metals, indicating a requirement of de novo RNA synthesis. The protein synthesis inhibitor cycloheximide significantly inhibited metal-mediated induction of Nqo1 and Gst ya mRNAs, which coincided with a decrease in the nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression, implying the requirement of Nrf2 protein synthesis for the induction of these genes. Furthermore, inhibition of Nrf2 protein degradation by carbobenzoxy-l-leucyl-l-leucyl-leucinal (MG-132), a 26S proteasome inhibitor, significantly reversed the cycloheximide-mediated inhibition of Nqo1 and Gst ya mRNAs, which coincided with an increase in the expression of Nrf2, confirming that a transcriptional mechanism is involved. Nqo1 and Gst ya mRNA and protein decay experiments revealed lack of post-transcriptional and post-translational mechanisms. This is the first demonstration that heavy metals regulate the expression of Nqo1 and Gst ya genes through a transcriptional mechanism.

The impact of experimental hyperlipidemia on the distribution and metabolism of amiodarone in rat

The tissue distribution and hepatic microsomal metabolism of amiodarone were studied in a hyperlipidemic rat model. Rats were rendered hyperlipidemic by the intraperitoneal injection of poloxamer 407. Other normolipidemic animals given saline in place of poloxamer 407 were used as control animals. After single intravenous injection of amiodarone HCl (25mg/kg) rats were anesthetized and plasma and tissue specimens were obtained. Liver microsomal protein was harvested and used to measure velocity of desethylamiodarone formation from amiodarone and cytochrome P450 (CYP) protein expression. Hyperlipidemia caused large increases in plasma concentrations of amiodarone. In tissues, however, concentrations of drug selectively increased, decreased or did not change. In heart, the site of action of the drug, as well as liver and spleen, amiodarone concentrations increased. In other tissues such as kidney, lung and brain, concentrations decreased. No changes were seen in fat or thyroid. Decreases were observed in liver metabolic efficiency, and expression of CYP3A1/2 and 2C11. No changes were seen in CYP2B1/2, 2C6, 2D1 or 1A2. This experimental hyperlipidemia caused a complex pattern of changes in tissue distribution of AM. In addition, there are decreases in the expression of some important rat CYP isoenzymes.

Benzo[a]Pyrene, 3-Methylcholanthrene and ß-Naphthoflavone Induce Oxidative Stress in Hepatoma Hepa 1c1c7 Cells by an AHR-dependent Pathway

Polycyclic aromatic hydrocarbons have been shown to cause oxidative stress in vitro and in vivo in various animal models but the mechanisms by which these compounds produce oxidative stress are unknown. In the current study we have investigated the role of the aryl hydrocarbon receptor (AHR) in the production of reactive oxygen species (ROS) by its cognate ligands and the consequent effect on cyp1a1 activity, mRNA and protein expressions. For this purpose, Hepa 1c1c7 cells wild-type (WT) and C12 mutant cells, which are AHR-deficient, were incubated with increasing concentrations of the AHR-ligands, benzo[a]pyrene (B[a]P, 0.25-25 μM), 3-methylcholanthrene (3MC, 0.1-10 μM) and β-naphthoflavone (βNF, 1-50 μM). The studied AHR-ligands dose-dependently increased lipid peroxidation in WT but not in C12 cells. However, only B[a]P and βNF, at the highest concentrations tested, significantly increased H2O2 production in WT but not C12 cells. The increase in lipid peroxidation and H2O2 production by AHR-ligands were accompanied by a decrease in the cyp1a1 catalytic activity but not mRNA or protein expressions, which were significantly induced in a dose-dependent manner by all AHR-ligands, suggesting a post-translational mechanism is involved in the decrease of cyp1a1 activity. The AHR-ligand-mediated decrease in cyp1a1 activity was reversed by the antioxidant N-acetylcysteine. Our results show that the AHR-ligands induce oxidative stress by an AHR-dependent pathway.

Metformin Rescues the Myocardium from Doxorubicin-Induced Energy Starvation and Mitochondrial Damage in Rats

Clinical use of doxorubicin (DOX) is limited by its cardiotoxic side effects. Recent studies established that metformin (MET), an oral antidiabetic drug, possesses an antioxidant activity. However, whether it can protect against DOX-induced energy starvation and mitochondrial damage has not been reported. Our results, in a rat model of DOX-induced cardiotoxicity, show that DOX treatment significantly increased serum levels of LDH and CK-MB, indicators of cardiac injury, and induced expression of hypertrophic gene markers. DOX also caused marked decreases in the cardiac levels of glutathione, CoA-SH and ATP, and mRNA expression of catalase and NQO-1. These biochemical changes were associated with myocardial histopathological and ultrastructural deteriorations, as observed by light and electron microscopy, respectively. Cotreatment with MET (500 mg/kg) eliminated all DOX-induced biochemical, histopathological, and ultrastructural changes. These findings demonstrate that MET successfully prevents DOX-induced cardiotoxicity in vivo by inhibiting DOX-induced oxidative stress, energy starvation, and depletion of intramitochondrial CoA-SH.

Regulation of TNF-α and NF-κB activation through the JAK/STAT signaling pathway downstream of histamine 4 receptor in a rat model of LPS-induced joint inflammation.

Histamine 4 receptor (H4R) is a novel target for the pharmacological modulation of histamine-mediated immune signals during inflammatory diseases. The purpose of this study was to assess the effects of the H4R agonist 4-methylhistamine dihydrochloride (4-MeH) and antagonist JNJ7777120 (JNJ) in the inflamed rat knee. Animals were fasted for 18h before a single dose of 4-MeH or JNJ (30mg/kg) was administered intraperitoneally (i.p.), both followed by intra-articular (i.a.) injection of LPS 2h later. Blood and synovial fluid were collected after a short incubation period and TNF-α, NF-κB, and IkB-α levels were measured via flow cytometry. Additionally, we assessed the effects of H4R engagement on the expression of IL-1β, TNF-α, and NF-κB mRNAs and the protein levels of TNF-α, NF-κB, JAK-1, and STAT-3 in the inflamed knee tissue. These results revealed increased TNF-α and NF-κB expression and decreased IkB-α levels in both the LPS alone and 4-MeH treated groups in whole blood and synovial fluid. Further, IL-1β, TNF-α, and NF-κB mRNA levels were significantly increased and western blot analysis confirmed increased expression of TNF-α, NF-κB, JAK-1, and STAT-3 in both LPS and 4-MeH treatment groups. Furthermore, these increases were completely inhibited in the inflamed knee tissue of the JNJ-treated group. Thus, the inhibition of inflammatory mediators and signaling pathways by the H4R antagonist JNJ suggests the anti-arthritic importance of this molecule.

The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model

Lead (Pb(2+)) is a naturally occurring systemic toxicant heavy metal that affects several organs in the body including the kidneys, liver, and central nervous system. However, Pb(2+)-induced cardiotoxicity has never been investigated yet and the exact mechanism of Pb(2+) associated cardiotoxicity has not been studied. The current study was designed to investigate the potential effect of Pb(2+) to induce cardiotoxicity in vivo and in vitro rat model and to explore the molecular mechanisms and the role of aryl hydrocarbon receptor (AhR) and regulated gene, cytochrome P4501A1 (CYP1A1), in Pb(2+)-mediated cardiotoxicity. For these purposes, Wistar albino rats were treated with Pb(2+) (25, 50 and 100mg/kg, i.p.) for three days and the effects on physiological and histopathological parameters of cardiotoxicity were determined. At the in vitro level, rat cardiomyocyte H9c2 cell lines were incubated with increasing concentration of Pb(2+) (25, 50, and 100 μM) and the expression of hypertrophic genes, α- and β-myosin heavy chain (α-MHC and β-MHC), brain Natriuretic Peptide (BNP), and CYP1A1 were determined at the mRNA and protein levels using real-time PCR and Western blot analysis, respectively. The results showed that Pb(2+) significantly induced cardiotoxicity and heart failure as evidenced by increase cardiac enzymes, lactate dehydrogenase and creatine kinase and changes in histopathology in vivo. In addition, Pb(2+) treatment induced β-MHC and BNP whereas inhibited α-MHC mRNA and protein levels in vivo in a dose-dependent manner. In contrast, at the in vitro level, Pb(2+) treatment induced both β-MHC and α-MHC mRNA levels in time- and dose-dependent manner. Importantly, these changes were accompanied with a proportional increase in the expression of CYP1A1 mRNA and protein expression levels, suggesting a role for the CYP1A1 in cardiotoxicity. The direct evidence for the involvement of CYP1A1 in the induction of cardiotoxicity by Pb(2+) was evidenced by the ability of AhR antagonist, resveratrol, to significantly inhibit the Pb(2+)-modulated effect on β-MHC and α-MHC mRNAs. It was concluded that acute lead exposure induced cardiotoxicity through AhR/CYP1A1-mediated mechanism.

Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2) and human breast (MCF7) cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways.