Saleh A. Bakheet

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.

REVERSAL OF CISPLATIN‐INDUCED CARNITINE DEFICIENCY AND ENERGY STARVATION BY PROPIONYL‐l‐CARNITINE IN RAT KIDNEY TISSUES

1 The present study examined whether propionyl‐l‐carnitine (PLC) could prevent the development of cisplatin (CDDP)‐induced acute renal failure in rats. 2 Forty adult male Wistar albino rats were divided into four groups. Rats in the first group were injected daily with normal saline (2.5 mL/kg, i.p.) for 10 consecutive days, whereas the second group received PLC (250 mg/kg, i.p.) for 10 consecutive days. Animals in the third group were injected daily with normal saline for 5 consecutive days before and after a single dose of CDDP (7 mg/kg, i.p.). Rats in the fourth group received a combination of PLC (250 mg/kg, i.p.) for 5 consecutive days before and after a single dose of CDDP (7 mg/kg, i.p.). On Day 6 following CDDP treatment, animals were killed and serum and kidneys were isolated for analysis. 3 Injection of CDDP resulted in a significant increase in serum creatinine, blood urea nitrogen (BUN), thiobarbituric acid‐reactive substances (TBARS) and total nitrate/nitrite (NOx), as well as a significant decrease in reduced glutathione (GSH), total carnitine, ATP and ATP/ADP in kidney tissues. 4 Administration of PLC significantly attenuated the nephrotoxic effects of CDDP, manifested as normalization of the CDDP‐induced increase in serum creatinine, BUN, TBARS and NOx and the CDDP‐induced decrease in total carnitine, GSH, ATP and ATP/ADP in kidney tissues. 5 Histopathological examination of kidney tissues from CDDP‐treated rats showed severe nephrotoxicity, in which 50–75% of glomeruli and renal tubules exhibited massive degenerative changes. Interestingly, administration of PLC to CDDP‐treated rats resulted in a significant improvement in glomeruli and renal tubules, in which less than 25% of glomeruli and renal tubules exhibited focal necrosis. 6 Data from the present study suggest that PLC prevents the development of CDDP‐induced acute renal injury by a mechanism related, at least in part, to the ability of PLC to increase intracellular carnitine content, with a consequent improvement in mitochondrial oxidative phosphorylation and energy production, as well as its ability to decrease oxidative stress. This will open new perspectives for the use of PLC in the treatment of renal diseases associated with or secondary to carnitine deficiency.

Effect of long-term human exposure to environmental heavy metals on the expression of detoxification and DNA repair genes.

The present study was designed to evaluate the influence of long-term environmental human exposure to three heavy metals, lead (Pb), cadmium (Cd), and mercury (Hg), on the expression of detoxifying, xenobiotic metabolizing, and DNA repair genes in Mahd Ad-Dahab city. The study groups consisted of 40 healthy male residents (heavy metal-exposed) and 20 healthy male from Riyadh city, 700 km away, and served as control group. The heavy metal-exposed group with high exposure to Pb, Cd, or Hg was divided into three subgroups Pb-, Cd-, and Hg-exposed groups, respectively. The mRNA expression levels of detoxifying, NQO1, HO-1, GSTA1, MT-1, and HSP70, were significantly decreased in all heavy metal-exposed group as compared to control group. This was accompanied with a proportional decrease in the expression of xenobiotic metabolizing gene, cytochrome P4501A1. On the other hand, the DNA repair gene OGG1 and the 8-OHdG level were dramatically inhibited in Cd-exposed group only.

Dysregulation of Th1, Th2, Th17, and T regulatory cell-related transcription factor signaling in children with autism

Autism is a neurodevelopmental disorder characterized by stereotypic repetitive behaviors, impaired social interactions, and communication deficits. Numerous immune system abnormalities have been described in individuals with autism including abnormalities in the ratio of Th1/Th2/Th17 cells; however, the expression of the transcription factors responsible for the regulation and differentiation of Th1/Th2/Th17/Treg cells has not previously been evaluated. Peripheral blood mononuclear cells (PBMCs) from children with autism (AU) or typically developing (TD) control children were stimulated with phorbol-12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A. The expressions of Foxp3, RORγt, STAT-3, T-bet, and GATA-3 mRNAs and proteins were then assessed. Our study shows that children with AU displayed altered immune profiles and function, characterized by a systemic deficit of Foxp3+ T regulatory (Treg) cells and increased RORγt+, T-bet+, GATA-3+, and production by CD4+ T cells as compared to TD. This was confirmed by real-time PCR (RT-PCR) and western blot analyses. Our results suggest that autism impacts transcription factor signaling, which results in an immunological imbalance. Therefore, the restoration of transcription factor signaling may have a great therapeutic potential in the treatment of autistic disorders.

Evaluation of Chromosomal Instability in Diabetic Rats Treated with Naringin

We used the bone marrow DNA strand breaks, micronucleus formations, spermatocyte chromosomal aberrations, and sperm characteristic assays to investigate the chromosomal instability in somatic and germinal cells of diabetic rats treated with multiple doses of naringin. The obtained results revealed that naringin was neither cytotoxic nor genotoxic for the rats at all tested doses. Moreover, naringin significantly reduced the diabetes-induced chromosomal instability in somatic and germinal cells in a dose-dependent manner. In addition, diabetes induced marked biochemical alterations characteristic of oxidative stress including enhanced lipid peroxidation, accumulation of oxidized glutathione, reduction in reduced glutathione, and accumulation of intracellular reactive oxygen species. Treatment with naringin ameliorated these biochemical markers dose-dependently. In conclusion, naringin confers an appealing protective effect against diabetes-induced chromosomal instability towards rat somatic and germinal cells which might be explained partially via diminishing the de novo free radical generation induced by hyperglycemia. Thus, naringin might be a good candidate to reduce genotoxic risk associated with hyperglycemia and may provide decreases in the development of secondary malignancy and abnormal reproductive outcomes risks, which seems especially important for diabetic patients.We used the bone marrow DNA strand breaks, micronucleus formations, spermatocyte chromosomal aberrations, and sperm characteristic assays to investigate the chromosomal instability in somatic and germinal cells of diabetic rats treated with multiple doses of naringin. The obtained results revealed that naringin was neither cytotoxic nor genotoxic for the rats at all tested doses. Moreover, naringin significantly reduced the diabetes-induced chromosomal instability in somatic and germinal cells in a dose-dependent manner. In addition, diabetes induced marked biochemical alterations characteristic of oxidative stress including enhanced lipid peroxidation, accumulation of oxidized glutathione, reduction in reduced glutathione, and accumulation of intracellular reactive oxygen species. Treatment with naringin ameliorated these biochemical markers dose-dependently. In conclusion, naringin confers an appealing protective effect against diabetes-induced chromosomal instability towards rat somatic and germinal cells which might be explained partially via diminishing the de novo free radical generation induced by hyperglycemia. Thus, naringin might be a good candidate to reduce genotoxic risk associated with hyperglycemia and may provide decreases in the development of secondary malignancy and abnormal reproductive outcomes risks, which seems especially important for diabetic patients.

Salubrious effects of dexrazoxane against teniposide-induced DNA damage and programmed cell death in murine marrow cells

The intention of the present study was to answer the question whether the catalytic topoisomerase-II inhibitor, dexrazoxane, can be used as a modulator of teniposide-induced DNA damage and programmed cell death (apoptosis) in the bone marrow cells in vivo. The alkaline single cell gel electrophoresis, scoring of chromosomal aberrations, micronuclei and mitotic activity were undertaken in the current study as markers of DNA damage. Apoptosis was analysed by the occurrence of a hypodiploid DNA peak and caspase-3 activity. Oxidative stress marker such as intracellular reactive oxygen species production, lipid peroxidation, reduced and oxidised glutathione were assessed in bone marrow as a possible mechanism underlying this amelioration. Dexrazoxane was neither genotoxic nor apoptogenic in mice at the tested dose. Moreover, for the first time, it has been shown that dexrazoxane affords significant protection against teniposide-induced DNA damage and apoptosis in the bone marrow cells in vivo and effectively suppresses the apoptotic signalling triggered by teniposide. Teniposide induced marked biochemical alterations characteristic of oxidative stress including accumulation of intracellular reactive oxygen species, enhanced lipid peroxidation, accumulation of oxidised glutathione and reduction in the reduced glutathione level. Prior administration of dexrazoxane ahead of teniposide challenge ameliorated these biochemical alterations. It is thus concluded that pretreatment with dexrazoxane attenuates teniposide-induced oxidative stress and subsequent DNA damage and apoptosis in bone marrow cells. Based on our data presented, strategies can be developed to decrease the teniposide-induced DNA damage in normal cells using dexrazoxane. Therefore, dexrazoxane can be a good candidate to decrease the deleterious effects of teniposide in the bone marrow cells of cancer patients treated with teniposide.

Proanthocyanidins Produce Significant Attenuation of Doxorubicin-Induced Mutagenicity via Suppression of Oxidative Stress

This study has been initiated to determine whether proanthocyanidins can protect against doxorubicin-induced mutagenicity in mice and to elucidate the potential mechanism of this protection. Pretreatment of mice with proanthocyanidins (100 mg/kg/day, orally) for 7 days and simultaneously with doxorubicin (12 mg/kg, i.p.) for another day, significantly reduced the frequency of bone marrow DNA strand breaks and micronucleated polychromatic erythrocytes compared to doxorubicin-treated mice alone. Furthermore, proanthocyanidins caused a reduction in bone marrow suppression induced by doxorubicin treatment. In male germline, orally administration of proanthocyanidins (100 mg/kg/day, orally) for 7 consecutive days before and 7 consecutive days after treatment with doxorubicin (12 mg/ kg, i.p.), significantly elevated the levels of sperm count and motility reduced by doxorubicin treatment. Furthermore, proanthocyanidins significantly decreased the elevated levels of spermatogonial and spermatocyte chromosomal aberrations and sperm head abnormality induced by doxorubicin. Prior administration of proanthocyanidins ahead of doxorubicin reduced the doxorubicin induced testicular lipid peroxidation and prevented the reduction in testicularnonprotein sulfhydryl significantly. Conclusively, this study provides for the first time that proanthocyanidins have a protective role in the abatement of doxorubicin-induced mutagenesis and cell proliferation changes in germinal cells of mice that reside, at least in part, in their radical scavengeractivity. Therefore, proanthocyanidins can be a promising chemopreventive agent to avert secondary malignancy and abnormal reproductive outcomes risks in cancer patients receiving doxorubicin-involved treatment.

Molecular cytogenetic evaluation of the mechanism of micronuclei formation induced by camptothecin, topotecan, and irinotecan

We used the conventional bone marrow micronucleus test complemented with the fluorescent in situ hybridization with the minor satellite DNA probe to investigate the mechanisms of induction of micronuclei in mice treated with camptothecin and its clinical antineoplastic analogues topotecan and irinotecan. All experiments were performed with male Swiss albino mice. Single doses of 1 mg/kg camptothecin or 0.6 mg/kg topotecan were injected intraperitoneally and bone marrow was sampled at 30 hr (camptothecin) or 24 hr (topotecan) after treatment. A dose of 60 mg/kg irinotecan was injected intravenously, once every fourth day for 13 days and bone marrow was sampled 24 hr after the last treatment. In animals treated with camptothecin, a total of 1.07% micronuclei were found and 70% of them were centromere‐negative, indicating their formation by DNA strand breaks and reflecting the predominant clastogenic activity of camptothecin. Exposure to topotecan and irinotecan yielded 1.71 and 0.83% micronuclei, respectively. About 52.7 and 48.8% of the induced micronuclei, respectively, were centromere‐positive, indicating their formation by whole chromosomes and reflecting the aneugenic activity of both compounds. Correspondingly, about 47.3 and 51.2% of the induced micronuclei, respectively were centromere‐negative, demonstrating that topotecan and irinotecan not only induce chromosome loss but also DNA strand breaks. Both the clastogenic and aneugenic potential of these drugs can lead to the development of secondary tumors and abnormal reproductive outcomes. Therefore, the clinical use of these agents must be weighed against the risks of secondary malignancies in cured patients and persistent genetic damage of their potential offspring. Environ. Mol. Mutagen. 2009.

Grape seed proanthocyanidin extract has potent anti-arthritic effects on collagen-induced arthritis by modifying the T cell balance.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation of the synovial joints, joint malformations, and disability. The continuous use of conventional anti-inflammatory drugs is associated with severe adverse effects. Grape seed proanthocyanidin extract (GSPE) is considered to have protective effects against several diseases. In this study based on the mouse adjuvant-induced-arthritis (AIA) model, we examined the effects of GSPE on the key mediators of arthritic inflammation, namely T cell subsets, glucocorticoid-induced tumour necrosis factor receptor (GITR) expressing cells, CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, Th17 cells, Th1/Th2 cytokines, and inflammatory mediator gene expression. We treated BALB/c mice with 25, 50, or 100 mg/kg GSPE or saline daily (14 days) per orally (p.o.) at the onset of AIA. At the peak phase of AIA (day 14), the heparinised whole blood and ankle joints of all groups were collected and tested. GSPE-treated mice showed a substantial reduction in the levels of T cell subsets, GITR-expressing cells, and Th1 cytokines as well as the inflammatory mediators (MCP-1, MIP-2, and ICAM-1) that induce them compared with the vehicle-treated (saline) and arthritic mice. However, GSPE significantly upregulated the number of Tregs and Th2 cytokine producing cell number or it also induced Th17/Treg rebalance and orchestrated various pro-inflammatory and anti-inflammatory cytokines and the gene expression of their mediators that mediate cellular infiltration into the joints. This might, contribute to its anti-arthritic activity. Our results suggest that p.o. treatment with GSPE attenuated AIA in mice might offer a promising alternative/adjunct treatment for RA.