Evaluation of the protective efficacy of immunoglobulin Y (IgY- antibodies) prepared against Walterinnesia aegyptia snake venom in Saudi Arabia

Four groups of eight chickens were immunized intramuscularly with Walterinnesia aegyptia snake venoms mixed with Freund's complete adjuvant during the period from 1st October 2009 to 1st October 2011 at the Center of Excellence in Biotechnology Research, King Saud University, Saudi Arabia. Three weeks later, the injections were repeated with the venoms in incomplete Freund's adjuvant. Three boosters were given with the venoms at three weeks intervals. The immunoglobulin Y (IgY)-antibodies was extracted by ammonium sulphate-caprylic acid method, the antibody titer were tested by enzyme linked immunosorbant assay and the protective efficacies of the extracted immunoglobulins were performed. IgY-preparation extracted by ammonium sulphate-caprylic acid method showed lack of low molecular weight bands (non-immunoglobulin proteins) and the bands representing IgY-antibodies, which have molecular weights ranging from 180 to 200 kDa, appeared sharp and clear. Moreover, evaluation of the protective value of the IgY - antibodies prepared revealed that, one milliliter of extracted IgY-antibodies containing 15 mg/ml anti-W. aegyptia venom specific IgY could produce 100% protection against 50 LD50 and 75% protection against 60 LD50. Laying hens could be used as an alternative source of polyclonal antibodies against W. aegyptia snake venoms due to several advantages as compared with mammals traditionally used for such purpose.Keywords: Snake venom, Walterinnesia aegyptia, immunoglobulins Y, protective efficacy, caprylic aci

Thymoquinone attenuates diethylnitrosamine induction of hepatic carcinogenesis through antioxidant signaling

Hepatocellular carcinoma accounts for about 80–90% of all liver cancer and is the fourth most common cause of cancer mortality. Although there are many strategies for the treatment of liver cancer, chemoprevention seems to be the best strategy for lowering the incidence of this disease. Therefore, this study has been initiated to investigate whether thymoquinone (TQ), Nigella sativa derived-compound with strong antioxidant properties, supplementation could prevent initiation of hepatocarcinogenesis-induced by diethylnitrosamine (DENA), a potent initiator and hepatocarcinogen, in rats. Male Wistar albino rats were divided into four groups. Rats of Group 1 received a single intraperitoneal (I.P.) injection of normal saline. Animals in Group 2 were given TQ (4 mg/kg/day) in drinking water for 7 consecutive days. Rats of Group 3 were injected with a single dose of DENA (200 mg/kg, I.P.). Animals in Group 4 were received TQ and DENA. DENA significantly increased alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin, thiobarbituric acid reactive substances (TBARS) and total nitrate/nitrite (NOx) and decreased reduced glutathione (GSH), glutathione peroxidase (GSHPx), glutathione-s-transferase (GST) and catalase (CAT) activity in liver tissues. Moreover, DENA decreased gene expression of GSHPx, GST and CAT and caused severe histopathological lesions in liver tissue. Interestingly, TQ supplementation completely reversed the biochemical and histopathological changes induced by DENA to the control values. In conclusion, data from this study suggest that: (1) decreased mRNA expression of GSHPx, CAT and GST during DENA-induced initiation of hepatic carcinogenesis, (2) TQ supplementation prevents the development of DENA-induced initiation of liver cancer by decreasing oxidative stress and preserving both the activity and mRNA expression of antioxidant enzymes

Pro-inflammatory and oxidative stress pathways which compromise sperm motility and survival may be altered by L-carnitine

The testis is an immunologically privileged organ. Sertoli cells can form a blood-testis barrier and protect sperm cells from self-immune system attacks. Spermatogenesis may be inhibited by severe illness, bacterial infections and chronic inflammatory diseases but the mechanism(s) is poorly understood. Our objective is to help in understanding such mechanism(s) to develop protective agents against temporary or permanent testicular dysfunction. Lipopolysaccaride (LPS) is used as a model of animal sepsis while L-carnitine (LCR) is used as a protective agent. A total of 60 male Swiss albino rats were divided into four groups (15/group). The control group received Saline; the 2nd group was given LCR (500 mg/kg i.p, once). The third group was treated with LPS (5 mg/kg i.p once) and the fourth group received LCR then LPS after three hours. From each group, five rats were used for histopathological examination. Biochemical parameters were assessed in the remaining ten rats. At the end of the experiment, animals were lightly anaesthetized with ether where blood samples were collected and testes were dissected on ice. Sperm count and motility were evaluated from cauda epididymis in each animal. Also, oxidative stress was evaluated by measuring testicular contents of reduced glutathione (GSH), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-HDG, the DNA adduct for oxidative damage) in testicular DNA. The pro-inflammatory mediator nitric oxide (NO) in addition to lactate dehydrogenase (LDHx) isoenzyme-x activity as an indicator for normal spermatozoal metabolism were assessed in testicular homogenate. Serum interlukin (IL)-2 level was also assessed as a marker for T-helper cell function. The obtained data revealed that LPS induced marked reductions in sperm's count and motility, obstruction in seminiferous tubules, hypospermia and dilated congested blood vessels in testicular sections concomitant with decreased testicular GSH content and LDHx activity. Moreover, the testicular levels of MDA, 8-HDG (in testicular DNA) and NO as well as serum IL-2 level were increased. Administration of LCR before LPS returned both sperm count and motility to normal levels. Also, contents of testicular GSH, MDA, 8-HDG and NO returned back to the corresponding control values. In addition, serum IL-2 level as well as histological abnormalities were markedly improved in LCR + LPS-treated rats. In conclusion, LPS increased proinflammatory and oxidative stress markers in the testis leading to a marked testicular dysfunction. L-carnitine administration ameliorates these effects by antioxidant and/or anti-inflammatory mechanisms suggesting a protective role against male infertility in severely infected or septic patients

Metallothionein induction reduces caspase-3 activity and TNFα levels with preservation of cognitive function and intact hippocampal neurons in carmustine-treated rats

Hippocampal integrity is essential for cognitive functions. On the other hand, induction of metallothionein (MT) by ZnSO4 and its role in neuroprotection has been documented. The present study aimed to explore the effect of MT induction on carmustine (BCNU)-induced hippocampal cognitive dysfunction in rats. A total of 60 male Wistar albino rats were randomly divided into four groups (15/group): The control group injected with single doses of normal saline (i.c.v) followed 24 h later by BCNU solvent (i.v). The second group administered ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v, once) then BCNU solvent (i.v) after 24 h. Third group received BCNU (20 mg/kg, i.v, once) 24 h after injection with normal saline (i.c.v). Fourth group received a single dose of ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v) then BCNU (20 mg/kg, i.v, once) after 24 h. The obtained data revealed that BCNU administration resulted in deterioration of learning and short-term memory (STM), as measured by using radial arm water maze, accompanied with decreased hippocampal glutathione reductase (GR) activity and reduced glutathione (GSH) content. Also, BCNU administration increased serum tumor necrosis factor-alpha (TNFα), hippocampal MT and malondialdehyde (MDA) contents as well as caspase-3 activity in addition to histological alterations. ZnSO4 pretreatment counteracted BCNU-induced inhibition of GR and depletion of GSH and resulted in significant reduction in the levels of MDA and TNFα as well as the activity of caspase-3. The histological features were improved in hippocampus of rats treated with ZnSO4 + BCNU compared to only BCNU-treated animals. In conclusion, MT induction halts BCNU-induced hippocampal toxicity as it prevented GR inhibition and GSH depletion and counteracted the increased levels of TNFα, MDA and caspase-3 activity with subsequent preservation of cognition

Downregulation of Oxidative and Nitrosative Apoptotic Signaling by L-Carnitine in Ifosfamide-Induced Fanconi Syndrome Rat Model

It is well documented that ifosfamide (IFO) therapy is associated with sever nephropathy in the form of Fanconi syndrome. Although oxidative stress has been reported as a major player in IFO-induced Fanconi syndrome, no mechanism for this effect has been ascertained. Therefore, this study has been initiated to investigate, on gene expression level, the mechanism of IFO-induce nephrotoxicity and those whereby carnitine supplementation attenuates this serious side effect of IFO. To achieve the ultimate goals of this study, adult male rats were assigned to one of four treatment groups, namely, control, L-carnitine, IFO, and IFO plus L-carnitine. Administration of IFO for 5 days significantly increased serum creatinine, blood urea nitrogen (BUN), and total nitrate/nitrite (NOx) production in kidney tissues. In addition, IFO significantly increased mRNA expression of inducible nitric oxide synthase (iNOS), caspase-9, and caspase-3 and significantly decreased expression of glutathione peroxides (GPx), catalase (CAT), and Bcl2 in kidney tissues. Administration of L-carnitine to IFO-treated rats resulted in a complete reversal of the all biochemical and gene expression changes, induced by IFO, to the control values. Data from this study suggest that L-carnitine prevents the development of IFO-induced nephrotoxicity via downregulation of oxidative and nitrosative apoptotic signaling in kidney tissues

Superoxide anion stress attenuates the contractile response of the guinea pig vas deferens to ATP and diadenosine tetraphosphate

Induction of endogenous superoxide anion stress by the use of the superoxide dismutase inhibitor diethylthiocarbamate (DETCA; 10 mmol/l) produced a potent inhibition of the ATP (0.3-10 mmol/l) and diadenosine tetraphosphate (AP(4)A) contractile activity in the isolated vas deferens by 29-92 and 24-90%, respectively. Pyrogallol (0.1 mmol/l), the exogenous superoxide anion generator, produced a significant inhibition on the contractile activity of the vas deferens induced by ATP and AP(4)A by 33-89 and 25-82%, respectively. DETCA (10 mmol/l) and pyrogallol (0.1 mmol/l) attenuated the contractile response of isolated guinea pig vas deferens strips to the selective P2X agonist alpha,beta-methyleneATP (alpha,beta-meATP; 50 mu mol/l) by 25 and 47%, respectively. In Ca2+-free high-K+ (80 mmol/l) Krebs solution, pyrogallol and DETCA produced inhibition of the contractile response to alpha,beta-meATP (50 mu mol/l) in similar way to that in normal Krebs solution. The further addition of CaCl2 (1 mmol/l) abolished the inhibitory effects exerted by pyrogallol and DETCA. The control contractile response to alpha,beta-meATP (50 mu mol/l) was not affected in Ca2+-free high-K+ (80 mmol/l) Krebs solution. It may be concluded that superoxide anion stress produces a significant inhibitory effect on both mono-and di-nucleotide purinergic contraction of the vas deferens. Superoxide anion appears to interrupt the P2X(1)-mediated transduction cascade at some step(s) of intracellular calcium handling. Copyright (C) 2008 S. Karger AG, Base

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

Desferrioxamine Attenuates Doxorubicin-Induced Acute Cardiotoxicity through TFG-β/Smad p53 Pathway in Rat Model

Interaction of doxorubicin DOX with iron and the consequent generation of reactive oxygen species (ROS) is a major player in DOX-induced cardiomyopathy. Accordingly, this study has been initiated to investigate the preventive effect of the iron chelator, desferrioxamine (DFX), against DOX-induced acute cardiotoxicity in rats. Male Wistar albino rats were divided into four groups and were injected intraperitoneally (I.P.) with normal saline, a single dose of DOX (15 mg/kg), a single dose of DFX (250 mg/kg) and a combined treatment with DFX (250 mg/kg) 30 min prior to a single dose of DOX, (15 mg/kg). A single dose of DOX significantly increased mRNA expression of TGF-β, Smad2, Smad4, CDKN2A and p53 and significantly decreased Samd7 and Mdm2 mRNA expression levels. Administration of DFX prior to DOX resulted in a complete reversal of DOX-induced alteration in cardiac enzymes and gene expression to normal levels. Data from this study suggest that (1) DOX induces its acute cardiotoxicity secondary to increasing genes expression of TGF-β/Smad pathway. (2) DOX increases apoptosis through upregulation of CDKN2A and p53 and downregulation of Mdm2 gene expression. (3) The preventive effect of DFX against DOX-induced cardiotoxicity is mediated via the TGF-β1/Smad pathway

Metallothionein Induction Reduces Caspase-3 Activity and Tnfα Levels with Preservation of Cognitive Function and Intact Hippocampal Neurons in Carmustine-Treated Rats

Hippocampal integrity is essential for cognitive functions. On the other hand, induction of metallothionein (MT) by ZnSO4 and its role in neuroprotection has been documented. The present study aimed to explore the effect of MT induction on carmustine (BCNU)-induced hippocampal cognitive dysfunction in rats. A total of 60 male Wistar albino rats were randomly divided into four groups (15/group): The control group injected with single doses of normal saline (i.c.v) followed 24 h later by BCNU solvent (i.v). The second group administered ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v, once) then BCNU solvent (i.v) after 24 h. Third group received BCNU (20 mg/kg, i.v, once) 24 h after injection with normal saline (i.c.v). Fourth group received a single dose of ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v) then BCNU (20 mg/kg, i.v, once) after 24 h. The obtained data revealed that BCNU administration resulted in deterioration of learning and short-term memory (STM), as measured by using radial arm water maze, accompanied with decreased hippocampal glutathione reductase (GR) activity and reduced glutathione (GSH) content. Also, BCNU administration increased serum tumor necrosis factor-alpha (TNFα), hippocampal MT and malondialdehyde (MDA) contents as well as caspase-3 activity in addition to histological alterations. ZnSO4 pretreatment counteracted BCNU-induced inhibition of GR and depletion of GSH and resulted in significant reduction in the levels of MDA and TNFα as well as the activity of caspase-3. The histological features were improved in hippocampus of rats treated with ZnSO4 + BCNU compared to only BCNU-treated animals. In conclusion, MT induction halts BCNU-induced hippocampal toxicity as it prevented GR inhibition and GSH depletion and counteracted the increased levels of TNFα, MDA and caspase-3 activity with subsequent preservation of cognition