Simultaneous Determination of Different Polyamines and their Mono-Acetylated Derivatives in Gastric Tissue by HPLC with Post-Column Derivatization

A simple and highly sensitive procedure is described enabling the simultaneous determination of biogenic polyamines (PAs) and their related monoacetyl derivatives in stomach tissue. The method is based on HPLC using octane sulfonate as an ion-pairing agent employed in acetate buffers at pH 4.5. The application is accompanied with fluorescence detection followed by post-column derivatization with o-phthaldialdehyde at room temperature (20±0.5°C). N1- and N8-acetylspermidines (ASPD) can be determined with this method in the same run without performing any special procedures or pre-purification in concentrations exceeding 8.5 pmoles. The variability in reproducibility of the day-today precision and duplicate determination, and simultaneous determination of standard mixture and biological samples were found < 2%. The mean (± s.e.mean) retention times (n=12) for putrescine (Put), N1-ASPD, N8-ASPD, spermidine (Spd) and spermine (Spm) are 8.97±0.025; 17.64±0.063; 18.99±0.133; 28.20±0.070 and 39.81±0.098 min, respectively. The method was applied to determine PAs and specifically N1- and N8-ASPD in glandular part of stomach tissue of fasting rats (STFR) without any interference with endogenous aminoacids, histamine, and other reactive moieties. PAs and both mono-ASPD have been successfully determined in the STFR and the values are as follows: Put 37.2±10.1; N1-ASPD 5.88±0.48; N8-ASPD 4.43±0.94; Spd 750.7±22.7 and Spm 618.2±37.4 nmole/g of wet tissue. Information on gastric tissue polyamines and their acetylated derivatives may be useful in understanding the mechanism of drugs or agents that play some part in gastric ulcer production or its repair mechanisms

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

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

Acacia senegal gum exudate offers protection against cyclophosphamide-induced urinary bladder cytotoxicity

Cylophosphamide (CYCL) is a strong anticancer and immunosuppressive agent but its urotoxicity presents one of the major toxic effects that limit its wide usage particularly in high dose regimens. Therefore, this study aimed to investigate Acacia Senegal gum exudate, Gum Arabic (GA), for its possible role as a natural, nontoxic agent against CYCL-induced urotoxicity. Male Swiss albino rats were exposed to CYCL (150 mg/kg BW, once i.p) with or without GA oral supplementation (7.5 g/kg/day for 6 days) through drinking water. Glutathione (GSH), Malondialdehyde (MDA) and Nitric oxide (NO) bladder contents were assessed. Responsiveness of the bladder rings to acetylcholine (ACh) in vitro, microscopic and macroscopic features are also investigated. CYCL produced pronounced harmful effects on bladder urothelial lining with significant increases in (MDA) and NO levels in the tissue homogenates. Bladder-GSH content is dropped by over 60% following CYCL injection. Bladder contractility, as measured by its responsiveness to ACh, recorded a marked reduction. The isolated bladders exhibited such macroscopic changes as severe edema, inflammation and extravasation. The bladder weight increased as well. Histological changes were evident in the form of severe congestion, petechial hemorrhage and chronic inflammatory reaction in the lamina propria accompanied with desquamated epithelia. GA, a potential protective agent, produced an almost complete reversal of NO induction, lipid peroxidation or cellular GSH bladder contents in the GA + CYCL-treated group. Likewise, bladder inflammation and edema were reduced. Bladder rings showed a remarkable recovery in their responsiveness to ACh. Bladder histological examination showed a near normal configuration and structural integrity, with a significant reduction in inflammation and disappearance of focal erosions. These remarkable effects of GA may be attributed to its ability to neutralize acrolein, the reactive metabolite of CYCL and/or the resultant reactive oxygen metabolites, through a scavenging action. GA may limit the cascading events of CYCL-induced damage, initiating a cytoprotective effect leading to structural and functional recovery of the bladder tissues

Association between Paraoxonases Gene Expression and Oxidative Stress in Hepatotoxicity Induced by CCl 4

Objectives. The purpose of the study is to evaluate the hepatoprotective effect of rutin in carbon tetrachloride- (CCl4-) induced liver injuries in rat model. Methods. Forty male Wistar albino rats were divided into four groups. Group I was the control group and received dimethyl sulphoxide (DMSO) and olive oil. Group II received rutin. Groups III was treated with CCl4. Group IV was administered rutin after 48 h of CCl4 treatment. Liver enzymes level, lipid profile, lipid peroxidation, and hydrogen peroxide were measured. The genes expression levels were monitored by real time RT-PCR and western blot techniques. Results. CCl4 group showed significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), thiobarbituric acid reactive substances (TBAR), hydrogen peroxide (H2O2), and lipid profile and a significant decrease in glutathione peroxidase (GPx), glutathione S transferase (GST), catalase (CAT), paraoxonase-1 (PON-1), paraoxonase-3 (PON-3), peroxisome proliferator activated receptor delta (PPAR-δ), and ATP-binding cassette transporter 1 (ABAC1) genes expression levels. Interestingly, rutin supplementation completely reversed the biochemical and gene expression levels induced by CCl4 to control values. Conclusion. CCl4 administration causes aberration of genes expression levels in oxidative stress pathway resulting in DNA damage and hepatotoxicity. Rutin causes hepatoprotective effect through enhancing the antioxidant genes

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

Anti-metastatic and anti-proliferative activity of eugenol against triple negative and HER2 positive breast cancer cells

Abstract Background Eugenol is a natural phenolic compound and possesses anticancer and antibacterial activities. Breast cancer is a major global health problem, and most of the chemotherapeutic agents are highly toxic with long-term side effects. Therefore, this study aimed to explore the possibility of using eugenol as an anti-metastatic and anti-proliferative agent against MDA-MB-231 and SK-BR-3 breast cancer cells. Methods Breast cancer cell lines MDA-MB-231 and SK-BR-3 were treated with eugenol and cell proliferation was measured using a real-time cell electronic sensing system. Annexin V analysis with flow cytometry was used to detect the effect of eugenol on cell death. In MDA-MB-231 and SK-BR-3 cells, metastatic potential after eugenol treatment was examined using a wound-healing assay. Real-time PCR was used to study the effect of eugenol on the expression of anti-metastatic genes such as MMP2, MMP9, and TIMP-1, and genes involved in apoptosis including Caspase3, Caspase7, and Caspase9. Results Treatment with 4 μM and 8 μM eugenol for 48 h significantly inhibited cell proliferation of MDA-MB-231, with an inhibition rate of 76.4%, whereas 5 μM and 10 μM of eugenol for 48 h significantly inhibited the proliferation of SK-BR-3 cells with an inhibition rate of 68.1%. Eugenol-treated cells showed significantly decreased MMP2 and MMP9 expression and an insignificant increase in TIMP1 expression in HER2 positive and triple negative breast cancer cells. Eugenol significantly increased the proportion of MDA-MB-231 and SK-BR-3 cells in late apoptosis and increased the expression of Caspase3, Caspase7, and Caspase9. Conclusion To the best of our knowledge, this is the first study to describe the anti-metastatic effect of eugenol against MDA-MB-231 and SK-BR-3 breast cancer cell lines

Antinociceptive Activity of Vigabatrin in Mice After Prolonged Treatment: Possible Development of Tolerance

We have evaluated vigabatrin (γ-vinyi-γ-aminobutyric acid), an irreversible inhibitor of γ-aminobutyric acid (GABA)-transarninase responsible for GABA degradation, for its effects on nociceptive response, changes in spontaneous locomotor activity and body temperature in mice after a prolonged treatment regimen. The mice received vigabatrin 0.26% w/v chronically in drinking water for 7, 14 and 21 days. Changes in locomotion, body temperature and nociception were recorded after 7, 14 and 21 days respectively in different groups. Also, possible withdrawal symptoms were determined up to three days after 7, 14 or 21 days of treatment. In another experiment the animals were given acutely, 250 mg/kg of vigabatrin by oral gavage and the changes in response to the said parameters were assessed 90 min after treatment. Acute treatment increased the latency in the hot-plate reaction time and a highly significant decline in locomotion and body temperature. In contrast to the acute treatment studies, there were essentially no effects of vigabatrin on nociception, locomotor activity or body temperature either on the last day of each treatment or upon withdrawal for the next consecutive three days. We conclude that the changes in nociception, locomotion and body temperature after acute treatment with vigabatrin are due to neuromediator interactions and a possible direct effect of GABA accumulation. After prolonged treatment tolerance to the pharmacological effects of vigabatrin develop that was evident by no change in nociception, locomotion and body temperature. This may be attributed to the possible failure in the maintenance of GABA pools resulting in a reduction in enhanced GABA release mediated by vigabatrin in acute treatment. Further studies of mechanisms by which vigabatrin tolerance develops to these pharmacological responses are warranted