Gemcitabine induced cytotoxicity, DNA damage and hepatic injury in laboratory mice.

The present study was conducted to demonstrate cytotoxicity, apoptosis and hepatic damage induced by gemcitabine in laboratory mice. Animals were treated with a single dose of gemcitabine (415 mg/kg body wt), equivalent to a human therapeutic dose, and sacrificed after 1, 2 and 3 weeks. A significant decrease in mean body weight and absolute liver weight was registered. The levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased as a result of this induced stress. Various structural changes were observed in the liver tissue of treated mice, as evident in the histological sections. Specifically, gemcitabine exposure was able to induce apoptosis in liver cells, and the incidence of TUNEL positive liver cells was increased compared to the control group. DNA fragmentation appeared on agarose gel and flow cytometry analysis confirmed the induction of apoptosis. These findings in gemcitabine-treated animal tissues suggest that inhibition or disruption of cells' DNA synthesis may be the mechanism by which this drug induces toxicity in the animal body.

Proteomic approach to identify molecular signatures during experimental hepatic fibrosis and resveratrol supplementation.

We document the changes in sera and liver proteome during experimental hepatic fibrosis (HF) and its protection by resveratrol (Rsvtrl) in rats. HF was induced by N'-Nitrosodimethylamine (NDMA) administration thrice a week for 21-days. Compared to the control group, significant (P < 0.05) elevations in ALT, AST, ALP, γGT and bilirubin occurred during fibrosis. H&E, IHC for α-SMA and reticulin stainings of liver demonstrated that collagen amassing and HF were concurrent. Rsvtrl-supplementation refurbished hepatic architecture and lessened collagen deposition. 2DE proteome analyses showed ~398 (pH, 3-10) and ~129 signatures (pH, 4-7) within 5-201 kDa range in liver and sera, respectively. Nearly, 45 spots in liver and 18 in sera were differentially expressed in fibrotic and Rsvtrl-supplemented animals. MALDI-TOF-MS/MS analysis revealed at least ten signatures as the potential biomarkers for HF, because of the significant changes (>2 fold) in their expression. Out of them, two signatures have not so far been characterized and we have accomplished it in this study. Our proteomics data provides new evidence on an overall involvement of carbohydrate, lipid, Ca2+ signaling and oxidative pathways during NDMA-induced HF. More importantly, the oxidative pathway appears to be the principal mediator in the NDMA-induced HF as well as the hepato-protection by Rsvtrl.

Resveratrol mitigate structural changes and hepatic stellate cell activation in N'-nitrosodimethylamine-induced liver fibrosis via restraining oxidative damage.

Resveratrol, a polyphenol, found in skin of red grapes, peanuts and berries possesses anti-inflammatory, anti-carcinogenic and lipid modulation properties. Here, we demonstrate in vivo antifibrotic activity of resveratrol in a mammalian model, wherein hepatic fibrosis was induced by N'-nitrosodimethylamine (NDMA) administration. Apart from being a potent hepatotoxin, NDMA is a known mutagen and carcinogen, as well. To induce hepatic fibrosis, rats were administered NDMA (i.p.) in 10mg/kgb.wt thrice/week for 21 days. Another group of animals received resveratrol supplement (10mg/kgb.wt) subsequent to NDMA administration and were sacrificed weekly. The changes in selected biomarkers were monitored to compare profibrotic effects of NDMA and antifibrotic activity of resveratrol. The selected biomarkers were: sera transaminases, ALP, bilirubin, liver glycogen, LPO, SOD, protein carbonyl content, ATPases (Ca(2+), Mg(2+), Na(+)/K(+)) and hydroxyproline/collagen content. Alterations in liver architecture were assessed by H&E, Masson's trichrome and reticulin staining of liver biopsies. Immuno-histochemistry and immunoblotting were employed to examine expression of α-SMA. Our results demonstrate that during NDMA-induced liver fibrosis transaminases, ALP, bilirubin, hydroxyproline and liver collagen increases, while liver glycogen is depleted. The decline in SOD (>65%) and ATPases, which were concomitant with the elevation in MDA and protein carbonyls, strongly indicate oxidative damage. Fibrotic transformation of liver in NDMA-treated rats was verified by histopathology, immuno-histochemistry and immunoblotting data, with the higher expressivity of α-SMA-positive HSCs being most established diagnostic immuno-histochemical marker of HSCs. Resveratrol-supplement refurbished liver architecture by significantly restoring levels of biomarkers of oxidative damage (MDA, SOD, protein carbonyls and membrane-bound ATPases). Therefore, we conclude that antifibrotic effect of resveratrol is due to restrained oxidative damage and down-regulation of α-SMA, which inhibits HSC activation to obstruct liver fibrosis.

Vitamin B12 supplement alleviates N'-nitrosodimethylamine-induced hepatic fibrosis in rats.

Abstract Context: Altered vitamin B12 levels have been correlated with hepatotoxicity; however, further evidence is required to establish its protective role. Objective: To evaluate the effects of vitamin B12 supplement in protecting N'-nitrosodimethylamine (NDMA)-induced hepatic fibrosis in Wistar rats. Materials and methods: Hepatic fibrosis was induced by administering NDMA in doses of 10 mg/kg body weight thrice a week for 21 days. Another group received equal doses (10 mg/kg body weight) of vitamin B12 subsequent to NDMA treatment. Animals from either group were sacrificed weekly from the start of the treatment along with their respective controls. Progression of hepatic fibrosis, in addition to the effect of vitamin B12, was assessed biochemically for liver function biomarkers, liver glycogen, hydroxyproline (HP) and B12 reserves along with histopathologically by hematoxylin and eosin (H & E) as well immunohistochemical staining for α-SMA expression. Results and discussion: Elevation in the levels of aminotransferases, SALP, total bilirubin and HP was observed in NDMA treated rats, which was concomitant with remarkable depletion in liver glycogen and B12 reserves (p < 0.05). Liver biopsies also demonstrated disrupted lobular architecture, collagen amassing and intense fibrosis by NDMA treatment. Immunohistochemical staining showed the presence of activated stellate cells that was dramatically increased up to day 21 in fibrotic rats. Following vitamin B12 treatment, liver function biomarkers, glycogen contents and hepatic vitamin B12 reserves were restored in fibrotic rats, significantly. Vitamin B12 administration also facilitated restoration of normal liver architecture. Conclusion: These findings provide interesting new evidence in favor of protective role for vitamin B12 against NDMA-induced hepatic fibrosis in rats.

Understanding the mechanism of hepatic fibrosis and potential therapeutic approaches.

Hepatic fibrosis (HF) is a progressive condition with serious clinical complications arising from abnormal proliferation and amassing of tough fibrous scar tissue. This defiance of collagen fibers becomes fatal due to ultimate failure of liver functions. Participation of various cell types, interlinked cellular events, and large number of mediator molecules make the fibrotic process enormously complex and dynamic. However, with better appreciation of underlying cellular and molecular mechanisms of fibrosis, the assumption that HF cannot be cured is gradually changing. Recent findings have underlined the therapeutic potential of a number of synthetic compounds as well as plant derivatives for cessation or even the reversal of the processes that transforms the liver into fibrotic tissue. It is expected that future inputs will provide a conceptual framework to develop more specific strategies that would facilitate the assessment of risk factors, shortlist early diagnosis biomarkers, and eventually guide development of effective therapeutic alternatives.

Effect of N'-nitrosodimethylamine on red blood cell rheology and proteomic profiles of brain in male albino rats.

We investigated the effects of N'-nitrosodimethylamine (NDMA) induced toxicity on red blood cell rheology in male rats and identified bands in proteomic profiles of brain which can be used as novel markers. Polyacrylamide gel electrophoresis (PAGE) profiles exhibited constitutive as well as induced expression of the polypeptides. Remarkably, the molecular weight range of the polypeptides (8-150 kDa) corresponded to that of the family of heat shock proteins. Our results revealed significant changes in blood parameters and showed the presence of acanthocytes, tear drop cells, spicules and cobot rings in the treated categories. Lactate dehydrogenase and esterase zymograms displayed a shift to anaerobic metabolism generating hypoxia-like conditions. This study strongly suggests that NDMA treatment causes acute toxicity leading to cell membrane destruction and alters protein profiles in rats. It is therefore recommended that caution should be exercised in using NDMA to avoid risks, and if at all necessary strategies should be designed to combat such conditions.