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Chronic tissue injury resulting in fibrosis of multiple organs, responsible for one-third of the death globally. Liver fibrosis is a common pathway/condition involved in all chronic liver diseases. Thioacetamide (TAA), a hepatotoxicant, was used to induce hepatic fibrosis. Anti-diabetic drug glibenclamide (GLB) possesses anti-inflammatory properties and inhibits NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation. Dimethyl fumarate (DMF), a multiple sclerosis drug, activates the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and maintains the antioxidant status in the cell. The present study was designed to investigate (i) role of NLRP3 inflammasome and Nrf2/ARE pathway in TAA-induced hepatotoxicity and liver fibrosis, (ii) mechanism involved in GLB and DMF mediated hepatoprotection against TAA-induced hepatotoxicity, and (iii) additional/synergistic hepatoprotective effect of combination treatment with NLRP3 inhibition + Nrf2 activation or GLB + DMF or MCC950 + 4OI to reverse/ameliorate the experimental liver fibrosis completely. TAA was administered intraperitoneally to mice for seven consecutive weeks, and treatments of GLB, DMF, GLB + DMF, MCC950, 4OI, and MCC950 + 4OI were provided for the last three consecutive weeks. The intervention with GLB, DMF, GLB + DMF, MCC950, 4OI, and MCC950 + 4OI significantly protected TAA-induced oxidative stress and inflammatory conditions by improving biochemical, histological, and immunoexpression changes in mice. The GLB, DMF, and GLB + DMF intervention exhibited a better protective effect compared with MCC950, 4OI, and MCC950 + 4OI, which revealed that this specific inhibitor/activator possesses only NLRP3 inflammasome inhibitory/Nrf2 activatory properties. In contrast, the clinical drug GLB and DMF have several other beneficial effects, which are independent of NLRP3 inhibition and Nrf2 activation.

Ethanol consumption increases the prevalence of gastric ulcer (GU) in rats with type II diabetes (T2D). Induction of GU by absolute ethanol (5 mL/kg or 3.94 g/kg) in the animal model resembles human ulcer characteristics. The aim was to investigate the role of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway in the treatment of GU in diabetic condition. The rats were exposed to absolute ethanol 1 h before sacrifice and T2D was induced by combined exposure of high-fat diet and low dose streptozotocin. Pretreatment of tert-butylhydroquinone (tBHQ) (25 and 50 mg/kg), metformin (500 mg/kg), and omeprazole (20 mg/kg) were given once daily for last three consecutive weeks. In ethanol-exposed diabetic rats, pretreatment with tBHQ, omeprazole, and metformin reduced gastric mucosal lesion, ulcer index, histological alterations, malondialdehyde level, and apoptosis. Furthermore, the intervention of tBHQ, omeprazole, and metformin improved the integrity of the stomach mucosa, glutathione, gastric pH, collagen, and goblet cells. tBHQ treatment improved ethanol-induced alterations of Nrf2, catalase, heat shock protein 70 (HSP70), NF-κB, and endothelin-1 expressions in diabetic rats. In diabetic conditions, the incidence of GU is increased due to elevated levels of reactive oxygen species, inflammatory mediators, depleted levels of cellular antioxidants, and altered gastric parameters. The tBHQ intervention could be a rational strategy to protect these changes.

Diabetes increases the possibility of germ cell damage, hypogonadism, and male infertility. Diabetic condition negatively impacts zinc (Zn) and selenium (Se) levels in the body. Zn and Se are among the most important trace elements involved in the regulation of redox reaction, antioxidants enzymes activities, and DNA expression in a germ cell. The present study aimed to elucidate the combined effects of Zn and Se treatment on diabetes-induced germ cell damage in male Sprague Dawley rats. Type 1 diabetes was induced by the single intraperitoneal (i.p.) injection of streptozotocin (55 mg/kg). Zn (3 mg/kg, i.p.) and Se (0.5 mg/kg, i.p.) were administered daily for 8 consecutive weeks. All the animals were provided with normal feed and water throughout the study. The effects on germ cell damage were evaluated by body weight, feed-water intake, organ weight, sperm count, motility, sperm head morphology, biochemical analysis, histology, immunohistochemistry, halo assay, germ cell comet assay, testes terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling (TUNEL) assay, sperm TUNEL assay, serum protein pattern analysis, and subcellular analysis using transmission electron microscopy. Further, the expressions of nuclear erythroid-derived related factor 2, catalase, glutathione peroxidase 4, and glutathione peroxidase 5 were carried out to ascertain the mechanism of protection. The present results demonstrated that 8 weeks combined treatment of Zn (3 mg/kg, i.p.) and Se (0.5 mg/kg, i.p.) reduced diabetes-induced germ cell damage. This study further highlighted that Zn and Se combination treatment might be a better strategy for the germ cell protection in diabetes and deserve further investigation.

Two-stage rat hepatocarcinogenesis model was used to induce early carcinogenesis in which thioacetamide (TAA) promotes diethylnitrosamine (DEN) initiated carcinogenesis. Dimethyl fumarate (DMF) used to treat multiple sclerosis, activates the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway during oxidative stress, and maintains antioxidant levels. Glibenclamide (GLB), a sulphonylurea drug used to treat type II diabetes, possesses anti-inflammatory properties and inhibits NLRP3 inflammasomes. The present study was designed to investigate the concurrent intervention of DMF and GLB on DEN + TAA-induced early hepatic carcinogenesis. DMF and GLB treatment improved DEN + TAA-induced decrease in body weight, increase in liver weight and plasma transaminases, histopathological alterations, DNA damage, and apoptosis. DMF and GLB intervention significantly ameliorated the DEN + TAA-induced alterations in the antioxidant (Nrf2, HO-1, SOD-1, catalase), inflammatory (NF-κB, NLRP3, ASC, caspase-1), fibrogenic (TGF-β1, collagen) and regenerative proliferative stress (GST-p, HGF, c-MET, TGFα, EGF, AFP) markers. The present results indicate that Nrf2/ARE activation and NLRP3 inhibition might be a rational approach to attenuate oxidative stress and chronic inflammation associated progression of hepatocarcinogenesis.

Gastric ulcer (GU) is the most common health concern that occurs due to alcohol consumption, smoking and physiological stress. Ethanol-induced GU in animal model resembles the pathophysiology of human ulcer. The present study was designed to investigate the cytoprotective and anti-inflammatory properties of tert-butylhydroquinone (tBHQ), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, against gastric mucosal damage induced by acute exposure of ethanol (5 ml/kg). The intervention of tBHQ (25 and 50 mg/kg, per os (po)) and omeprazole (20 mg/kg, po) was done for 10 consecutive days. Omeprazole was chosen as a standard drug because it is prescribed for the treatment of GU. Pretreatment of tBHQ decreased gastric mucosal lesion, ulcer index, apoptotic cells and lipid peroxidation level induced by ethanol. Furthermore, the intervention of tBHQ increased gastric mucosa integrity, pH, reduced glutathione, collagen and mucus-producing goblet cells. Intervention of tBHQ increased the expression of antioxidant markers such as Nrf2, haeme oxygenase-1 and catalase and decreased the expressions of inflammatory markers such as nuclear factor kappa-light-chain-enhancer of activated B cells and cyclooxygenase-2. The cytoprotective potential of tBHQ against gastric mucosal damage might be due to its ability to enhance cellular antioxidants and anti-inflammatory responses.

Zinc (Zn) plays an important role in maintaining the process of spermatogenesis and reproductive health. Bisphenol A (BPA), an endocrine disrupting chemical is known to be a reproductive toxicant in different animal models. The present study was designed to study the effect of two of the utmost determinative factors (Zn deficient condition and influence of toxicant BPA) on germ cell growth and overall male reproductive health in the testis, epididymis, and sperm using (a) biochemical, (b) antioxidant, (c) cellular damage, (d) apoptosis, and (e) protein expression measurements. Rats were divided into Control (normal feed and water), BPA (100 mg/kg/d), zinc deficient diet (ZDD; fed with ZDD), and BPA + ZDD for 8 weeks. Body and organ weights, sperm motility and counts, and sperm head morphology were evaluated. The histology of testes, epididymides, and prostate was investigated. Testicular deoxyribonucleic acid (DNA) damage was evaluated by Halo and Comet assay, apoptosis of sperm and testes were quantified by TUNEL assay. Serum protein electrophoretic patterns and testicular protein expressions such as Nrf-2, catalase, PCNA, and Keap1 were analyzed by Western blot analysis. The results showed that BPA significantly increased the testicular, epididymal, and prostrate toxicity in dietary Zn deficient condition due to testicular hypozincemia, hypogonadism, increased cellular and DNA damage, apoptosis, as well as perturbations in protein expression.

Cyclophosphamide (CP) is a widely used anticancer and immunosuppressant drug. Nevertheless, clinical utilization of CP is limited due to considerable adverse effects and toxicities. Nicotinamide (NMD) is a micronutrient and the effect of NMD against CP-induced hepatotoxicity is yet unexplored. The present study was designed to evaluate the chemoprotective effect of NMD against CP-induced hepatic injury in Sprague-Dawley rats. Hepatotoxicity was induced by the administration of CP (30 mg/kg/day) for 10 consecutive days by intraperitoneal injection. The chemoprotective effect of NMD treatment (200 mg/kg) against CP-induced hepatotoxicity was evaluated by the oxidative stress, liver function, histopathological changes, and DNA damage. NMD cotreatment significantly reduced CP-induced oxidative stress, histological changes, and apoptosis in the liver. The present study demonstrated that NMD treatment ameliorated CP-induced hepatic damage by improving the antioxidant system and reducing DNA damage. The present findings revealed that NMD supplementation might be useful to reduce CP-associated hepatotoxicity, and thereby can increase the therapeutic utility of CP.

Glibenclamide (GLB), most widely used in the treatment of type II diabetes mellitus, inhibits K+ATP channel in pancreatic-β cells and releases insulin, while thioacetamide (TAA) is a well-known hepatotoxicant and most recommended for the induction of acute and chronic liver disease. The purpose of this study was to evaluate the hepatoprotective potential of GLB against TAA-induced hepatic damage in Wistar rats. TAA (200 mg/kg, ip, twice weekly) and GLB (1.25, 2.5, and 5 mg/kg/day, po) were administered for 6 consecutive weeks. Different biochemical, DNA damage, histopathological, TEM, immunohistochemical, and western blotting parameters were evaluated. GLB treatment has no effects on the TAA-induced significant decrease in body and liver weights. TAA treatment significantly increased liver index and treatment with GLB has no effect the same. TAA treatment altered the liver morphology, whereas treatment with GLB normalized the alteration in morphology. Further, significant increase in oxidative stress, apoptosis, and DNA damage was found in TAA-treated animals and GLB treatment significantly reduced these effects. TAA-induced plasma transaminases and serum ALP levels were significantly restored by GLB. Furthermore, histopathological findings showed the presence of lymphocyte infiltration, collagen deposition, bridging fibrosis, degeneration of portal triad, and necrosis in TAA-treated animals and GLB intervention significantly reduced the same. TEM images revealed that GLB significantly normalized the hepatic stellate cell morphology as well as restored the number of lipid droplets. GLB treatment significantly downregulated the expressions of TGF-β1, α-SMA, NLRP3, ASC, caspase-1, and IL-1β, and upregulated MMP-2 and catalase against TAA-induced liver damage. The outcomes of the present study confirmed that GLB ameliorated the liver damage induced by TAA.

Zinc (Zn) was proved to be a germ cell protectant against various disease conditions and toxic insults. Besides other mechanisms, here we have explored the important role of Zn and Zn-dependent SOD1in methotrexate (MTX)-induced germ cell damage. MTX was given 5 mg/kg i.p. once a week for four consecutive weeks, while Zn was supplemented daily at the doses of 3 and 6 mg/kg i.p. for four consecutive weeks. After four weeks of treatment the animals were sacrificed and observed for various end points. There were several histopahtological alterations in the testes like desquamation and altered tubular structures. DNA damage was also increased by MTX as evident by TUNEL assay. Sperm head abnormalities were increased in case of MTX treated animals. Protein expressions of PCNA, BCl-2/Bax, SOD, catalase and GPX5 were found to be altered by the MTX treatment. To further investigate the role of Zn and Zn-dependent SOD1, rats were injected intratesticularly with diethyldithiocarbamate (DEDTC) for three days after MTX 20 mg/kg i.p. was given on the first day. DEDTC in combination with MTX was found to significantly decrease the protein expressions of SOD1, catalase, Nrf2 and GPX4, along with deranged histology. This study adds to the point that Zn might be a better germ cell protectant and deserve further investigation.

Zinc (Zn) is one of the most important trace elements in the body and is required for insulin secretion and release. Zn is also required for the growth and development of the reproductive system. Alteration in the Zn levels can cause moderate to severe damage to various organs, including the reproductive system. Most of type 2 diabetic patients have altered Zn levels/signaling. So, here we investigated the role of Zn-deficient diet (ZDD) in type 2 diabetes. Type 2 diabetes in the rat was induced by the combination of high-fat diet (HFD) and a single low dose of streptozotocin (STZ, 35 mg/kg, i.p.). Control animals were fed normal pellet diet throughout the study, while ZDD was given for four consecutive weeks to the diabetic rats, which were earlier kept on HFD for 16 weeks. The present findings showed that ZDD further decreased the serum Zn, plasma insulin and serum testosterone levels, whereas it increased cholesterol, triglycerides, BUN, %HbA1c in diabetic rats. Oxidative stress in testes was increased by ZDD as evidenced by decreased glutathione, catalase and SOD1 levels. ZDD-induced several abnormalities in sperm head morphology, altered sperm decondensation, sperm chromatin and protamine content, along with significant histopathological alterations in testes and epididymis. Further, ZDD altered protein levels of MT, MTF-1, Keap1, Nrf2, Nf-κB, GPX4 and GPX5 levels in the testes and epididymis of diabetic rat. The present results demonstrated that dietary Zn deficiency could exacerbate type 2 diabetes-induced germ cell damage.

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)-induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA-induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA-induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains-containing protein 3; NLRP3, apoptosis-associated speck like protein containing a caspase recruitment domain; ASC, caspase-1, nuclear factor-kappa B; NF-κB, interleukin-6), fibrogenic makers (α-smooth muscle actin; ɑ-SMA, transforming growth factor; TGF-β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid-derived 2)-like factor 2; Nrf2, superoxide dismutase-1; SOD-1, catalase). The present findings concluded that DMF protects against TAA-induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is much higher in diabetic and obese individuals. Combined exposure of high-fat diet (HFD) and single low-dose streptozotocin (STZ) was used to induce type II diabetes–associated NAFLD, as it better replicates the human pathology of fatty liver. Glibenclamide (GLB) is a potent NLRP3 inflammasome inhibitor and possesses anti-inflammatory and anti-oxidant properties. So it was pertinent to investigate its hepatoprotective potential against NAFLD in rat. HFD was provided to rat for 17 consecutive weeks and glibenclamide (GLB; 0.5 and 2.5 mg/kg/day, orally) was administered for the last 12 consecutive weeks. Establishment of NAFLD was clearly indicated by significant increase in liver weight, glucose, triglyceride, cholesterol, % glycosylated haemoglobin and insulin levels, and GLB intervention reduced the same. GLB restored HFD-induced significant increase in ROS, MDA and decrease in GSH. Histopathological studies revealed the macro- and micro-vascular steatosis and mild degree of inflammation in HFD-fed rat compared with control, and GLB intervention reduced the same. HFD exposure significantly increased the DNA damage and apoptosis compared with control, and GLB intervention reduced the same. Immunohistochemical and immunoblotting findings showed that GLB improved the hepatic expressions of inflammatory markers (NLRP3, ASC, caspase-1, IL-1β, NF-κB), anti-oxidant markers (SOD, catalase) and insulin signalling markers (p-AKT, p-GSK-3β, p-IRS). Hepatoprotective effects of GLB was mediated by decreasing the levels of glucose, triglycerides, cholesterol, DNA damage, apoptosis and inflammatory markers, and by improving the anti-oxidant status and insulin signalling pathway in HFD fed rat.

Recent evidences highlighted that histone deacetylases (HDACs) can deacetylate the histone, various transcription factors and regulatory proteins, which directly or indirectly affect glucose metabolism. The present study aimed to evaluate the comparative effects of sodium butyrate (NaB) and metformin on the glucose homeostasis, insulin-resistance, fat accumulation and dyslipidemia in type-2 diabetic rat. Diabetes was developed in Sprague-Dawley rats by the combination of high-fat diet (HFD) and low dose streptozotocin (STZ, 35 mg/kg). NaB at the doses of 200 and 400 mg/kg twice daily as well as metformin (as a positive control) 150 mg/kg twice daily for 10 consecutive weeks were administered by i.p. and oral route, respectively. NaB treatment significantly reduced the plasma glucose, HbA1c, insulin-resistance, dyslipidemia and gluconeogenesis, which are comparable to metformin treatment. Further, NaB treatment ameliorated the micro- and macro-vesicular steatosis in liver and fat deposition in brown adipose tissue, white adipose tissue (adipocytes hypertrophy) as well as pancreatic beta-cell damage. In the present study, both NaB and metformin inhibited the diabetes-associated increased HDACs activity, thereby increased the acetylation of histone H3 in liver. The present findings demonstrated that NaB and metformin reduced insulin-resistance, dyslipidemia, fat accumulation and gluconeogenesis thereby improved the glucose homeostasis in rat. Thus, NaB might be a promising molecule for the prevention and treatment of type-2 diabetes and dyslipidemia.

Zinc (Zn) is one of the most important trace elements required for several biological processes. Diabetes negatively affects many organs, and diabetic patients are often hypozincemic. The present study aims to investigate the role of Zn supplementation in the testes, epididymis, and sperms of streptozotocin (STZ)-induced diabetic rat. Serum, testicular, and sperm Zn contents were found to be altered in diabetic rat. Biochemical, histopathological, and protein expression profiles were determined to decipher the role of Zn in protecting the cellular perturbations. Further, histopathological analyses of testes and epididymis showed deranged architecture along with other noted abnormalities. Diabetic testes showed decreased Nrf2, HO-1, SOD1, PCNA, and Bcl-2 expressions whereas increased COX-2, NF-κB, MT, IL-6, and p-ERK levels. SOD1 and GPX5 were decreased in the epididymis of diabetic rat, whereas Zn supplementation attenuated these changes. The present results demonstrate the beneficial role of Zn supplementation in diabetes-associated testicular alterations of rat.

Oxidative stress and inflammation are the mediators of diabetes and related secondary complications. Oxidative stress arises because of the excessive production of reactive oxygen species and diminished antioxidant production due to impaired Nrf2 activation, the master regulator of endogenous antioxidant. It has been established from various animal models that the transcription factor Nrf2 provides cytoprotection, ameliorates oxidative stress, inflammation and delays the progression of diabetes and its associated complications. Whereas, deletion of the transcription factor Nrf2 amplifies tissue level pathogenic alterations. In addition, Nrf2 also regulates the expression of numerous cellular defensive genes and protects against oxidative stress-mediated injuries in diabetes. The present review provides an overview on the role of Nrf2 in type 1 diabetes and explores if it could be a potential target for the treatment of diabetes and related complications. Further, the rationality of different agent’s intervention has been discussed to mitigate organ damages induced by diabetes.

Several reports indicated that histone deacetylases (HDACs) play a crucial role in inflammation and fibrogenesis. Sodium butyrate (SB) is a short-chain fatty acid having HDAC inhibition potential. The present study aimed to evaluate the protective effect of SB against L-arginine (L-Arg)-induced pancreatic fibrosis in Wistar rats. Pancreatic fibrosis was induced by twice intraperitoneal (i.p.) injections of 20% L-Arg (250 mg/100 g) at 2-h interval on day 1, 4, 7, and 10, whereas SB (800 mg/kg/day) was administrated for 10 days. At the end of the study, biochemical estimations, histological alterations, DNA damage, and the expression of various proteins were evaluated. Posttreatment of SB decreased L-Arg-induced oxidative and nitrosative stress, DNA damage, histological alterations, and fibrosis. Interestingly, posttreatment of SB significantly decreased the expression of α-smooth muscle actin, interleukin-1β, inducible nitric oxide synthase, and 3-nitrotyrosine. The present study demonstrated that posttreatment of SB alleviates L-Arg-induced pancreatic damage and fibrosis in rat.

Epigenetic modifications are increasingly recognized to play a significant contribution in diabetic nephropathy (DN). Histone deacetylases (HDACs) are the emerging target in the pathogenesis and progression of DN. Valproic acid (VPA), a widely used anti-epileptic drug and has been proven as an HDAC inhibitor. This study was aimed to evaluate the protective roles of VPA on HDAC-mediated NF-κB/iNOS signaling and autophagy in DN. Diabetes was induced by a single injection of STZ (50 mg/kg), whereas VPA at the doses of 150 and 300 mg/kg/day for 8 weeks was administered by oral route in Sprague–Dawley rat. Blood and urine were collected before animal were sacrificed, while kidneys were dissected after sacrificed. The podocyte and renal injuries were assessed using biochemical markers, histology, podocyte effacement, DNA damage and apoptosis as well as protein expression evaluation. VPA treatment improves the plasma and urinary biomarkers of renal function, decreased expression of iNOS, 3-nitrotyrosine, NF-κB, p-NF-κB, HDAC4/5, calmodulin, calbindin, apoptosis and DNA damage. Further, VPA treatment increased histone acetylation and ameliorated the histological alterations and podocyte effacement. Interestingly, VPA treatment also restored diabetes-associated perturbations in autophagy by HDAC inhibition. To the best of our knowledge, this is the first report, which highlights the beneficial role of VPA in DN. The present results clearly exhibited that VPA treatment ameliorates the podocyte and renal injuries mainly by facilitating the autophagy and inactivation of NF-κB/iNOS signaling. The present findings demonstrated that VPA may be useful in the treatment of DN, since the present experimental doses are clinically relevant.

Colon carcinogenesis is long known to be associated with ulcerative colitis (UC), a chronic gastrointestinal disorder. Various pre-clinical and clinical studies have shown that melatonin (MEL) has beneficial effects in cancer. However, elucidation of the detailed molecular mechanisms involved in MEL-mediated protection against the colon carcinogenesis deserves further investigation. The present study was aimed at deciphering the effect of MEL on autophagy and Nrf2 signaling pathways in a mouse model of colitis-associated colon carcinogenesis (CACC). For the induction of CACC, male Swiss Albino mice were administered a single ip injection of 20 mg 1, 2-dimethylhydrazine dihydrochloride (DMH)/kg bw, followed by 3 cycles of 3% w/v dextran sulfate sodium (DSS) in drinking water treatment initiated 1 wk after DMH injection. One week after the initiation of DSS treatment, MEL was administered at the dose of 1 mg/kg, bw, po for 8 and 18 wk. Mice were sacrificed at 10 and 20 wk after DMH injection. MEL treatment decreased the progression of CACC by down regulating the process of autophagy as revealed by the expression pattern of various autophagy markers such as Beclin-1, LC3B-II/LC3B-I ratio and p62. These findings were accompanied with the increased expression of Nrf2 and the associated antioxidant enzymes, NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase-1 (HO-1) in the colon of mice with CACC. MEL intervention reduced autophagy by ameliorating inflammation and oxidative stress in the colon of mice with CACC. We conclude that MEL treatment attenuates the progression of CACC in mice by modulating autophagy and Nrf2 signaling pathways. © 2015 Wiley Periodicals, Inc.

Valproic acid (VPA) has been reported as inhibitor of histone deacetylases (HDACs). Several reports indicated that HDACs play a crucial role in the pathogenesis of fibrosis and hepatic stellate cell (HSC) activation. The present study was aimed to evaluate the anti-fibrotic effect of VPA against thioacetamide (TAA)-induced hepatic fibrosis and activation of the HSC in rat. VPA and TAA were administrated intraperitoneally at the dose of 400 and 200 mg/kg each at 2 days interval, respectively for a period of 6 weeks. Administration of TAA significantly increased the absolute and relative liver weight, aspartate aminotransferase and alanine aminotransferase levels, which were significantly decreased by VPA treatment as compared to TAA control. VPA treatment prevents the TAA-induced activation of HSC and decreases collagen deposition and infiltration of inflammatory cells as revealed by Sirius red and H&E staining. Interestingly, VPA co-treatment led to significantly increase the DNA damage and apoptosis in the activated HSC as compared to TAA control. Further, TAA decreased the expression of matrix metalloproteinase-2 (MMP-2), while VPA co-treatment significantly increased the expression of MMP-2 as compared to respective control. The present study clearly demonstrated that VPA treatment significantly alleviates TAA-induced activation of HSC and subsequent hepatic fibrosis.

Ulcerative colitis affects many people worldwide. Inflammation and oxidative stress play a vital role in its pathogenesis. Previously, we reported that ulcerative colitis leads to systemic genotoxicity in mice. The present study was aimed at elucidating the role of α-lipoic acid in ulcerative colitis-associated local and systemic damage in mice. Experimental colitis was induced using 3%w/v dextran sulfate sodium in drinking water for 2 cycles. α-Lipoic acid was administered in a co-treatment (20, 40, 80 mg/kg bw) and post-treatment (80 mg/kg bw) schedule. Various biochemical parameters, histological evaluation, comet and micronucleus assays, immunohistochemistry and western blot analysis were employed to evaluate the effect of α-lipoic acid in mice with ulcerative colitis. The protective effect of α-lipoic acid was mediated through the modulation of nuclear factor kappa B, cyclooxygenase-2, interleukin 17, signal transducer and activator of transcription 3, nuclear erythroid 2-related factor 2, NADPH: quinone oxidoreductase-1, matrix metalloproteinase-9 and connective tissue growth factor. Further, ulcerative colitis led to an increased gut permeability, plasma lipopolysaccharide level, systemic inflammation and genotoxicity in mice, which was reduced with α-lipoic acid treatment. The present study identifies the underlying mechanisms involved in α-lipoic acid-mediated protection against ulcerative colitis and the associated systemic damage in mice.

Ulcerative colitis (UC) is a chronic gastrointestinal disorder. Substantial research reveals that melatonin has beneficial effects in ulcerative colitis both experimentally and clinically. We have previously reported that ulcerative colitis was associated with local and systemic damage in mice. The purpose of this study was to reveal the novel targets of melatonin in its protective mechanism against ulcerative colitis in mice. We also wished to determine whether or not melatonin protected against ulcerative colitis-induced systemic damage in mice.Ulcerative colitis was induced in mice by use of 3% (w/v) dextran sulfate sodium for two cycles. One cycle comprised 7 days of DSS-treated water followed by 14 days of normal drinking water. Melatonin was administered at doses of 2, 4, or 8 mg/kg bw/day, po throughout. The effect of melatonin in mice with UC was evaluated by use of biochemical data, histological evaluation, comet and micronucleus assays, immunohistochemistry, and western blot analysis.The results indicated that melatonin treatment ameliorated the severity of ulcerative colitis by modulating a variety of molecular targets, for example nuclear factor kappa B, cyclooxygenase-2, interleukin 17, signal transducer and activator of transcription 3, nuclear erythroid 2-related factor 2, matrix metalloproteinase-9, and connective tissue growth factor. Further, ulcerative colitis increased gut permeability, plasma lipopolysaccharide level, systemic inflammation, and genotoxicity. Melatonin treatment led to mucosal healing and reduced ulcerative colitis-induced elevated gut permeability and reduced the plasma LPS level, systemic inflammation, and genotoxicity.Melatonin ameliorated ulcerative colitis-associated local and systemic damage in mice.

Micronucleus (MN) assay is widely used for the determination of the genotoxic potential of new chemical entities. Improvement in the sensitivity of MN assay will be advantageous for the successful detection of marginally active genotoxins. In the past, several improvements have been made in the automated scoring of micronuclei, while very few attempts have been taken to improve the sensitivity of manual micronuclei detection. The present study aims to validate the effect of valproic acid (VPA) pretreatment on the sensitivity of peripheral blood micronucleus (PBMN) assay using cyclophosphamide (CP, 50mg/kg), methotrexate (MTX, 20mg/kg) and zidovudine (AZT, 400mg/kg) in rodents. However, to find out the optimum VPA pretreatment time as well as to detect the effect of species and age difference, separate experiments were conducted on young Swiss albino mice (24-28 days) and Sprague-Dawley rats (21-24 days), in which significant increase in MN induction was observed with 3-day VPA pretreatment in both the species. Based on these results, studies on adult mice were conducted with 3-day VPA pretreatment along with CP or MTX or AZT. The results of the present study clearly demonstrate that the 3-day VPA pretreatment significantly enhances the sensitivity of PBMN assay in peripheral blood (PB) in adult mice. After validation with other standard genotoxins as well as other HDAC (histone deacetylase) inhibitors, this model may be useful for the detection of marginally active DNA damaging agents.

Recently, sodium valproate (VPA) has been proven as histone deacetylase (HDAC) inhibitor and potentiates the cytotoxicity of anticancer drugs, and also exhibit promising anti-cancer activity. Present study aimed to investigate the influence of pre- and post-treatment of VPA on cyclophosphamide (CP) induced genotoxicity and germ cell toxicity in mice. All the animals were treated with VPA at the dose of 500 mg/kg/day on alternate day thrice/week for a period of two weeks, CP at the dose of 200 mg/kg on 7(th) and 15(th) day and sacrificed 24 h after administration (i.p.) of the last dose. End point of evaluation includes sperm count, sperm head morphology, sperm comet assay and histology. VPA treatment significantly decreases CP induced sperm count, testes and epididymis weight; increased sperm head abnormality and sperm DNA damage. Both VPA pre- and post-treatment augmented CP induced DNA damage and the germ cell toxicity; however, pre-treatment induced more cytotoxicity and genotoxicity as compared to post-treatment.

a b s t r a c t Ulcerative colitis is a chronic gastrointestinal disorder eliciting the risk of colorectal cancer, the third most common malignancy in humans. The present study was aimed to characterize dextran sulfate sodium-induced ulcerative colitis and to elucidate its influence on the bone marrow cell proliferation and the subsequent stimulation of the systemic genotoxicity in mice. Experimental colitis was induced in Swiss mice using 3% (w/v) dextran sulfate sodium in drinking water. The severity of colitis was assessed on the basis of clinical signs, colon length, oxidative stress parameters, various pro-inflammatory markers, histopathological evaluation and immunohistochemical staining of 8-oxo7,8-dihydro-2� -deoxyguanosine in the colon of dextran sulfate sodium treated mice. Further, assessment of genotoxicity was carried out using alkaline and modified comet assays in the colon and lymphocytes and micronucleus assay in the peripheral blood of mice. For the evaluation of inflammation-induced cell proliferation in the bone marrow, proliferating cell nuclear antigen immunostaining was carried out in the bone marrow of mice. Dextran sulfate sodium induced severe colitis as evident from the elevated disease activity index, reduced colon length, increased oxidative stress, histological abnormalities and oxidative DNA damage in the colon of mice. Moreover, colitis-induced elevated prostaglandin-E2 level in the plasma of dextran sulfate sodium treated mice stimulated the cell proliferation in the bone marrow, which further triggered colitis-induced DNA damage in the peripheral blood of mice.

Furosemide (FS) is a potent loop diuretic widely used in the management of fluid retention associated with cardiac, renal, and hepatic failure as well as for the treatment of hypertension. FS is a well-characterized and known hepatotoxin in both human and animal test systems. In this study, an attempt has been made to investigate the in vivo genotoxicity of FS at the hepatotoxic equivalent doses using the chromosomal aberration and the comet assay in the bone marrow cells of mice as the endpoints of evaluation. The animals were treated with FS at the doses of 2.5, 5, 10, 20, 40, and 80 mg/kg/body weight (bw) intraperitoneal (ip) for both single (24 h) and repeated dose (seven consecutive days) toxicity studies. FS toxicity in the hepatocytes was evaluated using the parameters, such as, alanine-/aspartate-aminotransferase (ALT/AST), single cell gel electrophoresis (comet), tissue histology, DNA fragmentation, and TUNEL assay as the endpoints. The results clearly demonstrate that FS produced toxic responses in the hepatocytes as evident from increased ALT/AST level, DNA damage, TUNEL positive cells and increased DNA fragmentation in mice in vivo. However, it is interesting that in bone marrow cells, FS did not induced structural chromosomal abberations, but produced mild DNA strand breaks as observed by the comet assay. So it is considered as weak genotoxic toward the bone marrow cells when compared to the hepatocytes of mice.

Impairment in the insulin receptor signaling and insulin mediated effects are the key features of type 2 diabetes. Here we report that S961, a peptide insulin receptor antagonist induces hyperglycemia, hyperinsulinemia ( approximately 18-fold), glucose intolerance and impairment in the insulin mediated glucose disposal in the Sprague-Dawley rats. Further, long-term S961 treatment (15day, 10nM/kg/day) depletes energy storage as evident from decrease in the adiposity and hepatic glycogen content. However, peroxysome-proliferator-activated-receptor-gamma (PPARgamma) agonist pioglitazone significantly (P

Urotoxicity is one of the major problems associated with cyclophosphamide (CP) chemotherapy in cancer patients. Melatonin is a potent antioxidant and reduces CP-induced urotoxicity. However, the molecular mechanisms of protection offered by melatonin are not yet clear. The present study investigated the role of nuclear erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-kappaB) on melatonin-mediated protection against CP-induced urotoxicity. CP was administered intraperitoneally at the dose of 150 mg/kg to induce urotoxicity in male Sprague-Dawley rats. Melatonin treatment (10 mg/kg) was initiated 3 days before and continued for 1 day after the CP administration. Melatonin treatment reduced the CP-induced oxidative stress and DNA damage in the urinary bladder as observed by abrogation in thiobarbituric acid-reactive substances and glutathione levels as well as comet and modified comet assay parameters. Melatonin treatment reduced the bladder damage and apoptosis as observed by histological analysis and TUNEL assay. Melatonin increased the expression of transcription factor Nrf2 as well as associated phase-II enzymes NADPH: quinone oxidoreductase-1 and heme oxygenase-1. Further melatonin treatment reduced the expression of transcription factor NF-kappaB. The results of the present study provide evidence that melatonin treatment favorably alters Nrf2 and NF-kappaB expression and, this appears to be at least in part responsible for observed protection against CP-induced urotoxicity.

Cyclophosphamide, an alkylating agent, disturbs the oxidant and antioxidant balance that is associated with several unwanted toxic effects and induction of secondary cancers. Astaxanthin is a powerful antioxidant and possess several beneficial effects against various human diseases and physiological disorders. The present study was aimed to investigate the effects of astaxanthin against cyclophosphamide-induced oxidative stress, DNA damage, cell death and induction of GST-P foci in rat liver. Further attempt has been made to study the influence of astaxanthin on antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E(2)-related factor 2) in the induction of phase-II enzymes NAD(P)H: quinine oxidoreductase-1(NQO-1) and Hemoxygenase-1 (HO-1). Both pre- and post-treatment with astaxanthin (25mg/kg) decreased cyclophosphamide-induced oxidative stress and DNA damage in the liver as evident from the restoration in malondialdehyde and glutathione level as well as modified comet assay parameters. Significant decrease in the number as well as area of GST-P foci in rat hepatocytes was observed with astaxanthin post-treatment. Treatment with astaxanthin significantly decreased the expression of p53 and p38 as compared to cyclophosphamide treated group. It was further observed that the level of Nrf2 and phase-II enzymes, i.e. NQO-1 and HO-1 were increased with astaxanthin treatment. The present study confirms that astaxanthin is a potent antioxidant and attenuates oxidative stress, DNA damage, cell death as well as induction of early hepatocarcinogenesis in rat induced by cyclophosphamide. Our results provide the evidence that one of the mechanism of chemoprotection offered by astaxanthin is mediated through Nrf2-ARE pathway.

OBJECTIVES: To investigate the role of diethyldithiocarbamate (DEDTC) in cyclophosphamide (CP)-induced nephrotoxicity in Sprague–Dawley rat. DEDTC is a known chelating agent for copper and zinc. It is also used as a thiol protecting agent, as nuclear factor kappa-light-chain-enhancer of activated B-cells inhibitor and nitric oxide synthase inhibitor. It is also reported to inhibit superoxide dismutase (SOD) both in vitro and in vivo conditions. Considering this wide range of actions, current study investigated the role of DEDTC in CP-induced nephrotoxicity in experimental rat model. MATERIALS AND METHODS: Thirty-two male rats were randomized into four groups. Group 1, control received only saline ip; Group 2 and 4, received CP at the dose of 150 mg/kg body weight ip on the 4th day, while Group 3 and 4, received DEDTC at the dose of 250 mg/kg alternatively (fractionated dose of 1000 mg/kg). All the experimental animals were sacrificed on the 7th day and organs of interest were collected for biochemical, histopathological, DNA damage, and immunohistochemical assessments. RESULTS: DEDTC administration was found to further exacerbate the condition of CP-induced kidney damage as assessed by several biochemical and histological parameters. Further, the damage was also significantly reflected in the bladder in DEDTC-treated animals as compared to controls. SOD1 (Cu/Zn- dependent enzyme) expression was found to be decreased and this might be due to the action of DEDTC on SOD and other antioxidants. CONCLUSION: The present study indicates that DEDTC administration further exacerbated the CP-induced kidney damage in rat.

Methotrexate (MTX) is an anti-metabolite, widely used in the cancer chemotherapy and rheumatoid arthritis. However, its long-term clinical use is restricted on account of its severe intestinal toxicity. The present study was aimed to investigate the intestinal toxicity of MTX and the possible protective effect of α-lipoic acid (LA) on Sprague–Dawley rats. MTX-induced intestinal toxicity was evaluated at the dose of 2.5 mg/kg for short-term (5 days treatment) and 1 mg/kg for long-term (5 days in a week for four consecutive weeks treatment) study. The possible protective effect of LA was evaluated in both short- as well as long-term study in a dose-dependent manner. MTX treatment induced diarrhoea and mortality in rats, indicating its severe toxicity in the target organ of investigation, i.e. , intestine. Further, the intestinal toxicity of MTX was assessed by evaluating different parameters of oxidative stress, DNA damage, cytotoxicity as well as histological changes. Immunostaining for p53 revealed higher genotoxic assault in the intestinal cells due to MTX treatment. Pretreatment of rats with LA led to significant decrease in the oxidative stress, DNA damage, cellular damage, inflammatory changes and apoptosis as determined by malondialdehyde level, glutathione level, comet assay parameters, histological evaluation, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In the present investigation, we report that LA pretreatment ameliorates MTX-induced intestinal toxicity in rat as evident from the protection against oxidative stress, decrease in DNA damage and protection of cellular morphology as well as improvement in the stool consistency and animal survival rate.

Diethylnitrosamine (DEN), a potent hepatocarcinogen, is found in tobacco smoke, processed meat as well as in different food products. Quercetin (QC), a naturally occurring flavonoid has excellent antioxidant properties. The present study was aimed to investigate the chemoprotective potential of QC against DEN induced hepatotoxicity in Sprague-Dawley (SD) rats. Quercetin was administered (10, 30 and 100 mg/kg) for 5 consecutive days after DEN (200 mg/kg) treatment. The animals were killed 24 h after the last dose of QC/saline treatment. The DEN induced hepatotoxicity was evident by elevated malondialdehyde (MDA) and decreased glutathione (GSH) levels in the liver. A significant increase in the levels of plasma aspartate transaminase (AST) and plasma alanine transaminase (ALT) was observed in the DEN treated group. The DEN induced DNA damage was evaluated using a single cell gel electrophoresis (SCGE) assay. A significant increase in the number of TUNEL positive cells was observed in the DEN treated group. Quercetin restored AST, ALT and GSH levels at all the tested doses. Restoration of the MDA level and cellular morphology was observed at doses of 10 and 30 mg/kg of QC. Further, DEN induced DNA damage and apoptosis was ameliorated by QC. The results indicate that QC ameliorates the DEN induced hepatotoxicity in rats and can be a candidate for a good chemoprotectant.

Streptozotocin (STZ) is a naturally occurring compound isolated from Streptomyces achromogens. It is used extensively for inducing diabetes in experimental animals. Diabetes mellitus is known to have proven adverse effects on male sexual organs and their reproductive functions. The atrophy of prostate gland and other organs of the genitourinary tract were observed in experimental diabetic animals. STZ exhibits a structural resemblance to d -glucose due to the presence of sugar moiety in its structure. Pancreatic β-cells mainly contain GLUT1 and GLUT2 glucose transporters. Possibly due to structural resemblance, STZ and d -glucose, share a common recognition site for entry into the β-cells. The objective of the present study is to evaluate the effect of d -glucose on STZ-induced toxicity in accessory sex organs of male rats. Animals were kept on overnight fasting. One group received vehicle and served as negative control, while all other groups were given STZ (45 mg/kg). Animals that received only STZ served as positive control. The effect of d -glucose was studied on STZ treated animals with different dosage of d -glucose (250, 500, 1000 and 2000 mg/kg). Restoration of body weight, plasma glucose and plasma insulin was evident only at 1000 and 2000 mg/kg of d -glucose. The protective effect of d -glucose is evident only when it is administered simultaneously with STZ. In the present investigation, we report that simultaneous administration of d -glucose along with STZ ameliorates STZ-induced toxicity. This is evident from the restoration of accessory sex organ's weight, cellular morphology as well as insulin level.

DNA repair is an essential outcome of DNA damage, which may compromise the end point of various in vitro and in vivo test systems of the genotoxicity evaluation. poly(ADP-ribose) polymerase (PARP) enzymes have an essential role in DNA repair. Here, we investigated the effect of 3-AB, a PARP inhibitor on the sensitivity of comet and PBMN assays.This study aimed to enhance the sensitivity of the comet and peripheral blood micronucleus (PBMN) assays using 3-aminobenzamide (3-AB), a well-characterized PARP inhibitor.Cyclophosphamide (CP, 50 mg/kg), 5-flourouracil (5-FU, 25 mg/kg), zidovudine (AZT, 400 mg/kg) and furosemide (FUR, 60 mg/kg) were selected as genotoxins. 3-AB was given every 8 h with the first dose given 2 h before the genotoxin treatment. For the PBMN assay, small amount of blood was taken from the tail tip of each animal and smears were prepared. The comet assay was performed in PBL, bone marrow and liver.In the comet as well as PBMN assay, 3-AB pre-treatment enhanced the extent of DNA damage in all the combination groups (3-AB + CP, 3-AB + 5-FU and 3-AB + AZT) compared to CP, 5-FU and AZT per se. 3-AB also enhanced the DNA damage caused by FUR in the bone marrow and liver.This study results clearly demonstrate that the pretreatment with 3-AB (30 mg/kg) significantly enhances the sensitivity of the PBMN and comet assays. This model may be useful for the detection of marginally active DNA damaging agents.

Recent reports highlighted the role of histone deacetylases (HDACs) in the pathogenesis of diabetic nephropathy (DN), but the exact molecular mechanisms by which HDAC inhibitors ameliorate DN still remain unclear. The present study was aimed to investigate the renoprotective effects of sodium butyrate (NaB) in diabetes-induced renal damages, apoptosis and fibrosis in juvenile rats. Diabetes was induced by single injection of STZ (60mg/kg), whereas NaB (500mg/kg/day) was administrated for 21days by i.p. route in a pre- and post-treatment schedule. End-points of evaluation included biochemical estimation, histology, protein expression as well as apoptosis and DNA damage examinations. Post-treatment with NaB significantly decreased plasma glucose, creatinine, urea, histological alterations including the fibrosis and collagen deposition as well as decreased the HDACs activity, expression of eNOS, iNOS, α-SMA, collagen I, fibronectin, TGFβ-1, NFκB, apoptosis and DNA damage in the diabetic kidney. These results showed that NaB treatment improved the renal function and ameliorated the histological alterations, fibrosis, apoptosis and DNA damage in the kidney of juvenile rats.

The aim of the present study is to investigate whether nicotine augmented the testicular toxicity and angiotensin converting enzyme inhibitor, enalapril, can ameliorate the effects in diabetic rat. Male Sprague Dawley rats were randomized into five groups: control, nicotine, diabetic, Diab + Nico, and Diab + Nico + Enal. Animals were made diabetic by single injection of streptozotocin (55 mg/kg/intraperitoneally). Nicotine dissolved in drinking water at a concentration of 100 µg/ml was given ad libitum and enalapril was given orally at a dose of 10 mg/kg/day for four consecutive weeks. After 4 weeks of treatment, animals were killed and biochemical parameters glucose, glycosylated hemoglobin, cotinine, and the testosterone levels were measured. Testicular toxicity was evaluated using sperm count, sperm comet assay, histology, and immunohistochemical staining of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and the proinflammatory markers (nuclear factor kappa B (NF-κB), cyclooxygenase (COX-2), and tissue necrotic factor alpha (TNF-α)) evaluated by western blotting. Results showed that nicotine did not alter the blood glucose and glycosylated hemoglobin level, significantly decreased the sperm count and increased the sperm DNA damage. These changes were accompanied by significant increases in the 8-oxo-dG, NF-κB, COX-2, and TNF-α expression. Furthermore, the intervention of enalapril in nicotine-treated diabetic rat attenuated the testicular damage and restored sperm count, sperm DNA damage, as well as reduced the expression of NF-κB, COX-2, and TNF-α. These findings clearly suggest that nicotine not only augmented the testicular toxicity in the diabetic rat but also increases the risk of germ cell toxicity effects that were attenuated by enalapril treatment.

Testing new chemical entities for genotoxicity is an integral part of the preclinical drug-development process. Lowering the detection limit and enhancing the sensitivity of genotoxicity assays is required, as the standard test-battery fails to detect some carcinogens (non-genotoxic) and weak genotoxins. One of the mechanisms that affect the detection of weak genotoxins is related with the DNA-repair efficiency of the cell system used. In the present study, 3-aminobenzamide (3-AB, 30 mg/kg body-weight), a poly(ADP-ribose)polymerase inhibitor, was used to evaluate the DNA-damaging potential of zidovudine (AZT, 400 mg/kg bw), doxorubicin (DOX, 5 mg/kg bw) and cyclophosphamide (CP, 50 mg/kg bw, as a positive control) and sucrose (SUC, 3 g/kg bw, as a negative control) in Swiss female mice. The endpoints considered included micronucleus formation, DNA breakage (in peripheral blood lymphocytes, bone marrow and liver; comet assay) and chromosome aberrations, as well as immunohistochemistry of PARP-1 and phosphorylated histone H2AX (γ-H2AX). The results clearly indicate that the genotoxicity of zidovudine (AZT), doxorubicin (DOX) and cyclophosphamide (CP) was significantly increased in the combination treatments (3-AB + AZT, 3-AB + DOX, 3-AB + CP) as compared with the respective controls (treatment with AZT, DOX and CP alone). There was no increase in the genotoxicity per se after treatment with SUC, 3-AB or 3-AB + SUC, compared with the control (saline). Correlation analysis suggests that all genotoxicity parameters are well correlated with each other. The results clearly show that the genotoxicity of weak genotoxins can be enhanced and detected in the presence of 3-AB in mice. Thus, this approach can be used in the pre-clinical genotoxicity screening of weak genotoxins.

Type 1 diabetes (T1D) also known as juvenile diabetes is a chronic autoimmune disorder that precipitates in genetically susceptible individuals by environmental factors particularly during early age. Both genetic and epigenetic factors are implicated in the beta-cell development, proliferation, differentiation and function. Recent evidences suggested that there is a link between diabetes and histone deacetylases (HDACs), because HDAC inhibitors promote beta-cell development, proliferation and function as well as improve glucose homeostasis. Sodium butyrate (NaB) is a short chain fatty acid having HDAC inhibition activity. The present study was aimed to investigate the protective role of NaB treatment on the beta-cell proliferation, function and glucose homeostasis as well as apoptosis in juvenile diabetic rat. Diabetes was induced by single injection of STZ (60 mg/kg, i.p.) in chilled citrate buffer, while NaB (500 mg/kg/day) was administrated by i.p. route for 21 days as pre- and post-treatment schedule. Plasma glucose and insulin levels, HbA1c, glucose tolerance, apoptosis, and expression of proliferating cell nuclear antigen (PCNA), p38, p53, caspase-3, extracellular signal-regulated kinase-1/2 (ERK-1/2), forkhead box protein O1 (FOXO1) and insulin receptor substrate-1 (IRS-1) as well as histone acetylation were evaluated. NaB treatment decreased plasma glucose, HbA1c, beta-cell apoptosis and improved plasma insulin level and glucose homeostasis through HDAC inhibition and histone acetylation in diabetic animal as compared to control. NaB treatment improved the beta-cell proliferation, function and glucose homeostasis as well as reduced beta-cell apoptosis in juvenile diabetic rat by the modulation of p38/ERK MAPK and apoptotic pathway.

Testicular damage is a common clinical problem in diabetic individuals that severely affects the quality of life. The present study investigates the possible protective mechanisms of telmisartan, an angiotensin II-receptor antagonist in the germ cell of diabetic rat. Male SD rats were used and randomized into six groups: control, telmisartan control, diabetic control and diabetic group treated with telmisartan at the doses of 3, 6 and 12mg/kg/day, per oral for 4 weeks. Diabetes was induced by injecting a single dose of streptozotocin (STZ), (55mg/kg) dissolved in ice-cold 10mM citrate buffer; pH 4.4 and administered i.p. immediately after preparation to the SD rats. At the end of the study, rats were sacrificed and the levels of nitrite, superoxide, malondialdehyde (MDA), glutathione (reduced and peroxidase) and superoxide dismutase (SOD) were measured. Germ cell toxicity was evaluated by using sperm count, sperm comet assay, histology of testes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Further to confirm the oxidative and nitrosative damage, immunohistological quantification of 8-oxo-dG (8-oxo-7,8-dihydro-2'-deoxyguanosine) and 3-nitrotyrosine were evaluated respectively. Results showed that telmisartan significantly restored the levels of nitrite, superoxide, malondialdehyde, and glutathione and superoxide dismutase in diabetic testes. Further, telmisartan significantly increased the sperm counts, reduced apoptotic cell death, sperm DNA damage, oxidative and nitrosative damage in diabetic rat. Western blot analysis showed that telmisartan reduced the testicular inflammation and cell death by down-regulating the expression of NF-κB, IL-6, TNF-α, p-ERK1/2, iNOS, caspase-3 and increasing the PPAR-γ expression. Results clearly indicate that telmisartan significantly reduced the both oxidative and nitrosative stress, inflammation and cell death in diabetic testes. The present results confirmed that telmisartan exhibited beneficial role in the germ cell of diabetic rat.

There exists a close relationship between ulcerative colitis and various hepatic disorders. The present study was aimed to evaluate the hepatocellular damage in experimental colitis model. Ulcerative colitis was induced in Swiss mice by cyclic treatment with 3% w/v dextran sulfate sodium in drinking water. The severity of colitis was assessed on the basis of disease activity index and colon histology. The effect of ulcerative colitis on the liver was assessed using various biochemical parameters, histological evaluation, sirius red staining, immunohistochemical staining with peroxisome proliferator-activated receptor γ, 8-oxo-7,8-dihydro-2'-deoxyguanosine and placental glutathione S-transferase, comet assay (alkaline and modified), Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling assay and western blot analysis to detect the protein expression of nuclear factor kappa B, cyclooxygenase-2, nuclear erythroid 2-related factor 2 and NADPH: quinone oxidoreductase-1. Dextran sulfate sodium induced severe colitis in mice as evident from an elevated disease activity index and histological abnormalities. Ulcerative colitis increased the permeability of colon as apparent from a significant reduction in the expression of tight junction protein, occludin. Further, the bacterial translocation assay as well as the analysis of lipopolysaccharide level revealed the existence of various bacterial species in the liver of ulcerative colitis-induced mice. There was a significant increase in the plasma alanine and aspartate transaminases and liver triglyceride levels, expression of peroxisome proliferator-activated receptor γ, inflammatory markers, oxidative stress, fibrosis, oxidative DNA damage and apoptosis in the liver of mice. Moreover, there was an increase in the expression of nuclear factor kappa B and cyclooxygenase-2 and a reduction in the expression of nuclear erythroid 2-related factor 2 and NADPH: quinone oxidoreductase-1 in the liver of severe ulcerative colitis-induced mice. The results of the present study provide evidence that ulcerative colitis is accompanied with hepatic damage in mice.

The chromosome aberration assay (CA) in bone marrow cells and the micronucleus test (MNT) in both bone marrow and peripheral blood erythrocytes have been carried out for the evaluation of the clastogenic potential of acephate (asataf) in a chick in vivo test system. Technical grade acephate was administered to evaluate dose-responses (25, 50 and 100 mg/kg), route-responses (i.p. and p.o.) and time-responses (6, 24 and 48 h). A comparison of CA frequencies after acute and chronic dosing was also performed. Only 50 mg/kg of acephate induced significant bone marrow chromosome aberrations after 24 h exposure while all three doses induced significant increases in micronuclei in both bone marrow and peripheral blood erythrocytes in i.p. route only. The presented data confirm our earlier reports that the neonatal chicken test system is a convenient model and can be used as an alternative to mammalian systems for screening some environmental contaminants for genotoxicity.

Doxorubicin is a widely used chemotherapeutic agent; however, its clinical uses are limited due to its cardiotoxicity associated with an induction of oxidative stress. This study was aimed to investigate the protective effect of hesperetin against doxorubicin-induced cardiotoxicity in rats. Doxorubicin was administered at the dosage of 4 mg/kg bw/week, ip for a period of 5 consecutive weeks. Hesperetin was administered at the dosages of 25, 50 and 100 mg/kg bw, po by gavage for 5 consecutive days in a week for 5 weeks. The animals were killed 1 week after the last injection of doxorubicin. Hesperetin at the doses of 50 and 100 mg/kg bw significantly reduced MDA and increased GSH levels in the doxorubicin-treated animals. Further, hesperetin significantly reduced doxorubicin-induced DNA damage as well as apoptosis at 25, 50, and 100 mg/kg bw as evident from the comet and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assays, respectively. Thus, hesperetin ameliorated doxorubicin-induced cardiotoxicity by reducing oxidative stress, abnormal cellular morphology and DNA damage in rat. Moreover, nuclear factor-kappa B, p38, and caspase-3 play a role in the hesperetin-mediated protection against doxorubicin-induced cardiotoxicity. This study indicates the protective effect of hesperetin against doxorubicin-induced cardiotoxicity.

Quercetin is an antioxidant flavonoid, found ubiquitously in nature and extensively used in herbal medicines and food additives. This study aimed to investigate the effect of quercetin on diethylnitrosamine-induced preneoplastic lesions, using the medium-term rat liver bioassay. The γ-benzene hexachloride was used as promoter at the doses of 0.1, 1.0, and 10.0 mg/kg against a single dose of diethylnitrosamine (200 mg/kg) in male Sprague-Dawley rats. All the rats were subjected to 70% partial hepatectomy at Week 4. The protective effect of quercetin (5 and 25 mg/kg) was examined against the highest dose of γ-benzene hexachloride (10 mg/kg). A significant increase in the number as well as the mean area of glutathione S-transferase placental form (GST-P) positive foci, p53 positive hepatocytes, and the percentage of apoptotic cells were observed in the diethylnitrosamine-treated group. In the present investigation, both doses of QC (5 and 25 mg/kg) led to a significant decrease in the number as well as the mean area of GST-P positive foci, TUNEL positive apoptotic cells, p53 positive hepatocytes, and restoration of cellular morphology. These results clearly indicate that quercetin inhibits diethylnitrosamine-induced hepatic preneoplastic lesions in medium-term rat liver bioassay.

Prostatic hyperplasia is a common problem of the aged men population. Recent experimental and clinical studies provide sufficient evidence that apart from androgens, insulin also plays an important role in the pathogenesis of prostatic hyperplasia. The present study was aimed to investigate the relative influence of testosterone and insulin on the cellular proliferation and prostatic growth. Effect of testosterone on the prostate of hypoinsulinemic, and glandular injection of insulin-receptor antagonist S961 on the prostate of castrated Sprague-Dawley rat (220±10 g) was examined. Significant decrease in the weight of the ventral prostate was observed in the streptozotocin-induced hypoinsulinemic rats (~6 fold), which is restored by the intervention of testosterone. Although, glandular injection of S961 did not led to any change in the frequency of proliferating cell nuclear antigen (PCNA) positive cells in normal rats, significant decrease was observed in the castrated rats. Castration led to increase in the frequency of the caspase-3 and the TUNEL positive cells in the ventral prostate. Further, long-term (6 weeks) administration of S961 induced significant decrease in the weight of the ventral prostate. Results of the present study provide that both testosterone and insulin promote prostatic cell proliferation and change in the level of either of the hormone results in the destabilization of cellular equilibrium, and modulation of the insulin-receptor signaling in the prostate may provide an alternative strategy for the treatment of prostatic enlargement. Further, studies are required to better understand the interplay between these hormones in the regulation of prostatic growth.

Doxorubicin is a widely used chemotherapeutic agent causing serious dose-dependent toxicity to non-target tissues such as testis. Its testicular toxicity is mainly due to the induction of oxidative stress. Hesperetin exerts its beneficial effects against oxidative stress-induced cellular damage. In the present investigation, doxorubicin was administered intraperitoneally at the dose of 4 mg/kg bw/week for a period of 5 consecutive weeks. Hesperetin was administered at the doses of 25, 50 and 100mg/kg bw per oral by gavage for 5 consecutive days in a week for 5 weeks. Animals were sacrificed 1 week after the last injection of doxorubicin. The results of the present study clearly indicate the prevention of oxidative stress, DNA damage and the cellular toxicity by hesperetin treatment as evident from the analysis of biochemical parameters, comet assay, halo assay, Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling assay, immunohistochemistry and histology. Hesperetin protection against doxorubicin-induced germ cell toxicity was further evident from the sperm count and sperm head morphological evaluation. Moreover, the role of nuclear factor-kappa B, p38 and caspase-3 on hesperetin-mediated protection against doxorubicin-induced testicular toxicity was also investigated. The present study clearly revealed the amelioration of doxorubicin-induced testicular toxicity by the intervention with hesperetin.

Previous studies have demonstrated increased incidence of benign prostatic hyperplasia in insulin-resistant individuals. In addition to androgens, prostatic growth is sensitive to the peptide growth factors including insulin. Experimental studies employing intervention of selective β-cell toxin streptozotocin and castration suggest that depletion of either insulin or testosterone results in the severe prostatic atrophy (>80%). Exogenous testosterone and diet-induced experimental hyperinsulinemia induces prostatic enlargement in rats. Further, hyperinsulinemia sensitizes prostate towards the growth promoting effect of testosterone, and testosterone augments prostatic growth even in the hypoinsulinemic rats. However, in castrated rats diet-induced hyperinsulinemia fails to promote prostatic growth. Based on these evidences it is hypothesized that in the presence of testosterone insulin plays an important role in the prostatic growth. The epidemiological reports witnessing increased incidences of prostatic enlargement in men with metabolic syndrome, which are known to have increased level of insulin, provides a validating clue to the hypothesis. Further, the hypothesis suggests that targeting insulin signaling pathway could be a new objective for the treatment of prostatic enlargement.

Micronucleus (MN) and comet assay can successfully detect the genetic damage in two different cell population of peripheral blood i.e. erythrocytes and lymphocytes. The present study was aimed to investigate the kinetics of MN formation as well as to identify the time of maximum MN induction in the peripheral blood erythrocytes in juvenile rats and thereafter to examine its relationship with the observed DNA damage in the lymphocytes as determined by comet assay. The rat peripheral blood micronucleus (PBMN) assay is generally not preferred owing to the selective elimination of micronucleated cells from the circulation by spleen. However, inefficient splenic removal of micronucleated cells in the juvenile Sprague-Dawley (SD, 26 days) rats conferred advantage to be a suitable model for PBMN assay. The kinetics of MN formation and DNA damage in the peripheral blood were determined with cyclophosphamide (50mg/kg), chlorambucil (30mg/kg), methotrexate (20mg/kg), cisplatin (5mg/kg) and paclitaxel (0.5 and 1mg/kg). All the tested chemicals with different mechanisms of action have induced time-dependant changes in the MN frequency in the peripheral blood erythrocytes. Comet assay in the peripheral blood lymphocytes also revealed similar pattern of rise and fall in the DNA damage. MN frequency and the different comet assay parameters exhibited significant positive correlation with all the tested chemicals and in both of the assays the peak was observed in between 36 and 48h post-treatment. Results of the present study clearly demonstrates that MN frequency in the peripheral blood erythrocytes exhibits positive correlation with the DNA damage in the peripheral blood lymphocytes as evident from different comet assay parameters. Further, the study highlights the detection of DNA damage in two different cell population and establishes the complementary nature of these two bioassays for the genotoxicity testing by combining a conventional technique to the smarter one.

The role of selenium, a trace element in the human diet, has been extensively studied against cancer, immunity and infectious/inflammatory diseases. The purpose of the present study was to investigate the beneficial effect of ebselen, an organo-selenium compound, against cyclophosphamide-induced oxidative stress and DNA damage in mice. Malondialdehyde and total glutathione were estimated in the liver to detect the oxidative stress induced by cyclophosphamide. Standard and modified comet assays (the latter incorporated lesion-specific enzymes, formamidopyrimidine-DNA glycosylase and endonuclease-III) were used to detect the normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, a micronucleus assay capable of detecting DNA damage was also carried out in the mouse bone marrow and the peripheral blood reticulocytes induced by cyclophosphamide. The results confirm that pre-treatment with ebselen (2.5, 5 and 10 mg/kg) for 5 consecutive days decreased the oxidative stress induced by cyclophosphamide (100 mg/kg) based on the restoration in concentration of malondialdehyde and glutathione in the liver and decreased DNA damage and micronuclei count in the bone marrow and the peripheral blood. It is concluded that pre-treatment with ebselen attenuates cyclophosphamide-induced oxidative stress and the resultant DNA damage in mice.