Simultaneous intervention against oxidative stress and inflammation by targeting Nrf2/ARE and NLRP3 inflammasome pathway mitigates thioacetamide-induced liver fibrosis in rat.

Liver fibrosis is a typical pathological state/stage involved in most chronic liver diseases and its persistence results in cirrhosis. Inflammasomes are cytoplasmic sensors that induce inflammation in response to stress. Glibenclamide (GLB) is an USFDA-approved drug for type 2 diabetes and is reported to possess anti-inflammatory activity by inhibiting inflammatory cytokines. Dimethyl fumarate (DMF) is an USFDA-approved drug for multiple sclerosis and has been reported to activate the Nrf2/ARE pathway to maintain the cellular antioxidant balance. A total of 36 rats were randomized into six groups (n = 6 each). The rats were injected with thioacetamide (TAA) 200 mg/kg, intraperitoneally every third day for eight consecutive weeks to induce liver fibrosis and oral treatment of GLB 0.5 mg/kg/day and DMF 25 mg/kg/day, and their combinations were provided for the last four consecutive weeks. Treatment with GLB, DMF, and GLB+DMF significantly protected against TAA-mediated oxidative stress and inflammatory conditions by improving hepatic function test, triglycerides, hydroxyproline, and histopathological alterations, by inhibiting the NLRP3 inflammasome signaling and fibrogenic markers, and by activating Nrf2/ARE pathway in Wistar rats. The present results suggest that simultaneous Nrf2/ARE activation and NLRP3 inflammasome inhibition could significantly contribute to developing a novel therapy for patients with liver fibrosis.

Dimethyl fumarate-mediated Nrf2/ARE pathway activation and glibenclamide-mediated NLRP3 inflammasome cascade inhibition alleviate type II diabetes-associated fatty liver in rats by mitigating oxidative stress and inflammation.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is much higher in patients with type II diabetes (T2D). Inflammasomes are multimolecular complexes reported to involve inflammatory conditions. The nuclear factor (erythroid-derived 2)-like factor 2/antioxidant responsive element (Nrf2/ARE) pathway is an important regulator of antioxidant status in cells. Antidiabetic drug glibenclamide (GLB) is reported as  NACHT, leucine-rich repeat, and pyrin domain domains-containing protein 3 (NLRP3) inflammasome inhibitor, whereas anti-multiple sclerosis drug dimethyl fumarate (DMF) is reported as an Nrf2/ARE pathway activator. Both GLB and DMF possess anti-inflammatory and antioxidant properties, therefore, the hypothesis was made to look into the alone as well as the combination potential of GLB, DMF, and GLB + DMF, against NAFLD in diabetic rats. This study was aimed to investigate (1) the involvement of NLRP3 inflammasome and Nrf2/ARE signaling in diabetes-associated NAFLD (2) the effect of GLB, DMF, GLB + DMF, and metformin (MET) interventions on NLRP3 inflammasome and Nrf2/ARE signaling in diabetes-associated NAFLD. The rats were injected with streptozotocin (STZ) 35 mg/kg and fed a high-fat diet (HFD) for 17 consecutive weeks to induce diabetic NAFLD. The oral treatment of GLB 0.5 mg/kg/day, DMF 25 mg/kg/day, their combination and MET 200 mg/kg/day, were provided from the 6th to the 17th week. Treatment with GLB, DMF, GLB + DMF, and MET significantly alleviated HFD + STZ-induced plasma glucose, triglycerides, cholesterol, %HbA1c, hepatic steatosis, NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, CARD, caspase-1, interleukin-1ß (IL-1ß), nuclear factor-κB (NF-κB), Nrf2, superoxide dismutase 1, catalase, IGF 1, heme oxygenase 1, receptor for the advanced glycation end product (RAGE), and collagen-1 in diabetic rats. Further, a mechanistic molecular study employing other specific NLRP3 inhibitors and Nrf2 activators will significantly contribute to the development of novel therapy for fatty liver diseases.

Simultaneous Modulation of NLRP3 Inflammasome and Nrf2/ARE Pathway Rescues Thioacetamide-Induced Hepatic Damage in Mice: Role of Oxidative Stress and Inflammation.

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.

The intervention of tert-butylhydroquinone protects ethanol-induced gastric ulcer in type II diabetic rats: the role of Nrf2 pathway.

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.

Zinc and selenium combination treatment protected diabetes-induced testicular and epididymal damage in rat.

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.

NLRP3 inhibitor glibenclamide attenuates high-fat diet and streptozotocin-induced non-alcoholic fatty liver disease in rat: studies on oxidative stress, inflammation, DNA damage and insulin signalling pathway.

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-1ß, NF-κB), anti-oxidant markers (SOD, catalase) and insulin signalling markers (p-AKT, p-GSK-3ß, 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.

Diethylnitrosamine and thioacetamide-induced hepatic damage and early carcinogenesis in rats: Role of Nrf2 activator dimethyl fumarate and NLRP3 inhibitor glibenclamide.

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-ß1, 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.

Ethanol-induced gastric ulcer in rats and intervention of tert-butylhydroquinone: Involvement of Nrf2/HO-1 signalling pathway.

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.

Glibenclamide protects against thioacetamide-induced hepatic damage in Wistar rat: investigation on NLRP3, MMP-2, and stellate cell activation.

Glibenclamide (GLB), most widely used in the treatment of type II diabetes mellitus, inhibits K+ATP channel in pancreatic-ß 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-ß1, α-SMA, NLRP3, ASC, caspase-1, and IL-1ß, 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.

Implementation of Good Laboratory Practices (GLP) in basic scientific research: Translating the concept beyond regulatory compliance.

The principles of Good Laboratory Practices (GLPs) are mainly intended for the laboratories performing studies for regulatory compliances. However, today GLP can be applied to broad disciplines of science to cater to the needs of the experimental objectives, generation of quality data and assay reproducibility. Considering its significance, it can now be applied in academics; industries as well as government set ups throughout the world. GLP is the best way to promote the reliability, reproducibility of the test data and hence facilitates the international acceptability. Now it is high time to translate and implement the concept of GLP beyond regulatory studies. Thus, it can pave the way for better understanding of scientific problems and help to maintain a good human and environmental health. Through this review, we have made an attempt to explore the uses of GLP principles in different fields of science and its acceptability as well as looking for its future perspectives.

Methotrexate-induced germ cell toxicity and the important role of zinc and SOD1: Investigation of molecular mechanisms.

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.

Role of Zinc Supplementation in Testicular and Epididymal Damages in Diabetic Rat: Involvement of Nrf2, SOD1, and GPX5.

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.

Melatonin modulated autophagy and Nrf2 signaling pathways in mice with colitis-associated colon carcinogenesis.

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.

Mechanistic insight into beta-carotene-mediated protection against ulcerative colitis-associated local and systemic damage in mice.

PURPOSE: Ulcerative colitis (UC), a chronic gastrointestinal disorder, is a debilitating disease affecting many people across the globe. Research suggests that the levels of several antioxidants, including ß-carotene (ß-CAR), decrease in the serum of patients with UC. The present study was aimed at elucidating the molecular mechanisms involved in ß-CAR-mediated protection against UC in mice. METHODS: UC was induced in mice using 3%w/v dextran sulfate sodium in drinking water for two cycles; one cycle comprised of 7 days of dextran sulfate sodium-treated water followed by 14 days of normal drinking water. ß-CAR was administered at the doses of 5, 10 and 20 mg/kg bw/day, po throughout the experiment. The effect of ß-CAR in mice with UC was evaluated using biochemical parameters, histological evaluation, comet and micronucleus assays, immunohistochemistry and Western blot analysis. RESULTS: The results indicated that ß-CAR treatment ameliorated the severity of UC by modulating various molecular targets such as 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, ß-CAR treatment maintained the gut integrity by increasing the expression of a tight junction protein, occludin, which was decreased in the colon of mice with UC. Also ß-CAR treatment significantly reduced UC-associated elevated plasma lipopolysaccharide level, systemic inflammation and genotoxicity. CONCLUSION: ß-CAR ameliorated UC-associated local and systemic damage in mice by acting on multiple targets.

Influence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose)polymerase, in the evaluation of the genotoxicity of doxorubicin, cyclophosphamide and zidovudine in female mice.

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.

Pre-pubertal exposure of cytarabine-induced testicular atrophy, impaired spermatogenesis and germ cell DNA damage in SD rats.

CONTEXT: Cytarabine (Ara-C) is an effective chemotherapeutic drug for the treatment of acute leukaemias. It inhibits the DNA synthesis and repair, thereby causes cytotoxicity in the proliferating cells. OBJECTIVE: This study was aimed to investigate the effects of pre-pubertal exposure of Ara-C on testesticular development in juvenile SD rats and their function at puberty. MATERIALS AND METHODS: Ara-C was injected at the doses of 50, 100 and 200 mg/kg/day from postnatal day (PND) 29-42 (14 days) by intraperitoneal (i.p.) route. Half of the animals were sacrificed on PND 43 and remaining on PND 70. End points of the evaluation included gross pathological examination, histomorphometric analysis, sperm count and sperm head morphology, cell proliferation and DNA damage as well as apoptosis analysis. RESULTS: Ara-C treatment significantly decreased food and water intake, weight gain, testes and epididymis weight and increased histological alterations in the seminiferous tubule. Furthermore, Ara-C treatment significantly decreased the PCNA-positive cells and sperm count in a dose-dependent manner. Ara-C treatment also increased the DNA damage and apoptosis in testes and sperm as evident from the comet and TUNEL assays results. DISCUSSION: The present study results clearly indicated that Ara-C treatment impaired spermatogenesis and adversely affects the testicular development and its function in rats by reducing the germ cell proliferation and the inducing DNA damage and apoptosis.

Dextran sulfate sodium-induced ulcerative colitis leads to testicular toxicity in mice: Role of inflammation, oxidative stress and DNA damage.

Ulcerative colitis is associated with an alteration in gonadal hormones and affects testicular weight in rodents. However, association of ulcerative colitis with testicular damage is not clearly known. Ulcerative colitis was induced using 5% (w/v) dextran sulfate sodium in normal drinking water for 1, 7-day cycle in short-term study and 2.5% (w/v) dextran sulfate sodium in normal drinking water for 4 cycles with 2 weeks remission period between each cycle in long-term study. Ulcerative colitis was associated with a significant increase in inflammation, oxidative stress, DNA damage in testes and sperm DNA damage and a significant decrease in the epididymal sperm count and 3ß-HSD expression. No difference was observed in the plasma testosterone levels between control and treatment groups. In the present study, ulcerative colitis was associated with testicular damage, and juvenile mice were found to be more sensitive than adult mice.

Protective role of sodium butyrate, a HDAC inhibitor on beta-cell proliferation, function and glucose homeostasis through modulation of p38/ERK MAPK and apoptotic pathways: study in juvenile diabetic rat.

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.

Effects of nicotine on the testicular toxicity of streptozotocin-induced diabetic rat: intervention of enalapril.

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.

Protective role of atorvastatin against doxorubicin-induced cardiotoxicity and testicular toxicity in mice

Doxorubicin (DOX), a potent chemotherapeutic agent, is widely used for the treatment of various malignancies. However, its clinical uses are limited due to its dose-dependent adverse effects particularly cardiac and testicular toxicities. DOX-induced toxicity is mainly due to the induction of oxidative stress. Atorvastatin (ATV), a 3-hydroxy 3-methyl glutaryl coenzyme A reductase inhibitor, with lipid-lowering activity, acts as an antioxidant at lower doses. It possesses pleiotropic effects independent of cholesterol-lowering property usually shown at lower doses, which include antioxidant and anti-inflammatory activities. The present study was aimed to investigate the possible protection exerted by atorvastatin against oxidative stress and DNA damage induced by DOX in the heart and testes of mice. The protective role of ATV in the heart and testes of DOX-treated mice was evident from the amelioration of oxidative stress, DNA and cellular damage. The present study clearly indicates that ATV offers a significant protection against DOX-induced oxidative stress and DNA damage in the heart and testes of mice (AU)