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.

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 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)

Melatonin reduces ulcerative colitis-associated local and systemic damage in mice: investigation on possible mechanisms.

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Melatonin ameliorated ulcerative colitis-associated local and systemic damage in mice.

Role of α-lipoic acid in dextran sulfate sodium-induced ulcerative colitis in mice: studies on inflammation, oxidative stress, DNA damage and 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.

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.

Ulcerative colitis-induced hepatic damage in mice: studies on inflammation, fibrosis, oxidative DNA damage and GST-P expression.

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.

Dextran sulfate sodium-induced ulcerative colitis leads to increased hematopoiesis and induces both local as well as systemic genotoxicity in mice.

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-oxo-7,8-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.

Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes.

Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.

Cardioprotective effects of hesperetin against doxorubicin-induced oxidative stress and DNA damage in rat.

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.

Hesperetin protects testicular toxicity of doxorubicin in rat: role of NFκB, p38 and caspase-3.

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.

Evaluation of male germ cell toxicity in rats: correlation between sperm head morphology and sperm comet assay.

The present study was aimed to investigate the germ cell toxicity of doxorubicin and find out the possible correlation between sperm head morphological evaluation and sperm comet assay, which are used to assess male germ cell toxicity. The correlation between these two assays was validated using a potent germ cell toxicant, doxorubicin, in male Sprague-Dawley rats. Doxorubicin was administered intra-peritionally at the doses of 1.25, 2.5 and 5mg/kg weekly once for a period of 5 weeks and all the animals were sacrificed after 1 week of receiving the last dose. The germ cell toxicity of doxorubicin was assessed using oxidative stress parameters, sperm head morphology, sperm comet assay, halo assay and histology in testes as the end point of evaluation. A significant increase in the % abnormality in sperm head was found in the animals treated with 2.5 and 5mg/kg/week doxorubicin. Doxorubicin treatment significantly increased the DNA damage of sperm in a dose-dependent manner as observed by sperm comet assay parameters. A strong positive correlation was observed between the sperm head morphological evaluation and the sperm comet assay. Therefore, it can be concluded that the damage in genetic material of sperm may result into abnormalities in the sperm head morphology. The sperm head morphological evaluation is considered to be essential for the assessment of male germ cell toxicity by several regulatory bodies like the Organization for Economic Cooperation and Development (OECD) and the International Conference on Harmonization (ICH). However, acceptance of the sperm comet assay by regulatory authorities as a standard genotoxicity test for assessing male germ cell toxicity still requires further validation of the assay.