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Effects of insulin treatment on hepatic CYP1A1 and CYP2E1 activities and lipid peroxidation levels in streptozotocin-induced diabetic rats
Abstracts / Toxicology Letters 280S (2017) S131–S136
for future novel biomarkers over the pre-existing markers used to detect cholangiocyte DILI. The main aims of this study are to isolate murine cholangiocytes and hepatocytes and to characterise their global miRNA levels with a view to using the most abundant miRNAs as detectable biomarkers of DILI in patients. Hepatocytes were isolated from male 8 to 12-week CD-1 mice with a modified version of the two-step collagenase perfusion procedure. Cholangiocytes were also isolated from this procedure following a positive immunoaffinity selection using an anti-EpCAM hybridoma antibody. The hybridoma antibody specificity for cholangiocytes was confirmed by positive immunohistochemistry staining of bile ducts, and absence of staining in the parenchyma. Isolated cholangiocytes and hepatocytes were shown to have >95% purity shown by positive expression of CK19 and albumin respectively, by immunofluorescence and Western blot. Cell isolations (n = 4) for both cells types were prepared and subjected to global miRNA expression profiling using Agilent microarray technology. Analysis of this data will identify top candidate biomarkers of cholangiocyte damage, which have the potential to be detected in DILI patient biofluids. http://dx.doi.org/10.1016/j.toxlet.2017.07.375 P-04-03-14 Effects of insulin treatment on hepatic CYP1A1 and CYP2E1 activities and lipid peroxidation levels in streptozotocin-induced diabetic rats Benay Can Eke 1 , Gökc¸e Kuzgun 1 , Rahman Bas¸aran 1 , Ebru Ario˘glu Inan 2 1 2
Pharmaceutical Toxicology, Ankara University, Ankara, Turkey Pharmacology, Ankara University, Ankara, Turkey
Diabetes mellitus is a metabolic disorder caused by insulin deficiency or inadequate use of produced insulin. In many studies, the increased reactive oxygen species (ROS) leading to oxidative stress have been shown to play a role in the etiology and progression of diabetes. Cytochrome P450 monooxygenases (CYP450) are one of the sources in the production of ROS and the activities of these enzymes may be affected in the case of diabetes. This study was therefore undertaken to investigate how hepatic CYP2E1 and CYP1A1 influence the liver oxidative stress in diabetes and whether insulin regulates the both enzymes. For this purpose, we studied the effects of diabetes on the activities of hepatic CYP2E1 and CYP1A1, and the levels of liver lipid peroxidation, which is an important indicator of oxidative stress, in control, streptozotocin-induced and insulin-treated groups of rats. Our findings indicated that hepatic CYP2E1 and CYP1A1 activities increased in diabetic conditions and the increased activities of both enzymes were restored to normal levels with insulin treatment, but there was no significant change in the liver lipid peroxidation levels in diabetic rats compared to control group. The results obtained suggest that insulin may modulate streptozotocin-induced alterations in the levels of liver lipid peroxidation. http://dx.doi.org/10.1016/j.toxlet.2017.07.376 P-04-03-15 Dose effect and time effect of CdTe quantum dots on antioxidant capacities of liver and kidneys in mice Pei Li Huang, Lin Tian
S135
Capital Medical University, Beijing, China Although quantum dot (QD)-induced toxicity occurs due to free radicals, generation of oxidative stress mediated by ROS formation is considered an important mechanism. However, free-radical mechanisms are essentially difficult to elucidate at the molecular level because most biologically relevant free radicals are highly reactive and short-lived, making them difficult to directly detect, especially in vivo. Antioxidants play an important role in preventing or, in most cases, limiting the damage caused by ROS. Healthy people and animals possess many endogenous antioxidative substances that scavenge free radicals in vivo to maintain the redox balance and genome integrity. The antioxidant capacity of an organism is highly important but seldom studied. In this study, the dose and time effects of CdTe QDs on the antioxidant capacities of the liver and kidneys were investigated in mice using the EPR spin tapping technique. We found that the liver and kidneys of health mice contain specific antioxidant capacities that scavenge • OH and • O2 − . Furthermore, oxidative stress markers (SOD, CAT, GPx, GSH and MDA) were examined. In dose-course studies, the free radical scavenging efficiencies of the liver and kidneys were found to gradually decrease with increasing concentration of CdTe QDs exposure. The activities and levels of SOD, CAT, GPx and MDA were observed to increase in treated groups, whereas those of GSH were reduced. The time-course studies revealed that the QD-induced antioxidant efficiency reduction was time dependent with GSH decrease and could recover after a period of time. http://dx.doi.org/10.1016/j.toxlet.2017.07.377 P-04-03-16 CAR-mediated tumor formation in rats induced by the herbicide metazachlor Christiane Wiemann 1 , Manuela Goettel 2 , Naveed Honarvar 3 , Audrey Vardy 4 , Ivana Fegert 2 1 ProductsSafety, Regulatory Toxicology Crop Protection, BASF Österreich GmbH, Wien, Austria 2 Product Safety, Regulatory Toxicology Crop Protection, BASF SE, Ludwigshafen, Germany 3 Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany 4 Concept Life Sciences (former CXR Biosciences), Dundee, United Kingdom
Metazachlor induces liver tumors in female Wistar rats. To elucidate the mode of action (MoA) and potential human relevance a set of mechanistic studies in rodent liver tissue and hepatocytes as well as human hepatocytes has been conducted. Metazachlor does not give indication of a genotoxic effects. In order to address the suggested MoA via the activation of the Constitutive Androstane Receptor (CAR) a series of mechanistic studies were performed also excluding other possible mechanisms. A Metazachlor-induced CAR-mediated MoA was confirmed by showing the nuclear translocation of CAR and induction of CAR-regulated Cytochrome P450 isoenzymes (CYP) of the CYP 2B family. Centrilobular hypertrophy and liver cell proliferation, considered as further characteristic key events for this MoA, were also observed in the Metazachlor-induced liver tissue. It could be shown that cell proliferation determined in rat liver tissue and in primary rat hepatocytes after treatment with metazachlor did not occur, when primary hepatocytes of CAR-knockout rats were treated with metazachlor. An arylhydrocarbon receptor-mediated mechanism and a peroxisome proliferator activated receptor ␣-mediated mechanism were excluded by the absence of the respective mRNA and enzyme
for future novel biomarkers over the pre-existing markers used to detect cholangiocyte DILI. The main aims of this study are to isolate murine cholangiocytes and hepatocytes and to characterise their global miRNA levels with a view to using the most abundant miRNAs as detectable biomarkers of DILI in patients. Hepatocytes were isolated from male 8 to 12-week CD-1 mice with a modified version of the two-step collagenase perfusion procedure. Cholangiocytes were also isolated from this procedure following a positive immunoaffinity selection using an anti-EpCAM hybridoma antibody. The hybridoma antibody specificity for cholangiocytes was confirmed by positive immunohistochemistry staining of bile ducts, and absence of staining in the parenchyma. Isolated cholangiocytes and hepatocytes were shown to have >95% purity shown by positive expression of CK19 and albumin respectively, by immunofluorescence and Western blot. Cell isolations (n = 4) for both cells types were prepared and subjected to global miRNA expression profiling using Agilent microarray technology. Analysis of this data will identify top candidate biomarkers of cholangiocyte damage, which have the potential to be detected in DILI patient biofluids. http://dx.doi.org/10.1016/j.toxlet.2017.07.375 P-04-03-14 Effects of insulin treatment on hepatic CYP1A1 and CYP2E1 activities and lipid peroxidation levels in streptozotocin-induced diabetic rats Benay Can Eke 1 , Gökc¸e Kuzgun 1 , Rahman Bas¸aran 1 , Ebru Ario˘glu Inan 2 1 2
Pharmaceutical Toxicology, Ankara University, Ankara, Turkey Pharmacology, Ankara University, Ankara, Turkey
Diabetes mellitus is a metabolic disorder caused by insulin deficiency or inadequate use of produced insulin. In many studies, the increased reactive oxygen species (ROS) leading to oxidative stress have been shown to play a role in the etiology and progression of diabetes. Cytochrome P450 monooxygenases (CYP450) are one of the sources in the production of ROS and the activities of these enzymes may be affected in the case of diabetes. This study was therefore undertaken to investigate how hepatic CYP2E1 and CYP1A1 influence the liver oxidative stress in diabetes and whether insulin regulates the both enzymes. For this purpose, we studied the effects of diabetes on the activities of hepatic CYP2E1 and CYP1A1, and the levels of liver lipid peroxidation, which is an important indicator of oxidative stress, in control, streptozotocin-induced and insulin-treated groups of rats. Our findings indicated that hepatic CYP2E1 and CYP1A1 activities increased in diabetic conditions and the increased activities of both enzymes were restored to normal levels with insulin treatment, but there was no significant change in the liver lipid peroxidation levels in diabetic rats compared to control group. The results obtained suggest that insulin may modulate streptozotocin-induced alterations in the levels of liver lipid peroxidation. http://dx.doi.org/10.1016/j.toxlet.2017.07.376 P-04-03-15 Dose effect and time effect of CdTe quantum dots on antioxidant capacities of liver and kidneys in mice Pei Li Huang, Lin Tian
S135
Capital Medical University, Beijing, China Although quantum dot (QD)-induced toxicity occurs due to free radicals, generation of oxidative stress mediated by ROS formation is considered an important mechanism. However, free-radical mechanisms are essentially difficult to elucidate at the molecular level because most biologically relevant free radicals are highly reactive and short-lived, making them difficult to directly detect, especially in vivo. Antioxidants play an important role in preventing or, in most cases, limiting the damage caused by ROS. Healthy people and animals possess many endogenous antioxidative substances that scavenge free radicals in vivo to maintain the redox balance and genome integrity. The antioxidant capacity of an organism is highly important but seldom studied. In this study, the dose and time effects of CdTe QDs on the antioxidant capacities of the liver and kidneys were investigated in mice using the EPR spin tapping technique. We found that the liver and kidneys of health mice contain specific antioxidant capacities that scavenge • OH and • O2 − . Furthermore, oxidative stress markers (SOD, CAT, GPx, GSH and MDA) were examined. In dose-course studies, the free radical scavenging efficiencies of the liver and kidneys were found to gradually decrease with increasing concentration of CdTe QDs exposure. The activities and levels of SOD, CAT, GPx and MDA were observed to increase in treated groups, whereas those of GSH were reduced. The time-course studies revealed that the QD-induced antioxidant efficiency reduction was time dependent with GSH decrease and could recover after a period of time. http://dx.doi.org/10.1016/j.toxlet.2017.07.377 P-04-03-16 CAR-mediated tumor formation in rats induced by the herbicide metazachlor Christiane Wiemann 1 , Manuela Goettel 2 , Naveed Honarvar 3 , Audrey Vardy 4 , Ivana Fegert 2 1 ProductsSafety, Regulatory Toxicology Crop Protection, BASF Österreich GmbH, Wien, Austria 2 Product Safety, Regulatory Toxicology Crop Protection, BASF SE, Ludwigshafen, Germany 3 Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany 4 Concept Life Sciences (former CXR Biosciences), Dundee, United Kingdom
Metazachlor induces liver tumors in female Wistar rats. To elucidate the mode of action (MoA) and potential human relevance a set of mechanistic studies in rodent liver tissue and hepatocytes as well as human hepatocytes has been conducted. Metazachlor does not give indication of a genotoxic effects. In order to address the suggested MoA via the activation of the Constitutive Androstane Receptor (CAR) a series of mechanistic studies were performed also excluding other possible mechanisms. A Metazachlor-induced CAR-mediated MoA was confirmed by showing the nuclear translocation of CAR and induction of CAR-regulated Cytochrome P450 isoenzymes (CYP) of the CYP 2B family. Centrilobular hypertrophy and liver cell proliferation, considered as further characteristic key events for this MoA, were also observed in the Metazachlor-induced liver tissue. It could be shown that cell proliferation determined in rat liver tissue and in primary rat hepatocytes after treatment with metazachlor did not occur, when primary hepatocytes of CAR-knockout rats were treated with metazachlor. An arylhydrocarbon receptor-mediated mechanism and a peroxisome proliferator activated receptor ␣-mediated mechanism were excluded by the absence of the respective mRNA and enzyme