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Evaluation of cholestatic potential of endothelin receptor antagonists
Abstracts / Toxicology Letters 258S (2016) S62–S324
aRG spheroids when the drug and BA co-exposures were prolonged to 14 days. This effect was also observed when hepatic spheroids derived from pooled PHH from 10 individual donors were used. We also evaluated mechanisms behind the drug induced cholestasis using chlorpromazine as an example. Toxicity of chlorpromazine was associated with intracellular BA accumulation, disruption of the F-actin cytoskeleton and repression of BSEP mRNA expression. Furthermore, an increased oxidative stress and modulation of death receptor signaling was seen in the presence of both BAs and chlorpromazine in the systems. In conclusion, we provide proof of principle that the 3D hepatic spheroid models can identify cholestatic drugs and allow mechanistic studies of drug induced cholestasis. http://dx.doi.org/10.1016/j.toxlet.2016.06.1528 P06-036 Evaluation of cholestatic potential of endothelin receptor antagonists M.G. Burbank 1,∗ , A. Sharanek 1 , A. Burban 1 , H. Mialanne 2 , H. Aerts 2 , C. Guguen Guillouzo 4 , R.J. Weaver 3 , A. Guillouzo 1 1
Inserm UMR 991, Rennes 1 University, 35043 Rennes, France Biologie Servier, 45520, Gidy, France 3 International Research Institute Servier, Suresnes, 92150, France 4 Biopredic International, St Grégoire, 35760 Rennes, France 2
Several endothelin receptor antagonists have been developed for the treatment of pulmonary arterial hypertension. Some of them have been related to clinical cases of hepatotoxicity (sitaxentan) and/or cholestasis (bosentan). Recently, we showed that like other cholestatic drugs, bosentan impaired in vitro bile canaliculi dynamics typified by loss of spontaneous contractions and dilatation of bile canaliculi (BC) associated with deregulation of the ROCK/MLCK pathway (Sharanek, Burban et al. Sci Rep, 2016). We aimed to determine whether other endothelin receptor antagonists, i.e. ambrisentan, macitentan and sitaxentan, could cause similar BC alterations, using differentiated HepaRG cells. Our results showed that only macitentan similarly induced cholestatic disorders characterized by BC dilatation. This drug also strongly inhibited taurocholic acid canalicular efflux after 2 h, compared to a limited effect of bosentan and sitaxentan; however it had a much lower inhibitory effect than these two drugs on influx activity after either 2 or 24 h. Moreover, the three drugs caused decreased intracellular accumulation and parallel increased levels in culture media of total bile acids. In addition, after 24 h macitentan and bosentan down-regulated BSEP and together with sitaxentan, NTCP expression. Only bosentan down-regulated OATP-B and MRP3. These results suggest that the recently marketed macitentan, structurally similar to bosentan, can cause in vitro major BC alterations. By contrast, ambrisentan appears as a safe drug and sitaxentan that has been withdrawn from the market for hepatotoxic cases, does not impair BC dynamics. Financial support: European Community [Contract MIP-DILI115336] and ANRT [n◦ 2013/0112] to MB. http://dx.doi.org/10.1016/j.toxlet.2016.06.1529
S135
P06-037 Evaluation of the reproductive toxicity of levetiracetam in male rats M. Baysal 1,∗ , S. Ilgin 1 , V. Kilic 2 , G. Kilic 2 , S. Ucarcan 2 , O. Atli 1 1
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey 2 Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey Levetiracetam (LEV) is a commonly used antiepileptic drug in adolescence which is an important period for reproductive development. In this study, our goal was to investigate the potential toxicity of LEV on male reproductive system in rats. 50, 150 and 300 mg/kg LEV was administered to rats orally for 70 consecutive days for a complete spermatogenesis process. Sperm concentration, motility and morphology were investigated by a computer assisted sperm analysis system. Serum testosterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by ELISAs and histopathological examination of testis tissue was performed. Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels were measured in testes tissue and sperm DNA damage was determined by Comet assay to elucidate possible underlying mechanisms. According to our results, sperm parameters were shown to get significantly worse with LEV treatment. Serum testosterone, FSH, and LH levels were significantly decreased in 300 mg/kg LEV group. SOD and GSH levels were significantly decreased while MDA levels were significantly increased in 150 and 300 mg/kg LEV groups. CAT levels were significantly decreased in LEV-administered groups. Histopathology of the testes showed that LEV especially resulted in injury of the tubular and cellular structure in 300 mg/kg LEV group. The results of sperm comet assay showed that LEV induced significant DNA damage 150 and 300 mg/kg LEV groups. In conclusion, LEV, which affected sperm parameters negatively and induced DNA damage and oxidative stress in male rats, is considered to be a drug exerting reproductive toxicity risk. http://dx.doi.org/10.1016/j.toxlet.2016.06.1530 P06-038 Gatifloxacin-induced alterations in the expression of apoptotic, DNA methylating and chromatin modifying genes in Rat liver T.I. Olugbemi, S. Rotimi ∗ , O.J. Ebebeinwe, C.U. Duru, O.A. Rotimi Department of Biological Sciences, Covenant University, Ota, Nigeria
Gatifloxacin, a fourth generation fluoroquinolone, is used for the treatment of severe and life threatening bacterial infection. It has however been reported that the use of fluoroquinolone may result in some non-antibiotic effects which include epigenetic modifications in the host. In other to study this in the liver, we exposed rats to 10 mg/kg, 20 mg/kg, 40 mg/kg and 80 mg/kg gatifloxacin for 5days orally. Reverse transcription polymerase chain reaction was used to assess the effects of gatifloxacin on the expression of genes involved in apoptosis, histone modification as well as DNA methyltransferase I. The results showed that a dose-dependent increase in the expression of BCL2 and caspase 8 with a concomitant dose-dependent decrease in caspase 9 expression. Gatifloxacin treatment also resulted in significant (p < 0.05) increase in the expression level of DNA methyltransferase as well as Ehmt, Kat2 and Suv39, which are enzymes involved in histone modification. These changes observed at the lowest dosage of 10 mg/kg showed
aRG spheroids when the drug and BA co-exposures were prolonged to 14 days. This effect was also observed when hepatic spheroids derived from pooled PHH from 10 individual donors were used. We also evaluated mechanisms behind the drug induced cholestasis using chlorpromazine as an example. Toxicity of chlorpromazine was associated with intracellular BA accumulation, disruption of the F-actin cytoskeleton and repression of BSEP mRNA expression. Furthermore, an increased oxidative stress and modulation of death receptor signaling was seen in the presence of both BAs and chlorpromazine in the systems. In conclusion, we provide proof of principle that the 3D hepatic spheroid models can identify cholestatic drugs and allow mechanistic studies of drug induced cholestasis. http://dx.doi.org/10.1016/j.toxlet.2016.06.1528 P06-036 Evaluation of cholestatic potential of endothelin receptor antagonists M.G. Burbank 1,∗ , A. Sharanek 1 , A. Burban 1 , H. Mialanne 2 , H. Aerts 2 , C. Guguen Guillouzo 4 , R.J. Weaver 3 , A. Guillouzo 1 1
Inserm UMR 991, Rennes 1 University, 35043 Rennes, France Biologie Servier, 45520, Gidy, France 3 International Research Institute Servier, Suresnes, 92150, France 4 Biopredic International, St Grégoire, 35760 Rennes, France 2
Several endothelin receptor antagonists have been developed for the treatment of pulmonary arterial hypertension. Some of them have been related to clinical cases of hepatotoxicity (sitaxentan) and/or cholestasis (bosentan). Recently, we showed that like other cholestatic drugs, bosentan impaired in vitro bile canaliculi dynamics typified by loss of spontaneous contractions and dilatation of bile canaliculi (BC) associated with deregulation of the ROCK/MLCK pathway (Sharanek, Burban et al. Sci Rep, 2016). We aimed to determine whether other endothelin receptor antagonists, i.e. ambrisentan, macitentan and sitaxentan, could cause similar BC alterations, using differentiated HepaRG cells. Our results showed that only macitentan similarly induced cholestatic disorders characterized by BC dilatation. This drug also strongly inhibited taurocholic acid canalicular efflux after 2 h, compared to a limited effect of bosentan and sitaxentan; however it had a much lower inhibitory effect than these two drugs on influx activity after either 2 or 24 h. Moreover, the three drugs caused decreased intracellular accumulation and parallel increased levels in culture media of total bile acids. In addition, after 24 h macitentan and bosentan down-regulated BSEP and together with sitaxentan, NTCP expression. Only bosentan down-regulated OATP-B and MRP3. These results suggest that the recently marketed macitentan, structurally similar to bosentan, can cause in vitro major BC alterations. By contrast, ambrisentan appears as a safe drug and sitaxentan that has been withdrawn from the market for hepatotoxic cases, does not impair BC dynamics. Financial support: European Community [Contract MIP-DILI115336] and ANRT [n◦ 2013/0112] to MB. http://dx.doi.org/10.1016/j.toxlet.2016.06.1529
S135
P06-037 Evaluation of the reproductive toxicity of levetiracetam in male rats M. Baysal 1,∗ , S. Ilgin 1 , V. Kilic 2 , G. Kilic 2 , S. Ucarcan 2 , O. Atli 1 1
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey 2 Department of Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey Levetiracetam (LEV) is a commonly used antiepileptic drug in adolescence which is an important period for reproductive development. In this study, our goal was to investigate the potential toxicity of LEV on male reproductive system in rats. 50, 150 and 300 mg/kg LEV was administered to rats orally for 70 consecutive days for a complete spermatogenesis process. Sperm concentration, motility and morphology were investigated by a computer assisted sperm analysis system. Serum testosterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by ELISAs and histopathological examination of testis tissue was performed. Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels were measured in testes tissue and sperm DNA damage was determined by Comet assay to elucidate possible underlying mechanisms. According to our results, sperm parameters were shown to get significantly worse with LEV treatment. Serum testosterone, FSH, and LH levels were significantly decreased in 300 mg/kg LEV group. SOD and GSH levels were significantly decreased while MDA levels were significantly increased in 150 and 300 mg/kg LEV groups. CAT levels were significantly decreased in LEV-administered groups. Histopathology of the testes showed that LEV especially resulted in injury of the tubular and cellular structure in 300 mg/kg LEV group. The results of sperm comet assay showed that LEV induced significant DNA damage 150 and 300 mg/kg LEV groups. In conclusion, LEV, which affected sperm parameters negatively and induced DNA damage and oxidative stress in male rats, is considered to be a drug exerting reproductive toxicity risk. http://dx.doi.org/10.1016/j.toxlet.2016.06.1530 P06-038 Gatifloxacin-induced alterations in the expression of apoptotic, DNA methylating and chromatin modifying genes in Rat liver T.I. Olugbemi, S. Rotimi ∗ , O.J. Ebebeinwe, C.U. Duru, O.A. Rotimi Department of Biological Sciences, Covenant University, Ota, Nigeria
Gatifloxacin, a fourth generation fluoroquinolone, is used for the treatment of severe and life threatening bacterial infection. It has however been reported that the use of fluoroquinolone may result in some non-antibiotic effects which include epigenetic modifications in the host. In other to study this in the liver, we exposed rats to 10 mg/kg, 20 mg/kg, 40 mg/kg and 80 mg/kg gatifloxacin for 5days orally. Reverse transcription polymerase chain reaction was used to assess the effects of gatifloxacin on the expression of genes involved in apoptosis, histone modification as well as DNA methyltransferase I. The results showed that a dose-dependent increase in the expression of BCL2 and caspase 8 with a concomitant dose-dependent decrease in caspase 9 expression. Gatifloxacin treatment also resulted in significant (p < 0.05) increase in the expression level of DNA methyltransferase as well as Ehmt, Kat2 and Suv39, which are enzymes involved in histone modification. These changes observed at the lowest dosage of 10 mg/kg showed