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A review of developmental toxicity screening using zebrafish larvae
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1179 A review of developmental toxicity screening using zebrafish larvae A. Hill ∗ , M. Jones, A. Dodd, H. Diekmann Biology/Admet, Evotec (UK) Ltd, Abingdon, UK The zebrafish has gained increasing popularity for drug testing over the past decade due to numerous advantages over other vertebrate in vivo models. In particular, zebrafish larvae are virtually transparent, thereby allowing internal organs to be assessed without dissection, and their small size enables an in vitro-like assay format requiring only single milligrams of test compound to obtain a swift, full dose response readout. In recent years, a zebrafish developmental toxicity assay has been validated with 90 compounds, including those with known teratogenic potential. Fertilized eggs (n = 14 per concentration) were exposed to a six point standard aqueous concentration range from 4 h until 96 h post fertilisation, followed by morphological assessment (28 endpoints) using a dissecting stereomicroscope. Since the uptake into the zebrafish larva is compound dependent and can vary significantly even between similar compounds, the actual body burden was determined by LCMS at the NOEC and LOEC. The majority of drugs testing positive in traditional mammalian tests produced similar results in zebrafish larvae, although the specific phenotypes observed were different for some compounds. False negatives could be identified due to lack of compound getting into the larvae. Taking into account the interpretation from bioanalysis results, the overall predictivity for all compounds tested was 89%. Based on these results, the zebrafish assay shows potential as a screening tool for developmental toxicity. Due to the higher throughput and lower costs compared to mammalian developmental toxicity assays, the larval zebrafish screen would be suitable for testing large numbers of compounds in early drug discovery. doi:10.1016/j.toxlet.2011.05.413
P1180 Probing reactivation of tabun phosphylated cholinesterases by mutagenesis and new oximes Z. Kovarik 1,∗ , M. Katalinic´ 1 , N. Maˇcek 1 , J. Kalisiak 2 , Z. Radic´ 3 , V.V. Fokin 2 , P. Taylor 3 , K.B. Sharpless 2 1
Institute for Medical Research and Occupational Health, Zagreb, Croatia, 2 The Scripps Research Institute, La Jolla, USA, 3 Skaggs Scholll of Pharmacy and Pharmaceutical Sciences, UCSD, La Jolla, USA The copper-catalyzed azide-alkyne cycloaddition reaction enabled fast and reliable synthesis of libraries of oximes that were screened for the reactivation activity of tabun-inhibited human recombinant AChE, wild type, the AChE single mutant Y337A, and human BChE. Out of 100 oximes, 53 were able to reactivate wild type AChE, but only 14 oximes restored full activity. For the remaining oximes, the maximal reactivation was below 50%. It appears that a distance of 8 atoms between two quaternary nitrogens is optimal to achieve high level of AChE activity. Phosphorylated cholinebinding site Y337A AChE mutant was reactivated (>80%) with 13 oximes only. For three most efficient oximes (2PAM analogs) the observed reactivation rate was 4-times faster than for the most efficient reactivator of AChE wild type. Since all oximes were designed as reactivators of phosphorylated AChE, a limited reactivation capacity for BChE was expected. However, 37 oximes reactivated tabun-inhibited BChE more efficiently than 2PAM, and five of them
S115
reached 70% of maximal reactivation. In conclusion, although rank order of the rates differs for reactivation of three tabun phosphorylated enzymes, our findings may provide a platform for further modifications and development of more potent antidotes in tabun poisoning. doi:10.1016/j.toxlet.2011.05.414
P1181 Evaluation the effect of crocin (the major pigment of Crocus sativus) on cisplatin-induced renal toxicity B. Naghizadeh 1,∗ , S.M.T. Mansouri 1 , N. Vahdati Mashhadian 2 1
Pharmacology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, 2 Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Purpose: Reactive oxygen species (ROS) and oxidative damage are the most important factors in cisplatin-induced acute renal failure. This study examined the protective effects of crocin against cisplatin-induced renal oxidative stress in rat. Methods: Animals were divided into five groups (n = 6). Group 1 received normal saline (2 ml/day, i.p.). Group 2 received a single dose of cisplatin (5 mg/kg, i.p.). Groups 3–5 received crocin (100, 200, and 400 g/kg, i.p., respectively) for four consecutive days beginning 1-h before a single dose of cisplatin (5 mg/kg) on day 1. On day 5, blood samples were drawn and kidneys were removed for histopathological, biochemical and RT-PCR examinations. Twenty-four hours urinary chemistries were measured. Results: Blood urea and creatinine and urinary glucose and protein concentrations in crocin-treated groups were significantly lower compared to the cisplatin-treated group. Histopathological studies showed massive damage in the S3 segment of proximal tubules in cisplatin-treated group but not in crocin-treated groups. Crocin treatment resulted in a significant reduction in malondialdehyde (MDA) concentration and produced a significant elevation in total thiol and glutathione peroxidase concentrations. There was a significant elevation in the mRNA expression of glutathione peroxidase in crocin-treated groups. The results suggest that crocin attenuates cisplatin-induced renal oxidative stress in rats. doi:10.1016/j.toxlet.2011.05.415
P1182 Doxorubicin induced toxicity in mesenchymal stem cells M.S. Oliveira 1,∗ , J.L. Carvalho 2 , M.M. Melo 1 , A.M. Goes 2 1
College of Veterinary School, Minas Gerais Federal University, Belo Horizonte, Brazil, 2 Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil Purpose: Doxorubicin (DOX) is one of the most effective chemotherapeutic agents; however it causes dose-dependent cardiotoxicity. An alternative treatment for DOX cardiomyopathy that is being investigated is cell therapy using autologous stem cell transplantation. However it remains unclear if DOX may have deleterious effects on mesenchymal stem cell (MSC) from subjects receiving such antineoplasic agent. This study purpose was to evaluate DOX toxicity upon MSC. Methods: Adipose tissue derived mesenchymal stem cells (ATMSC) were isolated from Wistar rats, cultured up to the fourth passage and incubated with 5 mol/L of DOX for 24 h. Neonatal ventricular cardiomyocytes and breast cancer MDA-MB 231 cell cultures were also incubated at the same
P1179 A review of developmental toxicity screening using zebrafish larvae A. Hill ∗ , M. Jones, A. Dodd, H. Diekmann Biology/Admet, Evotec (UK) Ltd, Abingdon, UK The zebrafish has gained increasing popularity for drug testing over the past decade due to numerous advantages over other vertebrate in vivo models. In particular, zebrafish larvae are virtually transparent, thereby allowing internal organs to be assessed without dissection, and their small size enables an in vitro-like assay format requiring only single milligrams of test compound to obtain a swift, full dose response readout. In recent years, a zebrafish developmental toxicity assay has been validated with 90 compounds, including those with known teratogenic potential. Fertilized eggs (n = 14 per concentration) were exposed to a six point standard aqueous concentration range from 4 h until 96 h post fertilisation, followed by morphological assessment (28 endpoints) using a dissecting stereomicroscope. Since the uptake into the zebrafish larva is compound dependent and can vary significantly even between similar compounds, the actual body burden was determined by LCMS at the NOEC and LOEC. The majority of drugs testing positive in traditional mammalian tests produced similar results in zebrafish larvae, although the specific phenotypes observed were different for some compounds. False negatives could be identified due to lack of compound getting into the larvae. Taking into account the interpretation from bioanalysis results, the overall predictivity for all compounds tested was 89%. Based on these results, the zebrafish assay shows potential as a screening tool for developmental toxicity. Due to the higher throughput and lower costs compared to mammalian developmental toxicity assays, the larval zebrafish screen would be suitable for testing large numbers of compounds in early drug discovery. doi:10.1016/j.toxlet.2011.05.413
P1180 Probing reactivation of tabun phosphylated cholinesterases by mutagenesis and new oximes Z. Kovarik 1,∗ , M. Katalinic´ 1 , N. Maˇcek 1 , J. Kalisiak 2 , Z. Radic´ 3 , V.V. Fokin 2 , P. Taylor 3 , K.B. Sharpless 2 1
Institute for Medical Research and Occupational Health, Zagreb, Croatia, 2 The Scripps Research Institute, La Jolla, USA, 3 Skaggs Scholll of Pharmacy and Pharmaceutical Sciences, UCSD, La Jolla, USA The copper-catalyzed azide-alkyne cycloaddition reaction enabled fast and reliable synthesis of libraries of oximes that were screened for the reactivation activity of tabun-inhibited human recombinant AChE, wild type, the AChE single mutant Y337A, and human BChE. Out of 100 oximes, 53 were able to reactivate wild type AChE, but only 14 oximes restored full activity. For the remaining oximes, the maximal reactivation was below 50%. It appears that a distance of 8 atoms between two quaternary nitrogens is optimal to achieve high level of AChE activity. Phosphorylated cholinebinding site Y337A AChE mutant was reactivated (>80%) with 13 oximes only. For three most efficient oximes (2PAM analogs) the observed reactivation rate was 4-times faster than for the most efficient reactivator of AChE wild type. Since all oximes were designed as reactivators of phosphorylated AChE, a limited reactivation capacity for BChE was expected. However, 37 oximes reactivated tabun-inhibited BChE more efficiently than 2PAM, and five of them
S115
reached 70% of maximal reactivation. In conclusion, although rank order of the rates differs for reactivation of three tabun phosphorylated enzymes, our findings may provide a platform for further modifications and development of more potent antidotes in tabun poisoning. doi:10.1016/j.toxlet.2011.05.414
P1181 Evaluation the effect of crocin (the major pigment of Crocus sativus) on cisplatin-induced renal toxicity B. Naghizadeh 1,∗ , S.M.T. Mansouri 1 , N. Vahdati Mashhadian 2 1
Pharmacology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, 2 Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Purpose: Reactive oxygen species (ROS) and oxidative damage are the most important factors in cisplatin-induced acute renal failure. This study examined the protective effects of crocin against cisplatin-induced renal oxidative stress in rat. Methods: Animals were divided into five groups (n = 6). Group 1 received normal saline (2 ml/day, i.p.). Group 2 received a single dose of cisplatin (5 mg/kg, i.p.). Groups 3–5 received crocin (100, 200, and 400 g/kg, i.p., respectively) for four consecutive days beginning 1-h before a single dose of cisplatin (5 mg/kg) on day 1. On day 5, blood samples were drawn and kidneys were removed for histopathological, biochemical and RT-PCR examinations. Twenty-four hours urinary chemistries were measured. Results: Blood urea and creatinine and urinary glucose and protein concentrations in crocin-treated groups were significantly lower compared to the cisplatin-treated group. Histopathological studies showed massive damage in the S3 segment of proximal tubules in cisplatin-treated group but not in crocin-treated groups. Crocin treatment resulted in a significant reduction in malondialdehyde (MDA) concentration and produced a significant elevation in total thiol and glutathione peroxidase concentrations. There was a significant elevation in the mRNA expression of glutathione peroxidase in crocin-treated groups. The results suggest that crocin attenuates cisplatin-induced renal oxidative stress in rats. doi:10.1016/j.toxlet.2011.05.415
P1182 Doxorubicin induced toxicity in mesenchymal stem cells M.S. Oliveira 1,∗ , J.L. Carvalho 2 , M.M. Melo 1 , A.M. Goes 2 1
College of Veterinary School, Minas Gerais Federal University, Belo Horizonte, Brazil, 2 Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil Purpose: Doxorubicin (DOX) is one of the most effective chemotherapeutic agents; however it causes dose-dependent cardiotoxicity. An alternative treatment for DOX cardiomyopathy that is being investigated is cell therapy using autologous stem cell transplantation. However it remains unclear if DOX may have deleterious effects on mesenchymal stem cell (MSC) from subjects receiving such antineoplasic agent. This study purpose was to evaluate DOX toxicity upon MSC. Methods: Adipose tissue derived mesenchymal stem cells (ATMSC) were isolated from Wistar rats, cultured up to the fourth passage and incubated with 5 mol/L of DOX for 24 h. Neonatal ventricular cardiomyocytes and breast cancer MDA-MB 231 cell cultures were also incubated at the same