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Is oxidative stress involved in OTA renal toxicity?
Abstracts / Toxicology Letters 258S (2016) S62–S324
P16-037 Is oxidative stress involved in OTA renal toxicity? J. Enciso Gadea ∗ , L. Pastor Castro, A. Azqueta Oscoz, A. López de Cerain Salsamendi, A. Vettorazzi Armental Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and proposed as a possible etiological agent of the Balkan Endemic Nephropathy (BEN) in humans. Thereby, OTA has been classified as a possible human carcinogen (group 2B). Though its mechanism of action is still unknown, many authors consider oxidative stress as a possible hypothesis. Moreover, large sex-differences have been observed in different carcinogenicity studies. Therefore, the objective of this study was to evaluate the sex-dependent kidney oxidative stress response to OTA in F344 rats. For that purpose, male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 mg OTA/kg b.w. or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) levels, total glutathione Stransferase (GST) and superoxide dismutase (SOD) activities were analysed in kidney tissue. Besides, the standard alkaline comet assay was used in combination with formamidopyrimidine DNA glycosylase (FPG) in order to detect oxidative DNA damage. In the 7-days study, male tGSH levels were significantly lower both in the control and the OTA-treated group. In the 21-days study, tGSH levels were significantly lower in control males, and female GST activity decreased as OTA dose increased. However, we could not find an increase of oxidative DNA damage in the kidneys of male or female F344 rats treated for 7 or 21 days. Our results are not able to link the higher nephrotoxicity that OTA exhibits in male rats with an increase in oxidative stress. http://dx.doi.org/10.1016/j.toxlet.2016.06.1876 P16-038 In vitro neurotoxicity of ‘Legal X’: The combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) induces mitochondrial impairment and disrupts thiol homeostasis in dopaminergic SH-SY5Y cells D. Dias Da Silva ∗ , A. Ferreira, M. Martins, M. Valente, F. Remião, F. Carvalho, M. Bastos, H. Carmo UCIBIO/REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Portugal In the drug abuse scene, 1-benzylpiperazine (BZP) and 1(m-trifluoromethylphenyl)piperazine (TFMPP) are concomitantly consumed under the name ‘Legal X’. As the central nervous system is one of the main targets of toxicity of these drugs, we assessed the detrimental effects of BZP:TFMPP combination (at three different mixture ratios) in human SH-SY5Y cells differentiated to induce a dopaminergic phenotype. The toxicity data obtained in the MTT assay for individual exposure of BZP (EC50 3.74 mM) and TFMPP (EC50 208.67 M) after 24 h were used to predict combined effects of the drugs, by employing the concentration addition (CA) and independent action (IA) models. The predictions and the effects obtained experimentally were compared. As the mixture effects did not deviate from
S247
CA, they were considered additive. IA concept overestimated the effects of two mixtures. Single ineffective concentrations of BZP and TFMPP additively provoked remarkable neurotoxicity when combined (p < 0.01, versus control). Cellular and mitochondrial (dys)function was further investigated. BZP disturbed oxidative defences (intracellular GSH/GSSG) to a higher extent than TFMPP, which in turn more severely disrupted mitochondria functioning (by hyperpolarizing the membrane and increasing Ca2+ efflux of the organelle). Additional oxidative and energetic homeostasis biomarkers, including intracellular ROS and ATP levels, were not significantly disturbed. The results obtained for the mixture agreed with the individual data, showing consistency with additivity. The data described herein clearly shows that the toxicological assessment of single drugs may considerably underestimate the real risks associated with the consumption of drug combinations in the corresponding abuse scenarios. http://dx.doi.org/10.1016/j.toxlet.2016.06.1877 P16-039 DNA damage after long-term exposure of rabbits to Imidacloprid and sodium tungstate A.I. Vardavas 1,∗ , E. Ozcagli 2 , P. Fragkiadaki 1 , P.D. Stivaktakis 1 , M.N. Tzatzarakis 1 , K. Kaloudis 1 , M. Tsardi 3 , G. Datseri 3 , J. Tsiaoussis 4 , C. Tsitsimpikou 5 , F. Carvalho 6 , A.M. Tsatsakis 1 1
Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece 2 Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazit–Istanbul 34116, Turkey 3 Department of Pathology, Medical School Voutes-Stavrakia, Heraklion, Crete, Greece 4 Laboratory of Anatomy, Medical School, University of Crete, Voutes, 71110 Heraklion, Crete, Greece 5 General Chemical State Laboratory of Greece, Department of Hazardous Substances, Mixtures and Articles, 16 An. Tsocha Str, 1152 Athens, Greece 6 UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal Purpose: Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified by EPA as both a toxicity class II and class III agent and metabolized in vitro by aldehyde oxidase (AOX) by the nitro-imino group reduction and from CYPs via oxidation reactions. AOX metabolizes many xenobiotics in vitro, but its importance in vivo is usually unknown in relation to cytochrome P450s (CYPs) and other detoxification mechanisms. In the present study the AOX activity reduction in the IMI metabolism is examined by using substances such as sodium tungsten (ST) and induced damage via the comet assay technique is evaluated. Methods: Nine New Zealand rabbits were used in this study separated equally into three groups, corresponding to the control group (CON) that received tap water, the IMI group that received imidacloprid and the co-exposed group with a combination of IMI and ST receiving imidacloprid + sodium tungstate dihydrate. The experiment lasted about 2.5 months. The dosage for the IMI group was 360 mg Imidacloprid/500 ml water/rabbit/exposure day and for the addition of ST, the dosage was 360 mg imidacloprid + 1000 mg sodium tungstate dihydrate/500 ml water/rabbit/exposure day. Peripheral blood samples were collected and the lymphocytes were used for the assay. Results: In our study an increase in tail intensity was observed in both exposure groups compared to the CON groups although not statistically. The IMI group reveals the highest achieved tail
P16-037 Is oxidative stress involved in OTA renal toxicity? J. Enciso Gadea ∗ , L. Pastor Castro, A. Azqueta Oscoz, A. López de Cerain Salsamendi, A. Vettorazzi Armental Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and proposed as a possible etiological agent of the Balkan Endemic Nephropathy (BEN) in humans. Thereby, OTA has been classified as a possible human carcinogen (group 2B). Though its mechanism of action is still unknown, many authors consider oxidative stress as a possible hypothesis. Moreover, large sex-differences have been observed in different carcinogenicity studies. Therefore, the objective of this study was to evaluate the sex-dependent kidney oxidative stress response to OTA in F344 rats. For that purpose, male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 mg OTA/kg b.w. or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) levels, total glutathione Stransferase (GST) and superoxide dismutase (SOD) activities were analysed in kidney tissue. Besides, the standard alkaline comet assay was used in combination with formamidopyrimidine DNA glycosylase (FPG) in order to detect oxidative DNA damage. In the 7-days study, male tGSH levels were significantly lower both in the control and the OTA-treated group. In the 21-days study, tGSH levels were significantly lower in control males, and female GST activity decreased as OTA dose increased. However, we could not find an increase of oxidative DNA damage in the kidneys of male or female F344 rats treated for 7 or 21 days. Our results are not able to link the higher nephrotoxicity that OTA exhibits in male rats with an increase in oxidative stress. http://dx.doi.org/10.1016/j.toxlet.2016.06.1876 P16-038 In vitro neurotoxicity of ‘Legal X’: The combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) induces mitochondrial impairment and disrupts thiol homeostasis in dopaminergic SH-SY5Y cells D. Dias Da Silva ∗ , A. Ferreira, M. Martins, M. Valente, F. Remião, F. Carvalho, M. Bastos, H. Carmo UCIBIO/REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Portugal In the drug abuse scene, 1-benzylpiperazine (BZP) and 1(m-trifluoromethylphenyl)piperazine (TFMPP) are concomitantly consumed under the name ‘Legal X’. As the central nervous system is one of the main targets of toxicity of these drugs, we assessed the detrimental effects of BZP:TFMPP combination (at three different mixture ratios) in human SH-SY5Y cells differentiated to induce a dopaminergic phenotype. The toxicity data obtained in the MTT assay for individual exposure of BZP (EC50 3.74 mM) and TFMPP (EC50 208.67 M) after 24 h were used to predict combined effects of the drugs, by employing the concentration addition (CA) and independent action (IA) models. The predictions and the effects obtained experimentally were compared. As the mixture effects did not deviate from
S247
CA, they were considered additive. IA concept overestimated the effects of two mixtures. Single ineffective concentrations of BZP and TFMPP additively provoked remarkable neurotoxicity when combined (p < 0.01, versus control). Cellular and mitochondrial (dys)function was further investigated. BZP disturbed oxidative defences (intracellular GSH/GSSG) to a higher extent than TFMPP, which in turn more severely disrupted mitochondria functioning (by hyperpolarizing the membrane and increasing Ca2+ efflux of the organelle). Additional oxidative and energetic homeostasis biomarkers, including intracellular ROS and ATP levels, were not significantly disturbed. The results obtained for the mixture agreed with the individual data, showing consistency with additivity. The data described herein clearly shows that the toxicological assessment of single drugs may considerably underestimate the real risks associated with the consumption of drug combinations in the corresponding abuse scenarios. http://dx.doi.org/10.1016/j.toxlet.2016.06.1877 P16-039 DNA damage after long-term exposure of rabbits to Imidacloprid and sodium tungstate A.I. Vardavas 1,∗ , E. Ozcagli 2 , P. Fragkiadaki 1 , P.D. Stivaktakis 1 , M.N. Tzatzarakis 1 , K. Kaloudis 1 , M. Tsardi 3 , G. Datseri 3 , J. Tsiaoussis 4 , C. Tsitsimpikou 5 , F. Carvalho 6 , A.M. Tsatsakis 1 1
Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece 2 Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazit–Istanbul 34116, Turkey 3 Department of Pathology, Medical School Voutes-Stavrakia, Heraklion, Crete, Greece 4 Laboratory of Anatomy, Medical School, University of Crete, Voutes, 71110 Heraklion, Crete, Greece 5 General Chemical State Laboratory of Greece, Department of Hazardous Substances, Mixtures and Articles, 16 An. Tsocha Str, 1152 Athens, Greece 6 UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal Purpose: Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified by EPA as both a toxicity class II and class III agent and metabolized in vitro by aldehyde oxidase (AOX) by the nitro-imino group reduction and from CYPs via oxidation reactions. AOX metabolizes many xenobiotics in vitro, but its importance in vivo is usually unknown in relation to cytochrome P450s (CYPs) and other detoxification mechanisms. In the present study the AOX activity reduction in the IMI metabolism is examined by using substances such as sodium tungsten (ST) and induced damage via the comet assay technique is evaluated. Methods: Nine New Zealand rabbits were used in this study separated equally into three groups, corresponding to the control group (CON) that received tap water, the IMI group that received imidacloprid and the co-exposed group with a combination of IMI and ST receiving imidacloprid + sodium tungstate dihydrate. The experiment lasted about 2.5 months. The dosage for the IMI group was 360 mg Imidacloprid/500 ml water/rabbit/exposure day and for the addition of ST, the dosage was 360 mg imidacloprid + 1000 mg sodium tungstate dihydrate/500 ml water/rabbit/exposure day. Peripheral blood samples were collected and the lymphocytes were used for the assay. Results: In our study an increase in tail intensity was observed in both exposure groups compared to the CON groups although not statistically. The IMI group reveals the highest achieved tail