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Effects of carbamates as oxidative stressors on glutathione levels and lipid peroxidation in CHO-K1 cells
Abstracts / Toxicology Letters 164S (2006) S1–S324
S235
tration was very transient after a dose of 50 mg kg−1 and the paraoxon-induced respiratory effects reappeared. Our data suggest that cholinesterase reactivation is a necessary but not sufficient condition for the antidotal effect of pralidoxime. Some mechanisms of toxicity, not related to the inhibition of cholinesterase activity, could explain these discrepancies.
of respiration at rest. As evidenced by the complete and sustained effects of atropine, the respiratory effects, at these doses, only involved muscarinic receptors, with no evidence of any depressant effect on the respiratory centres and nicotinic effect as evidenced by an increase in tidal volume with the lack of modification of the minute ventilation.
doi:10.1016/j.toxlet.2006.07.147
doi:10.1016/j.toxlet.2006.07.148
P17-04 Toxic doses of paraoxon alters respiratory pattern without causing respiratory failure in the rats
P17-05 Effects of carbamates as oxidative stressors on glutathione levels and lipid peroxidation in CHO-K1 cells
Villa Antoine, Houz´e Pascal, Monier Claire, Ris`ede Patricia, Baud Fr´ed´eric INSERM U705, Paris CEDEX 10, France Respiratory failure through a combination of muscarinic, nicotinic and central effects is considered the primary cause of death in acute organophosphate poisoning. However, the literature is unclear as to the mechanisms involved. The aim of this study was to assess the mechanisms of paraoxon-induced respiratory toxicity. We studied in rats poisoned subcutaneously with paraoxon, at doses near the LD50 , the pattern of respiration by whole body plethysmography in awake rats and the occurrence of acute respiratory failure using arterial blood gases over 4 and 3 h, respectively. Thereafter, we assessed the effects of atropine on paraoxon-induced effects on ventilation and arterial blood gases. The tested doses of paraoxon were, respectively, 10 (0.043 mg kg−1 ), 50 (0.215 mg kg−1 ) and 75% (0.322 mg kg−1 ) of the subcutaneous LD50 of paraoxon (plethysmography study) and, 50% and 75% LD50 (blood gas study). The effects of atropine (10 mg kg−1 ) were studied at the 75% dose. All results are expressed as mean ± S.E.M. Statistical analysis was performed using Student’s test and ANOVA tests with p < 0.05 as significant value. The rats were frankly symptomatic at the two highest doses of paraoxon. At 30 min post injection and throughout the study, there was a significant decrease in respiratory rate, an increase in expiratory time with no modifications of the inspiratory time. There was a significant increase of the tidal volume for the 50% and 75% LD50 doses. Apnea was not detected. For the two lowest doses of paraoxon, no effect on arterial blood gases was observed. For the 75% dose, paraoxon had no effects on PaO2 , PaCO2 and HCO3 − but there was a significant decrease in pH at 30 min. Atropine reversed all the paraoxon-induced respiratory pattern alterations. We conclude that paraoxon, at doses equal to 50% and 75% of the LD50 , alters the pattern
M.J. Ruiz 1 , E. Maran 2 , H. Berrada 1 , M. Fern´andez 2 1 Laboratorio de Toxicologia, Facultad de Farmacia, Universidad de Valencia, Spain; 2 Sezione di Chimica Analitica e Ambientale, Dip. Scienze Chimiche, Universita degli Studi di Trieste, Italy
The toxic effect of four carbamates, aldicarb and its metabolites (aldicarb sulfone and aldicarb sulfoxide) and propoxur in CHO-K1 cells was investigated. After 24 h exposure of 10 g/ml of each carbamate in cells, intracellular reduced glutathione (GSH) content was depleted. The rank order for GSH decreased as follows: aldicarb sulfone > aldicarb and aldicarb sulfone > propoxur. On the other hand, oxidized glutathione (GSSG) level appeared almost unchanged. The GSH:GSSG ratio was significantly reduced with aldicarb, aldicarb sulfone and aldicarb sulfoxide compared to control cells. Glutathione reductase (GR), glutathione peroxidasa (GPx) and glutathione-S-transferase (GST) contribute to antioxidant defence mechanisms in cells after carbamate exposure. Alteration of GSH levels was accompanied by induction of GR activity for all carbamates. The highest increase of GR activity was observed in cells exposed to aldicarb. Some differences between carbamates in GPx and GST activity were observed. GPx activity was increased after aldicarb sulfone, aldicarb sulfoxide and propoxur treatment, while GST activity was increased only after aldicarb sulfone and propoxur exposure. Lipid peroxidation in CHO-K1 cells exposed to carbamates was studied by thiobarbituric acid reactive substances (TBARS) production. Addition of aldicarb sulfone, aldicarb sulfoxide and propoxur to cells resulted in an increase in TBARS levels compared with control cells. For a further characterization of the nature of carbamate toxicity, cells were pre-treated before carbamate exposure with d-l-buthionine-(S,R)-sulfoximine (BSO).
S236
Abstracts / Toxicology Letters 164S (2006) S1–S324
Aldicarb sulfone, aldicarb sulfoxide and propoxur cytotoxicity was exacerbated by the previous depletion of cellular GSH by BSO. Results suggest that carbamates induce GSH depletion, leading to oxidative stress. However, the induction of the antioxidant enzyme GST produced by aldicarb sulfone and propoxur in CHO-K1 cells, suggests that the enzyme provides adequate protection to mammals cells through the detoxification of these carbamates.
tration (MCHC), leukocyte count (Leuko), triacylglycerols (TRIG), aspartate aminotransferase (AST), alanin aminotransferase (ALT), cholinesterase (ChE), calcium (Ca2+ ) and inorganic phosphate (PHOS) were comparable in the two groups during the study. Changes in the erythrocyte and biochemical profile after exposure to cypermethrin-based preparation may be referred to possible disruption of haematopoiesis and mild damage of liver.
Acknowledgement: This work was supported by the Generalitat Valenciana (GV2005-98).
Acknowledgements: This research was supported by the GACR Project No. 523/03/H076, USB RIFCH Project No. MSM 6007665809, MSM Project No. 621571240 and WMU Poland grant No. 0804 205.
doi:10.1016/j.toxlet.2006.07.149 P17-06 Effects of cypermethrin on hematological and biochemical profile on common carp (Cyprinus carpio) Vel´ısˇek 1,2 ,
Wlasow 4 ,
J. T. bodov´a 2,3 , R. Dobˇs´ıkov´a 3
P.
Gomulka 4 ,
Z.
Svo-
1 University of South Bohemia Ceske Budejovice, Czech
Republic; 2 Research Institute of Fish Culture and Hydrobiology Vodnany, University of South Bohemia Ceske Budejovice, Czech Republic; 3 University of Veterinary and Pharmaceutical Science Brno, Czech Republic; 4 University of Warmia and Mazury Olsztyn, Poland The aim of this study was to assess the effect of cypermethrin [(R,S)-␣-cyano-3-phenoxybenzyl (1RS)-cis,tra3-(2,2-dicholorovinyl)-2,2-dimethyl cyclopropane carboxylate] on common carp (Cyprinus carpio). The effect was assessed on the basis of the results of haematological and biochemical examination of a control and an experimental group exposed to Alimethrin 10 EC pesticide preparation (active substance 100 g l−1 of cypermethrin) in a concentration of 29.1 g l−1 . The experimental group showed significantly higher values (p < 0.01) of erythrocyte count (RBC), glucose (GLU), creatinkinase (CK), lactate (LACT) and significantly lower values (p < 0.01) of mean erythrocyte volume (MCV), erythrocyte haemoglobin (MCH), total protein (TP), albumins (ALB), total globulins (GLOB), ammonia (NH3 ), lactate dehydrogenase (LDH) and alkalic phosphatase (ALP) compared to the control group. Also, the relative and absolute count of lymphocyte was a significant (p < 0.01) decreased and a significant increased in both the relative and absolute count of developmental forms of neutrophile granulocytes, and both the band- and segmented neutrophile granulocytes in the experimental group. Values of haemoglobin content (Hb), haematocrit (PCV), mean colour concen-
doi:10.1016/j.toxlet.2006.07.150 P17-07 Acute renal failure alters the kinetics of pralidoxime in rats Maya Kayouka, Chantal Martin, Claire Monier, Patricia Risede, Jean-Michel Warnet, Frederic J. Baud, Pascal Houz´e INSERM U705; CNRS, UMR 7157; Universit´e Paris 5, France Pralidoxime methylsulfate (Contrathion® ) (PRX) is used as an antidote to organophosphate poisoning. The efficiency of the antidote depends on its plasma concentrations that should be greater than 4 mg L−1 . The pharmacokinetics of PRX is characterized by complete absorption, no protein binding, no metabolism and fast renal excretion resulting in an extremely short halflife (<60 min). Therefore, the aim of this study was to assess the effect of renal failure on the kinetics of PRX. We developed a rat model with acute renal failure (ARF) induced by potassium bichromate administration. On the first day, male Sprague–Dawley rats (250–350 g) were intoxicated by potassium bichromate subcutaneously administered (15 mg kg−1 ) and kept for 4 days in metabolic cages. On the second day, animals were treated with PRX (50 mg kg−1 ) by the intramuscular route. Blood samples were collected during 180 min after PRX injection and urine were collected 48 h post injection. Plasma PRX concentrations were measured by liquid chromatography with electrochemical detection. Results are expressed as mean ± S.E.M. Statistic analysis were performed using to Mann–Whitney and Anova tests ARF was observed 48 h following bichromate injection. Simultaneously, PRX kinetics were significantly different between the control and the treated groups (alpha and beta half-life: increment × 2 in ARF
S235
tration was very transient after a dose of 50 mg kg−1 and the paraoxon-induced respiratory effects reappeared. Our data suggest that cholinesterase reactivation is a necessary but not sufficient condition for the antidotal effect of pralidoxime. Some mechanisms of toxicity, not related to the inhibition of cholinesterase activity, could explain these discrepancies.
of respiration at rest. As evidenced by the complete and sustained effects of atropine, the respiratory effects, at these doses, only involved muscarinic receptors, with no evidence of any depressant effect on the respiratory centres and nicotinic effect as evidenced by an increase in tidal volume with the lack of modification of the minute ventilation.
doi:10.1016/j.toxlet.2006.07.147
doi:10.1016/j.toxlet.2006.07.148
P17-04 Toxic doses of paraoxon alters respiratory pattern without causing respiratory failure in the rats
P17-05 Effects of carbamates as oxidative stressors on glutathione levels and lipid peroxidation in CHO-K1 cells
Villa Antoine, Houz´e Pascal, Monier Claire, Ris`ede Patricia, Baud Fr´ed´eric INSERM U705, Paris CEDEX 10, France Respiratory failure through a combination of muscarinic, nicotinic and central effects is considered the primary cause of death in acute organophosphate poisoning. However, the literature is unclear as to the mechanisms involved. The aim of this study was to assess the mechanisms of paraoxon-induced respiratory toxicity. We studied in rats poisoned subcutaneously with paraoxon, at doses near the LD50 , the pattern of respiration by whole body plethysmography in awake rats and the occurrence of acute respiratory failure using arterial blood gases over 4 and 3 h, respectively. Thereafter, we assessed the effects of atropine on paraoxon-induced effects on ventilation and arterial blood gases. The tested doses of paraoxon were, respectively, 10 (0.043 mg kg−1 ), 50 (0.215 mg kg−1 ) and 75% (0.322 mg kg−1 ) of the subcutaneous LD50 of paraoxon (plethysmography study) and, 50% and 75% LD50 (blood gas study). The effects of atropine (10 mg kg−1 ) were studied at the 75% dose. All results are expressed as mean ± S.E.M. Statistical analysis was performed using Student’s test and ANOVA tests with p < 0.05 as significant value. The rats were frankly symptomatic at the two highest doses of paraoxon. At 30 min post injection and throughout the study, there was a significant decrease in respiratory rate, an increase in expiratory time with no modifications of the inspiratory time. There was a significant increase of the tidal volume for the 50% and 75% LD50 doses. Apnea was not detected. For the two lowest doses of paraoxon, no effect on arterial blood gases was observed. For the 75% dose, paraoxon had no effects on PaO2 , PaCO2 and HCO3 − but there was a significant decrease in pH at 30 min. Atropine reversed all the paraoxon-induced respiratory pattern alterations. We conclude that paraoxon, at doses equal to 50% and 75% of the LD50 , alters the pattern
M.J. Ruiz 1 , E. Maran 2 , H. Berrada 1 , M. Fern´andez 2 1 Laboratorio de Toxicologia, Facultad de Farmacia, Universidad de Valencia, Spain; 2 Sezione di Chimica Analitica e Ambientale, Dip. Scienze Chimiche, Universita degli Studi di Trieste, Italy
The toxic effect of four carbamates, aldicarb and its metabolites (aldicarb sulfone and aldicarb sulfoxide) and propoxur in CHO-K1 cells was investigated. After 24 h exposure of 10 g/ml of each carbamate in cells, intracellular reduced glutathione (GSH) content was depleted. The rank order for GSH decreased as follows: aldicarb sulfone > aldicarb and aldicarb sulfone > propoxur. On the other hand, oxidized glutathione (GSSG) level appeared almost unchanged. The GSH:GSSG ratio was significantly reduced with aldicarb, aldicarb sulfone and aldicarb sulfoxide compared to control cells. Glutathione reductase (GR), glutathione peroxidasa (GPx) and glutathione-S-transferase (GST) contribute to antioxidant defence mechanisms in cells after carbamate exposure. Alteration of GSH levels was accompanied by induction of GR activity for all carbamates. The highest increase of GR activity was observed in cells exposed to aldicarb. Some differences between carbamates in GPx and GST activity were observed. GPx activity was increased after aldicarb sulfone, aldicarb sulfoxide and propoxur treatment, while GST activity was increased only after aldicarb sulfone and propoxur exposure. Lipid peroxidation in CHO-K1 cells exposed to carbamates was studied by thiobarbituric acid reactive substances (TBARS) production. Addition of aldicarb sulfone, aldicarb sulfoxide and propoxur to cells resulted in an increase in TBARS levels compared with control cells. For a further characterization of the nature of carbamate toxicity, cells were pre-treated before carbamate exposure with d-l-buthionine-(S,R)-sulfoximine (BSO).
S236
Abstracts / Toxicology Letters 164S (2006) S1–S324
Aldicarb sulfone, aldicarb sulfoxide and propoxur cytotoxicity was exacerbated by the previous depletion of cellular GSH by BSO. Results suggest that carbamates induce GSH depletion, leading to oxidative stress. However, the induction of the antioxidant enzyme GST produced by aldicarb sulfone and propoxur in CHO-K1 cells, suggests that the enzyme provides adequate protection to mammals cells through the detoxification of these carbamates.
tration (MCHC), leukocyte count (Leuko), triacylglycerols (TRIG), aspartate aminotransferase (AST), alanin aminotransferase (ALT), cholinesterase (ChE), calcium (Ca2+ ) and inorganic phosphate (PHOS) were comparable in the two groups during the study. Changes in the erythrocyte and biochemical profile after exposure to cypermethrin-based preparation may be referred to possible disruption of haematopoiesis and mild damage of liver.
Acknowledgement: This work was supported by the Generalitat Valenciana (GV2005-98).
Acknowledgements: This research was supported by the GACR Project No. 523/03/H076, USB RIFCH Project No. MSM 6007665809, MSM Project No. 621571240 and WMU Poland grant No. 0804 205.
doi:10.1016/j.toxlet.2006.07.149 P17-06 Effects of cypermethrin on hematological and biochemical profile on common carp (Cyprinus carpio) Vel´ısˇek 1,2 ,
Wlasow 4 ,
J. T. bodov´a 2,3 , R. Dobˇs´ıkov´a 3
P.
Gomulka 4 ,
Z.
Svo-
1 University of South Bohemia Ceske Budejovice, Czech
Republic; 2 Research Institute of Fish Culture and Hydrobiology Vodnany, University of South Bohemia Ceske Budejovice, Czech Republic; 3 University of Veterinary and Pharmaceutical Science Brno, Czech Republic; 4 University of Warmia and Mazury Olsztyn, Poland The aim of this study was to assess the effect of cypermethrin [(R,S)-␣-cyano-3-phenoxybenzyl (1RS)-cis,tra3-(2,2-dicholorovinyl)-2,2-dimethyl cyclopropane carboxylate] on common carp (Cyprinus carpio). The effect was assessed on the basis of the results of haematological and biochemical examination of a control and an experimental group exposed to Alimethrin 10 EC pesticide preparation (active substance 100 g l−1 of cypermethrin) in a concentration of 29.1 g l−1 . The experimental group showed significantly higher values (p < 0.01) of erythrocyte count (RBC), glucose (GLU), creatinkinase (CK), lactate (LACT) and significantly lower values (p < 0.01) of mean erythrocyte volume (MCV), erythrocyte haemoglobin (MCH), total protein (TP), albumins (ALB), total globulins (GLOB), ammonia (NH3 ), lactate dehydrogenase (LDH) and alkalic phosphatase (ALP) compared to the control group. Also, the relative and absolute count of lymphocyte was a significant (p < 0.01) decreased and a significant increased in both the relative and absolute count of developmental forms of neutrophile granulocytes, and both the band- and segmented neutrophile granulocytes in the experimental group. Values of haemoglobin content (Hb), haematocrit (PCV), mean colour concen-
doi:10.1016/j.toxlet.2006.07.150 P17-07 Acute renal failure alters the kinetics of pralidoxime in rats Maya Kayouka, Chantal Martin, Claire Monier, Patricia Risede, Jean-Michel Warnet, Frederic J. Baud, Pascal Houz´e INSERM U705; CNRS, UMR 7157; Universit´e Paris 5, France Pralidoxime methylsulfate (Contrathion® ) (PRX) is used as an antidote to organophosphate poisoning. The efficiency of the antidote depends on its plasma concentrations that should be greater than 4 mg L−1 . The pharmacokinetics of PRX is characterized by complete absorption, no protein binding, no metabolism and fast renal excretion resulting in an extremely short halflife (<60 min). Therefore, the aim of this study was to assess the effect of renal failure on the kinetics of PRX. We developed a rat model with acute renal failure (ARF) induced by potassium bichromate administration. On the first day, male Sprague–Dawley rats (250–350 g) were intoxicated by potassium bichromate subcutaneously administered (15 mg kg−1 ) and kept for 4 days in metabolic cages. On the second day, animals were treated with PRX (50 mg kg−1 ) by the intramuscular route. Blood samples were collected during 180 min after PRX injection and urine were collected 48 h post injection. Plasma PRX concentrations were measured by liquid chromatography with electrochemical detection. Results are expressed as mean ± S.E.M. Statistic analysis were performed using to Mann–Whitney and Anova tests ARF was observed 48 h following bichromate injection. Simultaneously, PRX kinetics were significantly different between the control and the treated groups (alpha and beta half-life: increment × 2 in ARF