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296 Morphological changes in mouse tests after treatment with flutamide
s82
Poster Session P12. Drug toxicology
Thus, we concluded in establishing the risk criteria of toxic impact, which include: for E – changes of the generative function, and for L-T – activation of LP and hypertoxinemia. The extent of such risk depends on the effective dose (concentration) of medicines. 296
MORPHOLOGICAL CHANGES IN MOUSE TESTS AFTER TREATMENT WITH FLUTAMIDE
R. Anahara 1 , Y. Ono 1 , Y. Toyama 2 , C. Mori 1,3 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 2 Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Japan Flutamide (Flu) inhibits androgen uptake and/or nuclear binding of androgen in target tissues and is used as a medicine for prostate cancer. The purpose of the present study was to investigate the effect of Flu on testes of newborn and adult mice. Newborn ICR male mice were subcutaneously injected with 0.005, 0.05, 0.5, 5, 50 or 500µg/mouse/shot of Flu on Days 2, 4, 6, 8, 10 and 12. The day of birth was regarded as Day 1. Also, ICR adult male mice were subcutaneously injected with 0.05, 0.5, 5 or 50µg/mouse/day of Flu for sequential 5 days. Control animals received vehicle (corn oil). The testes taken from newborn until adult mice (Day 7, 14, 21, 28, 35, 42, 56, 63 and 84) were processed for electron microscopy. Some newborn animals were kept until the age of 84 days, and housed with normal females for fertility test. Spermatids with deformed nuclei and/or acrosomal caps were often observed in the seminiferous epithelium of Flu-treated mice. In addition, complete or partial deletion in the ectoplasmic specialization between the Sertoli cell and spermatids was observed. These abnormalities were found at all doses. The specialization between adjoining Sertoli cells, or the blood testis barrier, was not affected. The newborn animals were shown to be fertile when they reached 84 days of age. Since similar observations were reported after treatment with β-estradiol 3-benzoate, the presence of Flu, a potent anti-androgen, may create an ‘oestrogenic environment’ in mouse testes. (This work was supported by the fund for endocrine disrupters from the Ministry of the Environment, Japan.) 297
LONG-TERM METABOLIC AND ENDOCRINE ACTIVITY CHANGES IN RESPONSE TO CLENBUTEROL IN MATURE FEMALE PIGS
T. Gojmerac 1 , B. Mandi´c 2 , M. Korši´c 3 , A. Tomaši´c 1 , B. Vinkovi´c 1 . 1 Croatian Veterinary Institute, Zagreb, Croatia; 2 Vuk Vrhovac Institute for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine, University of Zagreb, Croatia; 3 University Department of Medicine-Rebro, Zagreb, Croatia Clenbuterol, a β2 -adrenergic agonist, has been illegally used as a repartitioning agent to improve the performance of meat-producing animals, since its abuse may cause risk for consumer health. The direct and indirect effect of clenbuterol especially depends on the mode and duration of administration, i.e. acute or long-term. In the present study, the effect of intravenously repeated administration of clenbuterol in a growth-promoting dose on the metabolic and endocrine status of mature female pigs was evaluated on the basis of biochemical findings. The growth-promoting dose of clenbuterol was administered intravenously daily for 20 days of estrous cycle to cross-bred mature female pigs (80–100 kg, n=12). Before (0) and at 30,60,90,120,180,240 and 300 min after the last drug dosage, blood samples were collected by jugular venipuncture and analyzed for serum insulin, nonesterified fatty acid (NEFA) and blood glucose concentration. Serum 17β-estradiol (17β-E) and progesterone (P) concentration were measured in the samples collected 2 times daily at 5-h intervals on the first 4 days after last drug dosage. Serum NEFA concentrations moderately increased and reached highest level, and serum insulin levels moderately decreased and reached lowest level at 30 min preprandially after the last clenbuterol dosage, but the changes did not differ significantly from those in control animals. During the same period, the blood glucose concentration
reached the highest level at 90 min postprandially after the last dosage and showed no significant difference from those in control animals. Serum 17β-E and P concentrations on the days around the onset of the next expected estrous were lower but not significantly, resulting in normal occurrence of estrous in the pigs. On the basis of the data obtained, we suggest that long-term administration of clenbuterol in a growth-promoting dose to female pigs provoked only subtle metabolic and endocrine activity changes, indicating down-regulation of the specific β2 -adrenergic receptors in target tissues. 298
A STUDY OF HEPARIN EFFECTS ON HEMATOBIOCHEMICAL FACTORS AND BILIARYBILIRUBIN SECRETION IN SHEEP
M. Pourjafar, K. Mostaghni. School of Veterinary Medicine, Shahrekord University, Shahrekord, Iran This study was carried out to find out the effects of heparin on hemogram, biochemical factors in serum, biliary secretion and the concentration of biliary bilirubin in sheep. In this study, 10 healthy Iranian crossbred male sheep, aged between 2–3 years and weighting 66–78 kg (71±4.06) were randomly selected. Before the experiment, blood samples were obtained in 3 consecutive days and the results of the tests for hematological, serum enzymes and serum bilirubin concentration were determined. These normal values were accepted as a control measure. In a preliminary study, to find out the abnormal effects of heparin to the above mentioned factors, heparin was administered at the rate of 107 IU/Kg b.w., IV as a loading dose, and 320 IU/KG b.w.,S.C every 12 hours for 5 consecutive days as a maintenance dose. The finding of the tests from the blood samples, during treatment periods and after that it has been noticed that the heparin did not have any significant effects on the factors mentioned above at this dose levels and also in increased dose by 1.5 times. Following anesthesia by using thiopental sodium (16 mg /Kg b.w., LV) and finding a cannula in the duodenum and the gall bladder, the volume of bile and the concentration of biliary bilirubin, were measured. The bile was collected and reinjected into the duodenum through the existing cannula, for maintaining the enterohepatic circulation throughout the experiment. Before using heparin, blood and bile samples were collected for 3 days. The samples were used for the determination of hemogram, enzymes, the concentration of bilirubin in serum, the volume of bile secretion, and the concentration of biliary bilirubin as normal values. Then, heparin was given at the rate of 214 IU/Kg b.w.LV, as a loading dose and 640 IU/Kg b.w., SC. every 12 hours as a maintenance dose for 5 consecutive days. The blood samples were taken in every hour, during treatment period and also three days following of treatment. At the same time biliary samples were taken every half an hour through the cannula within the gall bladder. Having measured the volume and sampled, the remaining bile was reinjected into the intestine through the existing cannula. The blood samples were used to determine the levels of Hb and HCT, red and white blood cell counts (and WBC differentiate counts), measurement of enzymes (AST, ALT, ALP) and bilirubin in serum. Also, from the bile samples, the concentration of bilirubin was measured. The results of the measurements prior to injection, during and after injection were analyzed by using the pair T-test with p=0.01. The released data from these experiments indicate that levels of Hb concentration and HCT and also red blood cell counts in the second day following the injection of heparin were significantly lowered in comparison with the control values (p<0.01). However, no significant changes were observed in white blood cell counts, its differentiate counts and the level of enzymes AST,ALT and ALP (p<0.01). On the other hand, a significant increase was observed in serum bilirubin concentration, biliary secretion and biliary bilirubin concentration on the second post injection day (p<0.01). From this study, in addition to the determination of treatment dose of heparin, it can be concluded that the toxic level of heparin injection can cause extravascular hemolysis in sheep. Obviously, reduction of red blood cells by the reticuloendothelial system, catabolism of heme, increased bile volume, increased concentration
Poster Session P12. Drug toxicology
Thus, we concluded in establishing the risk criteria of toxic impact, which include: for E – changes of the generative function, and for L-T – activation of LP and hypertoxinemia. The extent of such risk depends on the effective dose (concentration) of medicines. 296
MORPHOLOGICAL CHANGES IN MOUSE TESTS AFTER TREATMENT WITH FLUTAMIDE
R. Anahara 1 , Y. Ono 1 , Y. Toyama 2 , C. Mori 1,3 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 2 Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Japan Flutamide (Flu) inhibits androgen uptake and/or nuclear binding of androgen in target tissues and is used as a medicine for prostate cancer. The purpose of the present study was to investigate the effect of Flu on testes of newborn and adult mice. Newborn ICR male mice were subcutaneously injected with 0.005, 0.05, 0.5, 5, 50 or 500µg/mouse/shot of Flu on Days 2, 4, 6, 8, 10 and 12. The day of birth was regarded as Day 1. Also, ICR adult male mice were subcutaneously injected with 0.05, 0.5, 5 or 50µg/mouse/day of Flu for sequential 5 days. Control animals received vehicle (corn oil). The testes taken from newborn until adult mice (Day 7, 14, 21, 28, 35, 42, 56, 63 and 84) were processed for electron microscopy. Some newborn animals were kept until the age of 84 days, and housed with normal females for fertility test. Spermatids with deformed nuclei and/or acrosomal caps were often observed in the seminiferous epithelium of Flu-treated mice. In addition, complete or partial deletion in the ectoplasmic specialization between the Sertoli cell and spermatids was observed. These abnormalities were found at all doses. The specialization between adjoining Sertoli cells, or the blood testis barrier, was not affected. The newborn animals were shown to be fertile when they reached 84 days of age. Since similar observations were reported after treatment with β-estradiol 3-benzoate, the presence of Flu, a potent anti-androgen, may create an ‘oestrogenic environment’ in mouse testes. (This work was supported by the fund for endocrine disrupters from the Ministry of the Environment, Japan.) 297
LONG-TERM METABOLIC AND ENDOCRINE ACTIVITY CHANGES IN RESPONSE TO CLENBUTEROL IN MATURE FEMALE PIGS
T. Gojmerac 1 , B. Mandi´c 2 , M. Korši´c 3 , A. Tomaši´c 1 , B. Vinkovi´c 1 . 1 Croatian Veterinary Institute, Zagreb, Croatia; 2 Vuk Vrhovac Institute for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine, University of Zagreb, Croatia; 3 University Department of Medicine-Rebro, Zagreb, Croatia Clenbuterol, a β2 -adrenergic agonist, has been illegally used as a repartitioning agent to improve the performance of meat-producing animals, since its abuse may cause risk for consumer health. The direct and indirect effect of clenbuterol especially depends on the mode and duration of administration, i.e. acute or long-term. In the present study, the effect of intravenously repeated administration of clenbuterol in a growth-promoting dose on the metabolic and endocrine status of mature female pigs was evaluated on the basis of biochemical findings. The growth-promoting dose of clenbuterol was administered intravenously daily for 20 days of estrous cycle to cross-bred mature female pigs (80–100 kg, n=12). Before (0) and at 30,60,90,120,180,240 and 300 min after the last drug dosage, blood samples were collected by jugular venipuncture and analyzed for serum insulin, nonesterified fatty acid (NEFA) and blood glucose concentration. Serum 17β-estradiol (17β-E) and progesterone (P) concentration were measured in the samples collected 2 times daily at 5-h intervals on the first 4 days after last drug dosage. Serum NEFA concentrations moderately increased and reached highest level, and serum insulin levels moderately decreased and reached lowest level at 30 min preprandially after the last clenbuterol dosage, but the changes did not differ significantly from those in control animals. During the same period, the blood glucose concentration
reached the highest level at 90 min postprandially after the last dosage and showed no significant difference from those in control animals. Serum 17β-E and P concentrations on the days around the onset of the next expected estrous were lower but not significantly, resulting in normal occurrence of estrous in the pigs. On the basis of the data obtained, we suggest that long-term administration of clenbuterol in a growth-promoting dose to female pigs provoked only subtle metabolic and endocrine activity changes, indicating down-regulation of the specific β2 -adrenergic receptors in target tissues. 298
A STUDY OF HEPARIN EFFECTS ON HEMATOBIOCHEMICAL FACTORS AND BILIARYBILIRUBIN SECRETION IN SHEEP
M. Pourjafar, K. Mostaghni. School of Veterinary Medicine, Shahrekord University, Shahrekord, Iran This study was carried out to find out the effects of heparin on hemogram, biochemical factors in serum, biliary secretion and the concentration of biliary bilirubin in sheep. In this study, 10 healthy Iranian crossbred male sheep, aged between 2–3 years and weighting 66–78 kg (71±4.06) were randomly selected. Before the experiment, blood samples were obtained in 3 consecutive days and the results of the tests for hematological, serum enzymes and serum bilirubin concentration were determined. These normal values were accepted as a control measure. In a preliminary study, to find out the abnormal effects of heparin to the above mentioned factors, heparin was administered at the rate of 107 IU/Kg b.w., IV as a loading dose, and 320 IU/KG b.w.,S.C every 12 hours for 5 consecutive days as a maintenance dose. The finding of the tests from the blood samples, during treatment periods and after that it has been noticed that the heparin did not have any significant effects on the factors mentioned above at this dose levels and also in increased dose by 1.5 times. Following anesthesia by using thiopental sodium (16 mg /Kg b.w., LV) and finding a cannula in the duodenum and the gall bladder, the volume of bile and the concentration of biliary bilirubin, were measured. The bile was collected and reinjected into the duodenum through the existing cannula, for maintaining the enterohepatic circulation throughout the experiment. Before using heparin, blood and bile samples were collected for 3 days. The samples were used for the determination of hemogram, enzymes, the concentration of bilirubin in serum, the volume of bile secretion, and the concentration of biliary bilirubin as normal values. Then, heparin was given at the rate of 214 IU/Kg b.w.LV, as a loading dose and 640 IU/Kg b.w., SC. every 12 hours as a maintenance dose for 5 consecutive days. The blood samples were taken in every hour, during treatment period and also three days following of treatment. At the same time biliary samples were taken every half an hour through the cannula within the gall bladder. Having measured the volume and sampled, the remaining bile was reinjected into the intestine through the existing cannula. The blood samples were used to determine the levels of Hb and HCT, red and white blood cell counts (and WBC differentiate counts), measurement of enzymes (AST, ALT, ALP) and bilirubin in serum. Also, from the bile samples, the concentration of bilirubin was measured. The results of the measurements prior to injection, during and after injection were analyzed by using the pair T-test with p=0.01. The released data from these experiments indicate that levels of Hb concentration and HCT and also red blood cell counts in the second day following the injection of heparin were significantly lowered in comparison with the control values (p<0.01). However, no significant changes were observed in white blood cell counts, its differentiate counts and the level of enzymes AST,ALT and ALP (p<0.01). On the other hand, a significant increase was observed in serum bilirubin concentration, biliary secretion and biliary bilirubin concentration on the second post injection day (p<0.01). From this study, in addition to the determination of treatment dose of heparin, it can be concluded that the toxic level of heparin injection can cause extravascular hemolysis in sheep. Obviously, reduction of red blood cells by the reticuloendothelial system, catabolism of heme, increased bile volume, increased concentration