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397 Menstrual function in female workers professionally exposed to cytostatic drugs: Model of toxicological evaluation
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Poster Session P17. Reproductive and developmental toxicology
common external abnormalities. Results of this study show the hazards of ranitidine used during early pregnancy. 396
THE EFFECTS OF A SINGLE TERATOGENIC DOSE OF VALPROIC ACID ON PLASMA ZINC CONCENTRATION IN THE FEMALES RAT
P. Pasbakhsh, M. Barbarestani, F. Abolhassani, M. Abozaripour. Department of Anatomy,Tehran university of medical science,Tehran,Iran Valproic acid with good anticonvulsant activity and comparatively low central nervous sedation has become accepted as one of the most important anti epileptic drugs.For this reason it has been estimated that, in the United States, 700–1000 Pregnant women take valproic acid each year.The potential teratogenicity of valproic acid on neural tube in infants born to mothers who have taken this drug has been described and dismissed in several letters.The incidence of neural tube defect following exposure to valproic acid (VPA) during the first weeks of pregnancy can increase to 1–2%.VPA readily binds zinc. For this reason and for a relationship between zinc deficiency and neural tube defects and highest incidences of these abnormalities in areas of the world where human zinc deficiency exists we investigated the effect of VPA on plasma zinc concentration in females rat.For this purpose, we selected 12 rats in 2 groups: 1. Control (normal saline 4CC/kg-intra peritoneal). 2. Experimental (VPA 350Mg/kg-intra peritoneal). All the dams were sacrificed with cervical cutting and plasma prepared. Plasma zinc concentration was measured with flameless atomic absorption method.All statistical evaluations of data compared treatment group with control at P<0.05 levels of significance.Quantitative data were presented as a mean±SD and were analized by ANOVA. Results: plasma zinc concentration (mean±SD) in experimental group was lower than control group. the results of this study indicate that VPA reduces plasma zinc concentration. Regarding the role of zinc in embryonic development this reduced plasma zinc concentration is a possible mechanism of VPA teratogenesis. 397
MENSTRUAL FUNCTION IN FEMALE WORKERS PROFESSIONALLY EXSPOSED TO CYTOSTATIC DRUGS: MODEL OF TOXICOLOGICAL EVALUATION
G.F. Desogus. Study Reports of Toxicology, Azienda USL 7 di Carbonia, Italy Toxic effects, that are linked to the use of cytostatic drugs are known both on the gonadic function and at endocrine level in professionally exposed nurses, with reported alterations in sexual hormonal secretions and adverse effects on the oogenesis and the fertility. In this research work, an experimental model in order to value the associated risk of alterations concerning the menstrual cycle in nurses that manipulate cytostatic drugs. The epidemiological study is carried out on the staff exposed to the manipulation of the cytostatic drugs, taking into consideration some strictly correlated factors (age, smoke, alcohol, diet, pregnancy) and such data must be homogeneously distributed. Data referring to alterations of the menstrual cycle must be collected and analyzed for every worker. Data include changes about the duration of the cycle, variations of the menstrual flow, specific troubles and pathologies linked to irregular ovulations and mixed disfunctions of ovarian nature or linked to the activity of the adrenal gland. In order to improve the validity of data about the menstrual cycle, a series of information must be acquired. These are linked to the hormonal dosage (hypophysial gonadotropin, prolactine, estrogens): they are valid functional index in the diagnostic of female infertility. Data referring to personal, physiological and pathological anamnesis, must be collected and analysed, with the presetting of a series of generic questions (fertility) and specific ones (pregnancy), included the etiological factors of physiological and anatomical nature, observed births, waiting time of pregnancy, spontaneous abortions and reproductive pathologies. The correlation between the data of the observed female fertility and the aspected ones will define the relations of standardized fertility (RFS): In the demographic-statistic field, the fertility will
be associated to the concepts of fertilization and to the absence of correlated pathologies to the professional exposure on the nurses exposed to cytostatic drugs, normally active, with regular ovulation, with the exception of steril, pregnant and post-partum women. From the examination of the data the cases of infertility will be emphasized for non conception in the presence of sexual relations and absence of contraception and those cases of sterility for the presence of reproductive pathologies. Moreover, it will be necessary to consider some factors of individual susceptibility, studying the biochemical and hormonal variations, with the use of suitable epidemiological indicators of clinical type (fertility of couple, analysis of the menstrual cycle, malformations, reproductive troubles), in order to supplement the acquisition of specific knowledges around the mechanism linked to the environmental exposure to cytostatic drugs. 398
STUDY OF THE EFFECTS OF MATERNAL ADMINISTRATION OF MORPHINE ON THE EMBRYONIC LIVER SINUSOID AND KUPFFER CELLS
Gh. Kaka 1 , H. Sahraei 2 , S.H. Sadraei 1 , H. Bahadoran 1 , H. Dashtnavard 1 . 1 Departement of anatomy; 2 Departement of Physiology, Baghyatallah University of Medical Sciences, Tehran, Iran The hazardous and adverse effects of drug consumption in pregnancy on development of embryonic tissues is an obvious fact. In the present study, the effect of morphine consumption on the structure and number of Kupffer cells and liver sinusoids in the embryos were investigated. Sexually mature female Wistar rats (w:250–300 gr) were treated with 0.1 mg/ml of morphine sulfate solution orally. Addicted female rats were then caged overnight with non-addicted male rats and the day that sperm was detected was considered day zero of pregnancy. The female rats were killed by chloroform on gestation day 17 and their emberyos were taken out rapidly. The liver of embryos were removed, fixed and prepared for histological studies. The paraffin sections were then stained with H&E technique. Quantitative computer-assisted histomorphology study were done on the Kupffer cells and sinosoidal dilatation, as well as arrengment of the hepatocytes were examined. Our results showed that the number of kupffer cells and the widening of liver sinusoids were increased significantly in experimental group when compared to control group (p<0.05). In addition, the arrengment of the hepatocytes was more irregular in experimental group than control group. The conclusion of this study revealed the hazardous and risk of morphine addiction of female rats on the development of liver of their embryos. 399
IN VIVO MICROINJECTION OF ANTISENSE MORPHOLINO OLIGOS TO PREDICT THE TERATOGENIC POTENTIAL OF NEW DRUGS: AN INITIAL VALIDATION WORK ON VEGF
D. Manera, M. Longo, S. Zanoncelli, P. Meroni, K. Gunnarsson. Department of Discovery and Development Toxicology, Pharmacia S.p.a, Milan, Italy Angiogenesis is fundamental vertebrate developmental process that requires signaling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A is an important regulator of angiogenesis in humans and it is a target for anti-cancer drugs. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish loosing VEGF-A function. This is done by injecting specific morpholino phosphorodiamidate oligonucleotides into the fertilized eggs, at the one- to eightcell stage. Results obtained will be presented. Concomitantly, compounds with anti VEGF activity were tested in a Zebrafish Teratogen assay which is a 72-hour whole embryo developmental toxicity screening. After fertilization, 1000-cell stage blastulae are cultured up to the larval stage of “protruding mouth”, corresponding to the end of the embryogenetic and morphogenetic periods, in the presence of the substances to be tested. Phenotype of VEGF-A knock-down zebrafishes and those obtained with anti VEGF-A compounds will be compared to evaluate
Poster Session P17. Reproductive and developmental toxicology
common external abnormalities. Results of this study show the hazards of ranitidine used during early pregnancy. 396
THE EFFECTS OF A SINGLE TERATOGENIC DOSE OF VALPROIC ACID ON PLASMA ZINC CONCENTRATION IN THE FEMALES RAT
P. Pasbakhsh, M. Barbarestani, F. Abolhassani, M. Abozaripour. Department of Anatomy,Tehran university of medical science,Tehran,Iran Valproic acid with good anticonvulsant activity and comparatively low central nervous sedation has become accepted as one of the most important anti epileptic drugs.For this reason it has been estimated that, in the United States, 700–1000 Pregnant women take valproic acid each year.The potential teratogenicity of valproic acid on neural tube in infants born to mothers who have taken this drug has been described and dismissed in several letters.The incidence of neural tube defect following exposure to valproic acid (VPA) during the first weeks of pregnancy can increase to 1–2%.VPA readily binds zinc. For this reason and for a relationship between zinc deficiency and neural tube defects and highest incidences of these abnormalities in areas of the world where human zinc deficiency exists we investigated the effect of VPA on plasma zinc concentration in females rat.For this purpose, we selected 12 rats in 2 groups: 1. Control (normal saline 4CC/kg-intra peritoneal). 2. Experimental (VPA 350Mg/kg-intra peritoneal). All the dams were sacrificed with cervical cutting and plasma prepared. Plasma zinc concentration was measured with flameless atomic absorption method.All statistical evaluations of data compared treatment group with control at P<0.05 levels of significance.Quantitative data were presented as a mean±SD and were analized by ANOVA. Results: plasma zinc concentration (mean±SD) in experimental group was lower than control group. the results of this study indicate that VPA reduces plasma zinc concentration. Regarding the role of zinc in embryonic development this reduced plasma zinc concentration is a possible mechanism of VPA teratogenesis. 397
MENSTRUAL FUNCTION IN FEMALE WORKERS PROFESSIONALLY EXSPOSED TO CYTOSTATIC DRUGS: MODEL OF TOXICOLOGICAL EVALUATION
G.F. Desogus. Study Reports of Toxicology, Azienda USL 7 di Carbonia, Italy Toxic effects, that are linked to the use of cytostatic drugs are known both on the gonadic function and at endocrine level in professionally exposed nurses, with reported alterations in sexual hormonal secretions and adverse effects on the oogenesis and the fertility. In this research work, an experimental model in order to value the associated risk of alterations concerning the menstrual cycle in nurses that manipulate cytostatic drugs. The epidemiological study is carried out on the staff exposed to the manipulation of the cytostatic drugs, taking into consideration some strictly correlated factors (age, smoke, alcohol, diet, pregnancy) and such data must be homogeneously distributed. Data referring to alterations of the menstrual cycle must be collected and analyzed for every worker. Data include changes about the duration of the cycle, variations of the menstrual flow, specific troubles and pathologies linked to irregular ovulations and mixed disfunctions of ovarian nature or linked to the activity of the adrenal gland. In order to improve the validity of data about the menstrual cycle, a series of information must be acquired. These are linked to the hormonal dosage (hypophysial gonadotropin, prolactine, estrogens): they are valid functional index in the diagnostic of female infertility. Data referring to personal, physiological and pathological anamnesis, must be collected and analysed, with the presetting of a series of generic questions (fertility) and specific ones (pregnancy), included the etiological factors of physiological and anatomical nature, observed births, waiting time of pregnancy, spontaneous abortions and reproductive pathologies. The correlation between the data of the observed female fertility and the aspected ones will define the relations of standardized fertility (RFS): In the demographic-statistic field, the fertility will
be associated to the concepts of fertilization and to the absence of correlated pathologies to the professional exposure on the nurses exposed to cytostatic drugs, normally active, with regular ovulation, with the exception of steril, pregnant and post-partum women. From the examination of the data the cases of infertility will be emphasized for non conception in the presence of sexual relations and absence of contraception and those cases of sterility for the presence of reproductive pathologies. Moreover, it will be necessary to consider some factors of individual susceptibility, studying the biochemical and hormonal variations, with the use of suitable epidemiological indicators of clinical type (fertility of couple, analysis of the menstrual cycle, malformations, reproductive troubles), in order to supplement the acquisition of specific knowledges around the mechanism linked to the environmental exposure to cytostatic drugs. 398
STUDY OF THE EFFECTS OF MATERNAL ADMINISTRATION OF MORPHINE ON THE EMBRYONIC LIVER SINUSOID AND KUPFFER CELLS
Gh. Kaka 1 , H. Sahraei 2 , S.H. Sadraei 1 , H. Bahadoran 1 , H. Dashtnavard 1 . 1 Departement of anatomy; 2 Departement of Physiology, Baghyatallah University of Medical Sciences, Tehran, Iran The hazardous and adverse effects of drug consumption in pregnancy on development of embryonic tissues is an obvious fact. In the present study, the effect of morphine consumption on the structure and number of Kupffer cells and liver sinusoids in the embryos were investigated. Sexually mature female Wistar rats (w:250–300 gr) were treated with 0.1 mg/ml of morphine sulfate solution orally. Addicted female rats were then caged overnight with non-addicted male rats and the day that sperm was detected was considered day zero of pregnancy. The female rats were killed by chloroform on gestation day 17 and their emberyos were taken out rapidly. The liver of embryos were removed, fixed and prepared for histological studies. The paraffin sections were then stained with H&E technique. Quantitative computer-assisted histomorphology study were done on the Kupffer cells and sinosoidal dilatation, as well as arrengment of the hepatocytes were examined. Our results showed that the number of kupffer cells and the widening of liver sinusoids were increased significantly in experimental group when compared to control group (p<0.05). In addition, the arrengment of the hepatocytes was more irregular in experimental group than control group. The conclusion of this study revealed the hazardous and risk of morphine addiction of female rats on the development of liver of their embryos. 399
IN VIVO MICROINJECTION OF ANTISENSE MORPHOLINO OLIGOS TO PREDICT THE TERATOGENIC POTENTIAL OF NEW DRUGS: AN INITIAL VALIDATION WORK ON VEGF
D. Manera, M. Longo, S. Zanoncelli, P. Meroni, K. Gunnarsson. Department of Discovery and Development Toxicology, Pharmacia S.p.a, Milan, Italy Angiogenesis is fundamental vertebrate developmental process that requires signaling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A is an important regulator of angiogenesis in humans and it is a target for anti-cancer drugs. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish loosing VEGF-A function. This is done by injecting specific morpholino phosphorodiamidate oligonucleotides into the fertilized eggs, at the one- to eightcell stage. Results obtained will be presented. Concomitantly, compounds with anti VEGF activity were tested in a Zebrafish Teratogen assay which is a 72-hour whole embryo developmental toxicity screening. After fertilization, 1000-cell stage blastulae are cultured up to the larval stage of “protruding mouth”, corresponding to the end of the embryogenetic and morphogenetic periods, in the presence of the substances to be tested. Phenotype of VEGF-A knock-down zebrafishes and those obtained with anti VEGF-A compounds will be compared to evaluate