published_or_final_version / Community Medicine / Master / Master of Public Health

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Raymond D. Semlitsch Vita. Includes bibliographical references.

With meiosis spanning from embryonic development to the end of reproductive life in females, scientists have faced considerable limitations in studying female meiosis and the effects of toxicants on the developing oocyte. Over the last half century, various culture methods have been developed with the aim of studying the mechanisms of early ovary development, as well as for use in reproductive toxicology. However, very few of the established embryonic ovary culture systems have been used to investigate potential reproductive toxicants on the embryonic ovary, in particular when compared with the vast number of in vitro reproductive toxicity studies on the post-natal ovary. Here, a novel test compound, a topoisomerase II inhibitor: AstraZeneca Test Compound (AZTC), was used to investigate the efficacy and validity of ovarian culture methods when compared with in vivo reprotoxicity studies. AZTC was selected due to preliminary in vivo studies demonstrating its detrimental effects on spermatogenesis in male rats. AZTC targets bacterial type II topoisomerases that might have mammalian homologues involved in meiosis. Topoisomerase-II α was expressed within the female germ cells pre-natally, but became localised to the granulosa and stroma cells post-natally. This occurred both in vivo and in vitro. Ovaries from female rats exposed pre-natally to AZTC in vivo were analysed histologically and a significant increase in the number of primordial follicles was observed within the ovaries, as well as an increase in the number of unhealthy follicles. A novel mouse embryonic ovary culture system was developed by adapting, improving and bridging existing available culture techniques. The culture system supported growth of pre-meiotic mouse germ cells through prophase I of meiosis, the formation of primordial follicles and initiation of follicle growth. Cultured ovaries contained follicles at stages in comparable ratios to those in vivo and appeared morphologically normal and healthy. The culture also supported meiotic progression of oocytes to the pachytene stage, albeit with a slight delay. AZTC was used to validate the novel embryonic ovary culture by comparing the results with those from the in vivo study, where AZTC exposure had also occurred during embryonic development. Similar results were consistently observed between the in vivo and in vitro studies. In vitro effects of AZTC on the post-natal mouse ovary were also investigated, where neonatal mouse ovaries cultured with AZTC had fewer primordial follicles and more unhealthy follicles than did control ovaries. AZTC therefore demonstrated different effects when exposure occurred pre-natally vs. post-natally. The embryonic ovary culture was then used to examine the effects of another topoisomerase II inhibitor, etoposide, on the pre-natal ovary. Etoposide is a chemotherapy agent and has previously been prescribed to pregnant women. A significant reduction in the size of the follicle pool was observed in exposed cultured embryonic ovaries, where primordial and transitional follicles were targeted. Overall, establishment of post-natal culture systems have become a useful addition to in vivo reproductive toxicology studies. The embryonic ovary culture system developed here could become a valuable and powerful tool to screen potential reproductive toxicants, as well as to study the dynamics and regulation of early ovary development.

Quaternary ammonium compounds (QACs) are antimicrobial disinfectants commonly used in commercial and household settings. While these compounds have been used for decades, reproductive toxicity has not been thoroughly evaluated. Extensive use of QACs results in ubiquitous human exposure to potentially toxic compounds. Reproductive toxicity of two common QACs, alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC), was investigated to determine gender-specific toxicity with an emphasis on male reproductive function. Breeding pairs of mice exposed for six months to ADBAC+DDAC exhibited decreases in fertility and fecundity, with fewer pregnancies and decreased numbers of pups over a six month period. Females proceeded through significantly fewer estrus cycles, and both ovulation and implantation rates were reduced. Males exhibited declines in both sperm concentration and motility. Male reproductive toxicity was further assessed in a series of in-vitro and in-vivo experiments. ADBAC+DDAC were cytotoxic to testicular Sertoli cells in culture at concentrations greater than or equal to 0.0005%. Changes in blood-testis-barrier integrity (BTB) were observed at 0.01% ADBAC+DDAC using a two-compartment culture system that measures transepithelial electrical resistance (TER). Sertoli cell cytotoxicity correlated with decreased TER at ADBAC+DDAC concentrations above 0.001%. In-vitro fertilization capacity of epididymal sperm was reduced in males given a 10-day rest period following ADBAC+DDAC exposure. Multigenerational changes in sperm parameters and in mRNA expression of enzymes involved with epigenetic modifications were evaluated across three generations. Sperm concentration and motility were reduced in F0 males exposed directly to ADBAC+DDAC. In F1 males, sperm concentration was increased and motility decreased, while there was no change in the F2 progeny. Genes involved in epigenetic modifications were altered in the exposed F0, with upregulation of two histone acetyltransferases (Hat1 and Kat2b) and downregulation of one lysine-specific demethylase (Kdm6b). F1 and F2 generations were not different from controls except for downregulation of the methyltransferase Dnmt1 in F1 progeny. The reproductive toxicity of ADBAC+DDAC identified in these studies, particularly to the male, compels further investigation into the potential effects that these compounds may have on human reproduction. / Ph. D.

O objetivo do presente estudo foi o de determinar os efeitos da exposição prolongada ao KCN e ao KSCN em ratos, no período perinatal. Inicialmente, realizou-se o estudo toxicocinético do tiocianato em ratas, em três estados fisiológicos diferentes: fase estrogênica, gestacional e de lactação. Para tal, estes animais receberam, por via oral, na água de bebida ou gavage, a dose única de 3 mg/kg de KCN. Os resultados obtidos mostraram aumento significante nos níveis séricos, lácteos e no líquido amniótico, de tiocianato, após a administração do KCN. A partir destes dados, foram calculados os parâmetros toxicocinéticos. Na segunda etapa, foram utilizadas ratas, as quais foram divididas em 3 grupos controles e 18 experimentais, que receberam as diferentes doses de KCN (1; 3 e 30 mg/kg) ou KSCN (0,8; 2,4 e 24 mg/kg), na água de bebida, do 6º ao 20º dia de gestação e submetidas à eutanásia no 20º dia da gestação ou no 22º dia pós-parto; e do 1º ao 19º dia da lactação, e submetidas à eutanásia no 19º dia deste período. Ao final de cada experimento, os animais foram submetidos à eutanásia, no período apropriado, bem como os fetos e filhotes, coletando-se soro para análise dos níveis de tiocianato, glicose, colesterol, uréia, creatinina, alanina aminotransferase, aspartato aminotransferase, fosfatase alcalina, bem como foram retirados fragmentos do sistema nervoso central, do rim, do pâncreas, da tireóide, do fígado, do pulmão e do baço para estudo histopatológico. Ainda, realizou-se a dosagem de tiocianato no líquido amniótico e no leite, bem como a análise óssea e visceral dos fetos. Dos parâmetros bioquímicos analisados, em relação às mães, verificou-se que os níveis séricos de tiocianato estiveram aumentados, significantemente, em diversos grupos experimentais, no entanto, poucas foram as alterações nas enzimas e outras substâncias avaliadas. O estudo histológico revelou, tanto naquelas gestantes quanto nas lactantes, nefrose, hemorragia e hemossiderose renal; congestão, neuronofagia e gliose no SNC; congestão, vacuolização e proliferação dos ductos biliares e aumento no número de vacúolos de reabsorção no colóide dos folículos tireoidianos, de forma dose-dependente, nos diferentes grupos experimentais. Ratas gestantes, submetidas à eutanásia no 20º dia da gestação apresentaram, ainda, depleção de células das ilhotas de Langerhans. Em relação aos filhotes, tanto aqueles provenientes de fêmeas tratadas durante a gestação quanto da lactação, verificou-se que, embora a avaliação bioquímica tenha revelado alteração apenas no tiocianato sérico de filhotes de mães provenientes dos grupos experimentais, o estudo histológico, nestas proles, mostrou várias lesões, a saber: congestão, neuronofagia e gliose no SNC, congestão renal e hepática, vacuolização e proliferação dos ductos biliares. Por outro lado, não foram detectadas alterações na performance reprodutiva bem como na análise visceral e óssea dos fetos. Portanto, pode-se sugerir que o cianeto e/ou seu metabólito promovam efeito tóxico diretamente sobre o feto, no entanto estas alterações são passíveis de detecção apenas na fase pós-natal. Além disto, verificou-se, que o cianeto e/ou o tiocianato são carreados para o leite, podendo comprometer, também, a saúde do neonato. / The aim of the present study was to determinate the toxic effects of long-term exposure to KCN and KSCN in rats, during perinatal period. Initially, it was achieved the toxicokinetics study of the thiocyanate in female rats, in three different physiological states: estrogenic, gestation and lactation. These animals received, per os, in the drinking water or by gavage, the unique dose of 3.0 mg KCN/kg. The results showed significantly increased in the thiocyanate levels in the serum, milk and amniotic fluid, after the administration of the KCN. The toxicokinetics parameters were calculated based on these data. In a second part, female rats were distributed in 18 experimental and 3 control groups. The experimental groups were dosed with 1, 3 and 30 mg KCN/kg or 0.8; 2.4 and 24 mg KSCN/kg, daily, in the drinking water, from days 6 to 20 of gestation and euthanized on day 20 of gestation or on day 22 postpartum; or received from days 1 to 19 of lactation and euthanized on day 19 of lactation. At the end of each experiment, the animals were euthanized, in the appropriated period, as well as the fetuses and pups. The serum samples were obtained in order to determine the thiocyanate, glucose, cholesterol, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels as well as samples of the following organs for the histopathological study: central nervous system, kidney, pancreas, thyroid, liver, lung and spleen. Yet, the levels of thiocyanate were evaluated in the amniotic fluid and milk, from the mothers, as well as the skeletal and visceral analysis of the fetuses. From the biochemical parameters, in relation to the dams, it was verified that the thiocyanate levels were significantly increased, in several experimental groups; otherwise, too few alterations were observed in the enzymes and others substances analyzed. The histopathological study revealed, in the pregnant and lactating rats, nephrosis, hemorrhage and hemosiderosis in the kidneys; congestion, neuronophagia and gliosis in the CNS; vacuolization and proliferation of the biliar ducts in the liver and increase in the number of reabsorption vacuoles in follicular colloid, in a dose-dependent manner, in some experimental groups. At the pregnant rats that were euthanized on day 20 of gestation, it was verified depletion of cells from the islets of Langerhans. In relation to the pups, from mothers exposed during the gestation and lactation, it was verified alteration in the thiocyanate levels from experimental groups and the histopathological study revealed: congestion, neuronophagia and gliosis in the CNS, renal and hepatic congestion, vacuolization and proliferation of biliar ducts. On the other hand, it was verified no alterations in the reproductive performance nor skeletal and visceral analysis of the fetuses. Thus, it was suggested that the cyanide and/or its metabolite promoted toxic effect straight to the fetuses; however, these alterations are susceptible of detection only at postnatal phase. Furthermore, the cyanide and/or thiocyanate are transferred to the breast milk of rats and can also compromise the health of the offspring.

A Ipomea carnea é uma planta tóxica encontrada por todo Brasil e em outros países tropicais. Esta se conserva verde durante a seca, podendo servir como fonte de matéria verde para bovinos, ovinos e, particularmente, caprinos. Os animais intoxicados por esta planta desenvolvem sintomatologia de origem nervosa, imputada ao principal princípio ativo desta planta, a suainsonina. A suainsonina é um alcalóide indolizidinico, potente inibidor da αmanosidase lisossomal, sendo que a inibição desta enzima produz o acúmulo lisossômico de oligossacarídeos não processados completamente, perda de função celular e morte celular. Além desta alteração, a suainsonina também inibe a manosidase II do complexo de Golgi, levando a alterações na síntese, no processamento e no transporte de glicoproteínas. Os principais achados histológicos nesta intoxicação são células com vacúolos lisossomais no sistema nervoso central, tireóide, fígado, pâncreas e rins. Pesquisas anteriores mostraram que a ingestão de I. carnea por cabras gestantes produz malformações. O presente estudo propôs-se a estudar os efeitos teratogênicos da I. carnea em caprinos. Para tanto, acrescentou-se ao protocolo de avaliação de teratogenicidade em caprinos, desenvolvido neste laboratório, a avaliação neurocomportamental dos neonatos. Assim, foram utilizadas 27 cabras gestantes, divididas em 4 grupos: 3 experimentais e 1 controle. As cabras dos grupos experimentais a partir do 35º dia de gestação até o final da prenhez receberam 1, 3 e 5g/kg/dia de I. carnea. Realizou-se o exame clinico periódico nas fêmeas gestantes, coletando-se sangue para o estudo bioquímico. Procedeu-se também exames fetais ultrassonográficos. As fêmeas foram assistidas no momento do parto, realizando-se, pelas duas horas subseqüentes, anotações de alguns comportamentos apresentados pela mãe e neonato. Além disto, nos dias posteriores os filhotes passaram por uma série de avaliações neurocomportamentais até a 6ª semana de vida. Os resultados obtidos mostram que nenhuma das fêmeas que ingeriram a planta apresentaram sintomatologia nervosa. Foi observado abortamento nas fêmeas dos grupos que receberam 3g/kg/dia e 5g/kg/dia de I. carnea. Também foram observadas duas mortes fetais naquelas gestantes do grupo que recebeu a maior dose da planta. Com relação à bioquímica sangüínea foram verificadas alterações na atividade das enzimas AST e FA nas fêmeas que ingeriram a I. carnea durante a gestação. As mães dos grupos que receberam a planta apresentaram significantemente menor atenção aos seus filhotes. Não foram detectadas alterações nos parâmetros ultrassonográficos, nem malformações físicas nos neonatos, em nenhum dos grupos avaliados, no entanto, aqueles filhotes de cabras que ingeriram a maior dose apresentaram redução do peso ao nascimento. As avaliações comportamentais mostraram que os filhotes dos grupos experimentais apresentaram dificuldade de manter-se em estação imediatamente após o parto, bem como de realizar a primeira mamada e de distinguir sua mãe. Além disso, estes filhotes apresentavam maior latência de tempo para chegar às suas mães, nos diferentes testes de labirinto realizados. Este estudo reforça pesquisas anteriores, apontando o efeito teratogênico promovido pela I. carnea e recomenda a inclusão das avaliações de neuroteratogenicidade no protocolo de avaliação de teratologia proposta para ruminantes. / Ipomoea carnea, a shrub plant, is a toxic plant largely distributed throughout Brazil and others topical countries. This plant possess swainsonine, an indolizidinic alkaloid as the most important active toxic principle, which promotes cellular accumulation of not metabolized oligossacarides, due to inhibition of acid or lisossomal αmanosidasis enzyme, causing cellular vacuolization. This alkaloid also inhibits manosidase II of the golgi complex, modifying the glycoprotein synthesis, processing and carrier. It is well known that the ingestion of the plant promotes toxic effects in the central nervous system, liver, kidney, thyroid and pancreas, particularly in goats the most susceptible species. Previous researches conducted in this laboratory had proposed a protocol to evaluate teratogenic effects of xenobiotics in ruminants, using goats as animal model. In relation to I. carnea, an earlier study using this protocol revealed the teratologic effect. The aim of this research is to add to this protocol the evaluation of the neonate´s behavior. Twenty seven female goats were divided into 4 groups: 3 experimental and 1 control. The experimental goats received from gestation day 35 to parturition day the following doses of I. carnea fresh leaves: 1, 3 and 5 g/kg/day. During the pregnancy females were clinically accompanied, evaluating behavior and general body status, and serum biochemistry were performed. Fetuses were evaluated during pregnancy using ultrasonographic measurements. The parturition of all dams was assisted and mother-offspring behaviour was examined during the two consecutive hours post partum. Kid´s development was examined using various neurobehavioral tests up to 6 weeks of age. The data obtained showed abortion (n=1) in the females treated with 3 and 5g/kg/day I. carnea. Fetal dead (n=2) were observed in the females that eating highest dose of I. carnea. None of the treated dam presented neurologic effects during all gestational period. Aspartate-amine transferase and alkaline phosphatase were increased in experimental females. Offspring body weights were affected by exposure to I. carnea. Behavioral study revealed that treated dams were less likely to stand for nursing. Kids from I. carnea-treated females were unable to stand, nurse and recognize their mothers. These kids were also slower than controls to arrive at the mother in the maze tests. The present study complements previous research, confirming that I. carnea promotes reproductive alteration effects. In addition, the neurobehavioral tests employed here showed to be an important tool to monitorize the toxic effects promoted by toxicants during postnatal period. This research also suggests the inclusion of neurobehavioral evaluations in the ruminant´s teratology protocols.

Orientador: Wellerson Rodrigo Scarano / Resumo: O arsênio é um metaloide associado ao desenvolvimento de algumas patologias, como doenças cardiovasculares, lesões dérmicas e diferentes tipos de câncer. Pouco se sabe sobre a ação do arsênio ou compostos de arsênio na próstata durante o período pré-puberal e puberdade, estágios essenciais para a morfogênese tardia da próstata. Nesse sentido, este estudo teve como objetivo estabelecer se a exposição ao arsenito de sódio (NaAsO2) interfere na morfofisiologia da próstata ventral de ratos púberes. Para isso, 30 ratos machos da linhagem Wistar, no dia pós-natal 23 (DPN23), foram distribuídos, aleatoriamente, em 3 grupos experimentais (n =10/grupo). O grupo controle (Ctrl) recebeu água filtrada (veículo); o grupo As1 recebeu 0.01 mg/L de NaAsO2; e o grupo As2 recebeu 10.0 mg/L de NaAsO2. Todas as soluções foram diluídas na água do bebedouro e estiveram disponíveis aos animais do DPN23 ao DPN53. Os hábitos alimentares e a evolução do peso corpóreo dos animais foram acompanhados durante todo o período experimental. Ao final deste período, os animais foram pesados e, em seguida, eutanasiados (DPN53). Coletou-se o sangue para mensurar os níveis de testosterona. O fígado, os rins e a próstata ventral (PV) foram coletados e pesados. Apenas a PV foi dissecada e destinada às análises histológicas (hemilobo esquerdo) e moleculares (hemilobo direito). Os resultados dos parâmetros analisados durante o período experimental revelaram que o NaAsO2 não foi capaz de causar toxicidade sistêmica em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Arsenic is an endocrine disruptor associated with the development of some pathologies such as cardiovascular diseases, dermal lesions and different types of cancer. Little is known about the action of arsenic or arsenic compounds in the prostate during the prepubertal and puberty period, an essential stage for late morphogenesis of the prostate. Therefore, this study aimed to establish whether exposure to sodium arsenite (NaAsO2) interferes in the morphophysiology of the ventral prostate of pubertal rats. In this study, thirty male Wistar rats, on the postnatal day 23 (PND23), were randomly distributed to 3 experimental groups (n = 10/group). The control group (Ctrl) received only saline solution; the second group (As1) received 0.01 mg/L of NaAsO2; and the third group (As2) received 10.0 mg/L of NaAsO2. All solutions were diluted in drinking water and were available to the animals from DPN23 to DPN53. The eating habits and the evolution of the body weight of the animals were evaluated throughout the experimental period. At the end of this period, the animals were weighed and then euthanized (DPN53). Blood was collected to measure testosterone levels. The liver, kidneys and ventral prostate (VP) were collected and weighed. Only VP was dissected for histological analysis (left hemilobo) and molecular (right hemilobo). The results of the parameters analyzed during the experimental period revealed that NaAsO2 was not able to cause systemic toxicity in both exposed groups nor cha... (Complete abstract click electronic access below) / Mestre

A Ipomea carnea é uma planta tóxica encontrada por todo Brasil e em outros países tropicais. Esta se conserva verde durante a seca, podendo servir como fonte de matéria verde para bovinos, ovinos e, particularmente, caprinos. Os animais intoxicados por esta planta desenvolvem sintomatologia de origem nervosa, imputada ao principal princípio ativo desta planta, a suainsonina. A suainsonina é um alcalóide indolizidinico, potente inibidor da αmanosidase lisossomal, sendo que a inibição desta enzima produz o acúmulo lisossômico de oligossacarídeos não processados completamente, perda de função celular e morte celular. Além desta alteração, a suainsonina também inibe a manosidase II do complexo de Golgi, levando a alterações na síntese, no processamento e no transporte de glicoproteínas. Os principais achados histológicos nesta intoxicação são células com vacúolos lisossomais no sistema nervoso central, tireóide, fígado, pâncreas e rins. Pesquisas anteriores mostraram que a ingestão de I. carnea por cabras gestantes produz malformações. O presente estudo propôs-se a estudar os efeitos teratogênicos da I. carnea em caprinos. Para tanto, acrescentou-se ao protocolo de avaliação de teratogenicidade em caprinos, desenvolvido neste laboratório, a avaliação neurocomportamental dos neonatos. Assim, foram utilizadas 27 cabras gestantes, divididas em 4 grupos: 3 experimentais e 1 controle. As cabras dos grupos experimentais a partir do 35º dia de gestação até o final da prenhez receberam 1, 3 e 5g/kg/dia de I. carnea. Realizou-se o exame clinico periódico nas fêmeas gestantes, coletando-se sangue para o estudo bioquímico. Procedeu-se também exames fetais ultrassonográficos. As fêmeas foram assistidas no momento do parto, realizando-se, pelas duas horas subseqüentes, anotações de alguns comportamentos apresentados pela mãe e neonato. Além disto, nos dias posteriores os filhotes passaram por uma série de avaliações neurocomportamentais até a 6ª semana de vida. Os resultados obtidos mostram que nenhuma das fêmeas que ingeriram a planta apresentaram sintomatologia nervosa. Foi observado abortamento nas fêmeas dos grupos que receberam 3g/kg/dia e 5g/kg/dia de I. carnea. Também foram observadas duas mortes fetais naquelas gestantes do grupo que recebeu a maior dose da planta. Com relação à bioquímica sangüínea foram verificadas alterações na atividade das enzimas AST e FA nas fêmeas que ingeriram a I. carnea durante a gestação. As mães dos grupos que receberam a planta apresentaram significantemente menor atenção aos seus filhotes. Não foram detectadas alterações nos parâmetros ultrassonográficos, nem malformações físicas nos neonatos, em nenhum dos grupos avaliados, no entanto, aqueles filhotes de cabras que ingeriram a maior dose apresentaram redução do peso ao nascimento. As avaliações comportamentais mostraram que os filhotes dos grupos experimentais apresentaram dificuldade de manter-se em estação imediatamente após o parto, bem como de realizar a primeira mamada e de distinguir sua mãe. Além disso, estes filhotes apresentavam maior latência de tempo para chegar às suas mães, nos diferentes testes de labirinto realizados. Este estudo reforça pesquisas anteriores, apontando o efeito teratogênico promovido pela I. carnea e recomenda a inclusão das avaliações de neuroteratogenicidade no protocolo de avaliação de teratologia proposta para ruminantes. / Ipomoea carnea, a shrub plant, is a toxic plant largely distributed throughout Brazil and others topical countries. This plant possess swainsonine, an indolizidinic alkaloid as the most important active toxic principle, which promotes cellular accumulation of not metabolized oligossacarides, due to inhibition of acid or lisossomal αmanosidasis enzyme, causing cellular vacuolization. This alkaloid also inhibits manosidase II of the golgi complex, modifying the glycoprotein synthesis, processing and carrier. It is well known that the ingestion of the plant promotes toxic effects in the central nervous system, liver, kidney, thyroid and pancreas, particularly in goats the most susceptible species. Previous researches conducted in this laboratory had proposed a protocol to evaluate teratogenic effects of xenobiotics in ruminants, using goats as animal model. In relation to I. carnea, an earlier study using this protocol revealed the teratologic effect. The aim of this research is to add to this protocol the evaluation of the neonate´s behavior. Twenty seven female goats were divided into 4 groups: 3 experimental and 1 control. The experimental goats received from gestation day 35 to parturition day the following doses of I. carnea fresh leaves: 1, 3 and 5 g/kg/day. During the pregnancy females were clinically accompanied, evaluating behavior and general body status, and serum biochemistry were performed. Fetuses were evaluated during pregnancy using ultrasonographic measurements. The parturition of all dams was assisted and mother-offspring behaviour was examined during the two consecutive hours post partum. Kid´s development was examined using various neurobehavioral tests up to 6 weeks of age. The data obtained showed abortion (n=1) in the females treated with 3 and 5g/kg/day I. carnea. Fetal dead (n=2) were observed in the females that eating highest dose of I. carnea. None of the treated dam presented neurologic effects during all gestational period. Aspartate-amine transferase and alkaline phosphatase were increased in experimental females. Offspring body weights were affected by exposure to I. carnea. Behavioral study revealed that treated dams were less likely to stand for nursing. Kids from I. carnea-treated females were unable to stand, nurse and recognize their mothers. These kids were also slower than controls to arrive at the mother in the maze tests. The present study complements previous research, confirming that I. carnea promotes reproductive alteration effects. In addition, the neurobehavioral tests employed here showed to be an important tool to monitorize the toxic effects promoted by toxicants during postnatal period. This research also suggests the inclusion of neurobehavioral evaluations in the ruminant´s teratology protocols.

O objetivo do presente estudo foi o de determinar os efeitos da exposição prolongada ao KCN e ao KSCN em ratos, no período perinatal. Inicialmente, realizou-se o estudo toxicocinético do tiocianato em ratas, em três estados fisiológicos diferentes: fase estrogênica, gestacional e de lactação. Para tal, estes animais receberam, por via oral, na água de bebida ou gavage, a dose única de 3 mg/kg de KCN. Os resultados obtidos mostraram aumento significante nos níveis séricos, lácteos e no líquido amniótico, de tiocianato, após a administração do KCN. A partir destes dados, foram calculados os parâmetros toxicocinéticos. Na segunda etapa, foram utilizadas ratas, as quais foram divididas em 3 grupos controles e 18 experimentais, que receberam as diferentes doses de KCN (1; 3 e 30 mg/kg) ou KSCN (0,8; 2,4 e 24 mg/kg), na água de bebida, do 6º ao 20º dia de gestação e submetidas à eutanásia no 20º dia da gestação ou no 22º dia pós-parto; e do 1º ao 19º dia da lactação, e submetidas à eutanásia no 19º dia deste período. Ao final de cada experimento, os animais foram submetidos à eutanásia, no período apropriado, bem como os fetos e filhotes, coletando-se soro para análise dos níveis de tiocianato, glicose, colesterol, uréia, creatinina, alanina aminotransferase, aspartato aminotransferase, fosfatase alcalina, bem como foram retirados fragmentos do sistema nervoso central, do rim, do pâncreas, da tireóide, do fígado, do pulmão e do baço para estudo histopatológico. Ainda, realizou-se a dosagem de tiocianato no líquido amniótico e no leite, bem como a análise óssea e visceral dos fetos. Dos parâmetros bioquímicos analisados, em relação às mães, verificou-se que os níveis séricos de tiocianato estiveram aumentados, significantemente, em diversos grupos experimentais, no entanto, poucas foram as alterações nas enzimas e outras substâncias avaliadas. O estudo histológico revelou, tanto naquelas gestantes quanto nas lactantes, nefrose, hemorragia e hemossiderose renal; congestão, neuronofagia e gliose no SNC; congestão, vacuolização e proliferação dos ductos biliares e aumento no número de vacúolos de reabsorção no colóide dos folículos tireoidianos, de forma dose-dependente, nos diferentes grupos experimentais. Ratas gestantes, submetidas à eutanásia no 20º dia da gestação apresentaram, ainda, depleção de células das ilhotas de Langerhans. Em relação aos filhotes, tanto aqueles provenientes de fêmeas tratadas durante a gestação quanto da lactação, verificou-se que, embora a avaliação bioquímica tenha revelado alteração apenas no tiocianato sérico de filhotes de mães provenientes dos grupos experimentais, o estudo histológico, nestas proles, mostrou várias lesões, a saber: congestão, neuronofagia e gliose no SNC, congestão renal e hepática, vacuolização e proliferação dos ductos biliares. Por outro lado, não foram detectadas alterações na performance reprodutiva bem como na análise visceral e óssea dos fetos. Portanto, pode-se sugerir que o cianeto e/ou seu metabólito promovam efeito tóxico diretamente sobre o feto, no entanto estas alterações são passíveis de detecção apenas na fase pós-natal. Além disto, verificou-se, que o cianeto e/ou o tiocianato são carreados para o leite, podendo comprometer, também, a saúde do neonato. / The aim of the present study was to determinate the toxic effects of long-term exposure to KCN and KSCN in rats, during perinatal period. Initially, it was achieved the toxicokinetics study of the thiocyanate in female rats, in three different physiological states: estrogenic, gestation and lactation. These animals received, per os, in the drinking water or by gavage, the unique dose of 3.0 mg KCN/kg. The results showed significantly increased in the thiocyanate levels in the serum, milk and amniotic fluid, after the administration of the KCN. The toxicokinetics parameters were calculated based on these data. In a second part, female rats were distributed in 18 experimental and 3 control groups. The experimental groups were dosed with 1, 3 and 30 mg KCN/kg or 0.8; 2.4 and 24 mg KSCN/kg, daily, in the drinking water, from days 6 to 20 of gestation and euthanized on day 20 of gestation or on day 22 postpartum; or received from days 1 to 19 of lactation and euthanized on day 19 of lactation. At the end of each experiment, the animals were euthanized, in the appropriated period, as well as the fetuses and pups. The serum samples were obtained in order to determine the thiocyanate, glucose, cholesterol, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels as well as samples of the following organs for the histopathological study: central nervous system, kidney, pancreas, thyroid, liver, lung and spleen. Yet, the levels of thiocyanate were evaluated in the amniotic fluid and milk, from the mothers, as well as the skeletal and visceral analysis of the fetuses. From the biochemical parameters, in relation to the dams, it was verified that the thiocyanate levels were significantly increased, in several experimental groups; otherwise, too few alterations were observed in the enzymes and others substances analyzed. The histopathological study revealed, in the pregnant and lactating rats, nephrosis, hemorrhage and hemosiderosis in the kidneys; congestion, neuronophagia and gliosis in the CNS; vacuolization and proliferation of the biliar ducts in the liver and increase in the number of reabsorption vacuoles in follicular colloid, in a dose-dependent manner, in some experimental groups. At the pregnant rats that were euthanized on day 20 of gestation, it was verified depletion of cells from the islets of Langerhans. In relation to the pups, from mothers exposed during the gestation and lactation, it was verified alteration in the thiocyanate levels from experimental groups and the histopathological study revealed: congestion, neuronophagia and gliosis in the CNS, renal and hepatic congestion, vacuolization and proliferation of biliar ducts. On the other hand, it was verified no alterations in the reproductive performance nor skeletal and visceral analysis of the fetuses. Thus, it was suggested that the cyanide and/or its metabolite promoted toxic effect straight to the fetuses; however, these alterations are susceptible of detection only at postnatal phase. Furthermore, the cyanide and/or thiocyanate are transferred to the breast milk of rats and can also compromise the health of the offspring.

A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action. This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F2ɑ (PGF2ɑ), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF2ɑ. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels. Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.