Exposição intrauterina e lactacional de ratos machos a doses suprafisiológicas de manganês: estudo de toxicidade reprodutiva, renal e hepática
Data
2023-04-14
Autores
Silva, Ana Priscila Gomes 
Orientadores
Perobelli, Juliana Elaine
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Considerando a relevância da contaminação ambiental por Managanês(Mn) no cenário brasileiro e mundial, bem como os dados anteriores que indicam as células de Sertoli (CS) como alvo da toxicidade do Mn em adultos, o presente estudo teve como objetivo investigar se a exposição ao Mn durante o período de formação e proliferação das células de Sertoli em os ratos prejudicarão persistentemente a função reprodutiva a longo prazo. Realizando o estudo em 3 idades distintas: Período infantil dia pós natal (DPN)15, quando ocorre o término da proliferação das CS; puberdade, DPN 50, quando as CS sofrem uma mudança acentuada em sua morfologia e função; e idade adulta, DPN 90, quando as CS, estão completamente ativas. Parâmetros gerais de toxicidade renal e hepática também foram estudados. Ademais, a toxicidade direta do Mn sobre as CS foi investigada in vitro. Para atingir esses objetivos, o Ensaio de toxicidade aguda foi realizado para definição das doses de Mn a serem utilizadas no presente estudo, com isso definiu-se as doses experimentais, sendo dose alta (90mg/kg) e dose baixa (9mg/Kg). Posteriormente, ratas prenhes no dia gestacional (DG) 13 foram distribuídas aleatoriamente em 3 grupos experimentais que receberam o tratamento experimental do DG 13(Quando ocorre o povoamento das células de Sertoli nas gônadas dos fetos do sexo masculino) até o dia lactacional 15 (dia pós natal -DPN 15 dos filhotes- quando sessa a atividade mitótica das CS), via oral por gavagem: Ctl-Controle: Recebeu apenas o veículo água destilada (n= 6); Mn-9: Recebeu MnCl2 na dose de 9mg/Kg (n= 8); Mn-90: Recebeu MnCl2 na dose de 90mg/Kg (n=9). As mães prenhes/lactentes no DPN 15, um ou dois filhotes machos de cada ninhada (Ctl: n=8; Mn-9:n=9: Mn-90;n=8) foram submetidos à eutanásia para avaliação de parâmetros reprodutivos no período neonatal. Da mesma forma, no DPN 50 e DPN 90 um ou dois filhotes machos de cada ninhada (totalizando n=10/grupo) foi submetido à eutanásia para avaliação de parâmetros reprodutivos no período peripuberal e adulto. No DPN 15, a avaliação histopatológica dos testículos mostrou aumento na ocorrência de vacúolos e células acidófilas no Mn-90, além das análises de imuno-histoquímicas que indicam dano oxidativo ao material genético das células germinativas e célula de Sertoli dos animais expostos ao Mn. A análise da imunomarcação para conexina 43, indicativo de integridade da célula de Sertoli, revelou uma diminuição na intensidade de marcação nos grupos expostos ao Mn. Não foram observadas alterações no ducto epididimário. Já nos animais do DPN 50, foram observados danos oxidativos ao material genético das células germinativas em conjunto com um aumento de células acidófilos germinativas do grupo Mn-90. Para os animais avaliados no DPN 90, a avaliação histopatológica testicular revelou descolamento e degeneração no epitélio seminífero, o que pode ser considerado um evento tardio e progressivo do que o encontrado aos 50 dias de idade. Além disso, a dinâmica da espermatogênese foi alterada, juntamente com uma diminuição nos níveis de LH e FSH. Alguns achados histológicos epididimários foram observados nas duas idades avaliadas, principalmente nas regiões distais, como o aspecto cribriforme do epitélio e hiperplasia de células claras. Em conclusão, a exposição ao Mn no início da vida causa efeitos adversos nos parâmetros testiculares. A disrupção endócrina no eixo hipotálamo-hipófise-gônadas parece ser um fator relevante para esses eventos de longo prazo.
Considering the relevance of environmental contamination by Managanese(Mn) in the Brazilian and world scenario, as well as previous data that indicate Sertoli cells (CS) as a target for Mn toxicity in adults, the present study aimed to investigate whether exposure exposure to Mn during the period of Sertoli cell formation and proliferation in mice will persistently impair long-term reproductive function. Carrying out the study at 3 different ages: Infant period postnatal day (PND)15, when the end of SC proliferation occurs; puberty, PND 50, when the SCs undergo a marked change in their morphology and function; and adulthood, PND 90, when the CS are fully active. General parameters of renal and hepatic toxicity were also studied. Furthermore, the direct toxicity of Mn on SC was investigated in vitro. To achieve these objectives, the Acute Toxicity Test was performed to define the doses of Mn to be used in the present study, with which the experimental doses were defined, being high dose (90mg/kg) and low dose (9mg/Kg) . Subsequently, pregnant rats on gestational day (GD) 13 were randomly distributed into 3 experimental groups that received the experimental treatment of GD 13 (When sertoli cells populate in the gonads of male fetuses) until lactational day 15 (day post natal - PND 15 of the pups- when the mitotic activity of the CS ceases), orally by gavage: CtlControl: Received only the vehicle distilled water (n= 6); Mn-9: Received MnCl2 at a dose of 9mg/Kg (n=8); Mn-90: Received MnCl2 at a dose of 90mg/Kg (n=9). Pregnant/lactating mothers at DPN 15, one or two male offspring from each litter (Ctl: n=8; Mn-9:n=9: Mn90;n=8) were euthanized for evaluation of reproductive parameters at neonatal period. Likewise, in PND 50 and PND 90, one or two male pups from each litter (totaling n=10/group) were euthanized for evaluation of reproductive parameters in the peripubertal and adult period. In PND 15, the histopathological evaluation of the testes showed an increase in the occurrence of vacuoles and acidophilic cells in Mn-90, in addition to immunohistochemical analyzes that indicate oxidative damage to the genetic material of germ cells and Sertoli cells of animals exposed to Mn. Analysis of immunostaining for connexin 43, indicative of Sertoli cell integrity, revealed a decrease in staining intensity in groups exposed to Mn. No changes were observed in the epididymal duct. In PND 50 animals, however, oxidative damage to the genetic material of germ cells was observed together with an increase in germ acidophilus cells of the Mn-90 group. For the animals evaluated in the PND 90, the testicular histopathological evaluation revealed detachment and degeneration in the seminiferous epithelium, which can be considered a late and progressive event than the one found at 50 days of age. Furthermore, the dynamics of spermatogenesis was altered, along with a decrease in LH and FSH levels. Some epididymal histological findings were observed at both ages evaluated, mainly in the distal regions, such as the cribriform aspect of the epithelium and clear cell hyperplasia. In conclusion, exposure to Mn early in life causes adverse effects on testicular parameters. Endocrine disruption in the hypothalamic-pituitary-gonadal axis appears to be a relevant factor for these long-term events.
Considering the relevance of environmental contamination by Managanese(Mn) in the Brazilian and world scenario, as well as previous data that indicate Sertoli cells (CS) as a target for Mn toxicity in adults, the present study aimed to investigate whether exposure exposure to Mn during the period of Sertoli cell formation and proliferation in mice will persistently impair long-term reproductive function. Carrying out the study at 3 different ages: Infant period postnatal day (PND)15, when the end of SC proliferation occurs; puberty, PND 50, when the SCs undergo a marked change in their morphology and function; and adulthood, PND 90, when the CS are fully active. General parameters of renal and hepatic toxicity were also studied. Furthermore, the direct toxicity of Mn on SC was investigated in vitro. To achieve these objectives, the Acute Toxicity Test was performed to define the doses of Mn to be used in the present study, with which the experimental doses were defined, being high dose (90mg/kg) and low dose (9mg/Kg) . Subsequently, pregnant rats on gestational day (GD) 13 were randomly distributed into 3 experimental groups that received the experimental treatment of GD 13 (When sertoli cells populate in the gonads of male fetuses) until lactational day 15 (day post natal - PND 15 of the pups- when the mitotic activity of the CS ceases), orally by gavage: CtlControl: Received only the vehicle distilled water (n= 6); Mn-9: Received MnCl2 at a dose of 9mg/Kg (n=8); Mn-90: Received MnCl2 at a dose of 90mg/Kg (n=9). Pregnant/lactating mothers at DPN 15, one or two male offspring from each litter (Ctl: n=8; Mn-9:n=9: Mn90;n=8) were euthanized for evaluation of reproductive parameters at neonatal period. Likewise, in PND 50 and PND 90, one or two male pups from each litter (totaling n=10/group) were euthanized for evaluation of reproductive parameters in the peripubertal and adult period. In PND 15, the histopathological evaluation of the testes showed an increase in the occurrence of vacuoles and acidophilic cells in Mn-90, in addition to immunohistochemical analyzes that indicate oxidative damage to the genetic material of germ cells and Sertoli cells of animals exposed to Mn. Analysis of immunostaining for connexin 43, indicative of Sertoli cell integrity, revealed a decrease in staining intensity in groups exposed to Mn. No changes were observed in the epididymal duct. In PND 50 animals, however, oxidative damage to the genetic material of germ cells was observed together with an increase in germ acidophilus cells of the Mn-90 group. For the animals evaluated in the PND 90, the testicular histopathological evaluation revealed detachment and degeneration in the seminiferous epithelium, which can be considered a late and progressive event than the one found at 50 days of age. Furthermore, the dynamics of spermatogenesis was altered, along with a decrease in LH and FSH levels. Some epididymal histological findings were observed at both ages evaluated, mainly in the distal regions, such as the cribriform aspect of the epithelium and clear cell hyperplasia. In conclusion, exposure to Mn early in life causes adverse effects on testicular parameters. Endocrine disruption in the hypothalamic-pituitary-gonadal axis appears to be a relevant factor for these long-term events.
Descrição
Citação
SILVA, Ana Priscila Gomes. Exposição intrauterina e lactacional de ratos machos a doses suprafisiológicas de manganês: estudo de toxicidade reprodutiva, renal e hepática. 2023. 119 f. Tese (Doutorado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2023.