Effect of trivalent arsenicals on cell proliferation in mouse and human microvascular endothelial cells

AbstractChronic exposure to high levels of inorganic arsenic (iAs) has been associated with cancerous and non-cancerous health effects, including cardiovascular effects. However, the mechanism for a presumed toxic effect of arsenic on vascular tissue is not clear. Our working hypothesis is that inorganic trivalent arsenic and its methylated metabolites react with cysteine-containing cellular proteins and alter their function leading to adverse events such as cytotoxicity or proliferation. In this study, human microvascular endothelial cells (HMEC1) and mouse microvascular endothelial cells (MFP-MVEC) were exposed to arsenite (iAsIII), monomethylarsonous acid (MMAIII), or dimethylarsinous acid (DMAIII) for 72h to evaluate cytotoxicity, and for 24, 48 or 72h to evaluate cell proliferation. Both cell lines showed similar LC50 values, from 0.1 to 2.4μM, for all three trivalent arsenicals. The endothelial cells treated with1nM to 1μM concentrations of the three trivalent arsenicals did not show increased cell proliferation at 24, 48 or 72h or increased rate of proliferation at 72h of exposure. Overall, cytotoxicity of trivalent arsenicals to microvascular endothelial cells is similar to their cytotoxicity to epithelial cells, and that these compounds are not mitogenic

Cryptorchidism in the rat: a possible model to study testicular dysgenesis syndrome (TDS)

Cryptorchidism is a congenital defect affecting 2-4% of newborn boys, and is a major risk factor for infertility and testicular germ cell tumors (TGCT). The surgery that reallocates the testis into the scrotum, ochiopexy, should be performed between 6-12 months of age in order to allow normal fertility and reduce the risk of malignancy. Poor semen quality, undescended testis, hypospadias and TGCT are elements that, isolated or combined, compose the so-called Testicular Dysgenesis Syndrome (TDS). The present study aimed to characterize the testicular changes induced by a cryptorchidism and orchiopexy experimental model in the rat with the eventual purpose of better understanding TDS. Improved mechanical methods of abdominal cryptorchidism induction and a variation in the orchiopexy installation by anchoring the testes, and not the cauda epididymis, into the internal surface of the scrotal wall and gently pulling them down to the scrotum, are presented. To accomplish that, Sprague-Dawley male rats were surgically submitted to cryptorchidism by anchoring the testicular albuginea to the abdominal wall - the critical step of the present method - at 3 weeks of age; some of them were euthanized 3, 6 or 11 weeks after this surgery in order to register the morphological progression of cryptorchidism-induced testicular alterations. Other cryptorchidic animals were submitted to orchiopexy 3, 5 or 9 weeks after cryptorchidism; these animals were euthanized 3 or 8 weeks after orchiopexy. Sham operated animals underwent to cryptorchidism and orchiopexy at the same times as the surgical groups. At least 10 and 5 animals were used in surgical and sham groups, respectively, at the respective euthanasia moments. Cryptorchidic testis showed decreased weights, germ cell loss, spermatogenesis disruption, apoptosis and eventually some tubules with a Sertoli cells-only pattern, findings already described with other mechanical methods used to induce ...O criptorquidismo é uma anomalia congênito que afeta de 2 a 4% de meninos recém-nascidos, sendo um importante fator de risco para a infertilidade e tumores testiculares de células germinativas (TTCG). A cirurgia que realoca o testículo no escroto, oquidopexia, deve ser realizada entre 6 e 12 meses de idade, a fim de permitir fertilidade normal e reduzir o risco de malignidade. má qualidade do sêmen, testículo não descido, hipospadia e TTCG são elementos que, isolados ou combinados, compõem a chamada síndrome de disgenesia testicular (SDT). O presente estudo teve como objetivo caracterizar as alterações testiculares induzidas por uma modelo experimental de criptorquidia e orquidopexia em ratos, com o eventual objetivo de melhor compreender a SDT. Método mecânico aperfeiçoado de indução criptorquidismo abdominal e uma variação na instalação da orquidopexia ancorando os testículos, e não a cauda do epidídimo, na superfície interna da parede do escroto e puxando-os para o escroto, são apresentados. Para isso, ratos machos Sprague-Dawley foram submetidos cirurgicamente a criptorquidismo, ancorando a tunica albugínea dos testículos à parede abdominal - o passo crítico do presente método - na 3a semana de idade; alguns deles foram sacrificados após 3, 6 ou 11 semanas desta cirurgia, a fim de registrar a progressão morfológica das alterações testiculares induzidas pela criptorquidia. Outros animais criptorquídicos foram submetidos à orquidopexia após 3, 5 ou 9 semanas da criptorquidia; estes animais foram sacrificados 3 ou 8 semanas após orquidopexia. Animais falsamente operados (sham) foram submetidos a criptorquidia e orquidopexia nos mesmos momentos que os grupos cirúrgicos. Pelo menos dez e cinco animais foram utilizados em grupos de cirurgia e sham, respectivamente. Testículos criptorquídicos mostraram diminuição do peso, perda de células germinativas, parada na espermatogênese, apoptose e,...Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Estudo da toxicidade de diferentes doses do Diuron (3-(3,4 -dichlorophenyl)-1,1-dimethylurea) no epitélio da bexiga de ratos wistas machos por microscopia eletrônica de varredura

Diuron (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide widely used on agricultural crops such as soy, cotton and sugar cane. In a previous long-term study this herbicide exerted carcinogenic activity on the urinary bladder and renal pelvis mucosa of Wistar rats and breast of mice. Also, it was shown to be carcinogenic to the mice skin in a initiation-promotion assay. In 1997, the northamerican EPA evaluated Diuron as a “known/likely” carcinogen for humans (USEPA, 2004). In a previous study developed at this laboratory, male Wistar rats treated with Diuron 2500 ppm during 20 weeks presented increased indices of cell proliferation and incidences of simple urothelial hyperplasia (HS) in the urinary bladder. Under scanning electron microscopy (SEM) severe urothelial necrosis and hyperplasia were observed. However, in that study the urinary bladders of animals exposed to lower doses of Diuron were not examined under SEM. Therefore, the possible dose-response influence of Diuron on the urothelium under SEM is not known. The present study aimed to analyze under SEM the urinary bladder of male Wistar rats exposed to 125 ppm, 500 ppm and 2500 ppm doses of Diuron through diet during 20 weeks and to compare to the previous histological findings in the same material. Under SEM, 125 ppm and 2500 ppm groups presented significantly (p<0,05) increased incidences of simple hyperplasia, i.e., 7/10 and 8/10 respectively, compared to control group and the 500 ppm group The sensitivity of SEM was higher since it detected a 45% incidence of hyperplasiaswhile the histological analysis found only 27%. Considering SEM as the gold-standard, histology showed a 44% sensitivity, 86.4% specificity, a positive predictive value of 72,7% and negative predictive value of 65,5% and accuracy of 67,5%. Scanning Electron Microscopy...(Complete abstract click electronic access below)O Diuron (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) é um herbicida derivado da uréia, muito utilizado no Brasil para controlar plantas daninhas nas culturas de soja, algodão e cana de açúcar, entre outras. Evidências experimentais indicam que o Diuron é cancerígeno para roedores; induz tumores na pele e mama de camundongos, e na bexiga e pelve renal de ratos, quando administrado pela ração em concentrações elevadas. O Diuron foi classificado pela agência ambiental norte-americana (EPA) como “provável cancerígeno para a espécie humana”. Em um estudo prévio desenvolvido por nosso grupo, ratos Wistar machos tratados com Diuron 2500 ppm durante 20 semanas apresentaram aumento dos índices de proliferação celular e da incidência de hiperplasia urotelial simples (HS) na bexiga. À microscopia eletrônica de varredura (MEV) foram observados focos de necrose grave e hiperplasia do urotélio vesical dos animais expostos ao herbicida. Porém, nesse estudo não foram avaliados possíveis alterações uroteliais nas bexigas de animais expostos a concentrações menores de Diuron, de modo que não se sabe se há efeito doseresposta do Diuron sobre o urotélio. O objetivo deste trabalho foi estudar por MEV a ação do Diuron sobre o epitélio da bexiga de ratos Wistar machos expostos as doses de 125, 500 e 2500 ppm durante 20 semanas e verificar a existência de eventual dose-resposta nos fenômenos de citotoxicidade e regeneração celular e ainda, comparar os achados da MEV com os histológicos verificados previamente nas mesma bexigas. Os grupos 125ppm e 2500ppm de Diuron apresentaram incidências significativamente maiores (p<0,05) de lesões na mucosa, i.e, 7/10 e 8/10 respectivamente, comparados ao grupo controle e ao grupo 500ppm. Ao compararmos ...(Resumo completo, clicar acesso eletrônico abaixo

Estudo dose-resposta do herbicida diuron[3-(3,4-diclorofenil)-1,1-dimetiluréia] no epitélio da bexiga de ratos Wistar machos

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a herbicide that in a previous longterm study with Wistar rats fed at 2,500 ppm concentration showed high incidence of urothelial tumors in both genders. Accordingly, USEPA categorized Diuron as a “known/likely” human carcinogen. The accepted non-genotoxic mode of action (MOA) of Diuron encompasses urothelial necrosis induced by direct cytotoxicity, followed by regenerative cell proliferation and sustained urothelial hyperplasia that may favor neoplasia development. Scanning electron microscopy (SEM), light microscopy and labeling index are essentials tools for identification and classification of cytotoxic and proliferative changes in the bladder. The present study evaluated the dose-response of Diuron regarding urothelial lesions. Sixty male Wistar rats were fed Diuron for 20 weeks mixed in the diet at 0, 60, 125, 500, 1,250, or 2,500 ppm. Simple hyperplasia was significantly increased in the Diuron 1,250 and 2,500 ppm groups, and the cell proliferation at 2,500 ppm group. By SEM, the incidences and severity of lesions were significantly greater in the 500 and 1,250 ppm. Although numerically increased, the incidence of lesions in the 2,500 ppm group did not differ significantly from the control. The present study documented a doseresponse influence of Diuron on the rat urothelium, with a no observed effect level (NOEL) of 125 ppm.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Evaluation of early changes induced by diuron in the rat urinary bladder using different processing methods for scanning electron microscopy

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substituted urea herbicide carcinogenic to the rat urinary bladder at high dietary levels. The suggested non-genotoxic mode of action (MOA) of diuron encompasses cytotoxicity and necrosis followed by regenerative hyperplasia. Prenecrotic swollen cells as observed under scanning electron microscopy (SEM) have been reported as early morphological alterations, putatively related to diuron cytotoxicity. However, these changes were not observed in a previous SEM study conducted in this laboratory. This study evaluated whether these early alterations are actually due to diuron cytotoxicity or artifacts related to different processing methods used for SEM analysis. Male Wistar rats were fed ad libitum with basal diet, 7.1% sodium saccharin (NaS) or 2.500 ppm diuron for seven days or 15 weeks. The urinary bladders were processed for histological and labeling indices examinations and for SEM using two different processing methods. The incidence of simple hyperplasia after 15 weeks of exposure to diuron or to NaS was significantly increased. By SEM, the incidences and severity of lesions were significantly increased in the diuron group independently of exposure time. The different SEM processing methods used allowed for visualization of swollen superficial cells after seven days of diuron exposure. Probably the absence these cells in a previous study was due to the use very few animals. Our results support the hypothesis that the swollen cell is an early key event due to diuron-induced cytotoxicity and is the result of a degenerative process involved in the non-genotoxic carcinogenic mode of action of high doses of diuron.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Dose-response of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] in the urothelial mucosa of Wistar rats

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a herbicide that induced urothelial tumors in the urinary bladder of Wistar rats fed 2500. ppm during a long-term study. The currently suggested non-genotoxic mode of action (MOA) of diuron encompasses in succession urothelial necrosis induced by direct cytotoxicity, regenerative cell proliferation and sustained urothelial hyperplasia that increases the likelihood of neoplasia development. This study evaluated the dose-response profile of urothelial histological and ultrastructural lesions induced by diuron. Sixty male Wistar rats were fed ad libitum diuron mixed in the diet at 0, 60, 125, 500, 1250, or 2500. ppm for 20 weeks. The incidences of urothelial simple hyperplasia and the cell proliferation index were significantly increased in the diuron-fed 1250 and 2500. ppm groups. By scanning electron microscopy, the incidences and severity of lesions were significantly increased in the 500 and 1250. ppm groups. The incidences of urothelial hyperplasia in the kidney pelvis were significantly increased in the 500, 1250 and 2500. ppm groups. The present study documents the dose-response influence of diuron on the rat urothelium, with a no observed effect level (NOEL) at 125. ppm; 1250. ppm was as effective as 2500. ppm at inducing urothelial lesions. © 2013 Elsevier B.V

Diuron-induced rat urinary bladder carcinogenesis: Mode of action and human relevance evaluations using the International Programme on Chemical Safety framework

Diuron, a high volume substituted urea herbicide, induced high incidences of urinary bladder carcinomas and low incidences of kidney pelvis papillomas and carcinomas in rats exposed to high doses (2500 ppm) in a 2-year bioassay. Diuron is registered for both occupational and residential uses and is used worldwide for more than 30 different crops. The proposed rat urothelial mode of action (MOA) for this herbicide consists of metabolic activation to metabolites that are excreted and concentrated in the urine, leading to cytotoxicity, urothelial cell necrosis and exfoliation, regenerative hyperplasia, and eventually tumors. We show evidence for this MOA for diuron using the International Programme on Chemical Safety (IPCS) conceptual framework for evaluating an MOA for chemical carcinogens, and the United States Environmental Protection Agency (USEPA) and IPCS framework for assessing human relevance.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES