Cytotoxicity and Regenerative Proliferation as the Mode of Action for Diuron-Induced Urothelial Carcinogenesis in the Rat

Abstract

Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

Avaliação do potencial cancerígeno do Diuron [3-(3,4-Diclorofenil)-1,1-Dimetiluréia] no modelo de iniciação-promoção cutânea em camundongos swiss

“…genotoxic agent, what is in line with information from this laboratory that reported nongenotoxicity of the herbicide under two different comet assay protocols (Nascimento et al, 2006;Rocha et al, 2009).…”

“…Previous studies from this laboratory reported absence of diuron genotoxicity in the standard alkaline version of the comet assay with urothelial and peripheral blood cells (Nascimento et al, 2006). Besides, a modified version of the comet assay developed in vitro with CHO cells indicated that diuron also does not induce DNA cross-links (Rocha et al, 2009). …”

“…A hiperplasia e displasia epidérmicas são indicadores precoces da atividade cancerígena do composto testado, por serem consideradas precursoras de papilomas e carcinomas de células escamosas (Williams, 1999 Embora não seja geneticamente modificada, esta linhagem foi selecionada durante algumas gerações para expressar suscetibilidade elevada à carcinogênese cutânea de duas etapas, apresentando menor período de latência dos tumores (Slaga, 1986 Os modelos descritos são considerados ferramenta valiosa na identificação de cancerígenos químicos, principalmente quando a rota de exposição cutânea é a mais prevalente em humanos (Enzmann, 1998 (Carpenter et al, 2002;Perry et al, 2002). Além disso, EPIs podem ser insuficientes para cobrir toda a superfície cutânea e o contato com o exterior contaminado do equipamento é possível (Brower et al, 2005 (Rocha et al, 2009). …”

“…Estudos prévios em nosso laboratório indicaram ausência de genotoxicidade do Diuron pelo teste do cometa em células uroteliais e do sangue periférico de ratos Wistar machos (Nascimento et al, 2006). Além disso, uma versão modificada do ensaio desenvolvida in vitro em células CHO sugeriu que o produto também não é capaz de induzir ligações cruzadas (crosslinks) no DNA (Rocha et al, 2009). Frame, S., Crombie, R., Liddell, J., Stuart, D., Linardopoulos, S., Nagase, H., Portella, G., Brown, K., Street, A., Akhurst, R., Balmain, A.…”

“…Since diuron is released for agricultural use, it is necessary to clarify the mode of action (MOA) through which this product has caused rodent neoplasia and verify whether it is relevant to humans. In a recent study from this laboratory it was demonstrated that diuron and/or its metabolites induce urinary bladder carcinogenesis in Wistar rats through direct chemical cytotoxicity followed by regenerative cell proliferation; the specific mechanism of action, however, has not been clarified yet (Rocha et al, 2009). …”