Adverse outcome pathways – development and potential regulatory application

Introduction: Over the last two decades, chemical safety assessment and regulatory toxicology have progressed from empirical science based on direct observation of apical adverse outcomes in whole organisms to a predictive practice that infers outcomes and risks on the basis of accumulated understanding of toxicological mechanisms and modes of action. Objective: To provide general  concepts on how Adverse Outcome Pathways (AOPs) are developed and examples related to skin sensitization, endocrine, disruption, and mitochondrial dysfunction. Method: Narrative review based on data of the scientific literature relevant to the theme addressed and on the experience of the authors. Results: An AOP framework provides a systematic approach to organize knowledge about mechanisms of toxicity that may inform analytical domains in regulatory decision-making. AOPs are open structures that may indicate not only data gaps in the understanding of a toxicity process, but also testing procedures that will generate the necessary knowledge to fill those gaps. Every AOP should be continuously refined through the collaborative efforts of the scientific community. Depending on the amount and detail of information that is successively inserted, AOP may progress from the stage of a putative AOP to the stages of qualitative and quantitative AOPs, which are more fit-for-purpose to support regulatory decision-making. Conclusions: Continuous collaboration between AOP developers within the scientific community and the  regulatory corps toward the development of this mechanistic structure will support the advancement of toxicological sciences, regardless of its immediate application for regulatory purposes.TÍTULO PT: Vias de desfecho adverso – desenvolvimento e potencial aplicação regulatória Introdução: Durante as duas últimas décadas, a avaliação da segurança química e a toxicologia regulatória evoluíram de uma ciência empírica em grande parte baseada na observação de desfechos adversos em órgãos e ou organismos inteiros para uma prática preditiva que infere desfechos e riscos a partir do conhecimento acumulado sobre mecanismos e modos de ação toxicológicos. Objetivo: Discorrer sobre como as AOPs são desenvolvidas e fornecer exemplos relacionados à sensibilização cutânea, desregulação endócrina e disfunção mitocondrial. Método: Revisão narrativa baseada em dados da literatura científica relevantes para o tema abordado e na experiência dos autores. Resultados: A estrutura conceitual denominada AOP (do inglês, Adverse Outcome Pathway) permite uma abordagem sistemática do conhecimento disponível sobre mecanismos de toxicidade que pode subsidiar a tomada de decisões regulatórias. AOPs são estruturas abertas que podem indicar lacunas de dados para a compreensão de determinada via patogenética de toxicidade, de modo que podem ser continuamente aperfeiçoadas por esforços da comunidade científica. Dependendo das informações sucessivamente inseridas, as AOPs podem passar do estágio de uma AOP hipotética para os estágios de AOP qualitativa e AOP quantitativa, sendo este último o mais adequado para subsidiar decisões regulatórias. Conclusões: A colaboração contínua entre os desenvolvedores de AOPs dentro da comunidade científica e os corpos regulatórios para o desenvolvimento dessa estrutura mecanicista apoiará o avanço das ciências toxicológicas, independente de sua aplicação imediata para fins normativos.

O Risco Genético das Terapias do Câncer

O presente artigo descreve as conseqüências de algumas terapias utilizadas para o câncer sobre o material genético de pacientes com neoplasias. O objetivo é ressaltar a importância da avaliação da relação risco/ benefício dos diferentes tratamentos utilizados para o câncer, e da continuidade de pesquisas para o desenvolvimento de novas formas de terapia e de novas drogas antineoplásicas. Faremos breve exposição sobre o processo de carcinogênese de múltiplas etapas, destacando o conceito geral de que o câncer é uma doença genética. São apresentados os efeitos mutagênicos das radiações ionizantes e de algumas classes de quimioterápicos sobre o material genético. Além dos efeitos deletérios desses agentes para o paciente, são também relatados efeitos nocivos para os indivíduos que manipulam ou que são responsáveis pela condução dos tratamentos. Abordaremos de modo geral, os danos genéticos induzidos por agentes antineoplásicos mutagênicos tanto para o paciente como para os indivíduos que os manipulam, predispondo-os ao desenvolvimento de um segundo tumor ou de um tumor primário, respectivamente

Chemical carcinogenesis - mode of action to inform quantitative human risk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Brazilian experience with the medium-term multi-organ bioassay: Scientific and regulatory developments

In 1996 the Brazilian Institute for the Environment (IBAMA) officially adopted a variation of the multiorgan initiation-promotion DMBDD bioassay as a valid source of evidence of the carcinogenic potential of pesticides. The protocol adopted by IBAMA was a modification of the one originally proposed by researchers led by Nobuyuki Ito, from the Nagoya City University Medical School. Among the modifications established in the Brazilian protocol were the use of both sexes of the outbreed Wistar strain of rats and two positive control test chemicals. The adoption of the modified DMBDD protocol was instrumental during the last decade for qualifying technical people and to spread knowledge on chemical carcinogenesis in Brazil

Desenvolvimento e validação de técnica quantitativa de análise de imagem para avaliação do teste do cometa corado pela prata

INTRODUÇÃO: O ensaio do cometa ou técnica da eletroforese de células isoladas é largamente empregado para avaliação de danos e reparo do DNA em células individuais. O material pode ser corado por técnicas de fluorescência ou por sal de prata. Este último apresenta vantagens técnicas, como o tipo de microscópio utilizado e a possibilidade de armazenamento das lâminas. A análise dos cometas pode ser feita de modo visual, porém há a desvantagem da subjetividade dos resultados, que pode ser minimizada por análise digital automatizada. OBJETIVOS: Desenvolvimento e validação de método de análise digital de cometas corados por sal de prata. MÉTODOS: Cinquenta cometas foram fotografados de maneira padronizada e impressos em papel. Além de medidas manualmente, essas imagens foram classificadas em cinco categorias por três avaliadores, antes e depois de pré-processadas automaticamente pelo software ImageJ 1.38x. As estimativas geradas pelos avaliadores foram comparadas quanto sua correlação e reprodutibilidade. em seguida, foram desenvolvidos algoritmos de análise digital das medidas, com base em filtros estatísticos de mediana e de mínimo. Os valores obtidos foram comparados com os estimados manual e visualmente após o pré-processamento. RESULTADOS: As medidas manuais das imagens pré-processadas apresentaram maior correlação intraclasse do que as imagens preliminares. Os parâmetros automatizados apresentaram alta correlação com as medidas manuais pré-processadas, sugerindo que este sistema aumenta a objetividade da análise, podendo ser utilizado na estimativa dos parâmetros dos cometas. CONCLUSÃO: A presente análise digital proposta para o teste do cometa corado pela prata mostrou-se factível e de melhor reprodutibilidade que a análise visual.BACKGROUND: Comet assay or single cell gel electrophoresis is a useful and widely applied technique for the assessment of DNA damage and repair in individual cells. Nuclei can be stained with fluorescence methods or silver salts. The latter has technical advantages such as the type of microscope used and the possibility of slide storage. Comet analysis may be performed visually, however, there is the disadvantage of subjective results, which can be minimized by automated digital analysis. OBJECTIVES: Development and validation of digital analysis method for silver stained comet assays. METHODS: Fifty comets were photographed in a standardized way and printed on paper. Before and after being automatically preprocessed by ImageJ 1.38x software, the images were manually measured and classified into five categories by three evaluators. Their estimates were compared as to their correlation and reproducibility. Afterwards, an algorithm for automated digital analysis of the comet measurements was developed based on statistical filters of median and minimum. These results were compared with those manually and visually estimated after preprocessing. RESULTS: Manual measurements of preprocessed images showed higher intraclass correlation than the original ones. Automated results had high correlation with the pre-processed manual measurements, suggesting that this system increases objectivity and can be used in the estimation of comet parameters. CONCLUSIONS: Digital analysis of silver stained comet assay proved to be feasible and better reproducible than the visual analysis

Evaluation of Carcinogenic Potential of Diuron in a Rat Mammary Two-Stage Carcinogenesis Model

This study aimed to evaluate the carcinogenic potential of the herbicide Diuron in a two-stage rat medium-term mammary carcinogenesis model initiated by 7,12-dimethylbenz(a)anthracene (DMBA). Female seven-week-old Sprague-Dawley (SD) rats were allocated to six groups: groups G1 to G4 received intragastrically (i.g.) a single 50 mg/kg dose of DMBA; groups G5 and G6 received single administration of canola oil (vehicle of DMBA). Groups G1 and G5 received a basal diet, and groups G2, G3, G4, and G6 were fed the basal diet with the addition of Diuron at 250, 1250, 2500, and 2500 ppm, respectively. After twenty-five weeks, the animals were euthanized and mammary tumors were histologically confirmed and quantified. Tumor samples were also processed for immunohistochemical evaluation of the expressions of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, estrogen receptor-alpha (ER-alpha), p63, bcl-2, and bak. Diuron treatment did not increase the incidence or multiplicity of mammary tumors (groups G2 to G4 versus Group G1). Also, exposure to Diuron did not alter tumor growth (cell proliferation and apoptosis indexes) or immunoreactivity to ER-alpha, p63 (myoephitelial marker), or bcl-2 and bak (apoptosis regulatory proteins). These findings indicate that Diuron does not have a promoting potential on mammary carcinogenesis in female SD rats initiated with DMBA.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Effects of Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the urinary bladder of male Wistar rats

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 mucosa of male Wistar rats. In general, the genotoxic and mutagenic potentials of Diuron are considered to be negative. The present study aimed to evaluate the mode of action of Diuron on the urinary bladder mucosa of male Wistar rats. Six-week old male Wistar rats were fed pelleted Nuvilab diet mixed with Diuron at 125, 500 and 2500 ppm. As a positive control, 8.3% sodium saccharin (NaS) was fed in the diet. Preceding the sacrifice of the animals at the 20th week, urinary pH was measured and the genotoxic potential of Diuron was evaluated by the comet assay. Histological urothelial lesions in the urinary bladder and in the renal pelvis mucosa, cell proliferation/apoptosis evaluations, and scanning electron microscopy (SEM) of the urinary bladder mucosa were also performed. No DNA changes were found in urothelial or peripheral blood cells, and urinary pH was comparable to controls in all Diuron groups. In the urinary bladder urothelium, the incidence of simple hyperplasia (SH) by light microscopy was significantly increased (7/10; p < 0.005) in the 2500 ppm Diuron group but not at the lower doses. By SEM, three of five animals treated with 2500 ppm Diuron showed urothelial cell necrosis and hyperplasia. In the renal pelvis, the incidence of SH was significantly increased in the Diuron 500 and 2500 ppm and in the NaS 8.3% groups. Cell proliferation was significantly increased in the Diuron 2500 ppm, (p < 0.05) and NaS 8.3% (p < 0.05) groups. The results indicate that a high dietary concentration of Diuron is associated with urothelial necrosis and continuous regenerative cell proliferation that leads to urothelial hyperplasia. (c) 2006 Elsevier B.V.. All rights reserved