RESUMO
The medium-term multiorgan initiation-promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.
Assuntos
2-Acetilaminofluoreno/toxicidade , Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Neoplasias Experimentais/induzido quimicamente , Fenobarbital/toxicidade , Lesões Pré-Cancerosas/induzido quimicamente , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Bioensaio , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP2B1/biossíntese , Feminino , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Neoplasias Experimentais/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Especificidade de Órgãos , Lesões Pré-Cancerosas/enzimologia , Ratos Wistar , Esteroide Hidroxilases/biossínteseRESUMO
Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21-105 µg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21-21 µg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ~6-fold and ~2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105 µg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.
Assuntos
Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/biossíntese , Dietilnitrosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Fenobarbital/toxicidade , Regulação para Cima/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Morte Celular/efeitos dos fármacos , Células Cultivadas , Aberrações Cromossômicas/efeitos dos fármacos , Cocarcinogênese , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/genética , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática/efeitos dos fármacos , Feminino , Hepatócitos/patologia , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Índice Mitótico , RNA Mensageiro/metabolismo , Ratos , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/genéticaRESUMO
The effect on liver cytochrome P450 (CYP) by i.p. injections of methoxychlor (MXC) in corn oil at 0, 100, 150, 200 or 250 mg/kg twice daily for 3 days was investigated in adult male and female Wistar rats. The MXC injection (100 mg/kg b.w.) caused a similar increase of total CYP content in males and females as compared with controls who received the vehicle only. In males, this increase continued up to 250 mg/kg. As to the induction of specific CYP activities, the effect of MXC was found to be sex dependent with three different patterns. Males showed the greatest increases of ethoxy- and methoxyresorufin-O-dealkylase (EROD and MROD, respectively), two CYP1A1/1A2-related activities. On the contrary, females were more responsive than males for pentoxyresorufin-O-dealkylase (PROD) and benzyloxyresorufin-O-dearylase (BROD), two CYP2B-related activities. Finally, p-nitrophenol hydroxylase (PNPH), a CYP2E1-related activity, showed a similar small, although statistically significant, increase for both sexes. As to CYP apoprotein levels, CYP1A1 and CYP2B1/2B2 showed greater increases in females than in males; whereas, interestingly, CYP2E1 induction was higher in males than in females. These results indicate overall that gender modulates CYP expression after MXC injection both qualitatively and quantitatively, and, therefore, this pesticide is not a pure PB inducer. Moreover, the statistically significant increase of CYP3A2 apoprotein expression observed in females and also, to a lower extent, in males, and the decrease of CYP2C11 apoprotein found in males, two sex-related enzymes, may explain the reported endocrine disrupting effect of MXC. The relevance of the different patterns of rat liver CYP induction observed after MXC treatment, in relationship to the speculated endocrine disrupting potential of MXC in humans potentially exposed to this pesticide, needs further investigation.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inseticidas/farmacologia , Fígado/enzimologia , Metoxicloro/farmacologia , Animais , Western Blotting , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/biossíntese , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Feminino , Indicadores e Reagentes , Inseticidas/toxicidade , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Metoxicloro/toxicidade , Oxirredutases/biossíntese , Oxirredutases/metabolismo , Ratos , Ratos Wistar , Caracteres SexuaisRESUMO
Beta-Myrcene (MYR) is an acyclic monoterpene found in the essential oils of a variety of useful plants such as lemongrass (Cymbopogon citratus), hop, verbena, bay and others. MYR and essential oils containing this olefinic monoterpene are widely used as flavoring food additives, as fragrances in cosmetics and as scents in household products. The present study was undertaken to investigate the induction of liver monooxygenases by MYR. Female Wistar rats were treated by gavage with MYR (1000 mg/kg body weight) or corn oil (vehicle) for 1 or 3 consecutive days. Activities of ethoxycoumarin-O-deethylase (ECOD) and alkoxy-resorufin O-dealkylases (methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD) and benzyloxy-resorufin-O-dealkylation (BROD)) were determined fluorimetrically in the hepatic microsomal fraction. Exposure to MYR, either for 1 or 3 days, produced marked (13-34-fold) increases in the activities of PROD and BROD and only minor changes in ECOD, EROD and MROD. Since PROD and BROD are metabolized mainly by CYP2B isoenzymes, these results suggest that MYR induces this phenobarbital-inducible P450 subfamily. The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting. Levels of apoproteins CYP2B1/2B2 were increased 8.2-fold after treatment with MYR (1000 mg/kg body wt, 3 days). Results from this study therefore indicate that MYR is an inducer of isoenzymes belonging to CYP2B subfamily.