RESUMO
With the aim to assess the involvement of distinct forms of cytochrome P450 enzymes in the activation of procarcinogens, we have developed by means of retroviral infection a series of NIH/3T3 cell lines stably expressing human CYP1A1, CYP1A2, CYP2C10, CYP2D6, and CYP2E1 cDNA. The levels of cytochrome P450 enzyme activities were determined using specific substrates. An increase in specific catalytic activity could be observed in all cell lines compared to background activity in vector-infected cells. Furthermore, we developed a test system in which we are able to combine P450-expressing cells with a shuttle vector containing the lacZ' gene, which serves as a reporter gene for mutations. Using this system, we investigated the cytotoxicity and mutagenicity of the mycotoxin ochratoxin A. Natural occurrence of ochratoxin A in food commodities has been linked to an increased incidence of urinary tract tumors in certain geographic regions. Although biotransformation seems to play a crucial role in ochratoxin A toxicity, the possible contribution of metabolites to genotoxicity and carcinogenicity remained unelucidated. We have demonstrated that the mutation frequency of ochratoxin A was increased dependent upon concentration in NIH/3T3 cell lines, stably expressing human CYP1A1, CYP1A2, CYP2C10, and CYP3A4. In contrast, neither in vector-infected NIH/3T3 cells nor in CYP2D6- and CYP2E1-expressing cells was an increase of mutation frequency observed.
Assuntos
Células 3T3/enzimologia , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Testes de Mutagenicidade/métodos , Micotoxinas/toxicidade , Ocratoxinas/toxicidade , Animais , Carcinógenos/metabolismo , Análise Mutacional de DNA , Vetores Genéticos , Humanos , Óperon Lac , Camundongos , Mutação , Micotoxinas/metabolismo , Ocratoxinas/metabolismoRESUMO
Polychlorinated aromatic hydrocarbons modulate the proliferation and differentiation of human epidermal cells in vivo and in culture. One of the earliest events in the process of terminal differentiation is the increase in cell size. In this report the usefulness of morphometric cell size analysis as a quantifiable marker for chemical-induced differentiation was examined. Concentration-related increases in cell size distribution were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,4,7,8-pentachlorodibenzofuran in normal human keratinocytes and cells from an SV40-transformed keratinocyte cell line (SVK14) whereas the analog 1,2,3,4-tetrachlorodibenzo-p-dioxin did not affect the cell size distribution up to a concentration of 100 nM. The minimal effective concentrations of five 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins/dibenzofurans and a coplanar polychlorbiphenyl necessary to induce an increase in cell size distribution were determined in SVK14 cells. It was found that the potency of these compounds relative to that of 2,3,7,8-TCDD correlated well with the toxicity equivalency factors observed in other test systems. This indicates that the keratinocyte cell assay is a useful method for establishing the relative potency of various "dioxins" and their mixtures.