Frequencies of X-ray induced chromosome aberrations in lymphocytes of xeroderma pigmentosum and Fanconi anemia patients estimated by Giemsa and fluorescence in situ hybridization staining techniques

Linfócitos sanguíneos de pacientes com xeroderma pigmentosum (XP) e anemia de Fanconi (FA) foram avaliados quanto à sensibilidade, à ionizaçäo radiante estimando-se a freqüência de aberraçöes cromossômicas (CA) induzidas por raios-X (1 e 2 Gy). As freqüências de aberraçöes no genoma inteiro foram estimadas em preparaçöes de linfócitos irradiados nas fases G0 e G2 coradas com Giemsa. As freqüências de translocaçöes e dicêntricos envolvendo os cromossomos 1 e 3 e o cromossomo X foram determinadas em lâminas coradas por hibridizaçäo fluorescente in situ (FISH). Um aumento em todos os tipos de CA foi observado em linfócitos XP e FA irradiados na fase G0 quando comparados a controles. A freqüência de dicêntricos e anéis foi 6-27 por cento maior (com 1 e 2 Gy) em linfócitos XP e 37 por cento maior (com 2 Gy) em linfócitos FA do que em controles, enquanto que as deleçöes cromossômicas foram mais freqüentes em linfócitos XP irradiados (30 por cento com 1 Gy e 72 por cento com 2 Gy) do que em controles e 28-102 por cento mais freqüentes em linfócitos FA. Em linfócitos irradiados na fase G2 a freqüência total de CA foi 24-55 por cento mais elevada em linfócitos XP do que em controles. Na maior parte dos casos as freqüências de translocaçöes foram maiores do que as freqüências de dicêntricos (21/19).

Development And Application Of Tests With Human Derived Cells For The Detection Of Environmental Genotoxins.

Two novel approaches are described, in which metabolically competent human derived cells were used for the detection of genotoxic effects of environmental carcinogens. In the first, human hepatoma (Hep G2) cells were used for micronucleus and single cells were used for micronucleus and single cell gel electrophoresis (SCGE) assays. These cells have retained the activities of phase I and phase II enzymes which are usually lost during cultivation. We demonstrated that these cells are suitable for the detection of the genotoxic effects of representatives of various classes of DNA-reactive procarcinogens such as benzo(a) pyrene (B(a)P), 2-amino-3-methylimidazo-[4,5-f]-quinoline (IQ), cyclophosphamide (CP), and N-nitrosodimethylamine (NDMA), isatidine and aflatoxin B1 (AFB1). Furthermore, we found that these tests also detect the mutagenic effects of rodent carcinogens such as safrole and hexamethylphosphoramide (HEMPA), which give negative results in conventional in vitro procedures. Additional experimental series showed that genotoxicity assays with Hep G2 cells are also useful for the detection of co- and antimutagens, in particular for compounds which act via induction of activating and detoxifying enzymes. In the second approach, a protocol for stable co-cultivation sandwich cultures with primary human hepatocytes was used. The cultivation of the cells under organotypical conditions leads to an extension of their life span and results in an improved expression of drug metabolising enzymes. Two different experimental models were developed: In the first, the induction of HPRT mutations in V-79 cells was used as an endpoint, in the seconds, single strand breaks were measured in human K562 cells in SCGE assays. Experiments which were carried out with B(a)P and 7,12-diemethylbenz(a)anthracene as model compounds indicate that in both systems positive results are obtained. In conclusion, our data show that tests with human Hep G2 cells as well as sandwich cultures with primary human liver cells are useful for the detection of environmental carcinogens and probably reflect their effects in humans better than conventional in vitro assays with metabolically incompetent cells which are currently used in most mutagenicity studies.