Monitoring of genotoxic exposure of humans by the sister chromatid exchange test. Methodology and confounding factors.

During the last decades several in vitro assays for measurement of genotoxic exposure have been developed. Exposure of humans to genotoxic products in their workplaces and the environment may be evaluated by means of the sister chromatid exchange test (SCE), which visualize exchange of DNA material between sister chromatids of a chromosome. This thesis deals with the development of SCE methodology and screening of confounding factors to make the method useful in population monitoring of genotoxic exposure of chemicals in vivo. Human lymphocytes are cultured under standardized conditions. The cells are prepared for chromosome analysis by means of new-developed apparatuses treating 12 samples simultaneously. By this method the preparation is speeded up and breathing of glacial acetic acid is minimized. Slides are prepared and stained. The staining procedure includes irradiation of the slides with long wave UV light. The light source has been optimized with regard to simplicity and spectral distribution to fit the absorption spectrum of the dye bisbenzimid. The irradiation time and dose to produce distinct harlequin stained chromosomes has been found. Population screening demands analysis in microscope of a great number of chromosomes. The working time at microscope is long and improvement of ergonomics and automation to speed up the procedure was obtained by development of a special microscope which projects the picture of SCEs at table level. The scanning table was motorized. These changes gave excellent ergonomic proporties as fixed positions of the eyes and hands were avoided. The system is fast and flexible and may semiautomatically produce karyotypes. By pointing out SCEs the system automatically registers SCE number, cell coordinates, mitosis quality and perform statistics. The statistical model which best describe the distributions of SCE data has been analysed. This was a negative binomial distribution, which is close to the Poisson distribution. The transformation y = (S).5 + (S + 1).5 follows a normal distribution. Variances of the transformation of the sum of SCEs in 30 cells within subjects and between subjects have been calculated, and on that basis the demands to sample size have been calculated. To avoid to make false statements about a connection between an exposure and SCE a detailed knowledge of confounding factors is essential. Of endogenous factors gender, age, race, and heredity have been considered. Of these gender and age contribute to the variation in SCE. Very high SCE level has been found in healthy members of a family and such cases have to be excluded from exposure studies.(ABSTRACT TRUNCATED AT 400 WORDS)