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A Fluorescence Microscopic Study of Methodologic Effects on the Intranuclear Distribution of Benzo()pyrene

Cancer Section, Oklahoma Medical Research Foundation, and Departments of Anatomy and Urology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104

Various technics for cellular fixation and fractionation were studied for their effect on the intranuclear distribution of benzo()pyrene using fluorescence microscopy. The pattern of carcinogen distribution in live cell nuclei was studied as the control for potential methodologic effects. In L-929 fibroblasts and rat hepatocytes, the great majority of nuclei showed no demonstrable accumulation of fluorescent hydrocarbon except possibly in the nuclear membrane. However, in some fibroblast cultures intranuclear fluorescence was encountered associated with inclusions of unknown origin and with some micronuclei.

Fixation with a variety of agents widely employed in cytology was tested on L-cells. Unlike most individual or groups of agents, ethanol and osmium tetroxide did not preserve the live cell pattern of hydrocarbon. Dehydration of formalin-fixed cells in ethanol led to nucleolar uptake of carcinogen.

Nuclear fractions from liver were isolated in a number of sucrose and citric acid solutions. Citric acid, but never sucrose, allowed general intranuclear uptake of hydrocarbon. Resuspension of sucrose nuclei in various buffers caused no redistribution of benzo()pyrene except in the case of acetate buffer.

In the procedures which led to nuclear relocation of hydrocarbon, the dominant factor appeared to be the involved agent per se, such as ethanol, citric acid, or acetate. The effects of concentration, pH, or electrolyte content of solutions were not apparently important. The operation of these agents is discussed in terms of their known effect on nuclear lipids.

The results of the study are offered as evidence that fluorescence microscopy gives reliable qualitative, but probably not quantitative, information on the nuclear localization of the polycyclic hydrocarbons.

¹ Present Address: McArdle Cancer Research Institute, University of Wisconsin, Madison, Wisconsin.

Received 7/24/68. Accepted 1/20/69.