The differential effects of dimethylnitrosamine and ethionine on mitochondrial and extramitochondrial dehydrogenases in single intact cells

Abstract

In studying the bioenergetics of living cells, the microfluorometric analysis of coenzyme (NAD(P)H) responses to microinjected respiratory and glycolytic substrates enables, in principle, a search for qualitative/quantitative differences in normal versus carcinogen-treated (short-term, long-term) and malignant cells. Responses are compared in L-cells, same adapted to hypertonic media (i.e. L255, L355) and highly malignant rhabdomyosarcoma (CCL 136) cells. The largest responses to respiratory substrate (malate, isocitrate) and the lowest responses to glycolytic substrate (glucose-6- P) are in the L255, 355 cells which exhibit structural rearrangement and dense packing of mitochondria possibly due to high energy requirement for ion pumping. The converse is observed in the CCl 136 where there is no lack of these organelles, but they could be functionally deficient, as suggested by a predominant response to glucose-6- P compared to malate. In the control L-cell, the malate and glucose-6- P responses are relatively well balanced. Upon addition of dimethylnitrosamine to L-cells, there is an initial acceleration in the rate of glucose-6- P-induced NAD(P) reduction (? NADPH requirement for dimethylnitrosamine metabolization), followed by an upsurge of the malate response. In L355 cells, addition of the carcinogens dimethylnitrosamine or ethionine is followed by a strong reductive response to malate, and minimal response to glucose-6- P. The dramatic intensification of the NAD(P)H response to malate in L355 cells pretreated with an ATP trap (ethionine) or an uncoupler (dinitrophenol) strongly points to a requirement for ATP depletion. Weaker enhancement of NAD(P)H response (preferentially after glucose-6- P) is observed in the CCL 136 upon treatment with ethionine. The findings indicate the need for further study on differences in respiratory/glycolytic pathways and efficiency of ATP cycle in malignant cells exhibiting graded differences of structural/functional specialization.