Effects of 4-nitroquinoline-N-oxide on RNA synthesis.

rearrangement of the nucleolonema and pars amorpha of the nucleolus. In addition to being carcinogenic, 4-nitroquinoline-N-oxide and several of its derivatives have been shown to be potent mutagenic agents (18). Thus, 4-nitroquinoline-N-oxide has been reported to exert mutagenic effects in bacterial systems and in viruses (4). Yeast cells (18) after exposure to 4-nitro quinoline-N-oxide gave rise to mutants with defects in respira tory function. From a biochemical point of view, 4-nitroquinoline-N-oxide has been shown to exert diverse effects in a variety of systems. Among these are inhibition of glycolysis, inhibition of protein synthesis (21), reaction with sulfhydryl groups (34), and inter ference with the electron transport mechanism in photosyn thesis (1 1). Early studies in this laboratory indicated that, after exposure to 4-nitroquinoline-N-oxide, Chang liver cells and HeLa cells exhibited a decrease in acid-insoluble RNA in both isolated nuclei (24) and intact tissue culture cell systems (23). More recent studies have indicated that 4-nitroquinoline N-oxide can form complexes with DNA in solution (20, 22, 23, 25), and this laboratory has demonstrated an inhibition of the RNA polymerase reaction in isolated nuclei (derived from Chang liver cells and HeLa cells) after exposure to 4-nitroquin oline-N-oxide (24). In view of the morphology of the nucleolar caps in tissue culture cells exposed to 4-nitroquinoline-N-oxide and of the various biochemical actions which it is known to have, the effects of 4-nitroquinoline-N-oxide on RNA synthesis were in vestigated by studying the cytoplasmic fractions of RNA iso lated by sucrose density centrifugation and MAK column chro matography. MATERIALS AND METHODS HeLa cells were grown as monolayers in Eagle's medium with fetal calf serum and in spinner culture (13) using a modi fled Eagle's suspension medium with 5% fetal calf serum. 4-Nitroquinoline-N-oxide was added at a concentration of 10†" †• M in Hanks' balanced salt solution. The final concentra tion of 4-nitroquinoline-N-oxide used in these experiments varied from 0.5 to 10 jig/ml. Control cultures received volumes of Hank's balanced salt solution equivalent to that received by cultures receiving 4-nitroquinoline-N-oxide. The cells were labeled with uridine-3H (8000 mc/mmole), uridine-2-14C (100 mc/mmole), leucine-14C (200 mc/mmole), or 14C-labeled yeast protein hydrolysate (1500 jic/mg). The cells were chilled SUMMARY 4-Nitroquinoline-N-oxide, a potent mutagenic, carcinogenic, and carcinostatic agent, has been studied with respect to its effects on RNA synthesis using sucrose gradients, methylated albumin Kieselguhr columns, and the electron microscope. An analysis of polyribosomal amino acid incorporation in tissue culture …