Acetaminophen-induced proliferation of breast cancer cells involves estrogen receptors.

Previous studies have shown that acetaminophen, a common analgesic/antipyretic, induces proliferation of cultured breast cancer cells containing both estrogen and progesterone receptors (ER+/PR+). The main objective of this study was to evaluate the involvement of ERs in this effect. First, the effects of therapeutic acetaminophen concentrations were compared in breast cancer cells with high ERs and in T47Dco cells with lower ERs, to determine if acetaminophen-induced proliferation depends on ER levels. Second, the effects of two antiestrogens (ICI 182,780 and 4'-hydroxytamoxifen) on acetaminophen-induced proliferation were determined in three human breast cancer cell lines: two ER+/PR+ (MCF7, T47D) and one ER-/PR- (MDA-MB-231). Third, ER binding assays were performed in MCF7 cells to determine if acetaminophen competed with estradiol for binding to ERs. Proliferation endpoints monitored included percent cells in the DNA synthesis phase of the cell cycle, 3H-thymidine incorporation into DNA, and cell number. Acetaminophen did not induce DNA synthesis in T47Dco cells, but did in cells with higher ER levels, suggesting high ER levels are necessary for acetaminophen to induce proliferation. Antiestrogens inhibited acetaminophen-induced proliferation in ER+/PR+ cells while no effects were observed in ER-/PR- cells, further supporting ER involvement. However, acetaminophen did not compete with estradiol for binding to ERs in ER+/PR+ cells. Collectively, these data suggest that acetaminophen induces breast cancer cell proliferation via ERs without binding to ERs like estradiol. The second purpose of this study was to determine if acetaminophen is estrogenic/antiestrogenic in vivo (uterotrophic assays). Acetaminophen has no antiestrogenic/estrogenic activity in mice or rats uteri.

Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat.

Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-p-benzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity.

Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: multiple modes of action of nitric oxide synthase inhibitors.

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.