Cytotoxicity and Apoptosis-inducing Activity of Bisphenol A and Hydroquinone in HL-60 Cells

BPA (bisphenol A or 2,2-bis(4-hydroxy- phenol)propane) and hydroquinone (HQ, 1,4-benzenediol) are present in dental resin materials, and small quantities of these substances may be eluted from the resins. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA and HQ. Thus, it is important to investigate the cytotoxicity and apoptosis-inducing activity of these compounds. BPA and HQ reduced the viable cell number of human promyelocytic leukemia (HL-60), human oral squamous cell carcinoma (HSC-2) and human submandibular gland (HSG) cell lines in a concentration- dependent manner. The cytotoxic activity of HQ, but not of BPA, was significantly reduced by the addition of N-acetyl- L-cysteine (NAC). In biomimetic studies of the pro- oxidant/antioxidant activity of thiols during oxidation of BPA or HQ, the radical-scavenging activities of mixtures of BPA or HQ and 2-mercapto-1-methylimidazole (MMI, a thiol) were investigated by the induction period method. BPA without MMI showed a higher induction period (antioxidant activity) than did HQ, but BPA with MMI did not cause oxygen uptake. In contrast, HQ with MMI caused oxygen uptake, suggesting formation of MMI thiyl radicals during oxidation of HQ followed by reaction with molecular oxygen. This indicates that HQ may produce intracellular reactive oxygen species (ROS) and provides an explanation for the decrease in the cytotoxicity of HQ by NAC. BPA induced internucleosomal DNA fragmentation, a biochemical marker of apoptosis, only in HL-60 cells. BPA activated caspase-9 and caspase-3, suggesting induction of apoptosis via caspase activation by the caspase recruitment domain. The cytotoxicity of BPA was 2-fold less than that of HQ, whereas the apoptosis-inducing activity of BPA was 10-fold less than that of HQ. Bisphenol A or 2,2-bis(4-hydroxyphenol)propane (BPA) is an estrogen-like substance and an environmentally significant chemical. Very small amounts of benzenediols such as BPA and HQ (1,4-benzenediol) are present in dental restorative or sealant resin materials. Such dental resin materials containing BPA and HQ are introduced into the oral environment in an uncured state, although they are immediately cured by visible light irradiation. Consequently, BPA and HQ together with uncured methacrylate monomers may be incorporated into the human body. BPA is also the monomer for the production of polycarbonate plastics used in babies' bottles and is a major component of epoxy resins for the lining of food cans. Recently, BPA has been shown to have a number of disease-promoting effects, including carcinogenicity (1) and estrogen-like activity (2). Radical interactions between HQ/BPA mixtures and antioxidants (3), as well as the cytotoxicity of HQ and its apoptosis-inducing activity in HL-60 cells (4, 5), have been previously reported. The apoptotic effects of BPA have previously been studied (6, 7), but the mechanisms involved remain poorly understood. In the present study, the comparative cytotoxicities of BPA and HQ in the absence or presence of NAC against HL-60, HSC-2 and HSG cells were investigated with special attention to the mechanism of induction of apoptosis by BPA in HL-60 cells, as judged by DNA fragmentation and caspase activation. The mutagenicity and apoptosis-inducing activity of BPA and HQ are believed to be related to the effects of these compounds on glutathione (GSH) levels, probably via interactions between thiols and quinone intermediates derived from BPA or HQ oxidation (8, 9). Thus, in order to perform biomimetic studies on the pro-oxidant/antioxidant activity of thiols during BPA or HQ oxidation, the radical- scavenging properties of mixtures of BPA or HQ and 2- mercapto-1-methylimidazole (MMI) were also investigated