Rogers L K, Moorthy B, Smith C V
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA.
Chem Res Toxicol. 1997 Apr;10(4):470-6. doi: 10.1021/tx960159i.
Alkylation of DNA by acetaminophen metabolites has been reported previously, but has received little attention, and the biological impact of this alkylation is essentially unknown. In the present study, apparent covalent binding of acetaminophen metabolites to DNA in male ICR mice was observed at levels of 2.0 +/- 0.4 to 18.5 +/- 5.5 pmol of acetaminophen/mg of DNA in liver and 0.6 +/- 0.1 to 26.9 +/- 2.6 pmol of acetaminophen/mg of DNA in kidney with doses ranging from 10 to 400 mg/kg. Investigations of the reaction of [3H]-N-acetyl-p-benzoquinone imine (NAPQI) or [ring-14C]NAPQI with DNA in vitro yielded low levels of DNA alkylation. Greater apparent binding of [3H]NAPQI to DNA occurred in reactions containing nuclear proteins, such as by using chromatin or whole nuclei. The binding of NAPQI to purified DNA also was enhanced by the presence of 0.1 mM cysteine, but not by 1.0 mM cysteine. Increased binding of NAPQI to DNA in the presence of cysteine or nuclear protein is in contrast to the effects of alternate sulfhydryls on the binding of NAPQI to proteins, which implies that the mechanisms responsible for binding to DNA may be different than the mechanisms that mediate alkylation of protein. The alkylation of DNA by [ring-14C]NAPQI was enhanced markedly at buffer pH < 4.0, suggesting participation of a protonated form of NAPQI in binding to DNA under these conditions. Acetaminophen binding to DNA also was assessed in metabolic activation systems, including microsomes with cumene hydroperoxide or NADPH, and with horseradish peroxidase (HRP) and H2O2. Measurable binding was obtained in all systems, but HRP and H2O2 produced binding levels 200-fold greater than was observed with the microsomal systems. The 32P-postlabeling of DNA from acetaminophen-treated mice, and of DNA reacted with acetaminophen, HRP, and H2O2, produced unique spots that were not identical. The present data further support the hypothesis that acetaminophen metabolites bind covalently to DNA and demonstrate that this apparent binding is observed in experimental animals in vivo at doses that mimic therapeutic doses in humans.
对乙酰氨基酚代谢产物对DNA的烷基化作用此前已有报道，但很少受到关注，且这种烷基化的生物学影响基本未知。在本研究中，在雄性ICR小鼠体内观察到对乙酰氨基酚代谢产物与DNA的明显共价结合，剂量范围为10至400mg/kg时，肝脏中对乙酰氨基酚与DNA的结合水平为2.0±0.4至18.5±5.5pmol对乙酰氨基酚/mg DNA，肾脏中为0.6±0.1至26.9±2.6pmol对乙酰氨基酚/mg DNA。体外研究[3H]-N-乙酰对苯醌亚胺（NAPQI）或[环-14C]NAPQI与DNA的反应产生的DNA烷基化水平较低。在含有核蛋白的反应中，如使用染色质或完整细胞核时，[3H]NAPQI与DNA的明显结合更强。0.1mM半胱氨酸的存在增强了NAPQI与纯化DNA的结合，但1.0mM半胱氨酸则没有。在半胱氨酸或核蛋白存在下，NAPQI与DNA结合增加，这与其他巯基对NAPQI与蛋白质结合的影响相反，这意味着与DNA结合的机制可能不同于介导蛋白质烷基化的机制。在缓冲液pH<4.0时，[环-14C]NAPQI对DNA的烷基化作用明显增强，表明在这些条件下，质子化形式的NAPQI参与了与DNA的结合。在代谢活化系统中也评估了对乙酰氨基酚与DNA的结合，包括用异丙苯过氧化氢或NADPH处理的微粒体，以及用辣根过氧化物酶（HRP）和H2O2处理的系统。在所有系统中都获得了可测量的结合，但HRP和H2O2产生的结合水平比微粒体系统观察到的高200倍。对乙酰氨基酚处理小鼠的DNA以及与对乙酰氨基酚、HRP和H2O2反应的DNA进行32P后标记，产生了不同的独特斑点。目前的数据进一步支持了对乙酰氨基酚代谢产物与DNA共价结合这一假说，并表明在模拟人类治疗剂量的实验动物体内观察到了这种明显的结合。
