Free radical generation from heterocyclic amines by cytochrome b5 reductase in the presence of NADH
Abstract
We previously reported findings that NADPH/cytochrome P450 reductase can generate superoxide anion radical (O2
−) from heterocyclic amines (HCA) and from many anticancer agents in vitro. Here we present more evidence in which O2− is generated when recombinant human cytochrome b5 reductase (rh-Cytb5Rd) was incubated with HCAs such as IQ and MeIQ in the presence of NADH in vitro. This indicates that free radical generation by rh-Cytb5Rd in the presence of HCA may add new insight into the damage of DNA in addition to the previously known mechanism: interaction of activated HCA-intermediates to form DNA adduct.
Introduction
A classical theory of carcinogenesis involves, (a) radiation, (b) chemicals, and (c) infections. Firstly, (a) has been known for many decades; its capacity to generate highly reactive free radicals such as singlet oxygen and hydroxyl radical (OH) is well established. Secondly for (b), many chemicals are known to have potent carcinogenic activity such as benzo[a]pyrenes and heterocyclic amines (HCA), and they are usually metabolized by cytochrome P450 enzymes to the more reactive intermediates, which consequently form DNA adducts. In this connection, we have previously reported that a system involving NADPH cytochrome P450 reductase and heterocyclic amines or some anti-cancer agents such as mitomycin C, adriamycin and neocarzinostatin, can generate superoxide anion (O2−) [1], [2], [3], which can directly or indirectly damage DNA or other vital molecules. Thirdly for (c), infection-induced cancer was initially thought particularly related to tumor viruses. However, more recent studies have shown that many chronic infections, which are free of oncogenes, are now considered as important etiological agents in carcinogenesis such as hepatitis virus type B/C, Helicobacter pylori and herpes virus type 2 infections. Namely, some viral infections are now known to induce both O2− [4], [5], [6] and nitric oxide (NO) at the site of infection [7], [8], [9]. We and others have shown these facts in the model using influenza virus in mice [4], [5], [6], [7], [8], [9], herpes simplex virus in rats [10], Borna disease virus in rats [11], and bacterial infection with Salmonella typhimurium in mice [12], [13]. Experimental granuloma produced by the inhalation of silica particles and zymozan (yeast) in rats also showed involvement of both O2− and NO, forming peroxynitrite (ONOO−) [14]. Other researchers have also shown that Helicobacter pylori-infected gastric specimens of either humans or mice exhibited the presence of nitrotyrosine [15], [16], indicating the reaction product of O2− and NO, i.e. ONOO− was formed, which is a well known potent nitrating agent [17]. As a consequence, ONOO–nitrated tyrosine in protein as well as DNA in the corresponding tissues, not to mention hydroxylation of guanosine at the site of inflammation, can be detected readily. These accounts are discussed in view of infection-induced cancers involving free radical generations, in a review article in this context recently [9].
In this report, we describe that recombinant human cytochrome b5 reductase (rh-Cytb5Rd) in the presence of NADH and HCAs can generate O2− similar to cytochrome P450 reductase with NADPH.
Section snippets
Materials
2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were obtained from Wako Pure Chemical Industries, Osaka, Japan. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethyl-4-hydroxypiperidine (Tempol), the spin trap agents for radical species in electron spin resonance (ESR) measurement, were obtained from Dojindo Laboratories, Kumamoto, Japan, and from Sigma Chemical Co., St. Louis, MO, respectively.
Results and discussion
Fig. 1 demonstrates that the reaction mixture consisting of rh-Cytb5Rd together with NADH and HCAs (IQ and MeIQx) yielded typical ESR signals of DMPO spin adducts of O2− having a hyperfine coupling constant of mT, mT, mT. These two HCAs yielded similarly DMPO-OOH signals, which were abrogated by SOD (Fig. 1C,E). Furthermore, the amount of O2− radical captured by spin-trapping agent was quantified by double integration of the ESR signals and the result is shown in Fig. 2.
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