Structure-activity relationships in the free-radical metabolism of xenobiotics.

Many xenobiotics, including naturally occurring compounds, drugs, and environmental agents, are metabolized both in vivo and in vitro to free-radical intermediates. The one-electron reduction of nitroaromatic compounds, quinones, and a wide variety of other chemicals is catalyzed enzymatically by a number of reductases and dehydrogenases. Structure-activity studies have shown that the cytotoxicities of nitroaromatic compounds and quinones are related to their one-electron reduction potentials (E1(7)). Other factors such as oil:water partition coefficients may also be important. Xenobiotics may also be oxidized to free radicals by peroxidases. Hammett's rules apply to the one-electron oxidation of simple meta- or para-substituted phenols and amines by horseradish peroxidase, compound I.


Introduction
A free radical may be defined as any molecule that has an odd number of electrons (1). While the vast majority of known free radicals are derived from organic molecules, many inorganic radicals, e.g. O2, S03, are known to exist. The transformation of an organic molecule into a free radical may be accomplished in three different ways (2). For example, the one-electron oxidation of benzene (by removal of one of the IT-electrons) produces the benzene cation radical: O_O~~~+ eT he addition of an electron to a benzene wr-orbital generates the benzene anion radical: +e-_ When the C-H bond of benzene is cleaved homolytically, a hydrogen atom and the neutral phenyl radical are formed: icals, even under the relatively mild conditions encountered in vivo. To date more than twenty different classes of compounds are known to be metabolized in biological systems to free-radical intermediates (2)(3)(4). These chemicals include naturally occuring compounds, drugs, and environmental agents.

Detection of Free Radicals
Free radicals may be detected by using the technique of electron spin resonance (ESR). An ESR spectrum gives information on both the structure and concentration of a free radical. For example, the ESR spectrum in Figure 1 was obtained by incubating the herbicide paraquat with rat hepatic microsomes in the presence of the cofactor NADPH under anaerobic conditions (5). The radical derived from paraquat is readily identified from its ESR spectrum as the paraquat monocation radical based on the complex pattern of lines which arise from the interaction (hyperfine coupling) of the unpaired electron with the protons present in the molecule. The herbicidal activity of paraquat results from the reaction of its cation radical with oxygen to form superoxide, which initiates a series of events that eventually lead to lipid peroxidation (6). The mechanism of paraquat poisoning in man and other mammals is also thought to be superoxide-mediated, although the role of lipid peroxidation has not been unequivocally established (7).
Not all free radicals are stable enough to be detected directly by ESR. For such radicals, a technique called "spin trapping" may be employed (8). Spin trapping is a technique that involves the addition of a reactive free radical (R ) to an organic diamagnetic nitrone or nitroso compound (spin trap) to form a more stable nitroxide free radical (spin adduct): The structure of the parent free radi Pal (R) may then be determined from the hyperfine couplings of the ESR spectrum of the spin adduct. For example, the ESR spectrum of the radical formed during the ultraviolet irradiation of sulfanilamide in the presence of the spin trap 2-methyl-2-nitrosopropane is shown in Figure 2. This spectrum is easily identified as that of 4-tert-butylbenzenesulfonamide (9), which suggests the following mechanism for the photolysis:

Free-Radical Pathways in Xenobiotic Metabolism
One-Electron Enzymatic Reduction of Xenobiotics A wide variety of chemicals can be reduced to form free radicals by a number of different enzyme systems (Table 1). While oxygen has been omitted from Table 1 it should be pointed out that several of the enzymes will also reduce oxygen to its free radical form, superoxide I ' l l .
(O2-). From the data in Table 1 it is clear that aromatic molecules with vastly different structures may undergo one-electron reduction. Furthermore, some chemicals, e.g., nitroaromatic compounds and quinones, will accept an electron from almost any redox flavoprotein. There have been relatively few studies that have attempted to relate structure to activity for compounds undergoing enzymatic one-electron reduction. Most of the work in this area has involved either the nitroaromatic compounds or the quinones. Nitroaromatic Compounds. Aromatic nitrocompounds may be reduced in a series of steps to the corresponding amine: The one-electron reduction of a nitro compound gives rise to a nitro anion-free radical. Under aerobic conditions the nitro anion-radical reacts with oxygen to form superoxide with the regeneration of the parent nitro compounds: R-NO + 02 -* R-NO2 +°2 Aromatic nitro compounds, particularly those that contain a heterocyclic moiety, are widely used as chemotherapeutic agents in protozoal, fungal, and bacterial infections (13) and as radiosensitizers in cancer chemotherapy (14). These drugs are also known to be mutagenic and toxic to mammalian cells (15). The enzymatic formation of the nitro anion free radical is thought to play a key role in both the biological activity and toxicity of nitroaromatic compounds (3,4). Since the ease of formation of the nitro anion radical is related to the oneelectron reduction potential (EW) ofthe parent compound attempts have been made to correlate biological activity with this parameter (15)(16)(17)(18)(19)(20).
Adams and co-workers have tested 15 nitroaromatic and nitroheterocyclic compounds that can act as radi-osensitizers for their toxicity to Chinese hamster V79 cells in vitro (16). They found that cytotoxicity increased markedly as the electron affinity, measured as a oneelectron reduction potential, increased. A structureactivity relationship of the form -log C = bo + b1E1 + b2 log P was derived, where C is the specific cytotoxicity (concentration), E' is the one-electron reduction potential, P is the partition coefficient, and b0, b1, and b2 are coefficients derived from the least-squares fit to the data.
The potential E' is a measure of the ease of the oneelectron reduction of a compound RNO2 to the corresponding nitro anion-free radical RNO22. Since the cells used in this study were hypoxic, it seems unlikely that the species involved in the toxic response is superoxide. Adams and co-workers suggest instead that the fourelectron reduction product of nitroaromatic compound, the corresponding hydroxylamine, may be the toxic species (16). Work by the same authors has shown that toxicity of nitroaromatic and nitroheterocyclic compounds towards the same cells under aerobic conditions is also related to their one-electron reduction potentials (15). However, in this case there was no correlation with octanol:water partition coefficient. It was found, however, that quinones having the same one-electron reduction potential as the nitroaromatic compounds exhibited similar toxicity. These findings suggest that under aerobic conditions the toxic species may indeed be superoxide.
Quinones. The one-electron reduction of quinones leads to the formation of the corresponding semiquinone radical:  (Table 1). For example semiquinone metabolites of anthracycline antibiotics have been detected by ESR in both anaerobic NADPH-microsomal incubations (21)(22)(23) and in Ehrlich ascites cells (21). Under aerobic conditions the semiquinone reacts with oxygen to give superoxide and regenerate the parent quinone (24,25) (Fig. 2). The generation of superoxide by the anthracycline antitumor agent adriamycin is thought to be responsible for the cardiotoxicity of this drug (26). Superoxide is known to give rise to other reactive oxygen species including hydrogen peroxide and the hydroxyl radical (27). The observation that vitamin E ameliorates adriamycin-induced cardiomyopathy (28) is consistent with the initiation of lipid peroxidation by oxygen radicals derived from the oxidation of the adriamycin semiqu.inone.
(29-31). Svingen and Powis have reported that the enzymic one-electron reduction of a series of quinone antitumor agents, including adriamycin, daunomycin and mitomycin C, by NADPH-cytochrome P-450 reductase increases at more positive values of El (Table 2). Powis and Appel have studied the aerobic and anaerobic metabolism of a series of benzoquinones by three flavoenzymes that catalyze single-electron reduction (30). They found that metabolism was more closely related to single-electron reduction potential than the structural features or lipid solubilities of the benzoquinones. The lower limit for reduction by NADH:ubiquinone oxidoreductase with NADPH as the cofactor and purified NADPHcytochrome P-450 reductase was a quinone single-electron reduction potential of -240 mV. However, the lower limit for quinone reduction with purified NADHcytochrome b5 reductase and NADH:ubiquinone oxidoreductase with NADH as a cofactor was a single electron reduction potential of 170 mV. In general it was found that more negative single-electron potentials resulted in decreased quinone metabolism. Incubation of simple benzoquinones with isolated hepatocytes results in the liberation of superoxide (31) formed by the reaction of the corresponding semiquinone radicals with oxygen. Powis and co-workers have found that superoxide formation by simple benzoquinones or the anthracyclines is maximal at a quinone oneelectron reduction potential of -70 mV. This pattern was qualitatively similar to that observed with mitochondrial NADH:ubiquinone oxidoreductase and microsomal NADH-cytochrome b5 reductase. The observation that superoxide production by NADPH-cytochrome P-450 reductase is maximal at a quinone E' of -200 mV suggests that this enzyme is not rate-limiting for quinonestimulated superoxide formation by hepatocytes.
One-Electron Enzymic Oxidation of Xenobiotics Xenobiotics may be oxidized to free radicals by both peroxidases and oxidases (Table 3). Among the peroxidases, horseradish peroxidase (HPR) has received most attention. However, it seems likely that the mammalian peroxidases, e.g., myeloperoxidase, lactoperoxidase, and  prostaglandin synthase, probably catalyze many of the same reactions. It is interesting to note that peroxidase activity is also exhibited by catalase and methemoglobin. Among the oxidases that are known to generate free radicals, ceruloplasmin is the best studied. There has been only one attempt to relate the oneelectron enzymic oxidation of xenobiotics to structure. Job and Dunford have studied the effect of different substituents on the oxidation of phenols and aromatic amines by horseradish peroxidase (32). The one-electron oxidation of organic compounds (AH) by horseradish peroxidase may be depicted as follows: HRP + H2027*Compound I (green) Compound I + AH2-Compound II (red) + A H Compound II + AH2-*HRP + A H Job and Dunford used stopped-flow spectrometry to measure the rate of reduction of HRP to compound I by a series of phenols and aromatic amines. They found that Hammett's rules apply for HRP Compound I reduction by monosubstituted phenols and anilines with different substituents in the meta or para positions. At pH 7.0, log kvlkH was found to be -6.92o and -7.00a for phenols and anilines, respectively, where kH and kx are the second-order rate constants for the reaction between compound I and an unsubstituted phenol or aromatic amine with the substituent X in the meta or para positions. This finding indicates that all phenols are oxidized by compound I by the same mechanism suggesting that in all cases the neutral forms of the phenols are involved. The same conclusion can also be drawn for the substituted anilines. Since the meta and para substituents produce nearly the same effect, it follows that the rate-determining step in the oxidation of a phenol or an aromatic amine by compound I is the removal of one electron.