Nitroaromatic compounds: Environmental toxicity, carcinogenicity, mutagenicity, therapy and mechanism.

Review Article Received: 12 September 2013,

Revised: 25 November 2013,

Accepted: 26 November 2013

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.2980

Nitroaromatic compounds: Environmental toxicity, carcinogenicity, mutagenicity, therapy and mechanism Peter Kovacica* and Ratnasamy Somanathana,b ABSTRACT: Vehicle pollution is an increasing problem in the industrial world. Aromatic nitro compounds comprise a significant portion of the threat. In this review, the class includes nitro derivatives of benzene, biphenyls, naphthalenes, benzanthrone and polycyclic aromatic hydrocarbons, plus nitroheteroaromatic compounds. The numerous toxic manifestations are discussed. An appreciable number of drugs incorporate the nitroaromatic structure. The mechanistic aspects of both toxicity and therapy are addressed in the context of a unifying mechanism involving electron transfer, reactive oxygen species, oxidative stress and antioxidants. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: nitro aromatic; pollution; electron transfer; radical mechanism; toxicity

Introduction Nitroaromatic compounds have attracted attention over the decades in relation to pollutants, toxicity, mutagenesis, carcinogenesis, therapeutic action and as intermediates in the synthesis of complex organic molecules. The environmental contamination has been a major focus. The nitroaromatic portion has been the center of extensive literature reports relative to toxicity. The mode of action has attracted attention entailing a variety of perspectives. This review presents a broad overview of diverse classes of nitroaromatic compounds and their mechanism involving single electron transfer leading to radical anion, nitroso, hydroxylamine and amine formation and their physiological actions. The focus on action mode entails electron transfer (ET), reactive oxygen species (ROS) and oxidative stress (OS). The preponderance of bioactive substances or their metabolites incorporate ET functionality, which, we believe, play an important role in physiological responses. These main groups include quinones (or phenolic precursors), metal complexes (or complexors), aromatic nitro compounds (or related hydroxylamine and nitroso derivatives) and conjugated imines (or iminium species) (Kovacic and Somanathan, 2011). In vivo cycling with oxygen can occur, giving rise to OS through generation of ROS, such as hydrogen peroxide, hydroperoxides, alkyl peroxides and diverse radicals (hydroxyl, alkoxyl, hydroperoxyl and superoxide). In some cases, ET results in interference with normal electrical effects, for example, in respiration or neurochemistry. Generally, active entities possessing ET groups display reduction potentials in the physiologically responsive range, that is, more positive than – 0.5V. ET, ROS and OS that have been increasingly implicated in the mode of action of drugs and toxins (toxicants), for example, anti-infective agents (Kovacic and Becvar, 2000), anticancer drugs (Kovacic, 2007; Kovacic and Osuna, 2000), carcinogens (Kovacic and Jacintho, 2001a), reproductive toxins (Kovacic and Jacintho, 2001b), nephrotoxins (Kovacic et al., 2002), hepatotoxins (Poli et al., 1989), cardiovascular toxins (Kovacic and Thurn, 2005), nerve toxins (Kovacic and Somanathan, 2005), mitochondrial toxins

J. Appl. Toxicol. 2014

(Kovacic et al., 2005), abused drugs (Kovacic and Cooksy, 2005), immunotoxins (Kovacic and Somanathan, 2003), pulmonary toxins (Kovacic and Somanathan, 2009a, 2009b), dermal toxins (Kovacic and Somanathan, 2010a, 2010b), ototoxins (Kovacic and Somanathan, 2009a, 2009b), eye toxins (Kovacic and Somanathan, 2008), thyroid toxins (Kovacic and Edwards, 2010) and various other categories (Halliwell and Gutteridge, 1999). There is a plethora of experimental evidence supporting the OS theoretical framework, including generation of the common ROS, lipid peroxidation, degradation products of oxidation, depletion of antioxidants (AOs), effect of exogenous AOs, DNA oxidation and cleavage products, as well as electrochemical data. This comprehensive, unifying mechanism is in keeping with the frequent observations that many ET substances display a variety of activities, for example, multiple drug properties, as well as toxic effects. Although our mechanistic emphasis is on ET-ROS-OS, it should be stressed that bioactivation is often multifaceted. Emphasis in this review is on the more recent literature, as previous reviews have addressed earlier reports. In contrast to the present broad, comprehensive approach, most of the previous reviews presented a narrower focus. Nitrobenzenes Nitroaromatic compounds are widely used as pesticides, explosives, pharmaceuticals and chemical intermediates in industrial

*Correspondence to: Peter Kovacic, Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182-1030, USA. Email: [email protected] a Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA, USA b Centro de Graduados e Investigación del Instituto Tecnológico de Tijuana, Apdo postal 1166, Tijuana, B.C. Mexico

Copyright © 2014 John Wiley & Sons, Ltd.

P. Kovacic and R. Somanathan synthesis. They are potent toxic or carcinogenic compounds, presenting a considerable danger to the human population, and are widely distributed environmental pollutants found in work places (e.g. chemical industry), and in emissions from diesel and gasoline engines. The toxicity and carcinogenicity of these compounds and their metabolic pathways in organisms have been examined (Garner et al., 1984; International Agency for Research in Cancer (IARC), 1989). However, the knowledge of the fate of several nitro aromatic compounds and their physiological effects in humans is still scarce. Reductive metabolism has been quite well investigated, as set forth in subsequent sections. The quantitative structure–activity relationship and mechanism were the subject of a report on nitrobenzene toxicity (Katritzky et al., 2003). An important contributing factor is nitro group reduction, which can occur by at least two mechanisms, namely, single step reduction by nitroreductase and redox cycling with back oxidation. Hydrophobicity appears to control transport to the action site where electron affinity is an intrinsic reactivity property. Like nitrobenzene, the dinitrobenzenes are also important starting materials in the chemical industry; many of the reduced diamines are used as azo dyes in the textile industry. Numerous nitroaromatics, such as trinitrotoluene, 2,4- and 2,6dinitrotoluene, nitrobenzene and 1,3-dinitrobenzene, have been shown to cause testicular toxicity in laboratory animals (Mikšanová et al., 2004). A study revealed that 1,3-dinitrobenzene induced testicular toxicity and was shown to produce histological alterations in the testis, decreased sperm numbers, and altered sperm motility and morphology (Reeve and Miller, 2002). The mechanism is believed to proceed via one electron reduction of the nitroaromatic compound to nitroxyl anion radical. Under aerobic conditions, this metabolite delivers an electron to molecular oxygen creating the superoxide anion radical. This process oxidizes the nitroxyl anion radical back to the parent compound. In the absence of oxygen, the nitroxyl anion radical is further reduced by an electron to form nitrosonitrobenzene, which accepts another two electrons to form nirophenylhydroxylamine (NPHA). In the final reduction step, the NPHA is converted to nitroaniline, by two electrons (Scheme 1). Subsequent work by the same authors revealed that, nitrosonitrobenzene and NPHA nonenzymatically reacted with nonprotein sulfhydryls and mitochondrial glutathione (GSH), leading to GSH depletion and adduct formation with proteins (Reeve and Miller, 2002; Reeve et al., 2002). 2-Methoxynitrobenzene (2-NA) is used as a precursor in the synthesis of 2-methoxyaniline, an intermediate in the manufacture of many azo dyes. 2-NA exhibits carcinogenic activity, causing

neoplastic transformation in the urinary bladder, spleen and kidneys in rodents (Mikšanová et al., 2004). A report indicates that the toxicity of 2-NA in rodents is due to the xanthine oxidase products N-(2-methoxyphenyl)hydroxylamine and 2-anisidine, which form adducts with DNA (Mikšanová et al., 2004). However, in the human liver the 2-NA is detoxified by cytochrome P450 to 2-nitrophenol and 2,6-hydroxy nitrobenzene (Scheme 2). Nitrotyrosine promotes the formation of substantial amounts of ROS when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor, glucose-6-phosphate/ glucose-6-phosphate dehydrogenase (Krainev et al., 1998). Spin adduct formation is inhibited by superoxide dismutase, which requires aerobic conditions. Leucine enkephalin, a tyrosinecontaining pentapeptide, results in a similar generation of O2 and OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferrichrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis–Menten kinetics. No stimulation of acetylated ferrichrome c reduction is observed in the presence of superoxide dismutase. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferrichrome c reduction by nitrotyrosine. Nitration of two of four tyrosines within the sequence of the 6.5 kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2 generation implying steric restrictions for BPTI reductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferrichrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical, which is then recycled by oxygen to yield ROS. Nitrotyrosine may act to promote OS by means of catalytic redox cycling, and increased ROS production, as well as contributing to cardiovascular pathogenesis (Mu et al., 2008). Long-term (2 years) exposure of mice to o-nitrotoluene showed chemical-specific alterations in ras, p53 and β-catenin genes in hemangiosarcomas leading to mutagenesis (Hong et al., 2003). The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. A study examined the possibility of another cytotoxicity mechanism of the aminoand hydroxylamino metabolites of TNT, namely their flavoenzyme-catalyzed redox cycling (Šarlauskas et al., 2004). The above compounds acted as redox-cycling substrates for single ET. The cytotoxicity of the amino-hydroxylamino metabolites of TNT was partly prevented by the AO N,N′-diphenyl-p-

Scheme 1. Nitroreductive metabolism.

Scheme 2. Pathways of 2-methoxynitrobenzene metabolism.

wileyonlinelibrary.com/journal/jat



Nitro aromatic pollutants phenylene diamine and desferrioxamine, thus pointing to the involvement of OS. The data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity. TNT has been used as an explosive for many years. Several epidemiological studies and animal experiments showed that TNT induced reproductive toxicity, through oxidative DNA damage in the testis of Fischer 344 rats (Homma-Takeda et al., 2002). Chinese researchers showed male workers exposed to TNT, found to have a significantly decreased volume of semen and testosterone in the serum (Li et al., 1993). Metabolic pathways for nitro aromatic compounds have been well delineated for members of anti-infective drugs, and carcinogens. Reductases participate in the bioprocesses that commonly follow the sequence: parent nitro (ArNO2) to nitroso (ArNO) to hydroxylamine (ArNHOH) to pri-amine (ArNH2). Several ET pathways for generation of ROS are possible. In the case of TNT, redox cycling was catalyzed by reductase involving high yields of the nitro radical anion. For various nitroaromatics, reaction was enhanced by an increase in reduction potential. Thus, these substances may be able to participate in ET reactions in the biosphere. The hydroxylamine metabolite was identified in addition to the pri-amine. It is well established that ArNHOH can redox cycle with ArNO giving rise to OS. Other conceivable ET entities are the azo and azoxy metabolites, most likely formed from condensation reactions entailing ArNO, ArNHOH and ArNH2. Aromatic azo and azoxy compounds generally display reduction potentials in the range amenable to in vivo ET. By use of a well-known precedent, the observed binding might be attributed to electrophilic attack on bionucleophiles by nitrenium ions formed from ArNHOH. Various fractions of urinary metabolites from rats exhibited mutagenicity, which is commonly associated with ROS. Nitroaromatic explosives such as TNT and 2,4,6-trinitrophenylN-methyl-nitramine (tetryl) comprise an important group of toxic environmental pollutants whose toxicity is mainly attributed to the redox cycling of their free radicals (oxidative stress) and formation of nitroso and/or hydroxylamine metabolites (Miliukienė and Čėnas, 2008). In general, the cytotoxicity of nitroaromatics increases with an increase in their single electron reduction potential. This points to the prevailing mechanism of OS-type cytotoxicity. The structure–activity relationship showed that the immunotoxicity potential of nitroaromatic explosives might decrease in the order: tetryl ≥ TNT ≥ hydroxylamino metabolites of TNT > amino and diamino metabolites of TNT. Biological degradation of TNT has been extensively studied using microorganisms such as Pseudomonas and fungi species. The mechanism involves single ET to form radical anion followed by transformations into nitroso, hydroxylamine and finally into amine (Esteve-Nùńez et al., 2001). Interestingly, the fate of the nitro compounds in animal cells also follows the same pathway leading to the formation of ROS and OS. Biological and electrochemical ET reactions have many things in common. Electrochemistry can mimic the main oxidative reactions that occur in the human body, providing good approximation of what occurs in vivo. Hence, explanations based on electrochemistry have played an important role in interpreting and understanding the biological phenomena. The electrochemical behavior of an antitumoral o-quinone and nitrobenzene derivative obtained from 3-bromo-nor-β-lapachone was studied. Cyclic voltammetric experiments revealed that both quinone and nitro functions are reduced independently as one-ET


processes (Hernández et al., 2008). Depending on the reduction potential, a radical anion or a biradical dianion is obtained. The cyclic voltammetric characteristics of two nitroamphetamine derivatives 2-nitro-4,5-dimethoxyamphetamine and 2-nitro-4,5methylenedioxyamphetamine analogs of amphetamine drugs have been investigated (Fig. 1) (Nuńez-Vergara et al., 1994). These analogs were synthesized to evaluate the structure–activity relationship and mechanism of action for hallucinogens. The electrochemical study revealed a reversible one-electron reduction occurs to give radical anion; the reaction is similar to what happens within a cell. At a more negative potential, a further three-electron reduction occurs to give the hydroxylamine derivative. Cyclic voltammetry showed the tendency of the nitro radical anions to undergo further chemical reactions. Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are derivatives of polycyclic aromatic hydrocarbons (PAHs), which contain two or more fused aromatic rings made of carbon and hydrogen atoms. Nitro-PAHs occur in the environment as a mixture together with parent PAHs. Nitro-PAHs are usually present in much smaller quantities than PAHs. Nitro-PAHs in the environment either occur in the vapor phase or are absorbed to particulate matter. Nitro-PAHs originate primarily as direct or indirect products of incomplete combustion. Only a few Nitro-PAHs are produced industrially as chemical intermediates, such as nitronaphthalene and 5-nitroacenaphthene. Nitro-PAHs originate from PAHs by at least two distinct processes: (1) through nitration during combustion processes, e.g. in vehicle exhaust emissions, particularly diesel, but also gasoline and aircraft emissions, domestic heating/cooking, and wood burning, and (2) through atmospheric formation from PAHs by either gas phase reactions involving hydroxyl radical and nitrogen dioxide.

4-Nitrobiphenyls This section is a continuation of the non-PAH types. This class is related to the benzene category of the previous section. Xanthine oxidase catalyzed mutagenicity of 4-nitrobiphenyl (NBP), a dog-bladder carcinogen, was investigated (Swaminathan and Hatcher, 1986). In vitro enzymatic reduction of NBP revealed the major product to be 4-aminobiphenyl (ABP). The reduction intermediate N-hydroxylaminobiphenyl showed equal mutagenic activity in contrast to NBP. NBP, an environmental pollutant, in vivo undergoes reduction to potent ABP. ABP, a bladder carcinogen found in cigarette smoke, results in DNA adduct formation (Culp et al., 1997). The data suggest that adducts in human lung result from environmental exposure to NBP. The bioactivation pathways appear to involve reduction to the hydroxylamine metabolite with subsequent O-acetylation, and metabolic reduction to ABP. In another study involving nitrobiphenyls, 2,4,2′,4′-tetranitrobiphenyl, its metabolites and its mutagenic products were identified (Hirayama et al., 1991). The most mutagenic metabolite was 2,4′-diamino-2′,4-dinitrobiphenyl.

Figure 1. Nitroamphetamine derivatives.



P. Kovacic and R. Somanathan Various types of chlorinated NBP ethers (4-nitrobiphenyl ether, p-NO2; 2′-, 3′- and 4′-chloro-NBP ethers, 2,3- and 4-ClNO2; 2′,4′-dichloro-4-nitrobiphenyl ether, 2,4-diClNO2; 2′,4′,6’-trichloro4-nitrobiphenyl ether, 2,4,6-triClNO2) and their nitroso- and amino derivatives have been tested for mutagenicity (Miyauchi et al., 1983). All of the chlorinated NBP ethers except for 2,4,5-triClNO2 induced mutations without metabolic activation, although their mutagenic activities were weak compared with the nitroso and amino derivatives. The monochloro compounds were more effective in inducing mutations than those with multiple chlorines, and their mutagenic activities were closely related to the rate of reduction of the nitro-moiety. After metabolic activation, the mutagenicity of these compounds was enhanced. The amino derivatives all induced mutations, but only after metabolic activation. The amino compounds having fewer chlorine atoms were more effective in inducing mutations than those having multiple chlorines. The mutagenic activity of the nitro compounds was lower than that of the analogous nitroso and amino compounds, which showed almost the same mutagenicity. A report deals with toxicity of chlorinated p-nitrobiphenyl ethers induced by its metabolites nitroso and amino derivatives, which interact with hemoglobin leading to methemoglobin formation (Fe2+ to Fe3+) (Miyauchi et al., 1981). Metabolites of NBP ether (4-NO2) tested for mutagenicity activity toward Salmonella typhimurium in the guinea pig. The 4-NO and 4NHOH metabolites showed high mutagenic activity, while that of 4-NO2 was very weak (Miyauchi et al., 1984). 4-NO showed antimicrobial action at high concentrations. Metabolic activation with guinea pig liver homogenates enhanced the mutagenic activities of 4-NO2 and 4-NO, and converted 4-NH2, 4-N(OH)Ac and 4-N(OAc)Ac to the products responsible for the mutagenic activity.

Nitrofluorenes This group is related to the biphenyls. In rats administered with 2-nitrofluorene or 9-hydroxy-2-nitrofuorene showed hepatic DNA adduct 8-[N-(2-aminofluorenyl)-2′-deoxyguanosine, derived from interaction with the reduced nitro compound (Ritter and Malejka-Giganti, 1998). Reduction of the nitro group can proceed via one- or two-electron mechanism as shown in Scheme 1. Results suggest that 8-hydroxy-2′-deoxyguanosine in DNA is involved in mutagenesis (Suzuki et al., 1997). Some nitroaromatics, such as 4-nitroquinoline N-oxide or 2-nitrofluorene can produce 8-hydroxy-2′-deoxyguanosine in DNA, thereby inducing mutations. Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels (Fig. 2) (Ritter et al., 2000, 2002). Nitroaromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. One study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene,

Figure 2. Nitrofluorenes.


a potent mammary carcinogen in rat (Ritter et al., 2002). Potential adducts of 2,7-dinitrofluorene were generated by reduction of 2-nitroso-7-nitrofluorene with ascorbate in the presence of DNA (Ritter et al., 2002). The major adduct was characterized as N-(deoxyguanosine-8-yl)-2-amino-7-nitrofluorene, and another one was a deoxadenosine adduct of 2-amino-7-nitrofluorene. Nitronaphthalenes This type represents the first member of the fused ring category. 1-Nitronaphthalene (1-NN) is a mutagenic compound that has been detected in emissions from diesel engines, as well as in urban airborne particles. Rats exposed to 1-NN and its metabolites, analyzed by proton nuclear magnetic resonance spectroscopy and mass spectrometry, showed glutathionyl hydroxyl naphthalene products in the tracheobronchial airways and liver (Scheme 3) (Watt et al., 1999). Data showed that conjugates detected in the lung were C7,C8-epoxide derived and metabolites from the liver were C5,C6-epoxide derived (Scheme 3). A similar study showed the increased vulnerability of neonatal rats and mice to 1-NN-induced pulmonary injury (Fanucchi et al., 2004). 1-NN causes lesions in both Clara and ciliated cells (Lin et al., 2009). The rat is more susceptible to administration of the compound than mice. The toxicity of pulmonary toxicants has been attributed to the presence of activating enzymes with high k(cat) in susceptible species (Schultz et al., 2001). The mouse is sensitive to various metabolically activated lung toxicants. Recombinant CYP2F2 was shown to metabolize the pulmonary toxicant naphthalene. The pulmonary toxicants 1-nironaphthalene and 2-methylnaphthalene are metabolized readily with high k(cat) values to potentially cytotoxic intermediates. The involvement of metabolite formation and adduction to proteins in toxicity is important. Studies with naphthalene and 1-nironaphthalene have demonstrated the importance of cytochrome P450 monooxygenase in generating reactive metabolites that produce Clara cell injury (Boland et al., 2004). Covalently bound metabolites accounted for the greatest percentage of 1-NN metabolites, particularly in the lower airways. 1-NNT and its isomer 2-nitronaphthalene (2-NNT) are both genotoxic in vitro, although they do not require metabolic activation (Kirkland and Beevers, 2006). There is evidence that 2-NNT may induce liver and bladder tumors (Neumann, 2005). 2-NNT induced a mutagenic response in liver, and a marginal

Scheme 3. Metabolites from 1-nitronaphthalene.



Nitro aromatic pollutants one in bladder, whereas 1-NNT was negative. 2-NNT is a directacting mutagen in vitro, but 1-NNT was negative (Kirkland and Beevers, 2006). A report evaluated the mutagenicity, metabolism and DNA adduct formation of 5-nitrobenzo[b]naphtha[2,1-d]thiophene (King et al., 2001). Data indicated that both ring oxidation and nitroreduction are involved in the metabolism, leading to DNA adduct formation and mutagenicity (Scheme 4).

Evidence suggests that 3-NBA is readily reduced and activated in vivo (Chen et al., 2008). High 3-NBA nitroreductase levels in the liver and colon are consistent with the elevated mutant frequency values, and previously noted inductions of hepatic DNA adducts.

Nitrated Polycyclic Aromatic Hydrocarbons Nitroanthracenes

3-Nitrobenzanthrone Naphthalene is a parent of this class. Another group of nitrated polycyclic compounds is the nitrobenzanthrones, with four fused rings. 3-Nitrobenzanthrone (3-NBA) occurs in the diesel exhaust (DE) and in airborne particulate matter. The compound exhibits extremely high mutagenic activity and is a genotoxic carcinogen causing lung tumors in rats. Possible mechanistic pathway is delineated in (Scheme 5) (Arlt et al., 2004; Stiborová et al., 2010). 3-NBA is a suspected human carcinogen. A study demonstrated that the N-hydroxy metabolite induces oxidative damage through H2O2 in human cells (Murata et al., 2006). Oxidative DNA damage may play an important role in the carcinogenesis of 3-NBA in addition to DNA adduct formation. Human health hazards have been associated with DE. DE increases the incidence of tumors and the induction of 8hydroxyguanosine adducts in mice (Hallberg et al., 2012). DE is composed of a complex mixture of PAHs and particulates. One such PAH, 3-NBA, is present in DE and in urban air. 3-NBA has been observed to induce micronucleus formation in the DNA of human hepatoma cells. The environmental contaminant, 3-NBA, is mutagenic and a possible human carcinogen (Hanse et al., 2007). The main metabolite of 3-NBA is the amine derivative (3-ABA). The possibility of 3-NBA and 3-ABA to increase the production of ROS was demonstrated. Results provide evidence for a genotoxicity of 3-ABA in lung cells. Both compounds increase ROS and create DNA damage, leading to cancer.

Scheme 4. 5-nitrobenzo[b]naphtho[2,1-d]thiophene metabolite.

This category includes lower members, namely three rings, of fused benzenoids. A study was made to determine the concentrations of PAHs and nitrated PAHs in outdoor air samples collected in urban areas, affected mainly by traffic emissions, and to estimate in vitro mutagenic action and toxicity (Ciganek et al., 2004). The most abundant nitrated PAH derivatives were nitronaphthalenes, which were present exclusively in the vapor phase; 9-nitroanthracene (9-NA), 9-nitrophenanthrene and 3nitrofluoroanthene were associated with particulate matter. Industrial anthraquinones often contained PAH contaminants, particularly 9-NA (Fig. 3) (Butterworth et al., 2004). Many toxicology studies on anthraquinone used the impure compound. The contaminated material was mutagenic and contained 9-NA and other contaminants. 9-NA exhibited potent mutagenic activity in mammalian cell assays. Nitrophenanthrenes The fused three-ring benzenoid phenanthrene is an isomer of anthracene. The natural product aristolochic acid (AA) is an important member. Air pollutants and their characterization was the subject of a report from the outskirts of Rome (DiFilippo et al., 2007). Both PAH and nitro-PAHs were used to assess the nature and relative importance of sources. A series of positional isomers of nitro-PAHs and other organic compounds associated with particulate matter were investigated. 1- and 3Nitrophenanthrenes were the most abundant nitro-PAHs. A report deals with nitro-PAH compounds in fish contaminated with PAH and exposed to nitrite (Fig. 4) (Shailaja et al., 2006). Two strongly genotoxic nitro-PAH metabolites were formed, namely phenanthrene-6-nitro-1,2-dihydrodiol-3,4-epoxide and dihydroxy acetylamino nitrophenanthrene. However, the route of PAH administration seems to determine the nature of metabolites. Almost all of the hepatic cells of the fish exposed to phenanthrene in the presence of NO2 were found to have suffered extensive DNA fragmentation. AAs (Fig. 5) are nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used for medicinal purposes

Figure 3. Nitroanthracene.

Scheme 5. 2-Nitrobenzanthrone metabolic adduct formation with DNA.



Figure 4. 3-Nitrophenanthrene.


P. Kovacic and R. Somanathan NPs are strongly mutagenic in bacterial cell assays. A study showed that 1,8- and 1,6-dinitropyrenes are much more potent mutagens than other NPs (Fig. 7) (Murata et al., 2004). Both NPs induced cancer and leukemia in rodents. Another mechanism is presumed to involve the formation of nitro radical anion and superoxide formation, finally, leading to DNA adduct formation. In a related report, structurally similar mutagen 3,6-dinitrobenzo[e]pyrene was isolated from surface soil in Osaka, Japan (Watanabe et al., 2005).

Figure 5. Aristocholic acid.

(Yun et al., 2012). AAs have been implicated in the etiology of nephropathy, which is associated with carcinomas of the urinary tract. Following metabolic activation, AA reacts with DNA to form aristolactam–DNA adducts. The mechanism involves formation of a cyclic nitrenium ion with delocalized charge leading to the formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine (Arlt et al., 2002; Levova et al., 2012; Schmeiser et al., 2009). The predominant DNA adduct is mutagenic. Findings suggest that DNA damage by AA is not only responsible for tumor development, but also for kidney toxicity. Nitrated PAHs contribute to environmental pollution since they are found in emission from diesel, airplane and other fossil fuel combustions, as well as in cigarette smoke. Their genotoxicity upon metabolic activation suggests a health risk to humans. Among more than 200 environmental nitro PAHs and 1-nitropyrene (1-NP), 2-nitrofluorene, chrysene and benzanthrone predominate (Ritter and Malejka-Giganti, 1998). The principal PAHs contain four fused benzenoid rings.

1-Nitropyrene 1-NP (Fig. 6) is one of the main components of DE particles (DEP), generated from auto engines. Although mutagenesis and carcinogenesis have been investigated considerably, noncarcinogenic toxicities of 1-NP have not been much studied (Park and Park, 2009). 1-NP induces the expression of genes associated with pro-inflammation. The compound generated ROS and decreased GSH. 1-NP may be a pivotal component of DEP in causing inflammatory diseases. 1-NP undergoes reduction and oxidation by mammalian enzymes. Research was performed to determine whether 1-NP produced superoxide (Nachtman, 1986). The data demonstrate that 1-NP catalyzes the formation of superoxide, and that the reaction obeys Michalis–Menten kinetics; 1-NP may possess toxic effects related to OS. A study compared the AO effect of ardisin and epigallocatechin 3-O-gallate (EGCG) in hepatocytes exposed to either nenomyl or 1-NP (Ramírez-Mares and de Mejía, 2003). Results suggest that ardisin is a better suppressor of lipid peroxidation induced by benomyl and 1-NP than EGCG. Ardisin and EGCG are shown to be AOs that can protect against diseases arising from ROS. A study detected DNA strand breakage by a range of agents, some of which require metabolic activation, such as 1-NP and nitrofurantoin (Mitchelmore et al., 1998). This may serve as a sensitive biomarker of genotoxicity.

6-Nitrochrysene 6-Nitrochrysene (6-NC) is a carcinogen and mutagen (Guttenplan et al., 2007). It is metabolized by ring oxidation and nitroreduction. The active mutagenic metabolite appears to be trans-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD6-NHOH-C), formed from ring oxidation and nitroreduction. Metabolite action arising from either process alone exhibited similarities to mutation by 6-NC. The major DNA adducts in rat tissue treated with 6-NC are products formed from 1,2-DHD-6NHOH-C with guanine and adenine; 1,2-DHD-6-NHOH-C appears to be the ultimate genotoxic metabolite. Although 6-NC is less abundant than other related compounds in the environment, it is the most potent mammary carcinogen in the rat; its potency is not only higher than that of benzo[a]pyrene, but also of heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Krzeminski et al., 2011). 6-NC is activated by simple nitroreduction leading to the formation of 6-hydroxylaminochrysene; this metabolite yielded DNA adducts in reduced amine form. 6-NC is an environmental pollutant, as well as a potent mammary carcinogen and mutagen in rats (Krzeminski et al., 2011; Sun et al., 2009, 2012). A study revealed that its metabolites, 6-hydroxylamine and 1,2-dihydroxy-1,2-dihydro-6-nitrochrysenes, showed mutagenicity (Scheme 6) (Sun et al., 2012). 5,7-Dihydroxy-8-nitrochrysin induces apoptosis in human cancer cells (Zhao et al., 2010) and possesses stronger cytotoxicity than chrysin. Mechanistic evidence shows that it induces apoptosis by the generation of ROS and dephosphorylation.

Nitro Heteroaromatic Compounds Nitro heteroaromatics are widely used as therapeutic agents against a variety of protozoan and bacterial infections, and these molecules are all man-made. The defense action of these molecules takes place through the reduction of the nitro group to radical anion, nitroso, hydroxylamine and finally into amine, and these metabolites are all toxic to humans.

Figure 6. Nitropyrene.


Figure 7. Mutagenic pyrenes.



Nitro aromatic pollutants

Scheme 7. Reductive pathway of benznidazole. Scheme 6. 6-Nitrochrysene metabolic pathway and DNA adduct formation.

4-Nitroquinoline-N-oxide Oxidative stress exerted by superoxide-generating (redox cycling) agents, such as paraquat, triggers the soxS regulation of Escherichia coli. 4-Nitroquinoline-N-oxide (4-NQO) is a powerful inducer of soxS (Nuoshiba and Demple, 1993). The transcriptional induction of the soxS gene was dependent on the presence of molecular oxygen. Two 4-NQO-related compounds were also shown to induce soxS, mainly 4-nitropyridine-N-oxide, only slightly less efficient than 4-NQO, and 4-hydroxylaminoquinolineN-oxide, at a lower potency than 4-NQO. Thus, considerable OS is induced in cells by 4-NQO, which might contribute to the carcinogenic potency. A goal was to determine the effects of 4-NQO-induced carcinogenesis on tongue levels of protein-bound and free GSH and related thiols in the rat (Huang et al., 2007). Results suggest that protein glutathionylation, together with GSH and oxidized GSH levels, are induced during oral carcinogenesis in the rat, possibly as a result of enhanced levels of OS. A new bioassay has been developed that allows a rapid, sensitive detection of chemicals, such as 4-NQO, which manifest their acute toxicity, mutagenicity or carcinogenicity by inducing a pro-oxidant state in vivo (Knobeloch et al., 1990). Dietary silymarin suppresses rat carcinogenesis by 4-NQO (Yanaida et al., 2002).

Other nitroheterocycles Cyclic voltammetry and electron spin resonance were used on potentially antiprotozoal nitro derivatives of dihydroquinoxaline and imidazole (Aravena et al., 2010). A self-protonation process involving the nitro group and an acidic proton in the structure was observed in the first step of the reduction mechanism. Electron spin resonance spectra of the free radicals were obtained. Benznidazole (BEZ), a 2-nitroimidazole, was used against trypanosomiasis (Hall and Wilkinson, 2012). It was proposed that BEZ activation leads to reductive metabolites that can cause deleterious effects, including DNA damage and thiol depletion (Scheme 7). The key step in drug activation involves a nitroreductase, which catalyzes an interaction of enzyme, reductant and prodrug occurring through a ping-pong mechanism. The major product involves a reductive dialdehyde glyoxal. The reduction of BEZ by nitroreductase leads to the formation of highly reactive metabolites. Nitroheterocyclic compounds are used against protozoan and bacterial infections (Buschimi et al., 2009). These drugs are


suspected of being carcinogens. In a study, the cytotoxic and genotoxic potential of nifurtimox (NFX), BEZ and metronidazole (MTZ) were re-evaluated (Fig. 8). In Salmonella, NFX and BEZ are more mutagenic than MTZ. BEZ and NFX-induced DNA damage. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage, but also may interact with DNA. NFX, BEZ and megazol are nitroheterocycles, like other nitro compounds, that exhibit activity against Trypanosoma cruzi, the causative agent of Chagas disease (Oliveira et al., 2003), which causes approximately 400 000 deaths in Central and South America per year. Their activity is due at least in part to the well-documented sensitivity of trypanosomes and particularly T. cruzi towards OS. Upon one electron reduction, these compounds generate radical anions, which interfere with oxygen metabolism. For BEZ, the generation of reduced reactive species that react with parasite macromolecules is also proposed. Differently from mammalian host cells, T. cruzi is deficient in AO enzymes, which are essential to prevent oxidative damage. Trypanothione reductase is the key enzyme involved in the protection of T. cruzi against the OS. The radical anions generated by the nitro compounds upon reduction lead to trypanothione reductase depletion and, consequently, toxicity to T. cruzi. The nitroheterocyclic NFX targets Trypanosoma brucei, the causative agent of trypanosomiasis (Bot et al., 2013). The mode of action involves a reaction catalyzed by a nitroreductase. The trypanocidal activity of a library of other 5-nitrofurans was evaluated. Other members of the 5-nitrofurans class have the potential to treat trypanosomiasis. Nitroreduction is an essential step for the biological activity of mono-, di- and tri-nitrothiophenes, and by comparison, some mononitro benzo[b]thiophenes and benz[f]furans (Fig. 9) (Boga et al., 2012). Their electroreduction behavior was investigated by various techniques: calculations, cyclic voltammetry and

Figure 8. Nitroheterocyclic compounds.



P. Kovacic and R. Somanathan A study deals with aromatic nitro derivatives in relation to the mechanism between chemical substances and the biological receptor (Amzoiu et al., 2011). The results suggest that the interaction mechanism is based on ET from biological receptors to the lowest unoccupied molecular orbital energy levels of the nitro groups.

Other nitroheteroaromatics

Figure 9. Heterocyclic nitroaromatic compounds of the O and S classes.

electrochemical electron spin resonance spectroscopy. Although, the first reduction process leads to radical anions, both the computations and experimental results indicate there are significant differences in the fate of their corresponding reductions, for example, formation of secondary radicals or dianions. A series of nitroheterocyclic compounds was designed with linkages to melanine (Stewart et al., 2004). One compound, a melanine-linked nitrofuran (Fig. 10), showed pronounced activity against parasites. Studies into the mode of action indicated that neither reductive, nor oxidative stress was related to its trypanocidal activity, distinguishing it from some other trypanocidal nitroheterocycles. Under aerobic conditions, inhibitors of cytochrome P450 prevented nitrofurantoin-induced cytotoxicity and ROS formation (Pourahmad et al., 2001). This suggests that nitrofurantoin was reductively activated by reduced cytochrome P450. Lipid peroxidation preceded cytotoxicity. Desferrioxamine (a ferric chelator), AOs or ROS scavengers (catalase, mannitol, tempol [4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl] or dimethyl sulfoxide) prevented neuropeptide Y cytotoxicity. Apparently, H2O2 reacts with Fe2+ to form ROS, which causes lysosomal lipid peroxidation, membrane disruption, protease release and cell death. Derivatives of benzolo[3,2-a]quinolinium salts (QSDs) (Fig. 11), incorporating a nitro group, are reductively activated by enzymatic agents (Alegria et al., 2004). Oxygen consumption rates correlate with QSD redox potentials when NADH dehydrogenase is used as a reducing agent. QSDs bind covalently to bovine serum albumin. The amount of reacted GSH increases and the relative amount of oxidized GSH formed decreases, with an increase in the QSD reduction potential, thus indicating that GSH reacts with reduced nitro-containing QSDs, presumably through the formation of the nitroso-QSD-GSH conjugate. QSDs are bioreductively activated to react with oxygen and thiols.

Figure 10. Melanine-based nitroheterocycle.

Figure 11. Quinolinium salt.


This class is found extensively in the medical literature. Various reports contain the unifying mechanistic theme of ET-ROS-OS, which is the focus of this section. The drug is accompanied by the therapeutic action: MTZ and entramin (amebicide) (Ames et al., 1987a, 1987b), acrintrazole, niridazole and MTZ (anthelminths) (Kovacic et al., 1989), nitrofurazone and nitrofurantoin (antibacterial) (Ames et al., 1986), nitrofuran and nitrothiazole classes (antiprotozoal) (Ames et al., 1987a, 1987b) and 2-nitroimidazoles (radiation sensitizers) (Kovacic and Osuna, 2000). Literature devoted to toxicity of this class provides evidence of a major drawback to clinical use. Several examples are provided: nitrofurantoin (reproductive) (Kovacic and Osuna, 2000), nitrofurans (mitochondria (Kovacic et al., 2005) and 4-NQO (carcinogen).

Additional Studies On Effects, Including Mixtures This section is concerned with studies involving various types of aromatic nitro compounds, both arene and heteroaromatic categories, in relation to diverse properties and in many cases involving multiple nitroaromatic compounds. Various endogenous and exogenous compounds exert cytotoxic effects via oxygen reduction (Kappus and Sies, 1981). In general, these are reduced by intracellular enzymes (reductase) in one-ET reactions, before they in turn reduce O2 to the superoxide anion radical. Thus, a cycle is formed. Structures capable of “redox cycling” include aromatic nitro compounds. Toxic effects involve membrane damage resulting from peroxidative reactions of polyunsaturated fatty acids (lipid peroxidation), as well as the attack of ROS on proteins and nucleic acids. Thus, O2 metabolism is linked to carcinogenicity and mutagenicity. Powerful AO systems maintain low steady concentrations of harmful oxygen metabolites, and toxic effects, in part, may be explained by the constant drain of reducing equivalents resulting from redox cycling. Redox chemistry of different nitro compounds of biological significance is focused on understanding how the reduction of the nitro group can play a role in various aspects involving biobehavior (Squella et al., 2005). Formation of the nitro radical anion from one electron reduction generates a series of important consequences from chemical to biological aspects. Electrochemical techniques were used to study the formation, stability and reactivity of the nitro radical anion. From cyclic voltammetric experiments, it is possible qualitatively to visualize the formation of the nitro radical anion through the oneelectron reversible couple due to the redox system nitro/nitro radical anion. A favorable reaction may occur between the nitro radical anion and acidic hydrogen present in the molecule. DNA damage by eight compounds found in DE, benzo[a] pyrene, 3-NBA, 1-NP, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8dinitropyrene, 6-NC and 3-nitrofluorene, was assessed (Kucab



Nitro aromatic pollutants et al., 2012). DNA adduct formation is the best measure of genotoxicity of the nitro-PAHs tested (Kucab et al., 2012). Day and night samples were taken at a roadside site and tunnel in China. Six nitro-PAHs, namely 9-NA, 2- and 3nitrofluoranthene, 1-NP, 7-nitro[a]anthracene and 6-nitrobenzo [a]pyrene, were examined as pollutants (Wu et al., 2012). The concentration of the six nitro-PAHs in the cold season was higher than that in the warm season. Significantly, the difference between daytime and night-time concentration was found during both seasons. The diurnal variations of nitro-PAHs were all influenced by variations of nitrogen dioxide, sunlight and temperature. The exposure to the six NAPHs in the tunnel was higher than roadside values. A study of PAH and nitro-PAH was carried out for a year at five sites in the Metropolitan Zone of Mexico Valley (Amador-Muňoz et al., 2011). The highest concentrations of target compounds occurred in the dry seasons. Benzo[gh]perylene was the most abundant PAH, with 2-nitrofluoranthene and 9-NA the most abundant nitro-PAH. Various nitro-PAHs, including 6-NC, 9-NA and 6-nitrobenzo[1] pyrene, irradiated both dissolved or absorbed on to a surface (Warner et al., 2004), showed a relation between relative and nitro group orientation. The nature of the particle had more of an influence than did the structure of nitro-PAH. The main product from degradation was generally a quinone. Photochemical degradation of 1-NP, 2-nitrofluorene, 2,7dinitrofluorene, 6-NC, 3-nitrofluoranthene, 5-nitroacenaphthene and 9-NA was examined (Stewart et al., 2010). 9-NA, which has a perpendicular nitro group, is the fastest, while the more compact 1-NP and 3-nitrofluoranthene are the slowest degrading compounds. Mutagenic and other properties were investigated for 1-, 2-, 3-, 4- and 9-nitrophenathrene (1-NP, 2-NP, 3-NP, 4-NP and 9-NP) (Alparone and Librando, 2013). Based on structural and vibrational properties, 4-NP has no mutagenicity activity, while mutagenic potency of 1-nitroanthracene is predicted to be between that of 9-NP and 3-NP. Diesel particulate filters (DPFs) are a promising technology to detoxify DEs (Heeb et al., 2008). However, the secondary combustion of diesel soot may induce formation of other pollutants. Diesel soot is rated as carcinogenic and acts as a carrier for genotoxic compounds, such as PAHs or nitro-PAHs. Emissions of all investigated four- to six-ring PAHs were reduced by about 40–90%, including carcinogenic ones. Emissions of 1and 2-NN increased by 20–100%. Among the three-ring nitroPAHs, emissions of 3-nitrophenanthrene decreased by 30%, whereas 9-nitrophenanthrene and 9-NA were found only after DPFs. In the case of four-ring nitro-PAHs, emissions of 3nitrofluoranthene, 1-NP and 4-NP decreased by 40–60% with DPFs. DPFs detoxified DE with respect to total aryl hydrocarbons, including the carcinogenic PAHs. Nitration reactions were stereoselective with preferential substitution of peri-hydrogen atoms. The distribution of nitro-PAH contained in particles in the atmosphere of Mexico City was studied (Valle-Hernández et al., 2010). The greatest concentration was 9-NA detected during the cold, dry season. The concentrations of nitro-PAH were higher than those reported in other countries, but lower than from Chinese cities. Knowledge of nitro-PAH air concentrations can aid during surveillance of diseases associated with these exposures. Japanese scientists studied the concentration of nitroarenes (nitro-PAHs) in marine organisms (Uno et al., 2011). Mussels and oysters were collected from Osaka Bay, Japan. Bivalves had


relatively higher residues of 1-NN, 2-NN, 3-nitrophenanthrene and 9-nitrophenanthrene. Residues of 2-nitrofluorene, 1-NP, 4-NP and 6-NC were much lower compared to nitronaphthalenes and nitrophenanthrenes.

Comparison of ArNO2 versus ArNO As presented above, aromatic nitroso compounds are reductive metabolites of the corresponding nitro parents. The nitroso derivative can display physiological activity and is capable of ET and generation of ROS. One report deals with the physiological activities of ArNO in relation to reduction potentials in comparison with ArNO2 (Kovacic et al., 1990). Electrochemical studies were performed on nitrosobenzene, 1-NP and 1-methyl-2-nitrosoimidazole, yielding values in the range of 0.2 to – 0.2 V, favorable for in vivo ET. In acidic solution, ease of electron uptake was increased. Reduction occurred with greater ease in comparison with the related nitro compound. Also discussed are therapeutic and toxic effects, which can be mechanistically related to ET-ROS-OS.

Drugs and Prodrugs An appreciable number of drugs and other bioactive agents incorporate the nitroaromatic structure. An example is chloramphenicol (Fig. 12), a derivative of nitrobenzene, which is a broadspectrum antibiotic (Gringauz, 1997). The drug is particularly useful against typhoid fever and bacterial meningitis. The structure includes functional groups, such as ArNO2, which are rarely if ever, encountered in natural products. A major drawback is the occurrence of toxic effects, which include aplastic anemia and agranulocytosis. In relation to mode of action, reversible binding occurs at the 50S ribosomal subunit. The mechanism involving reduced metabolites accompanied by ET-ROS-OS is addressed in the section on nitrobenzenes. 5-(Aziridine-1yl)-2,4-dinitrobenzamide (CB1954) showed excellent therapeutic activity against Walker rat 256 tumors (Helsby et al., 2003). It acts as a prodrug activated by two-electron nitroreductase DT-diaphorase (NAD(P)H) quinone oxidoreductase. The critical metabolite was shown to be 4-hydroxylamine, which appears to be further activated by acetyl coenzyme A to form a second reactive center that acts in concert with the aziridine moiety to form a cytotoxic bifunctional DNA interstrand cross-linking agent (Scheme 8). A subsequent report showed that nitric oxide synthases are involved in the nitroreduction of the drug (Chandor et al., 2008). This nitroreduction could be of interest for the selective activation of prodrugs by nitric oxide synthases overexpressed in tumor cells. Nitrogen mustard SN 23862, an analogue of CB 1954, showed selective toxicity to tumor cells under hypoxic conditions (Helsby et al., 2003). Data showed that E. coli aerobic reductase


Figure 12. Chloramphenicol.


P. Kovacic and R. Somanathan

Scheme 11. Flutamide metabolism to iminoquinone.

Scheme 8. Reductive activation of dinitrobenzamide aziridine.

reduced only the 2-nitro group, which is presumed to be responsible for its marked cytotoxic bioactivation (Scheme 9). Nilutamide is a non-steroidal antiandrogen derivative that behaves as a competitive antagonist of the androgen receptors (Ask et al., 2003). This nitroaromatic compound is used in the treatment of metastatic prostatic carcinoma. In oral administration in humans, it is extensively metabolized in the liver, undergoing mainly reduction of the nitro to nitroso, hydroxylamine and, finally, to amine, some of which bind to DNA (Scheme 10). In addition, nilutamide and the amine metabolite also undergo hydroxylation of the aromatic ring and of the methyl groups of the heterocycle. The therapeutic value of the drug is overshadowed by the occurrence of several adverse effects. Flutamide is an antiandrogen primarily used in the treatment of metastatic prostate cancer (Coe et al., 2007), which is an idiosyncratic hepatotoxin that leads to severe toxicity. Flutamide possesses a nitroaromatic group that is a contributor to its mechanism of toxicity via the formation of nitroso, hydroxylamine and amine, all which bind to protein and DNA in the liver. The intermediate hydroxylamine may undergo biological dehydration to give the highly reactive iminoquinone, which could function as an ET agent (Kovacic and Somanathan, 2010a, 2010b), thus increasing ROS and OS (Scheme 11). Nimesulide, a nonsteroidal anti-inflammatory drug, is a cyclooxygenase-2 inhibitor that exhibits anti-inflammatory and analgesic effects by adversely affecting formation of prostaglandins via COC-2. The drug induces severe hepatic failure that can

be attributed to the nitroreduction to nitroso, hydroxylamine and, finally, to amine. A report identified several toxic metabolites of the drug, including the potent diiminoquinone, which is an efficient ET agent, creating a cascade of reactions, which may lead to hepatotoxicity (Kovacic and Somanathan, 2010a, 2010b; Li et al., 2009) (Scheme 12). Pro-oxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of ROS or to their inhibition of AO enzyme GSH reductase (GR) (Grellier et al., 2001). A study examined the activity against Plasmodium falciparum of 24 pro-oxidant compounds of different structures (nitrobenzenes, nitrofurans, quinones, 1,1′-dibenzyl-4,4′-bipyridinium and methylene blue), which possess a broad range of single-electron reduction potentials. The redox cycling activity of nitroaromatic and quinoidal compounds increased with an increase in their reduction potentials. Findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form ROS and much less significantly by the GR inhibition. GR inhibitors have become popular due to antimalarial and anticancer activities (Çakmak et al., 2011). In a study, the synthesis and GR inhibitory capacities of novel nitroaromatic compounds (Fig. 13) were reported. Ledakrin, 1-nitro-9-[(dimethylamino)propylamino]acridine, is an antitumor drug that has been used clinically and that yields drug– DNA adducts. A report showed the presence of 1-aminoacridine, produced enzymatically. Scheme 13 shows the possible metabolic breakdown of Ledakrin (Gorlewska et al., 2001). The aromatic nitro functionality is transformed into nitroso, hydroxylamine and, finally, into an amine, all of which can covalently bind to proteins and DNA. The diiminoquinone, in particular, is an efficient ET agent, which can lead to ROS and OS (Kovacic and Somanathan, 2010a, 2010b). Idiopathic Parkinson’s disease is a progressive disorder that has a worldwide distribution and affects over one million

Scheme 9. Reductive activation of mustard analogue SN 23862.

Scheme 10. Reductive activation of nilutamide.


Scheme 12. Metabolism of nimesulide in humans.



Nitro aromatic pollutants Various drugs and other physiological active agents are included in the Merck Index, as follows (O’Neil, 2006): nitrazoxanide (anthelmintic, antiprotozoal); nithirazide (antiprotozoal); nitracrine (antineoplastic); nitrazepam (anticonvulsant, hypnotic); nitrendipine (antihypertensive); nitropdan (anthelmintic); nitrofen (herbicide); nitrofurantoin (antibacterial); nitromersol (disinfectant); nitromide (antibacterial, coccidiostat); nitroscanate (anthelmintic); dinobuton (miticide); dinocap (acaricide, fungicide); and dinodeb (herbicide, insecticide, milicide). Figure 13. Novel aromatic nitro compounds.

Scheme 13. Reductive transformation of ledakrin.

people in North America alone. Tolcapone is a catechol-Omethyltransferase inhibitor used to control motor fluctuations in the disease. Since its entry on to the market in 1998, tolcapone has been associated with numerous cases of hepatotoxicity, including fulminant hepatic failure. A report delineates the various metabolic products that may form covalent adducts to hepatic proteins, resulting in damage to liver tissues (Smith et al., 2003) (Scheme 14).

Nitroreductase These enzymes participate as catalysts in the reduction of nitroaromatic compounds. The nitroreductase family comprises a group of flavin enzymes that metabolize using the reducing power of nicotinamide adenine dinucleotide. These enzymes can be found in the nitroreductase proteins, which play a central role in the activation of nitro compounds and have received a lot of attention. In a review, relevant aspects of nitroreductase enzymes are discussed: occurrence, catalytic reduction mechanism, physiological role, mediating the toxicity of nitro compounds and their influence on human health, as well as medical applications (de Oliveira et al., 2010). Nitroreductase is a homodimeric flavoenzyme that catalyzes the four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT, RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6trinitrophenyl-N-nitroaminoethylnitrate) (Koder et al., 2001). The enzyme had been isolated from a weapons dump. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient reducing agent due to the high nitroaromatic compound concentrations in the environment. Nitroreductases are called oxygen insensitive when they catalyze the two-electron reduction of nitro compounds in the presence of oxygen (Mermod et al., 2010). Such enzymes are widespread in nature and are able to reduce a wide range of substrates, such as nitroaromatic compounds, using NADH or NADPH as the reductant. They are flavoproteins that form homodimers, although oxygen in-vivo function remains largely unknown. Thioredoxin reductase reduces MTZ and other nitro compounds (Leitsch et al., 2007). By reducing MTZ, the enzyme renders itself vulnerable to adduct formation. Because thioredoxin reductase is ubiquitous, similar processes could occur in other organisms.

Mutagenesis A 2002 review outlines mainly mutagenicity and carcinogenicity of various nitroaromatic compounds, including monocyclic, biphenyls, polycyclic and heterocyclic types, such as 4-NQO, NPs, 6-nitro chrysene, 3-nitrofluorene, 3-NBA, 2,4-dichlorophenyl-pnitrophenyl ether, nitrobenzene, nitrofurans and nitrophenanthrene carboxylic acids. There is discussion of a metabolism, DNA binding, mechanism and structure–activity relationship (Purohit and Basu, 2002).

Tumorigenesis Scheme 14. Metabolic pathway of tolcapone.


This topic involving aromatic compounds was reviewed in 2007 (Kovacic and Somanathan, 2007). Simple members, such as o- and p-nitrotoluene, demonstrate carcinogenic potential



P. Kovacic and R. Somanathan (Dunnick et al., 2003). The ortho-isomer gave clear evidence for cancer at multiple sites in rats and mice. The para-isomer gave a weaker response. In a study of nitroanilines, a tumorigenic effect was observed mainly in the dinitro types (Winkelmann, 1975). Both the nitro and amino groups possess carcinogenic potential. For the metabolism of 2-nitroanisole, human hepatic microsomes yielded 2-nitrophenol and dihydroxybenzenes (see Scheme 2) (Mikšanová et al., 2004). The risk to human health from air pollutants is an important issue (Hatanka et al., 2001). Many mutagenic and carcinogenic compounds, such as PAHs and nitrated derivatives, are the main constituents of these pollutants. Analyses have indicated that 1-NP and 1,3- and 1,8-dinitropyrene are major nitroarene components of airborne and soil particles, and DEP. Diesel engine emission appears to be a principal source of contaminants and DEP and its crude extracts are often used as models for the investigation of air pollutants. The relationship between chemical carcinogenesis by polycyclic hydrocarbons, such as benzo [a]pyrene, and drug metabolizing enzymes has been extensively studied. The induction of drug metabolizing enzymes and the activation of procarcinogens have also been examined. As nitro PAHs are widespread environmental contaminants, it can be assumed that humans will ordinarily be exposed to them. The N-hydroxylamino metabolite of 3-NBA caused Cu-mediated DNA damage, indicating involvement of hydrogen peroxide. NP facilitates Cu(II)-mediated DNA insult in the presence of NADH. Catalase and Cu(I) chelators attenuate DNA damage indicating involvement of hydroperoxide and Cu(I) as a Fenton catalyst. Hence, oxidative DNA attack and DNA adduct formation may play important parts in carcinogenesis. The position of the nitro substituent determines the relative tumor activity (Sun et al., 2004). Human breast cancer cells can activate DNA. Articles deal with DNA damage induced by dinitropyrene (Murata et al., 2004; Watanabe et al., 2005), 2,7-dinitrofluorene (Ritter and MalejkaGiganti, 1998) and 6-NC (El-Bayoumy et al., 2004; Krzeminski et al., 2000). The comparative tumorigenicity of 6-NC and its metabolites was ascertained (Krzeminski et al., 2002) (see Scheme 5). A review outlines carcinogenicity and mutagenicity of various nitroaromatic compounds, including monocyclic, polycyclic and heterocyclic types (Kovacic and Somanathan, 2007). Dietary silymarin suppresses rat carcinogenesis by 4-NQO (Gan et al., 2001).

Other Mechanisms and Physiological Influences A review presents a detailed treatment of mutagenicity of nitroaromatic compounds, including various mechanisms (Purohit and Basu, 2002). Data suggest that various nitroreductases may participate in metabolism. A specific arylhydroxylamine esterification enzyme may be necessary for mutagen expression. N-Acetyltransferase appears to play a role. The mutagenicity of most nitro compounds is greater in strains deficient in certain gene products, indicating that covalent adducts are undergoing repair by excision; with nitrofurans in E. coli, the mutagenic activity was similar to the toxicity. Evidence from mammalian cells demonstrates that 4-NQO gives rise to mutagenesis and chromosome aberrations in hamster ovaries. Data point to the importance of DNA excision repair in removing lesions. Conclusive evidence shows that the principal DNA adduct from 1-NP is responsible for the mutagenic response in mammalian cells. In hamster bone


marrow, 2-NF was found to produce sister chromatid exchange. Similarly, 3-NBA induced chromosome aberrations. Various factors contribute, e.g. DNA sequence, orientation, hydrophobicity, ability to intercalate and metabolism. A study suggests that perpendicular orientation of the nitro group gives rise to a profound reduction in tumor formation and mutagenicity. Structure–activity relationship data deal with the involvement of reduction potentials in relation to physiological activity. There is also inclusion of ROS-OS, which is a unifying theme of our review.

Conclusions This review mainly deals with the ET-ROS-OS mechanism of nitro aromatic compounds, which are pollutants derived from combustion. The ET aspect could involve the nitro group directly or the nitroso group formed from nitro reduction. Various ROS are derived from superoxide generated from ET. Harmful effects are discussed, in addition to therapy.

Future directions More research is needed on the topic of structure–activity relationships. The internal combustion engine involves oxidative conditions to which the aromatic hydrocarbon products are exposed. Additional research could be performed that focuses on quinone products, which lead to ET-ROS-OS and subsequent toxicity. Acknowledgments Editorial assistance by Thelma Chavez and Anna Andrade is acknowledged.

Conflict of Interest The Authors did not report any conflict of interest.

References Alparone A, Librando V. 2013. Prediction of mutagenic activity of nitrophenanthrene and nitroanthracene isomers by simulated IR and Raman spectra. Chemosphere 90: 158–163. Alegria AE, Flores W, Cordones E, Rivera L, Sanchez-Cruz P, Cordero M, Cox O. 2004. Reductive activation and thiol reactivity of benzazolo [3,2,-a]quinolinium salts. Toxicology 199: 87–96. Aravena M, Figueroa R, Olea-Azar C, Arán V. 2010. ESR, electrochemical and ORAC studies of nitro compounds with potential antiprotozoal activity. J. Chil. Chem. Soc. 55: 244–249. Amador-Muñoz O, Villalobos-Pietrini, R, Miranda J, Vera-Avilla LE. 2011. Organic compounds of PM2.5 in Mexico Valley: spatial and temporal patterns, behavior and source. Sci. Total Environ. 409: 1453–1465. Ames JR, Ryan MD, Kovacic P. 1986. Mechanism of antibacterial action: electron transfer and oxy radicals. J. Free Rad. Biol. Med. 2: 377–391. Ames JR, Hollstein U, Gagneux AR, Ryan MD, Kovacic P. 1987a. An integrated concept of amebicidal action: electron transfer and oxyradical. Free Rad. Biol. Med. 3: 85–96. Ames JR, Ryan MD, Kovacic P. 1987b. Mode of action of antiprotozoan agents. Electron transfer and oxy radicals. Life Sci. 41: 1895–1902. Amzoiu E, Anoaica PG, Lepádatu CI. 2011. QSAR study of toxicity of aromatic nitroderivatives using the electronegativity of OMO/UMO states as fingerprint descriptors. Rev. Roum. Chim. 56: 711–716. Arlt VM, Stiborova M, Schmeiser HH. 2002. Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17: 265–277. Arlt VM, Hewer A, Sorg BL, Schmeiser HH, Phillips DH, Stiborová M. 2004. 3-Aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for



Nitro aromatic pollutants activation by cytochrome P450 1A1 and P450 1A2. Chem. Res. Toxicol. 17: 1092–1101. Ask K, Dijols S, Giroud C, Casse L, Frapart Y-M, Sari M-A, Kim K-S, Stuehr DJ, Mansuy D, Camus P, Boucher J-L. 2003. Reduction of nilutamide by NO synthases: implications for the adverse effects of this nitroaromatic antiandrogen drug. Chem. Res. Toxicol. 16: 1547–1554. Boga C, Calvaresi M, Franchi P, Lucarini M, Fazzini S, Spinelli D, Tonelli D. 2012. Electron reduction processes of nitrothiophenes. A systematic approach by DFT computaions, cyclic voltammetry and ESR spectroscopy. Org. Biomol. Chem. 10: 7986–7995. Boland B, Lin CY, Morin D, Miller L, Plopper C, Buckpitt A. 2004. Site-specific metabolism of naphthalene and 1-nironaphthalene in dissected airways of rhesus macaques. J. Pharmacol. Exp. Ther. 310: 546–554. Bot C, Hall BS, Álvarez G, Di Maio R, González M, Cerecetto H, Wilkinson SR. 2013. Evaluating 5-nitrofurans as trypanocidal agents. Antimicrob. Agents Chemother. 57: 1638–1647. Buschimi A, Ferrarini L, Franzoni S, Galati S, Lazzaretti M, Mussi F, de Albuquerque CN, Zucchi TMAD, Poli P. 2009. Genotoxicity revaluation of three commercial nitroheterocyclic drugs: nifurtimox, benznidazole, and metronidazole. J. Parasitol. doi:10.1155/2009/463575. Butterworth BE, Mathre OB, Ballinger KE, Adalsteinsson O. 2004. Contamination is a frequent confounding factor in toxicology studies with anthraquinone and related compounds. Int. J. Toxicol. 23: 335–344. Çakmak R, Durdagi S, Ekinci D, Şentürk, M, Topal G. 2011. Design, synthesis and biological evaluation of novel nitroaromatic compounds as potent glutathione reductase inhibitors. Biorg. Med. Chem. Lett. 21: 5398–5402. Chandor A, Dijols S, Ramassamy B, Frapart Y, Mansuy D, Stuehr D, Helsby N, Boucher J-L. 2008. Metabolic activation of antitumor drug 5-(aziridine-1yl)-2,4-dinitrobenzamide (CB1954) by NO synthase. Chem. Res. Toxicol. 21: 836–843. Chen G, Gingerich J, Soper L, Douglas GR, White PA. 2008. Tissue-specific metabolic activation and mutagenicity of 3-nitrobenzanthrone in mutamouse. Environ. Mol. Mutagen. 49: 602–613. Ciganek M, Neca J, Adamec V, Janosek J, Machala M. 2004. A combined chemical and bioassay analysis of traffic-emitted polycyclic aromatic hydrocarbons. Sci. Total Environ. 334–335: 141–148. Coe KJ, Jia Y, Ho HK, Rademacher P, Bammler TK, Beyer RP, Farin FM, Woodke L, Plymate SR, Fausto N, Nelson SD. 2007. Comparison of the cytotoxicity of the nitroaromatic drug flutamide to its cyano analogue in the hepatocyte cell line TAMH: evidence for using toxicogenomic screening. Chem. Res. Toxicol. 20:1277–1290. Culp SJ, Roberts DW, Talaska G, Lang NP, Fu PP, Lay JO, Teitel H, Snawder JE, Von Tungein LS, Kadlubar FF. 1997. Immunochemical, 32Ppostlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation. Mutat. Res. 378: 97–112. de Oliveira IM, Bonatto D, Henriques JAP. 2010. Nitroreductase: enzymes with environmental, biotechnological and clinical importance. In Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Mendez-Villas A (eds). Formatex Badajoz 2: Spain; 1008–1019. DiFilippo P, Riccardi C, Gariazzo C, Incoronato F, Pomata D, Spicaglia S, Cecinato A. 2007. Air pollutants and the characterization of the organic content of aerosol particles in a mixed industrial/semi-rural area in central Italy. J. Environ. Monit. 9: 275–282. Dunnick JK, Burka LT, Mahler J, Sills R. 2003. Carcinogenic potential of onitritoluene and p-nitrotoluene. Toxicology 183: 221–234. El-Bayoumy K, Sharma AK, Lin J-M, Krzeminski J, Boyiri T, King LC, Lambert G, Padgett WS, Nesnow S, Amin S. 2004. Identification 2 of 5-(deoxyguanisine-N -yl)-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as the major DNA lesion in the mammary gland of rats treated with environmental pollutant 6-nitrochrysene. Chem. Res. Toxicol. 17: 1591–1599. Esteve-Nùńez A, Caballero A, Ramos JL. 2001. Biological degradation of 2,4,6-trinitrotoluene. Microbiol. Mol. Biol. Rev. 65: 335–352. Fanucchi MV, Day KC, Clay CC, Plopper CG. 2004. Increased vulnerability of neonatal rats and mice to 1-nitronaphthalene-induced pulmonary injury. Toxciol. Appl. Pharmacol. 201: 53–65. Garner RC, Martin CN, Clayson DB. 1984. Carcinogenic aromatic amines and related compounds. In Chemical Carcinogens, 2nd edn, ACS Monograph 182, Vol 1, Searle C (ed.). American Chemical Society: Washington, DC, 175–302.


Gan J, Skipper PL,Tannenbaum SR. 2001. Oxidation of 2,6dimethylaniline by recombinant human cytochrome P450s and human liver microsomes. Chem. Res. Toxicol. 14: 672–677. Gorlewska K, Mazerska Z, Sowiński P, Konopa J. 2001. Products of metabolic activation of the antitumor drug Ledakrin (nitracrine) in vitro. Chem. Res. Toxicol. 14: 1–10. Grellier P, Sarlauskas J, Anusevicius Z, Maroziene A, Houee-Levin C, Schrevel J, Cenas, N. 2001. Aniplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase. Arch. Biochem. Biophys. 393: 199–206. Gringauz A. 1997. An Introduction to Medicinal Chemistry -How Drugs Act and Why. Wiley-VCH: New York, 261–262. Guttenplan JB, Zhao ZL, Kosinska W, Norman RG, Krzeminski J, Sun YW, Amin S, El-Bayoumy K. 2007. Comparative mutational profiles of the environmental mammary carcinogen. 6-nitrochrysene and its metabolites in a lacl mammary epithelial cell line. Carcinogenesis 28: 2391–2397. Hall BS, Wilkinson SR. 2012. Activation of banznidazole by Trypanosomal type I nitroreductases results in glyoxal formation. Antimicrob. Agents Chemother. 56: 115–123. Hallberg LM, Ward JB, Hernandez C, Ameredes BT, Wickliffe JK. 2012. Part 3. Assessment of genotoxicity and oxidative stress after exposure to diesel exhaust from U.S. 2007-compliant diesel engines report on 1and 3-month exposures in the ACES bioassay. Res. Rep. Health Eff. Inst. 166: 163–184. Halliwell B, Gutteridge JMC. 1999. Free Radicals in Biology and Medicine. Oxford University Press: New York, 1–897. Hanse T, Seidel A, Borlak J. 2007. The environmental carcinogen 3nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells. Toxicol. Appl. Pharmacol. 221: 222–234. Hatanka N, Yamazaki H, Kizu R, Hayakawa K, Aoki Y, Iwanari M, Nakajima M, Yokoi T. 2001. Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust. Carcinogenesis 40: 1242–1249. Heeb NV, Schmid P, Kohler M, Gujer E, Zennegg M, Wenger D, Wichser A, Ulrich A, Gfeller U, Honegger P, Zeyer K, Emmenegger L, Petermann JL, Czerwinski J, Moismann T, Kasper M, Mayer A. 2008. Secondary effects of catalytic diesel particulate filters: conversion of PAHs versus formation of nitro-PAHS. Environ. Sci. Technol. 42: 3773–3779. Helsby N, Wheeler SJ, Pruijjn FB, Palmer BD, Yang S, Denny WA, Wilson WR. 2003. Effect of nitroreduction on the alkylating reactivity and cytotoxicity of the 2,4-dinitrobenzamide-5-aziridine CB 1954 and the corresponding nitrogen mustard SN 23862: distinct mechanisms of bioreductive activation. Chem. Res. Toxicol. 16: 469–478. Hernández DM, De Moura MA BF, Valencia DP, González FJ, González I, De Abreu FC, da Silva Júnior EN, Ferreira VF, Pinto AV, Goulart MOF, Frontana C. 2008. Inner reorganization during the radical-biradical transition in a nor-β-lapachone derivative possessing two redox centres. Org. Biomol. Chem. 6: 3414–3420. Hirayama T, Iguchi K, Yoshida S, Yamanaka Y, Watanabe T. 1991. Structural determination of a dietary mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2′,4′-tetranitrobiphenyl in Salmonella typhimurium TA98. Mutat. Res. 262: 203–207. Homma-Takeda S, Hiraku Y, Ohkuma S, Murata M, Ogawa K, Iwamuro T, Li S, Sun GF, Kumagai Y, Shimojo N, Kawanishi S. 2002. 2,4,6Trinitrotolune-induced reproductive toxicity via oxidative DNA damage by its metabolite. Free Radic. Res. 36: 555–566. Hong HL, Ton TV, Devereux TR, Moomaw C, Clayton N, Chan P, Dunnick JK, Sills RC. 2003. Chemical-specific alterations in ras, p53, and β-catenin genes in hemangiosarcomas from B6C3F1 mice exposed to o-nitrotoluene or riddelline for 2 years. Toxicol. Appli. Pharmacol. 191: 227–234. Huang Z, Komninou D, Kleinman W, Pinto JT, Gilhooly EM, Calcagnotto A, Richie JP. 2007. Enhanced levels of glutathione and protein glutathiolation in rat tongue epithelium during 4-NQO-induced carcinogenesis. Int. J. Cancer 120: 1396–1401. International Agency for Research in Cancer (IARC). 1989. Diesel and Gasoline Engine Exhaust and Some Nitroafrene, No. 46, IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Lyon. Kappus H, Sies H. 1981. Toxic drug associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia 37: 1233–1358. Katritzky AR, Oliferenko P, Oliferenko A, Lomaka A, Karelson M. 2003. Nitrobenzene toxicity: QSAR correlations and mechanistic interpretations. J. Phys. Org. Chem. 16: 811–817.



P. Kovacic and R. Somanathan King C, Kohan MJ, Brooks L, Nelson GB, Ross JA, Allison J, Adams L, Desai D, Amin S, Padgett W, Lambert GR, Richard AM, Nesnow S. 2001. An evaluation of the mutagenicity, metabolism, and DNA adduct formation of 5-nitrobenzo[b]naphtha[2,1-d]thiophene. Chem. Res. Toxicol. 14: 661–671. Kirkland D, Beevers C. 2006. Induction of LacZ mutations in Muta mouse can distinguish carcinogenic from non-carcinogenic analogues of diaminotolenes and nitronaphthalenes. Mutat. Res. 608: 88–99. Knobeloch LM, Blondin GA, Lyford SB, Harkin JM. 1990. A rapid bioassay for chemicals that induce pro-oxidant states. J. Appl. Toxicol. 10: 1–5. Koder RL, Oyedele O, Miller A-F. 2001. Retro-nitroreductase, a putative evolutionary precursor to Enterobacter cloacae strain 96-3 nitroreductase. Antioxid. Redox Signal. 3: 747–755. Kovacic P. 2007. Unifying mechanism for anticancer agents involving electron transfer and oxidative stress: clinical implications. Med. Hypotheses 69: 510–516. Kovacic P, Becvar LE. 2000. Mode of action of anti-infective agents: emphasis on oxidative stress and electron transfer. Curr. Pharm. Des. 6: 143–167. Kovacic P, Cooksy AL. 2005. Unifying mechanism for toxicity and addiction by abused drugs: electron transfer and reactive oxygen species. Med. Hypotheses 64: 366–367. Kovacic P, Edwards C. 2010. Integrated approach to the mechanisms of thyroid toxins: electron transfer, reactive oxygen species, oxidative stress, cell signaling, receptors, and antioxidants. J. Recept. Signal Transduct. 30: 133–142. Kovacic P, Jacintho JD. 2001a. Mechanisms of carcinogenesis: focus on oxidative stress and electron transfer. Curr. Med. Chem. 8: 773–796. Kovacic P, Jacintho JD. 2001b. Reproductive toxins: pervasive theme of oxidative stress and electron transfer. Curr. Med. Chem. 8: 863–892. Kovacic P, Osuna JA. 2000. Mechanisms of anticancer agents: emphasis on oxidative stress and electron transfer. Curr. Pharm. Des. 6: 277–309. Kovacic P, Somanathan R. 2003. Intergrated approach to immunotoxicity: electron transfer, reactive oxygen species, antioxidants, cell signaling and receptors. J. Recept. Signal Transduct. 28: 323–346. Kovacic P, Somanathan R. 2005. Neurotoxicity: The broad framework of electron transfer, oxidative stress and protection by antioxidants. Curr. Med. Chem. 5: 249–258. Kovacic P, Somanathan R. 2007. Mechanism of tumorigenesis: focus on oxidative stress, electron transfer and antioxidants. In Tumorigenesis Research and Advances, Wong DK. Nova Biomedical Books: New York; 23–65. Kovacic P, Somanathan R. 2008. Unifying mechanism for eye toxicity: electron transfer, reactive oxygen species, antioxidant benefits, cell signaling and cell membranes. Cell Membr. Free Radic. Res. 1: 56–69. Kovacic P, Somanathan R. 2009a. Ototoxicity and noise trauma; electron transfer, reactive oxygen species, cell signaling, electrical effects, and protection by antioxidants; practical medical aspects. Med. Hypotheses 70: 914–923. Kovacic P, Somanathan R. 2009b. Pulmonary toxicity and environmental contamination: radicals, electron transfer, and protection by antioxidants. In Reviews of Environmental Contamination and Toxicology, Vol. 201, Whitacre DM (ed.). New York: Springer, 41–69. Kovacic P, Somanathan R. 2010a. Mechanism of conjugated imine and iminium species, including marine alkaloids; electron transfer, reactive oxygen species, therapeutic and toxicity. Curr. Bioact. Compd 6: 46–59. Kovacic P, Somanathan R. 2010b. Dermal toxicity and environmental contamination: electron transfer, reactive oxygen species, oxidative stress, cell signaling, and protection by antioxidants. In Reviews of Environmental Contaminations and Toxicology, Vol. 203, Whitacre DM (ed.). Springer: New York; 119–138. Kovacic P, Somanathan R. 2011. Integrated approach to nitric oxide in animals and plants (mechanism and bioactivity): cell signaling and radicals. J. Recept. Signal Transduct. 31: 111–120. Kovacic P, Thurn LA. 2005. Cardiovascular toxins from the perspective of oxidative stress and electron transfer. Curr. Vasc. Pharmacol. 3: 107–117. Kovacic P, Ames JR, Rector DL, Jawdosiuk M, Ryan MD. 1989. Reduction potentials of anthelmitic drugs: possible relationship to activity. Free Rad. Biol. Med. 6: 131–139. Kovacic P, Kassel MA, Feinbery BA, Corbett MD, McCleilland RA. 1990. Reduction potentials in relation to physiological activities of benzenoid and heterocyclic nitroso compounds. Comparison with nitroso precursors. Biorg. Chem. 18: 265–275.


Kovacic P, Sacman A, Wu-Weis M. 2002. Nephrotoxins: widespread role of oxidative stress and electron transfer. Curr. Med. Chem. 9: 823–847. Kovacic P, Pozos RS, Somanathan R, Shangari R, O’Brien PJ. 2005. Mechanism of mitochondrial uncouplers, inhibitors, and toxins: focus on electron transfer, free radicals, and structure-activity relationships. Curr. Med. Chem. 5: 2601–2623. Krainev AG, Williams TD, Bigelow DJ. 1998. Enzymatic reduction of 3nitrotyrosine generates superoxide. Chem. Res. Toxicol. 11: 495–502. Krzeminski J, Desai D, Lin J-M, Serebryany V, El-Bayoumy K, Amin S. 2000. Anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-nitrochrysene and its reaction with 2′-deoxyadenosine-5′-monophosphate, and calf thymus DNA in vitro. Chem. Res. Toxicol. 13: 1143–1148. Krzeminski J, Desai D, Boyiri T, Rosa J, Krzeminski J, Sharma AK, Pittman B, Amin S. 2002. Comparative tumorigenicity of the environmental pollutant 6-nitrochrysene and its metabolites in the rat mammary gland. Chem. Res. Toxicol. 15: 972–978. Krzeminski J, Kropachev K, Kolbanovskiy M, Reeves D, Kolbanovskiy A, Yun B-H, Geacintov NE, Amin S, El-Bayoumy K. 2011. Inefficient nucleotide excision repair in human cell extracts of the N-)deoxyguanosin2 8-yl)-6aminocgrysene and 5-(deoxyguanosin-N -yl)-6-aminochrysene adducts derived from 6-nitrochrysene. Chem. Res. Toxicol. 24: 65–72. Kucab JE, Phillips DH, Arlt VM. 2012. Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock-in Hupkimouse embryo fibroblast. Environ. Mol. Mutagen. 53: 207–217. Leitsch D, Kolarich D, Wilson IBH, Altmann F, Duchéne M. 2007. Nitroimidazole action in Entamoeba histolytica: a central role for thioredoxin reductase. PloS Biol. 5(8): e211. Levova K, Moserova M, Nebert DW, Philips DH, Frei E, Schmeiser HH, Arlt VM, Stiborova M. 2012. NAD(H); quinone oxireductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid. Toxicol. Appl. Pharmacol. 265: 360–367. Li F, Chordia MD, Huang T, Macdonald TL. 2009. In vitro nimesulide toward understanding idiosyncratic hepatotoxicity: diiminoquinone formation and conjugation. Chem. Res. Toxicol. 22: 72–80. Li Y, Jiang QG, Yao SQ, Liu W, Tian GJ, Cui JW. 1993. Effects of exposure to trinitrotoluene on male reproduction. Biomed. Environ. Sci. 6: 154–160. Lin CY, Wheellock AM, Morin D, Baldwin RM, Lee MG, Taff A, Plopper C, Biuckpitt A, Rhode A. 2009. Toxicity and metabolism of methylnaphthalenes: comparison with naphthalene and 1-nitronaphthalene. Toxicology 260: 16–27. Mermod M, Mourlane F, Waltersperge S, Oberholzer AE, Baumann U, Solioz M. 2010. Structure and function of CinD (YtjD) of Lactococcus lactis, a coppe-induced nitroreductase involved in defense against oxidative stress. J. Bacteriol. 192: 4172–4180. Mikšanová M, Šulc M, Rýdlová H, Schmeiser HH, Frei E, Stiborová M. 2004. Enzymes involved in the metabolism of carcinogen 2-nitroanisole: evidence for its oxidative detoxification by human cytochromes P450. Chem. Res. Toxicol. 17: 663–671. Miliukienė V, Čėnas N. 2008. Cytotoxicity of nitroaromatic explosives and their biodegradation products in mice splenocytes: implications for their immunotoxicity. Z. Naturforsch. 63c: 519–525. Mitchelmore CL, Birmelin C, Livingston DR, Chipman JK. 1998. Detection of DNA strand breaks in isolated mussel (Mytilus edulis L) digestive gland cells using the “Comet” assay. Ecotoxicol. Environ. Saf. 41: 51–58. Miyauchi M, Koizumi M, Uematsu T. 1981. Studies on the toxicity of chlorinated p-nitrobiphenyl ether.1-Methemoglobin formation in vitro and vivo induced by nitroso and amino derivatives of chlorinated biphenyl ether. Biochem. Pharmacol. 30: 3341–3346. Miyauchi M, Haga M, Takou Y, Uematsu T. 1983. Mutagenic activity of chlorinated 4-nitrobiphenyl ethers and their nitroso- and aminoderivatives. Chem. Biol. Interact. 44: 133–141. Miyauchi M, Takou Y, Watanabe M, Uematsu T. 1984. Mutagenic activity of possible metabolites of 4-nitrobiphenyl ether. Chem. Biol. Interact. 51: 49–52. Mu H, Wang X, Lin P, Yao Q, Chen C. 2008. Nitrotyrosine promotes human aortic smooth muscle migration through oxidative stress and ERK1/2 activation. Biochem. Biophys. Acta 1783: 1576–1584. Murata M, Ohnishi S, Seike K, Fukuhara K, Miyata N, Kawanishi S. 2004. Oxidative DNA damage induced by carcinogenic dinitropyrene in the presence of P450 reductase. Chem. Res. Toxicol. 17: 1750–1756. Murata M, Tezuka T, Ohnishi S, Takamura-Enya T, Hisamatsu Y, Kawanishi S. 2006. Carcinogenic 3-nitrobenzanthrone induces oxidative damage to isolated and cellular DNA. Free Rad. Biol. Med. 40: 1242–1249.



Nitro aromatic pollutants Nachtman JP. 1986. Superoxide generation by 1-nitropyrene in rat lung microsomes. Res. Commun. Chem. Pharmacol. 51: 73–80. Neumann HG. 2005. Monocyclic aromatic amino and nitro compounds: Toxicity, genotoxicity and carcinogenicity, classification in a carcinogen category. In The MAK Collection for Occupational Health and Safety, Part I: MAK-Value Documentations, Vol. 21, DFG Deutsche Forschungsgemeinschaft, Greim H (ed.). Weinheim: Wiley-VCHVerlag; 3–45. Nuńez-Vergara LJ, Matus C, Alvarez-Lueje AF, Cassels BK, Squella JA. 1994. Nitro radical anion formation from nitro-substituted amphetamine derivatives. Electroanal. 6: 509–513. Nuoshiba T, Demple B. 1993. Potent intracellular oxidative stress exerted by the carcinogen 4-nitroquinoline-N-oxide. Cancer Res. 53: 3250–3052. Oliveira RB, Passos APF, Alves RO, Romanha AJ, Prado MAF, de Souza Filho JD, Alves RJ. 2003. In vitro evaluation of the activity of aromatic nitrocompounds against Trypanosoma cruzi. Mem. Inst. Oswaldo. Cruz, Rio de Janeiro 98: 141–144. O’Neil MJ, ed. 2006. The Merck Index, 14th edn. Merck Research Laboratories: Whitehouse Station, NJ; 1–556, 1136–1146. Park EJ, Park K. 2009. Induction of pro-inflammatory signals by 1nitropyrene in cultured BEAS-2B cells. Toxicol. Lett. 184: 126–133. Poli G, Cheeseman GH, Dianzani MU, Slater TF. 1989. Free Radicals in the Pathogenesis of Liver Injury. Oxford: Pergamon. Pourahmad J, Khan S, O’Brien PJ. 2001. Lysosomal oxidative stress cytotoxicity induced by nitrofurantoin redox cycling in hepatocytes. Adv. Exp. Med. Biol. 500: 261–265. Purohit V, Basu AK. 2002. Mutagenicity of nitroaromatic compounds. Chem. Res. Toxicol. 13: 674–692. Ramírez-Mares MV, de Mejía EG. 2003. Comparative study of the antioxidant effect of ardisin and epigallocatechin gallate in rat hepatocytes exposed to benomyl and 1-nitropyrene. Food Chem. Toxicol. 41: 1527–1535. Reeve IT, Miller MG. 2002. 1,3-Dinitrobenzene metabolism and protein binding. Chem ResToxicol 15: 352–360. Reeve IT, Voss JC, Miller MG. 2002. 1,3-Dinitrobenzene metabolism and GSH depletion. Chem. Res. Toxicol. 15: 361–366. Ritter CL, Malejka-Giganti D. 1998. Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro. Chem. Res. Toxicol. 11: 1361–1367. Ritter CL, Decker RW, Malejka-Giganti D. 2000. Reductions of nitro and 9oxo groups of environmental nitrofluorenes by the rat mammary gland in vitro. Chem. Res. Toxicol. 13: 793–800. Ritter CL, Culp SJ, Freeman JP, Marques MM, Beland FA, Malejka-Giganti D. 2002. DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver pr mammary gland cytosol. Chem. Res. Toxicol. 15, 536–544. Šarlauskas J, Nemeikaite-Čċniene A, Anusevičius Z, Misevičiene L, Martinez Julvez M, Medina M, Gomez-Moreno C, Čėnas N. 2004. Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity. Arch. Biochem. Biophys. 425:184–192. Schmeiser HH, Stiborovo M, Arlt VM. 2009. Chemical and molecular basis of the carcinogenicity of Aristolochia plants. Curr. Opin. Drug Discov. Dev. 12: 141–148. Schultz MA, Morin D, Chang AM, Buckpitt A. 2001. Metabolic capabilities of CYP2F2 with various pulmonary toxicants and its relative abundance in mouse lung subcompartments. J. Pharmacol. Exp. Ther. 296: 510–519. Shailaja MS, Rajamanikam R, Wahidulla S. 2006. Formation of genotoxic nitro-PAH compounds in fish exposed to ambient nitrite and PAH. Toxicol. Sci. 91: 440–447. Smith SS, Smith PL, Heady TN, Trugman JM, Harman WD, Macdonald TL. 2003. In vitro metabolism of tolcapone to reactive intermediates: relevance to tolcapone liver toxicity. Chem. Res. Toxicol. 16: 123–128. Squella JA, Bollo S, Núńez-Vergara LJ. 2005. Recent developments in the electrochemistry of some nitro compounds of biological significance. Curr. Org. Chem. 9: 565–581. Stewart ML, Bueno GJ, Baliani A, Klenke B, Brun R, Brock JM, Gilbert IH, Barrett M. 2004. Trypanocidal activity of melamine-based nitroheterocycles. Antimicrob. Agents Chemother. 48: 1733–1738.


Stewart G, Smith K, Chornes A, Harris T, Honeysucker T, Dasary SR, Yu H. 2010. Photochemical reaction of nitro polycyclic aromatic hydrocarbons: effect by solvent and structure. Environ. Chem. Lett. 8: 301–306. Stiborová M, Martínek V, Svobodová M, Šistoková J, Dvořak Z, Ulrichová J, Šimánek V, Frei E, Schmeiser HH, Phillips DH, Arlt VM. 2010. Mechanism of the different DNA adduct forming potentials of the urban air pollutants 2-nitrobenzanthrone and carcinogenic 3nitrobenzanthrone. Chem. Res. Toxicol. 23: 1192–1201. Swaminathan S, Hatcher JF. 1986. Xanthine oxidase-mediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl. Mutat. Res. 172: 37–45. Sun Y-W, Guengerich FP, Sharma AK, Boyiri T, Amin S, El-Bayoumy K. 2004. Human cytochromes P450 1B1 catalyze ring oxidation but not nitroreduction of environmental pollutant mononitropyrene isomers in primary cultures of human breast cells and cultured MCF-10A and MCF-7 cell lines. Chem. Res. Toxicol. 17: 1077–1085. Sun Y-W, Guttenplan JB,Khmelnitsky M, Krzeminski J, Boyiri T, Amin S, El-Bayoumy K. 2009. Stereoselective metabolism of the environmental mammary carcinogen 6-nitrochrysene to trans-1,2-dihydroxy-1,2dihydro-6-nitrochrysene by aroclor 1254-treated rat liver microsomes and their comparative mutation profiles in a lacl mammary epithelial cell line. Chem. Res. Toxicol. 22: 1992–1997. Sun Y-W, Guttenplan JB, Cooper T, Krzeminski J, Aliaga C, Boyiri T, Kosinka W, Zhao Z-L, Chen K-M, Berg A, Amin S, El-Bayoumy K. 2012. Mechanisms underlying the varied mammary carcinogenicity of the environmental pollutant 6-nitrochrysene and its metabolites (-)-[R,R]- and (+)-[S,S]-1,2-dihydroxy-1,2-dihydro-6-nitrochrysense in rat. Chem. Res. Toxicol. 26: 547–554. Suzuki M, Matsui K, Yamada M, Kasai H, Sofuni T, Nohmi T. 1997. Construction of mutants of Salmonella typhimurium deficient in 8hydroxyguanine DNA glycosylase and their sensitivities to oxidative mutagens and nitro compounds. Mutat. Res. 393: 233–246. Uno S, Tanaka H, Miki S, Kokushi E, Ito K, Yamamoto M, Koyama J. 2011. Bioaccumulation of nitroarenes in bivalves at Osaka Bay, Japan. Mar. Pollut. Bull. 63: 477–481. Valle-Hernández BL, Mugica-Alvarez V, Salinas-Talavera E, Amador-Muńoz O, Murillo-Tovar MA, Villalobos_Pietrini R, De Vizcaya-Ruíz A. 2010. Temporal variations of nitro-polycyclic aromatic hydrocarbons in PM10 and PM2.5 collected in Northern Mexico City. Sci. Total Environ. 408: 5429–5438. Warner SD, Farant JP, Butler IS. 2004. Photochemical degradation of selected nitropolycyclic aromatic hydrocarbons in solution and absorbed to solid particles. Chemosphere 54: 1207–1215. Watanabe T, Hasei T, Takahashi T, Asanoma M, Murahashi T, Hirayama T, Wakabayashi K. 2005. Detection of a novel mutagen 3,6dinitrobemzo[e]pyrene, as a major contaminant in surface soil in Osaka and Aichi prefecture, Japan. Chem. Res. Toxicol. 18: 283–289. Watt KC, Morin DM, Kurth MJ, Mercer RS, Plopper CG, Buckpitt AR. 1999. Glutathione conjugation of electrophilic metabolites of 1nitronaphthalene in rat tracheobronchial airways and liver: identification by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Chem. Res. Toxicol. 12: 831–839. Winkelmann VE. 1975. Chemotherapeutic effects of nitro compounds. Arzneimittelforschung 25: 681–708. Wu S, Yang B, Wang X, Hong H, Yuan C. 2012. Diurnal variations of nitrated polycyclic aromatic hydrocarbons in PM10 at a roadside site in Xiamen China. J. Environ. Sci. (China) 24: 1767–1676. Yanaida Y, Kohno H, Yoshida K, Hirose Y, Yamada Y, Mori H, Tanaka T. 2002. Dietary silymarin suppresses 4-nitroqunaline-1-oxide-induced tongue carcinogenesis in male F344 rats. Carcinogenesis 23: 787–794. Yun BH, Rosenquist TA, Sidorenko V, Iden CR, Chen CH, Pu YS, Bonala R, Johnson F, Dickman KG, Grollman AP, Turesky YS. 2012. Biomonitoring of aristolactam-DNA adducts in human tissues using ultra-performance liquid chromatography/ion-trap mass spectrometry. Chem. Res. Toxicol. 25: 1119–1131. Zhao XC, Tian L, Cao JG, Liu F. 2010. Induction of apoptosis by 5,7dihydroxy-8-nitrochrysin in breast cancer cells: the role of reactive oxygen species and Akt. Oncol. 37: 1345–1352.



Mutagenicity, carcinogenicity and teratogenicity of cobalt metal and cobalt compounds.

Comparative toxicity and carcinogenicity of soluble and insoluble cobalt compounds.

Mutagenicity, carcinogenicity and teratogenicity of procarbazine.

Mutagenicity, carcinogenicity and toxicity of beta-naphthoflavone, a potent inducer of P448.

Computational prediction of drug toxicity: the case of mutagenicity and carcinogenicity.

Mutagenicity-carcinogenicity as related to teratogenic activity.

Coordination chemistry and the carcinogenicity and mutagenicity of chromium(VI).

Vinyl chloride and vinyl benzene (styrene)--metabolism, mutagenicity and carcinogenicity.

Salmonella mutagenicity and rodent carcinogenicity: quantitative structure-activity relationships.

Synthesis of 1H-indazoles from N-tosylhydrazones and nitroaromatic compounds.

Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds.

QSAR screening of 70,983 REACH substances for genotoxic carcinogenicity, mutagenicity and developmental toxicity in the ChemScreen project.

carcinogenicity and Superfund decisions.

Mutagenicity of benzotrichloride and related compounds.

Mutagenicity testing of imidazole and related compounds.

Mutagenicity of nitrosourea compounds for Salmonella typhimurium.

Mutagenicity of nitroxyl compounds: structure-activity relationships.

Chronic oral toxicity and carcinogenicity study of stevioside in rats.

Toxicity and carcinogenicity of potassium bromate--a new renal carcinogen.

Mutagenicity and metabolism of vinyl chloride and related compounds.

Acute toxicity, genotoxicity, and dermal carcinogenicity assessment of isooctyl acrylate.

Toxicity and carcinogenicity studies of nalidixic acid in rodents.

Chronic toxicity and carcinogenicity of methylmercury chloride in B6C3F1 mice.

Dangerous and cancer-causing properties of products and chemicals in the oil refining and petrochemical industry--Part II: Carcinogenicity, mutagenicity, and developmental toxicity of 1,3-butadiene.