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 de-
cades in relation to pollutants, toxicity, mutagenesis, carcinogen-
esis, 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 in-
volving 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
(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, explo-
sives, 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: pkovacic@mail.sdsu.edu
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

J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd.

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 metabo-
lism has been quite well investigated, as set forth in subsequent
sections.
The quantitative structure–activity relationship and mecha-
nism 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 2,6-
dinitrotoluene, 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 nirophenylhy-
droxylamine (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 deple-
tion 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
Scheme 1. Nitroreductive metabolism.
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P. Kovacic and R. Somanathan
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 tyrosine-
containing 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 amino-
and 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 metabo-
lites of TNT was partly prevented by the AO N,N′-diphenyl-p-
Scheme 2. Pathways of 2-methoxynitrobenzene metabolism.
J. Appl. Toxicol. 2014
Nitro aromatic pollutants
phenylene diamine and desferrioxamine, thus pointing to the in-
volvement 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 delin-
eated 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 path-
ways 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 com-
monly associated with ROS.
Nitroaromatic explosives such as TNT and 2,4,6-trinitrophenyl-
N-methyl-nitramine (tetryl) comprise an important group of
toxic environmental pollutants whose toxicity is mainly attrib-
uted 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 reac-
tions that occur in the human body, providing good approxima-
tion of what occurs in vivo. Hence, explanations based on
electrochemistry have played an important role in interpreting
and understanding the biological phenomena. The electrochem-
ical 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
J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd.
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-4,5-
methylenedioxyamphetamine analogs of amphetamine drugs
have been investigated (Fig. 1) (Nuńez-Vergara et al., 1994). These
analogs were synthesized to evaluate the structure–activity rela-
tionship and mechanism of action for hallucinogens. The electro-
chemical 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 interme-
diates, 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 environ-
mental exposure to NBP. The bioactivation pathways appear to
involve reduction to the hydroxylamine metabolite with subse-
quent 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.
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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’-trichloro-
4-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 activa-
tion. 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 4-
NHOH 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 in-
volved 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 muta-
tions. Nitrofluorenes are mutagenic and carcinogenic environmen-
tal 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.
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P. Kovacic and R. Somanathan
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-methylnaph-
thalene are metabolized readily with high k(cat) values to poten-
tially cytotoxic intermediates. The involvement of metabolite
formation and adduction to proteins in toxicity is important.
Studies with naphthalene and 1-nironaphthalene have dem-
onstrated 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.
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Nitro aromatic pollutants
one in bladder, whereas 1-NNT was negative. 2-NNT is a direct-
acting 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).
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 demon-
strated 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 8-
hydroxyguanosine 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 me-
tabolite 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.
Scheme 5. 2-Nitrobenzanthrone metabolic adduct formation with DNA.
J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd.
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
This category includes lower members, namely three rings, of
fused benzenoids. A study was made to determine the concen-
trations 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 3-
nitrofluoroanthene were associated with particulate matter.
Industrial anthraquinones often contained PAH contaminants,
particularly 9-NA (Fig. 3) (Butterworth et al., 2004). Many toxicol-
ogy 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 im-
portant 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 3-
Nitrophenanthrenes 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 nitro-
phenanthrene compounds found in Aristolochia herbaceous
plants, many of which have been used for medicinal purposes
Figure 3. Nitroanthracene.
Figure 4. 3-Nitrophenanthrene.
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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 for-
mation 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, non-
carcinogenic 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 perox-
idation 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.
Figure 6. Nitropyrene.
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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 pre-
sumed 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).
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-DHD-
6-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-6-
NHOH-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 com-
pounds 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 environ-
mental 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 cytotoxic-
ity 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 mole-
cules 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 7. Mutagenic pyrenes.
J. Appl. Toxicol. 2014
Nitro aromatic pollutants

Scheme 7. Reductive pathway of benznidazole.

Scheme 6. 6-Nitrochrysene metabolic pathway and DNA adduct
formation.
suspected of being carcinogens. In a study, the cytotoxic and
genotoxic potential of nifurtimox (NFX), BEZ and metronidazole
4-Nitroquinoline-N-oxide (MTZ) were re-evaluated (Fig. 8). In Salmonella, NFX and BEZ are
more mutagenic than MTZ. BEZ and NFX-induced DNA damage.
Oxidative stress exerted by superoxide-generating (redox cycling)
The effects of MTZ, that shows comparatively low reduction
agents, such as paraquat, triggers the soxS regulation of
potential, seem to be strictly dependent on anaerobic/hypoxic
Escherichia coli. 4-Nitroquinoline-N-oxide (4-NQO) is a powerful
conditions. Both NFX and BNZ may not only lead to cellular
inducer of soxS (Nuoshiba and Demple, 1993). The transcriptional
damage, but also may interact with DNA.
induction of the soxS gene was dependent on the presence of
NFX, BEZ and megazol are nitroheterocycles, like other nitro
molecular oxygen. Two 4-NQO-related compounds were also
compounds, that exhibit activity against Trypanosoma cruzi, the
shown to induce soxS, mainly 4-nitropyridine-N-oxide, only
causative agent of Chagas disease (Oliveira et al., 2003), which
slightly less efficient than 4-NQO, and 4-hydroxylaminoquinoline-
causes approximately 400 000 deaths in Central and South
N-oxide, at a lower potency than 4-NQO. Thus, considerable OS
America per year. Their activity is due at least in part to the
is induced in cells by 4-NQO, which might contribute to the
well-documented sensitivity of trypanosomes and particularly
carcinogenic potency.
T. cruzi towards OS. Upon one electron reduction, these com-
A goal was to determine the effects of 4-NQO-induced carci-
pounds generate radical anions, which interfere with oxygen
nogenesis on tongue levels of protein-bound and free GSH
metabolism. For BEZ, the generation of reduced reactive species
and related thiols in the rat (Huang et al., 2007). Results suggest
that react with parasite macromolecules is also proposed.
that protein glutathionylation, together with GSH and oxidized
Differently from mammalian host cells, T. cruzi is deficient in
GSH levels, are induced during oral carcinogenesis in the rat,
AO enzymes, which are essential to prevent oxidative damage.
possibly as a result of enhanced levels of OS. A new bioassay
Trypanothione reductase is the key enzyme involved in the pro-
has been developed that allows a rapid, sensitive detection of
tection of T. cruzi against the OS. The radical anions generated
chemicals, such as 4-NQO, which manifest their acute toxicity,
by the nitro compounds upon reduction lead to trypanothione
mutagenicity or carcinogenicity by inducing a pro-oxidant state
reductase depletion and, consequently, toxicity to T. cruzi.
in vivo (Knobeloch et al., 1990). Dietary silymarin suppresses rat
The nitroheterocyclic NFX targets Trypanosoma brucei, the
carcinogenesis by 4-NQO (Yanaida et al., 2002).
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
Other nitroheterocycles
evaluated. Other members of the 5-nitrofurans class have the
Cyclic voltammetry and electron spin resonance were used on potential to treat trypanosomiasis.
potentially antiprotozoal nitro derivatives of dihydroquinoxaline Nitroreduction is an essential step for the biological activity of
and imidazole (Aravena et al., 2010). A self-protonation process mono-, di- and tri-nitrothiophenes, and by comparison, some
involving the nitro group and an acidic proton in the structure mononitro benzo[b]thiophenes and benz[f]furans (Fig. 9) (Boga
was observed in the first step of the reduction mechanism. et al., 2012). Their electroreduction behavior was investigated
Electron spin resonance spectra of the free radicals were obtained. by various techniques: calculations, cyclic voltammetry and
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, reduc-
tant 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 Figure 8. Nitroheterocyclic compounds.

J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat

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 signifi-
cant 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 indi-
cated 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 forma-
tion (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 sulf-
oxide) prevented neuropeptide Y cytotoxicity. Apparently, H2O2
reacts with Fe2+ to form ROS, which causes lysosomal lipid perox-
idation, 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 enzy-
matic 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 in-
crease 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.
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P. Kovacic and R. Somanathan
A study deals with aromatic nitro derivatives in relation to
the mechanism between chemical substances and the biolog-
ical receptor (Amzoiu et al., 2011). The results suggest that the
interaction mechanism is based on ET from biological recep-
tors to the lowest unoccupied molecular orbital energy levels
of the nitro groups.
Other nitroheteroaromatics
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 cyto-
toxic 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 one-
electron 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, 1,8-
dinitropyrene, 6-NC and 3-nitrofluorene, was assessed (Kucab
J. Appl. Toxicol. 2014
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 3-
nitrofluoranthene, 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 prod-
uct from degradation was generally a quinone.
Photochemical degradation of 1-NP, 2-nitrofluorene, 2,7-
dinitrofluorene, 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 com-
pact 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 1-
and 2-NN increased by 20–100%. Among the three-ring nitro-
PAHs, 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 3-
nitrofluoranthene, 1-NP and 4-NP decreased by 40–60% with DPFs.
DPFs detoxified DE with respect to total aryl hydrocarbons, includ-
ing 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
J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd.
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 physiolog-
ical 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 chloram-
phenicol (Fig. 12), a derivative of nitrobenzene, which is a broad-
spectrum antibiotic (Gringauz, 1997). The drug is particularly
useful against typhoid fever and bacterial meningitis. The struc-
ture 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 excel-
lent 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.
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 P. Kovacic and R. Somanathan

Scheme 11. Flutamide metabolism to iminoquinone.

be attributed to the nitroreduction to nitroso, hydroxylamine

and, finally, to amine. A report identified several toxic metabo-
lites 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).
Scheme 8. Reductive activation of dinitrobenzamide aziridine. 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 reduc-
reduced only the 2-nitro group, which is presumed to be respon-
tase (GR) (Grellier et al., 2001). A study examined the activity
sible for its marked cytotoxic bioactivation (Scheme 9).
against Plasmodium falciparum of 24 pro-oxidant compounds
Nilutamide is a non-steroidal antiandrogen derivative that
of different structures (nitrobenzenes, nitrofurans, quinones,
behaves as a competitive antagonist of the androgen receptors
1,1′-dibenzyl-4,4′-bipyridinium and methylene blue), which
(Ask et al., 2003). This nitroaromatic compound is used in the
possess a broad range of single-electron reduction potentials.
treatment of metastatic prostatic carcinoma. In oral administra-
The redox cycling activity of nitroaromatic and quinoidal com-
tion in humans, it is extensively metabolized in the liver, under-
pounds increased with an increase in their reduction potentials.
going mainly reduction of the nitro to nitroso, hydroxylamine
Findings imply that the antiplasmodial activity of nitroaromatic
and, finally, to amine, some of which bind to DNA (Scheme 10).
and quinoidal compounds is mainly influenced by their ability
In addition, nilutamide and the amine metabolite also undergo
to form ROS and much less significantly by the GR inhibition.
hydroxylation of the aromatic ring and of the methyl groups
GR inhibitors have become popular due to antimalarial and
of the heterocycle. The therapeutic value of the drug is
anticancer activities (Çakmak et al., 2011). In a study, the
overshadowed by the occurrence of several adverse effects.
synthesis and GR inhibitory capacities of novel nitroaromatic
Flutamide is an antiandrogen primarily used in the treatment
compounds (Fig. 13) were reported.
of metastatic prostate cancer (Coe et al., 2007), which is an
Ledakrin, 1-nitro-9-[(dimethylamino)propylamino]acridine, is an
idiosyncratic hepatotoxin that leads to severe toxicity. Flutamide
antitumor drug that has been used clinically and that yields drug–
possesses a nitroaromatic group that is a contributor to its
DNA adducts. A report showed the presence of 1-aminoacridine,
mechanism of toxicity via the formation of nitroso, hydroxyl-
produced enzymatically. Scheme 13 shows the possible metabolic
amine and amine, all which bind to protein and DNA in the liver.
breakdown of Ledakrin (Gorlewska et al., 2001). The aromatic nitro
The intermediate hydroxylamine may undergo biological dehy-
functionality is transformed into nitroso, hydroxylamine and, finally,
dration to give the highly reactive iminoquinone, which could
into an amine, all of which can covalently bind to proteins and DNA.
function as an ET agent (Kovacic and Somanathan, 2010a,
The diiminoquinone, in particular, is an efficient ET agent, which can
2010b), thus increasing ROS and OS (Scheme 11).
lead to ROS and OS (Kovacic and Somanathan, 2010a, 2010b).
Nimesulide, a nonsteroidal anti-inflammatory drug, is a
Idiopathic Parkinson’s disease is a progressive disorder
cyclooxygenase-2 inhibitor that exhibits anti-inflammatory and
that has a worldwide distribution and affects over one million
analgesic effects by adversely affecting formation of prostaglan-
dins via COC-2. The drug induces severe hepatic failure that can

Scheme 9. Reductive activation of mustard analogue SN 23862.

Scheme 10. Reductive activation of nilutamide. Scheme 12. Metabolism of nimesulide in humans.

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Nitro aromatic pollutants
Figure 13. Novel aromatic nitro compounds.
Scheme 13. Reductive transformation of ledakrin.
people in North America alone. Tolcapone is a catechol-O-
methyltransferase 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 hepato-
toxicity, 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).
Scheme 14. Metabolic pathway of tolcapone.
J. Appl. Toxicol. 2014 Copyright © 2014 John Wiley & Sons, Ltd.
Various drugs and other physiological active agents are in-
cluded in the Merck Index, as follows (O’Neil, 2006):
nitrazoxanide (anthelmintic, antiprotozoal); nithirazide (anti-
protozoal); 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).
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 en-
zymes are discussed: occurrence, catalytic reduction mechanism,
physiological role, mediating the toxicity of nitro compounds
and their influence on human health, as well as medical applica-
tions (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), tet-
ryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-
trinitrophenyl-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 wide-
spread 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-p-
nitrophenyl ether, nitrobenzene, nitrofurans and nitrophenan-
threne carboxylic acids. There is discussion of a metabolism, DNA
binding, mechanism and structure–activity relationship (Purohit
and Basu, 2002).
Tumorigenesis
This topic involving aromatic compounds was reviewed in
2007 (Kovacic and Somanathan, 2007). Simple members, such
as o- and p-nitrotoluene, demonstrate carcinogenic potential
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(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 en-
gine 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 chemi-
cal 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 Malejka-
Giganti, 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 esterifica-
tion 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 chromo-
some 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
wileyonlinelibrary.com/journal/jat Copyright © 2014 John Wiley & Sons, Ltd.
P. Kovacic and R. Somanathan
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 perpendicu-
lar orientation of the nitro group gives rise to a profound reduction
in tumor formation and mutagenicity. Structure–activity relation-
ship 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 relation-
ships. The internal combustion engine involves oxidative condi-
tions 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.
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