Review Article Received: 7 February 2014,

Accepted: 10 February 2014

Published online in Wiley Online Library: 22 May 2014

(wileyonlinelibrary.com) DOI 10.1002/jat.3005

Toxicity of imine–iminium dyes and pigments: electron transfer, radicals, oxidative stress and other physiological effects Peter Kovacica* and Ratnasamy Somanathana,b ABSTRACT: Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer–reactive oxygen species–oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer–reactive oxygen species–oxidative stress arising from imine–iminium. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: dyes; pigments; physiological effects; imine–iminium; electron transfer; radicals

Introduction

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*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

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Dyes are important, being widely used in the textile, leather, pharmaceutical, plastic, paint and food industries. Increasing attention has been devoted to the physiological effects, including toxicity of dyes and pigments. The relationship of conjugation to color is well established. However, scant attention has been paid to the relation of conjugated imine or iminium to physiological characteristics. The imine–iminium class of dyes addressed herein is composed of methylene blue (MB), rhodamine, malachite green, fuchsin, crystal violet (CV), auramine and cyanins. The pigment group consists of pyocyanine, phthalocyanine (Pcs) and pheophytin. Electron transfer (ET) is probably is the most important process in chemical transformations (Kovacic & Somanathan, 2013). A great impetus was provided to the area by the Marcus theory. The preponderance of bioactive substances or their metabolites incorporate ET functionalities, which, we believe, play an important role in physiological responses. The main groups include quinones (or phenolic precursors), metal complexes (or complexors), aromatic nitro compounds (or reduced hydroxylamine and nitroso derivatives) and conjugated imines (or iminium species). In vivo redox cycling with oxygen can occur, giving rise to oxidative stress (OS) through generation of reactive oxygen species (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 (e.g., in respiration or neurochemistry). Generally, active entities possessing ET groups display reduction potentials in the physiologically responsive range (i.e., more positive than about – 0.5 V). ET, ROS and OS have been increasingly implicated in the mode of action of drugs and toxins (toxicants), e.g., anti-infective agents (Kovacic & Becvar, 2000), anticancer drugs (Kovacic & Osuna, 2000), carcinogens

(Kovacic & Jacintho, 2000), reproductive toxins (Kovacic & Jacintho, 2001), nephrotoxins (Kovacic et al., 2002), hepatotoxins (Poli et al., 1989), cardiovascular toxins (Kovacic & Thurn, 2005), nerve toxins (Kovacic & Somanathan, 2005), mitochondrial toxins (Kovacic et al., 2005), abused drugs (Kovacic & Cooksy, 2005), ototoxins (Kovacic & Somanathan, 2008a), immunotoxins (Kovacic & Somanathan, 2008b), eye toxins (Kovacic & Somanathan, 2008c), pulmonary toxins (Kovacic & Somanathan, 2009), dermal toxins (Kovacic & Somanathan, 2010), gastrointestinal toxins (Kovacic and Jacintho, 2000), thyroid toxins (Kovacic & Edwards, 2010) and various other categories (Halliwell & Gutteridge, 1999). There is a plethora of experimental evidence supporting the ET-ROS theoretical framework, including generation of the common ROS, lipid peroxidation, degradation products of oxidation, depletion of antioxidants (AOs), effect of exogenous AOs, and 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 (e.g., multiple drug properties) as well as toxic effects. This review puts important focus on the imine–iminium ET agent whose action is depicted in Scheme 1. Superoxide can then act as precursor of other ROS as shown in Scheme 2. The iminium ET agent undergoes resonance stabilization after electron uptake (Scheme 3).

P. Kovacic and R. Somanathan

Scheme 1. Superoxide via electron transfer.

Scheme 2. Reactive oxygen species from superoxide.

Scheme 3. Resonance stabilization of conjugated iminium after electron uptake.

Nearly more than 800 000 tons of dyes are produced worldwide per year, and over 15% of the synthetic textile dyes used are lost during manufacturing or processing operations and released as effluents. The effluents will produce adverse effects on the ecoenvironments due to their nonbiodegradability, toxicity and potential carcinogenic and mutagenic nature. Most of the synthetic dyes have the potential to generate ROS leading to OS and toxicity. The adverse toxic effects are often countered by AOs. Some of the dyes also have potential AO and therapeutic values.

Methylene Blue A recent review article deals with the 120-year-long history of diverse application of MB, both in medical treatments and as a staining reagent (Schirmer et al., 2011). In recent years, there has been a surge of interest in MB as an antimalarial agent and as a potential treatment of neurodegenerative disorders, such as Alzheimer’s disease, possibly through its inhibition of the aggregation of tau protein. MB (Scheme 4) is a conjugated imine–iminium compound analogous to p-benzoquinone, and belonging to the phenothiazine class. There is appreciable literature dealing with the physiological activity of MB, including medicinal properties (Schirmer et al., 2011). However, there is scant attention paid to fundamental, mechanistic aspects. The unifying approach involving the conjugated imine–iminium function can be applied, as outlined in the Introduction. The action entails ET with generation of ROS resulting in OS. Various articles provide strong support for

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Scheme 4. Methylene blue.

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generation of ROS by MB, in line with the unifying theme of ET-ROS-OS. MB at concentrations above 5 μM increased intracellular ROS and OS as evidenced by oxidation of glutathione (GSH), vitamin C and dihydrofluorescein (May et al., 2003). D-Glucose, acting as AO, lessened the OS produced by the dye. MB can act as an acceptor similar to the p-benzoquinone analog, by removing electrons from the ET chain in mitochondria. Attention has also been centered upon the formation of singlet oxygen, usually in photosystems, e.g., activated MB. Thus, ROS is involved in many bioactions, such as phagocytosis, mutagenesis and genotoxicity (Berra et al., 2010). Singlet oxygen can cause DNA cleavage and base injury, which may play a role in aging and cancer. Another report deals with DNA damage induced by singlet oxygen formed by exposure to ultraviolet radiation and MB (DeFedericis et al., 2006). The dye, which forms singlet oxygen by a type II mechanism, causes oxidative damage to DNA predominantly with the formation of 8’-hydroxy-2’-deoxyguanosine (OH8dG) (Zhang et al., 2000). These processes may play a role in carcinogenesis (Oliver et al., 2003; Sturmey et al., 2009). MB been investigated in connection with photodynamic therapy (PDT) and apoptosis (Chen et al., 2008). MB-PDT generates ROS, which play a role in apoptosis, in addition to involvement of mitochondrial dysfunction. These properties may lead to use of MB-PDT therapy for melanoma treatment. A review relates the ET-ROS-OS hypothesis to the mechanism of carcinogenesis (Kovacic & Jacintho, 2000). There is considerable literature dealing with medicinal properties, one of the first being antimalarial action reported in the late nineteenth century. It was a successful treatment until WWII, when it was discontinued due to undesirable side effects. However, application has been recently revived. Existing literature supports a role for ET-ROS-OS in the mode of action of antimalarial agents (Halliwell & Gutteridge, 1999); malaria-infected erythrocytes are evidently under OS resulting in toxicity. Malaria parasites can be destroyed by exposure to ROS/RNS, including lipid peroxides and tert-butyl hydroperoxide. An antimalarial agent able to induce OS is the naturally occurring artemisinin, which is an endoperoxide. A mechanism involving iron and radical production has been reported (Kovacic & Becvar, 2000). There is literature on a variety of other medical applications. The dye is known to improve memory consolidation with an accompanying neuroprotective effect, and is a monoamine oxidase inhibitor. Much research is reported in combination with light for the treatment of psoriasis (Salah et al., 2009), Kaposi’s sarcoma (Tardivo et al., 2006) and viruses (Papin et al., 2005). There is inactivation of HIV (Floyed et al., 2004), hepatitis B and C (Müller-Breitkreutz & Mohr, 1998; Wagner et al., 2001), adenovirus and Staphylococcus aureus (Zolfaghari et al., 2009). Application has been made for the treatment of hypotension, sometimes associated with sepsis. As an antidote for methylenoglobinemia, MB is initially reduced to the leuco form, which subsequently reduces the heme group of methemoglobin to hemoglobin. A less wellknown use of MB is in the treatment of ifosamide neurotoxicity. Its action appears to involve the property of electron affinity. Clinical trials are reported for MB in the treatment of Alzheimer’s dementia. The drug may have an effect on mitochondrial function, which involves ET. Various reports deal with the impact of metabolism. MB is spontaneously or enzymatically reduced by NADPH to the colorless leuco MB, which in turn can be reoxidized by molecular oxygen or by iron(III)-containing compounds such as methemoglobin (Scheme 5) (Schirmer et al., 2011). The autoxidizing aspect results in ET to oxygen with generation of OS

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Toxicity of Imine-iminium dyes and pigments

Scheme 6. Azure B to iminoquinone.

Scheme 5. Methylene blue redox cycling in vivo and its metabolic products, Azure A and B.

(Culi et al., 1991), inhibition of malarial parasite propagation in culture (Vennerstrom et al., 1995), inhibition of Aβ-peptide aggregation and inhibition of tau-protein aggregation (Tanguchi et al., 2005; Wischik et al., 1993).

Rhodamine Dyes

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Rhodamine organic dyes with a xanthine ring framework contain iminium groups in extended conjugation (Fig. 1). Some literature deals with toxicology. A NIH report in 1989 indicated evidence of carcinogenicity of rhodamine 6G, when tested on female rats. Data showed increase in malignant pheochromocytomas of the adrenal gland, and that the dye caused embryotoxicity in mice (French, 1989). Rhodamine 6G is a potent inhibitor of mitochondrial oxidative phosphorylation (French, 1989). At low concentrations, ATP-dependent calcium ion uptake is blocked, at higher concentrations, respiration becomes uncoupled and respiration-dependent calcium uptake is inhibited. Rhodamine 6G also inhibits the import and processing of matrix-catalyzed mitochondrial proteins in cultured human fibroblasts (French, 1989). Other toxic manifestations have been noted. The dye inhibits heartbeat and kills Sprague–Dawley neonatal rat cardiac muscle cells in vitro (French, 1989). A study revealed the mutagenic activity of rhodamine dyes and their impurities in salmonellas and DNA damage in Chinese hamster ovary cells (Nestman et al., 1979).

Triphenylmethane Dyes Large numbers of synthetic dyes, which fall under this category, are brilliant and intensely colored having molecular structures based upon the hydrocarbon triphenylmethane. They have wide application in the textile industries for dying nylon, polyacrylonitrile, wool, silk, cotton and copying papers, and as printing inks. Representative dyes under this group bearing conjugated imine and iminium ions are malachite green (MG) (Scheme 7), CV and fuchsin (Fig. 2). The triphenylmethane derivatives are among the oldest man-made dyes, a practical process for the manufacture of fuchsin having been developed in 1859. Several other H3C H2C

CH2

H3C C H2

N

O

CH3

H3C H2C

CH2

N C H2

CH3

HN

O

CH3

NH

H3C COOH

COOEt

Figure 1. Structure of rhodamine B and rhodamine 6G.

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(see Introduction), which may be importantly involved in many biochemical and medicinal features of MB. Related to this is the wide use of MB as a redox indicator and use as a peroxide generator. It is intriguing that in addition to the considerable literature involving oxidative properties by MB, there are contrary reports dealing with AO action. For example, MB reduces OS in the kidney of rats exposed to ROS from treatment with cyclosporine A (Jena & Chainy, 2008). MB blunted surgery induced lipid and protein oxidation (Heydrick et al., 2007). It attenuates OS and decreases abdominal adhesions. MB reduces mitochondrial superoxide production and mitigates free radical generation, thus providing neuroprotection (Poteet et al., 2012; Zhang et al., 2006). The dye could reduce superoxide production by operating as an alternate ET carrier in mitochondria. MB is a redox agent that can function as a potent AO, acting as enhancer of the ET chain, thus preventing generation of mitochondrial ROS. This apparent dichotomy has been observed in the case of molecules such as vitamin C, vitamin E, thiols, selenium and flavonoids (Kovacic & Somanathan, 2006). In relation to MB, the AO action may arise from involvement of the dihydro metabolite or from ET in which the electron acts as a reducing agent, but not with oxygen. The behavior of methyl derivatives is also addressed. MB can also undergo mono-demethylation or di-demethylation to give a secondary amine, also referred to as Azure B and aniline Azure A, respectively (Scheme 5). Mono-demethylated Azure B can undergo deprotonation under basic conditions leading to the neutral iminoquinone, which can readily diffuse through membranes and could behave as an excellent ET agent as described in previous reviews (Scheme 6) (Kovacic & Somanathan, 2011; Schirmer et al., 2011). Furthermore, di-demethylated Azure A, bearing a primary amine group could also bind to DNA-forming adducts. Reports indicate that MB and its metabolites Azure A and B exhibit pharmacological activity, suggesting that MB could be a prodrug (Culi et al., 1991). In addition to the antitumoral and anti-inflammatory actions (Culi et al., 1991), other pharmacological effects include inhibition of GSH reductase (Buchhol et al., 2008), protection of mice from lethal lipopolysaccharide/endotoxic shock (Culi et al., 1991), suppression of the tumor necrosis factoralpha level, growth inhibition of transplanted tumors in mice

P. Kovacic and R. Somanathan

Scheme 7. Malachite green, its carbinol form and metabolite leucomalachite.

SO3

CH3

SO3

NH2

NH2 H2N

H2N

CH3

O3S NH2

NH2

Figure 2. Acid and basic fuchsin.

members of the class were discovered before their chemical constitutions were fully understood. CV, the most important of the group, was introduced in 1883.

adverse reactions, histopathology has revealed that MG causes detrimental effects in liver, gill, kidney, intestine, gonads and pituitary gonadotropic cells (Srivastava et al., 2004). It causes sinusoidal congestion and focal necrosis in liver, damaged mitochondria and causes nuclear alterations (Gerundo et al., 1991). One study showed the interactive effects of Ca2+ content and the presence of humic substance on MG in fish embryos and larvae (Meinelt et al., 2003). The results showed that the presence of higher Ca2+ causes the higher toxicity of MG. Exposure to MG in the stinging catfish, Heteropneus fossilis, caused significant depletion of serum calcium and protein levels (Srivastava et al., 1995). The total cholesterol level of blood was increased significantly at concentrations of MG in respect to all the time intervals. Rat FAO and L6 cell lines exposed to MG were studied and the results indicated that the primary mechanism of toxicity is the inhibition of mitochondrial and lysomal activity (Ding et al., 2012; Radko et al., 2011). Exposure of the striped dwarf catfish, Mystus vittatus, to MG lad to the closing of taste pores, disintegration of taste hairs and cellular components of taste buds and decline in glycoprotein moieties, followed by inability of the fish to sense the chemical nature of the surroundings (Kumar et al., 2007). There are additional reports on toxicity. Both clinical and experimental observations reveal that MG is a multi-organ toxin to mammals and other animals. Rabbits exposed to MG showed renal changes, decrease in food intake, damage to kidney, spleen and heart, lesions on skin, eyes, lungs and bones, and teratogenic effects in rats and mice (Culp et al., 1999; Desciens & Bablet, 1994; Fessard et al., 1999; Srivastava et al., 2004; Werth & Boiteaux, 1967). In an investigation of other toxic effects, MG is found to be carcinogenic to liver, thyroid and other organs of experimental animals (Mahudawala et al., 1999; National Toxicology Program, 2005; Rao, 1995; Rao & Fernandes, 1996; Sundararajan et al., 2000). Incidences of tumors in lungs, breast, ovary and thyroid follicle cells of rats are reported (Werth, 1958). MG also induces DNA damage in mice liver in a dose-dependent manner. The dye in low or high doses caused biochemical disturbances in the major glucolytic–gluconeogenic pathways and hepatic enzymes, depleted GSH and increased free radical formation and lipid peroxidation, followed by necrosis and cirrhosis (Donya et al., 2012).

Malachite Green

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MG, a conjugated iminium triphenylmethane dye, is used medicinally primarily as a therapeutic agent in aquaculture. It is available as the oxalate or hydrochloride salt. In solution, the dye exists as a mixture of the cation (chromatic MG) and its carbinol base, with the ratio depending on the pH of the solution; the dye also can undergo chemical and metabolic reduction to the leuco derivative (Scheme 7) (Culp & Beland, 1996). The conjugated malachite iminium salt could act as an excellent ET agent, in accord with the central theme of ET-ROS-OS. A study involving fungus, Cunninghamella elegans, showed the biodegradation of MG to the tridesmethyl-MG and tetradesmethyl-leucomalachite. The secondary and primary aromatic amine metabolites could bind to DNA-forming adducts (Wang et al., 2012). There are several reviews and a book addressing the therapeutic and toxicological properties of MG (Cascino, 2005; Culp & Beland, 1996; Srivastava et al., 2004; Sudova et al., 2007; Wang et al., 2012). The following paragraph deals with various toxic effects. MG has been extensively used as a topical fungicide and ectoparasiticide in fish farming throughout the world (Khodabakshi & Amin, 2012; Li et al., 2012; Srivastava et al., 2004). In a study of other

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Fuchsin Unlike MG, acid fuchsin (Fig. 2) exhibits several beneficial effects. Amyloid formation and aberrant protein aggregation play a role in a range of human diseases, including type 2 diabetes, Parkinson’s disease and Alzheimer’s disease. Human islet amyloid polypeptide is the major protein component of the pancreatic islet amyloid associated with type 2 diabetes. Reports showed that fuchsin inhibits glycosaminoglycan-mediated amyloid formation (Meng & Raleigh, 2011; Meng et al., 2010). A study also revealed fuchsin acid selectively inhibits HIV replication in vitro Baba et al., 1988). Imipramine blue (Fig. 3), a triphenylmethane dye, based on in vitro studies, effectively inhibited several cancer cell lines (Munson et al., 2012). The compound appears to halt the spread of cancer cells into normal brain tissue in animal models. Crystal Violet and Gentian Violet Investigations deal with both adverse and beneficial effects. CV dye was commonly used for the treatment of oral and vaginal

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Toxicity of Imine-iminium dyes and pigments H3C

CH3

N H2C N

CH2

CH2

H2 C

H2 C H3C

N CH2

N H2C

H3C

CH3 CH3

Figure 3. Imipramine blue.

candidiasis or for sterilization during operations up to the 1960s. CV is potentially toxic to mucosal membranes (Hashimoto et al., 2013). A study showed that CV instilled into the bladder of a 47-year-old Japanese woman to confirm the presence of vesicovaginal fistula, produced gross hematuria, frequent urination and lower abdominal pain (Hashimoto et al., 2013). Cystoscopy revealed desquamated epithelial cells and a hemorrhagic bladder wall. CV undergoes biodegradation by Nocardia corallina to give Michler’s ketone and p-dimethylaminophenol (Azmi et al., 1997), which in turn can undergo further degradation by demethylation leading to a potent primary amine (Scheme 8). A study revealed the chronic toxicity and carcinogenicity of gentian violet in mice (Littlefield et al., 1985). Auramine There is literature involved with toxicity. A review deals with physiological effects of auramine (Scheme 9) (IARC Monograph, 2010), including carcinogenicity in animals and likelihood in humans. An epidemiological study indicates an occupational bladder cancer risk. The dye is injurious to the human eye and induces sister chromatid exchange and DNA strand breaks in rodent cells. It brought about prophage and was mutagenic to

bacteria. In yeast, auramine caused DNA damage, aneuploidy and mitotic recombination. Mutation occurred in Drosophila, as well as chromosome damage to root tips and to hamster ovary cells. The mechanism of action was briefly treated. The mode of action may be due to electrophilicity at positively charged nitrogen atoms. In addition, hydroxylation of benzene rings may occur. An investigation was performed on the possible generation of free radicals by various carcinogens, including auramine (Brennan & Schiestl, 1998). The toxicity was reduced by addition of the AO N-acetylcysteine involving protection against OS by oxy radicals. Auramine, a diphenylmethane imine, is used in some countries as a food colorant and mostly used to color smoke in firework displays and in military applications. In another report on toxicology, a single landmark study in the United Kingdom revealed excess of bladder cancer in workers engaged in the manufacture of auramine (Case & Pearson, 1954). Oral administration of auramine to mice and rats showed hepatomas (Walpole, 1963; William & Bonser, 1962). A study involving rabbits revealed metaplasia of the urinary tract epithelium, suggestive of precancerous change (Bonser, 1962). There are various toxic manifestations. Auramine induced forward mutation in Salmonella typhimurium in the presence of metabolic activation (Skopek et al., 1981), and generated DNA strand breaks in primary cultures of rat hepatocytes (Martelli et al., 1998) and human cell line HuF22 in the absence of metabolic activation (Parodi et al., 1982). The dye also induced DNA fragmentation in the liver (Brambilla et al., 1985; Kitchin & Brown, 1994; Parodi et al., 1981), in the kidney (Parodi et al., 1982) and urinary bladder of rats (Martelli et al., 1998).

Cyanine Dyes This category is composed of three types: streptocyanines(I), hemicyanines(II) and closed cyanines(III) (Fig. 4). In II and III, the nitrogens compose part of a heteroaromatic ring. The class contains the iminium species composed of conjugated systems as illustrated in Fig. 4, acting as contributors to the resonance hybrid. The dyes, such as dithiazanine (Fig. 5a) and pyrvinium (Fig. 5b), are known to function as anthelmintic agents for treatment of ascaris, pinworm, nematodes and whipworm (Kovacic et al., 1989). Activity is destroyed by interruptions of conjugation. Increase in conjugated chain length results in increase in reduction potential. The electron involved in redox cycling with generation of ROS would undergo desirable delocalization. The more favorable reduction potential for (2a) vs (2b) H3C

Scheme 8. Crystal violet and its metabolites.

N

n

CH3

n

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CH3

III

Figure 4. Cyanines.

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Scheme 9. Auramine.

CH3

N

n

CH3

N CH3

II

N

CH3

CH3

I

N CH3

N

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Figure 8. Protonted pyocyanine.

Phthalocyanines Figure 5. Cyanine dyes.

may be due to increased stabilization of the delocalized electron by the sulfur orbitals. Toxicity of dithiazine has resulted in termination of widespread use. The adverse effects could be a reflection of ROS generation, in line with the proposed unifying theme (see Introduction). There are various toxic manifestations. A review entails the use of cyanine dyes as potential tau-protein aggregation inhibitors in neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington (Bulic et al., 2010). Cyanine dyes (Fig. 5, Fig. 6) showed mutagenic activity against the Salmonella cell strain TA98 and were toxic to mice and rats (Matselyukh et al., 2005).

Pigments Pyocyanine Pyocyanine (Fig. 7), a blue pigment present in bacterial secretion, is a highly conjugated iminoquinone, structurally related to the ET agent o-benzoquinone. Therefore, it is not surprising that there is participation in the electron transport chain. Ubiquinone and nicotinic acid, which are ET agents, are also affected by the pigment. Various reports address detrimental influences. Hydrogen peroxide produced by pyocyanine has adverse effects on the lung. ROS, believed to participate in various physiological effects, cause OS that damages DNA. The fungicidal mechanism is attributed to induction of a redox cascade that generates ROS. In the lung infected with cystic fibrosis, the pigment converts oxygen to superoxide, which can inhibit cytokines. The AO GSH is modulated by pyocyanine, and increased levels of catalase and SOD are generated to counter the increase in concentrations of radicals being generated. Protonation of the base in vivo can yield a conjugated iminium (Fig. 8), which should be even more effective as an ET species in redox cycling.

Pcs, derived from the parent (Fig. 9), play important roles in industry, medicines and biology (Jančula & Maršálek, 2012). The structure incorporates six imine groups in extended conjugation with themselves and with aromatic nuclei. As the Introduction designates conjugated imines as ET agents, it is not unexpected to find literature based on ET-ROS-OS. Hence, their ability to generate ROS is reported. In addition to their widespread use as dyes and pigments, various physiological effects are known to occur. Their role as natural toxins is in keeping with numerous findings linking toxicity with the unifying mechanism of ET-ROS-OS (see Introduction). Their property as biocides, which inhibit bacteria, is in accord with the designation of phagomimetic (Gutteridge et al., 1999; Kovacic & Jacintho, 2000). The ability to function as antitumor (Jančula & Maršálek, 2012; Kovacic & Osuna, 2000) and antifungal agents (Jančula & Maršálek, 2012; Kovacic et al., 1990) also is consistent with the unifying, mechanistic framework. Pcs produce toxic effects on aquatic plants, including phytotoxicity. There is discussion of Pcs as cyanocides and herbicides (Jančula & Maršálek, 2012). In most cases, the compounds studied are in the form of metal derivatives (Fig. 10). Thus, the metal may also participate as an ET agent. The same may apply to the protonated form. Another investigation deals with the ability of Pcs tetrasulfonate to combat scrapie in mice (Priola et al., 2003). A report that dealt with the toxic effects of water-soluble Pcs dyes was by Kurliandskiǐ et al. (1988).

Pheophytin Pheophytin (Pheo) (Fig. 11), a dark bluish waxy pigment, can be derived from chlorophyll by removal of the magnesium. In photosynthesis, it is the first electron acceptor in the ET chain of photosystem II, containing imine groups as part of a long, conjugated system. A reduction potential of – 0.5 V is reported

Figure 9. Phthalocyanine. Figure 6. Toxic carbocyanines.

830

Figure 7. Pyocyanine.

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Figure 10. Metallic phthalocyanine.

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Toxicity of Imine-iminium dyes and pigments

Figure 11. Pheophytin.

for Pheo (Kovacic et al., 1991) (see Introduction). As expected, the protonated iminium form of Pheo reduced at a more positive value of – 0.2 V. In a study of physiological activity, Pheo was more cytostatic/cytotoxic than pheophorbide (Fig. 11; R = methyl), a closely related compound (Chernomorsky et al., 1999). Food sources that yield such chlorophyll derivatives may play a significant role in cancer prevention. Evidence points to Pheo as a possible antimutagen (Yoshikawa et al., 1996).

Other Reports on Toxicity

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Conclusion This review involves practical application of a unifying mechanism to the understanding of basic elements operating in the toxicity of an important industrial class of dyes and pigments. An integral, common structural element is the presence of imine or iminium ET agents, which produce ROS-OS. An understanding of reaction at the basic molecular level can have practical application and serve as a basis for future research. The multifaceted approach also includes AOs and therapy. Acknowledgments Editorial assistance by Thelma Chavez and copy editing by Anna Andrade are acknowledged.

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

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In addition to the studies presented in the present review, there is additional, extensive literature, including other classes of dyes and pigments. Examples, mostly reviews, are presented in the following material. An area that attracted media attention involves hair dyes. A 2005 review deals with the genotoxic/ mutagenic/carcinogenic properties of hair dyes (Kirkland et al., 2005). Potential genotoxicity may be determined by tests for mutation, chromosomal alterations and carcinogenicity. Another report addresses mutagenic and carcinogenic properties of hair dyes (Stardumov, 1991), in addition to a similar review (Marzulli et al., 1978). Controversy has surrounded food dyes because of safety concerns (Kobylewski & Jacobson, 2012). There are toxicity problems for the nine approved dyes, including Red3, which displays carcinogenic insults. Various other dyes exhibited the same toxicity. Genotoxicity was observed for Yellow 5. Questions concerning safety were also raised for Citru Red2 and Orange B. A recommendation was made for removal of all of the approved dyes. Various investigations focus on azo dyes. Toxins, industrial effluents with azo dye components, pose a threat to human and aquatic creatures (Puvaneswari et al., 2006). The toxicity involves genes, mutagens and carcinogens, including lethal effects. Bioremediation approaches are presented. The toxic impact of azo dye metabolism is treated in another report (Feng et al., 2012). Azoreductase bring about reductive cleavage of the azo bond forming aniline derivatives that apparently are genotoxic (Feng et al., 2012). The metabolites are carcinogenic even though the parent may not be. The review addresses mechanisms in addition to toxic effects. The toxicity of the aromatic primary amine metabolites appears to involve oxidative conversion to hydroxylamine, nitroso compounds and nitro compounds, which can undergo redox cycling with the generation of toxic ROS (Kovacic & Somanathan, 2011). The last section deals mostly with more recent, relevant material on toxicity. Amido 10B, an azo dye, displays extreme toxic properties (Mittal, 2013). There is eye and skin irritation as well as harm to the respiratory system. A study deals with retinal pigment in which OS is linked to pathogenesis of age-related

macular degeneration (Cao, 2013). Results point to the role of aging, and OS in cytokine modulation involving inflammation. In recent years, research on ROS-OS from pollutants has increased in toxicology entailing interaction of basic and applied toxicology (Demirci & Hamamci, 2013). The ROS are generated by a variety of pollutants, including dyes in wastewater from industrial plants. Levels of AOs, such as catalase and GSH, decreased because of exposure to the toxicant. Another report deals with detoxification of MG, a triarylmethane dye, by manganese peroxidase (Saravankumar, 2013). In samples treated with Remazol orange, an azo dye, toxicological scrutiny showed inhibition of AOs, such as catalase in lipid peroxidation and protein oxidation, pointing to enhanced OS (Jadhav et al., 2012). The toxicity of certain dyes can be enhanced by exposure to light rays (Wang et al., 2009). The ROS mechanism for photo-induced acute toxicity was studied involving an anthraquinone dye intermediate. Data indicate that superoxide and singlet oxygen can be formed via ET or energy transfer during photosensitization. In a related investigation, evaluation of toxicity from ultraviolet light was performed (Balaiya et al., 2010). Formation of ROS indicates involvement of OS in retinal and pigment epithelial cells. The mechanism of malarial pigment hemozoin was explored (Schwarzer et al., 1996). After hemozoin phagocytosis, lipid peroxidation was enhanced, involving increase in 4hydroxynonenal. A 2006 report indicates that 4-oxo-2-nonenal, a metabolite from further oxidation, may also participate (Kovacic, 2006). Reduced toxicity of neutral red, a textile dye, was observed on exposure to photocatalysis involving BiOCl (Sarwan et al., 2012). Toxicity was no longer observed following photocatalytic treatment. Human AO defenses are evidently involved in hemoglobin toxicity (Abraham et al., 1998). Heme oxygenase appears to play a favorable role. A study was made of the relationship of ocular toxicity to melanin in retinal pigment (Dayhaw-Barker, 2002). Radiation may play a part in the pathology. Toxicity of a violet dye of the anthraquinone class was evaluated after inhalation (Jasket, 1994). Death resulting from liver damage occurred. Severe liver toxicity was observed. Other possible mechanisms could pertain. Some of the findings may be the result of gene expression from adaptation. In addition, some AO responses may trigger molecular biological processes. It seems logical to adopt a multifaceted approach, although the main focus is on ET-ROS-OS.

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