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Evidence of Quinone Metabolites of Naphthalene Covalently Bound to Sulfur Nucleophiles of Proteins of Murine Clara Cells after Exposure to Naphthalene
Abstract
Naphthalene-induced Clara cell toxicity in the mouse is associated with the covalent binding of electrophilic metabolites to cellular proteins. Epoxide and quinone metabolites of naphthalene are proposed to be the reactive metabolites responsible for covalent binding to proteins. To identify the nature of reactive metabolites bound to proteins (cysteine residues), we alkaline-permethylated proteins obtained from mouse Clara cells incubated with 0.5 mM naphthalene in vitro. Alkaline permethylation of protein adducts produced (methylthio)naphthalene derivatives detected by GC-MS. 3,4-Dimethoxy(methylthio)naphthalene was observed to be a predominant (methylthio)naphthalene derivative formed in the alkaline-permethylated protein sample obtained from Clara cells after exposure to naphthalene. This indicates that 1,2-naphthoquinone is a major metabolite covalently bound to cysteine residues of the cellular proteins. We have developed an immunoblotting approach to detect 1,2-naphthoquinone covalently bound to cysteine residues of proteins [Zheng, J., and Hammock, B. D. (1996) Chem. Res. Toxicol. 9, 904-909]. To identify 1,2-naphthoquinone covalently bound to sulfur nucleophiles of proteins, homogenates obtained from naphthalene-exposed Clara cells were separated by SDS-PAGE followed by Western blotting and immunostaining with the antibodies. Two protein bands with 24 and 25 kDa were detected by the antibodies, further supporting the view that 1,2-naphthoquinone is a reactive metabolite of naphthalene which binds to Clara cell proteins in vitro.
... The toxicity of naphthalene has been studied previously, and the negative impacts on mammalian systems are believed to be due primarily to products of metabolic modification (Kawabata and White 1990;Wilson et al., 1996;Sasaki et al., 1997;Zheng et al., 1997;Bagchi et al., 1998a;Bagchi et al., 1998b;Hoeke and Zellerhoff 1998). The products of metabolic activation include 1-naphthol, and 1,2and 1,4-napthoquinones, which have been implicated in the indirect cytotoxicity of naphthalene (Tingle et al., 1993;Flowers-Geary et al., 1996;Wilson et al., 1996;Zheng et al., 1997;Bagchi et al., 1998a;Bagchi et al., 1998b). ...
... The toxicity of naphthalene has been studied previously, and the negative impacts on mammalian systems are believed to be due primarily to products of metabolic modification (Kawabata and White 1990;Wilson et al., 1996;Sasaki et al., 1997;Zheng et al., 1997;Bagchi et al., 1998a;Bagchi et al., 1998b;Hoeke and Zellerhoff 1998). The products of metabolic activation include 1-naphthol, and 1,2and 1,4-napthoquinones, which have been implicated in the indirect cytotoxicity of naphthalene (Tingle et al., 1993;Flowers-Geary et al., 1996;Wilson et al., 1996;Zheng et al., 1997;Bagchi et al., 1998a;Bagchi et al., 1998b). The toxicity of naphthalene to plants has also been found to increase on exposure to light, and it was suggested that the increase in toxicity was due to the oxidation products of naphthalene (Ren et al., 1994). ...
... The are reactive towards proteins as well, capable of direct arylation of protein sulfhydryl groups (Smith 1985). In particular, 1,2-naphthoquinone binds to cysteine residues (Zheng et al., 1997). ...
There is surprisingly little data on the photooxidation of polycyclic aromatic hydrocarbons (PAHs) under environmentally relevant lighting conditions. The aqueous photooxidation reactions of naphthalene (the simplest and most water soluble PAH) were investigated using natural sunlight as a light source.Six of the major reaction products were identified, including 1-naphthol, coumarin, and two hydroxyquinones. The reactionproducts were consistent with initial [2,2] or [2,4] photocycloaddition reactions, with subsequent oxidations and/or rearrangements. The oxidation reactions in aqueous phase favoredproducts different from those observed in atmospheric oxidation reactions. However, similar photoproducts were observed withtitanium catalysts or in the presence of hydrogen peroxide. Theproducts from aqueous photooxidation were also similar to the products resulting from naphthalene metabolism. The observedphotooxidation products were generated by mechanisms that areexpected to occur with other PAHs as well, and thus naphthaleneoxidation provides a model for possible photoreactions of largerPAHs.
... In a study published by Zheng et al. (1997) it was shown that 1,2-NQ can react with cysteine residues through covalent bonds, thereby altering the protein structure (Zheng et al., 1997). An important study by Ahn et al. (2002) shows that 1,2-NQ inhibits PTP1B (protein tyrosine phosphatase 1B), a major negative regulator of insulin signaling, which plays an important role in the development of diabetes type 2 (Ahn et al., 2002;Kennedy and Ramachandran, 2000). ...
... In a study published by Zheng et al. (1997) it was shown that 1,2-NQ can react with cysteine residues through covalent bonds, thereby altering the protein structure (Zheng et al., 1997). An important study by Ahn et al. (2002) shows that 1,2-NQ inhibits PTP1B (protein tyrosine phosphatase 1B), a major negative regulator of insulin signaling, which plays an important role in the development of diabetes type 2 (Ahn et al., 2002;Kennedy and Ramachandran, 2000). ...
Extensive literature regarding the health side effects of ambient pollutants (AP) are available, such as diesel exhaust particles (DEPs), but limited studies are available on their electrophilic contaminant 1,2-Naphthoquinone (1,2-NQ), enzymatically derived from naphthalene. This review summarizes relevant toxicologic and biological properties of 1,2-NQ as an environmental pollutant or to a lesser degree as a backbone in drug development to treat infectious diseases. It presents evidence of 1,2-NQ-mediated genotoxicity, neurogenic inflammation, and cytotoxicity due to several mechanistic properties, including the production of reactive oxygen species (ROS), that promote cell damage, carcinogenesis, and cell death. Many signal transduction pathways act as a vulnerable target for 1,2-NQ, including kappaB kinase b (IKKbeta) and protein tyrosine phosphatase 1B (PTP1B). Antioxidant molecules act in defense against ROS/RNS-mediated 1,2-NQ responses to injury. Nonetheless, its inhibitory effects at PTP1B, altering the insulin signaling pathway, represents a new therapeutic target to treat diabetes type 2. Questions exist whether exposure to 1,2-NQ may promote arylation of the Keap1 factor, a negative regulator of Nrf2, as well as acting on the sepiapterin reductase activity, an NADPH-dependent enzyme which catalyzes the formation of critical cofactors in aromatic amino acid metabolism and nitric oxide biosynthesis. Exposure to 1,2-NQ is linked to neurologic, behavioral, and developmental disturbances as well as increased susceptibility to asthma. Limited new knowledge exists on molecular modeling of quinones molecules as antitumoral and anti-microorganism agents. Altogether, these studies suggest that 1,2-NQ and its intermediate compounds can initiate a number of pathological pathways as AP in living organisms but it can be used to better understand molecular pathways.
... In a study published by Zheng et al. (1997) it was shown that 1,2-NQ can react with cysteine residues through covalent bonds, thereby altering the protein structure (Zheng et al., 1997). An important study by Ahn et al. (2002) shows that 1,2-NQ inhibits PTP1B (protein tyrosine phosphatase 1B), a major negative regulator of insulin signaling, which plays an important role in the development of diabetes type 2 (Ahn et al., 2002;Kennedy and Ramachandran, 2000). ...
... In a study published by Zheng et al. (1997) it was shown that 1,2-NQ can react with cysteine residues through covalent bonds, thereby altering the protein structure (Zheng et al., 1997). An important study by Ahn et al. (2002) shows that 1,2-NQ inhibits PTP1B (protein tyrosine phosphatase 1B), a major negative regulator of insulin signaling, which plays an important role in the development of diabetes type 2 (Ahn et al., 2002;Kennedy and Ramachandran, 2000). ...
Extensive literature regarding the health side effects of ambient pollutants (AP) are available, such as diesel exhaust particles (DEPs), but limited studies are available on their electrophilic contaminant 1,2-Naphthoquinone (1,2-NQ), enzymatically derived from naphthalene. This review summarizes relevant toxicologic and biological properties of 1,2-NQ as an environmental pollutant or to a lesser degree as a backbone in drug development to treat infectious diseases. It presents evidence of 1,2-NQ-mediated genotoxicity, neurogenic inflammation, and cytotoxicity due to several mechanistic properties, including the production of reactive oxygen species (ROS), that promote cell damage, carcinogenesis, and cell death. Many signal transduction pathways act as a vulnerable target for 1,2-NQ, including kappaB kinase b (IKKbeta) and protein tyrosine phosphatase 1B (PTP1B). Antioxidant molecules act in defense against ROS/RNS-mediated 1,2-NQ responses to injury. Nonetheless, its inhibitory effects at PTP1B, altering the insulin signaling pathway, represents a new therapeutic target to treat diabetes type 2. Questions exist whether exposure to 1,2-NQ may promote arylation of the Keap1 factor, a negative regulator of Nrf2, as well as acting on the sepiapterin reductase activity, an NADPH-dependent enzyme which catalyzes the formation of critical cofactors in aromatic amino acid metabolism and nitric oxide biosynthesis. Exposure to 1,2-NQ is linked to neurologic, behavioral, and developmental disturbances as well as increased susceptibility to asthma. Limited new knowledge exists on molecular modeling of quinones molecules as antitumoral and anti-microorganism agents. Altogether, these studies suggest that 1,2-NQ and its intermediate compounds can initiate a number of pathological pathways as AP in living organisms but it can be used to better understand molecular pathways.
... The naphthoquinones are highly reactive and are under discussion as the ultimate carcinogens in the naphthalene metabolism. They can bind to macromolecules such as proteins and the DNA (Bolton et al. 2000;Buckpitt and Warren 1983;Cho et al. 1994;Troester et al. 2002;Tsuruda et al. 1995;Waidyanatha et al. 2002Waidyanatha et al. , 2004Zheng et al. 1997 ...
... In animal studies, the respiratory tract, the eyes and the haematopoietic system are the target tissues of naphthalene toxicity (Abdo et al. 2001;Shopp et al. 1984;Van Heyningen 1979;Zheng et al. 1997). ...
The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has evaluated a BAR (“Biologischer Arbeitsstoff Referenzwert”) for naphthalene, considering 1‐ and 2‐naphthol in urine to characterize the internal exposure. Naphthalene is classified in category 2 for carcinogenic substances and designated with an “H” because of its contribution to the toxicological hazards due to penetration through the skin in vitro and dermal absorption in vivo.
In a number of biomonitoring studies, the excretion of 1‐ and 2‐naphthol in urine of persons occupationally not exposed to naphthalene was examined. Tobacco smoking affects the naphthol excretion significantly, thus non‐smokers and smokers have to be considered separately. Up to now, no data of representative collectives of the German general population are available. Therefore, the largest German study was considered for the evaluation, where urine samples of 95 non‐smokers were analysed and a 95th percentile for the sum of 1‐ and 2‐naphthol (after hydrolysis) of 33.6 µg/l (30.6 µg/g creatinine) was found. These results are in good accordance with other national and international studies. Therefore, a BAR of 35 µg 1‐ plus 2‐naphthol (after hydrolysis)/l urine was evaluated for non‐smokers. Sampling time is at the end of exposure or the end of the working shift and after long term exposure at the end of the working shift after several shifts. For the interpretation of the result, the smoking status of the persons has to be considered. In case of an occupational co‐exposure to other polycyclic aromatic hydrocarbons (PAH), the analysis of additional PAH‐metabolites is recommended (see MAK Documentation “Polycyclic Aromatic Hydrocarbons”).
... Die Naphthochinone weisen eine hohe Reaktivität auf und werden als die ultimalen Kanzerogene im Naphthalinmetabolismus diskutiert. Sie können an Makromoleküle wie Proteine und die DNA binden (Bolton et al. 2000;Buckpitt und Warren 1983;Cho et al. 1994;Troester et al. 2002;Tsuruda et al. 1995;Waidyanatha et al. 2002Waidyanatha et al. , 2004Zheng et al. 1997). ...
... (3) 2-Naphthol (4) 1-Naphthylglukuronid/-sulfat (5) 2-Naphthylglukuronid/-sulfat (6) 1,4-Dihydroxynaphthalin Zielgewebe von Naphthalin im Tierversuch sind der Atemtrakt, das Auge und das blutbildende System (Abdo et al. 2001;Shopp et al. 1984;Van Heyningen 1979;Zheng et al. 1997). ...
Naphthalene
The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has evaluated a BAR (“Biologischer Arbeitsstoff Referenzwert”) for naphthalene, considering 1‐ and 2‐naphthol in urine to characterize the internal exposure. Naphthalene is classified in category 2 for carcinogenic substances and designated with an “H” because of its contribution to the toxicological hazards due to penetration through the skin in vitro and dermal absorption in vivo.
In a number of biomonitoring studies, the excretion of 1‐ and 2‐naphthol in urine of persons occupationally not exposed to naphthalene was examined. Tobacco smoking affects the naphthol excretion significantly, thus non‐smokers and smokers have to be considered separately. Up to now, no data of representative collectives of the German general population are available. Therefore, the largest German study was considered for the evaluation, where urine samples of 95 non‐smokers were analysed and a 95th percentile for the sum of 1‐ and 2‐naphthol (after hydrolysis) of 33.6 µg/l (30.6 µg/g creatinine) was found. These results are in good accordance with other national and international studies. Therefore, a BAR of 35 µg 1‐ plus 2‐naphthol (after hydrolysis)/l urine was evaluated for non‐smokers. Sampling time is at the end of exposure or the end of the working shift and after long term exposure at the end of the working shift after several shifts. For the interpretation of the result, the smoking status of the persons has to be considered. In case of an occupational co‐exposure to other polycyclic aromatic hydrocarbons (PAH), the analysis of additional PAH‐metabolites is recommended (see MAK Documentation “Polycyclic Aromatic Hydrocarbons”).
... 1,2-NQ has been proposed to be a toxic metabolite of naphthalene because of its ability to covalently bind to nucleophilic amino acid residues such as cysteine and histidine in cellular proteins (Smithgall et al., 1988;Zheng and Hammock, 1996;Zheng et al., 1997;Penning et al., 1999;Munday et al., 2007;Iwamoto et al., 2007), as shown in Fig. 1. For example, our previous findings suggest that formation of covalent bonds between 1,2-NQ and nucleophilic centers on protein tyrosine phosphatase 1B and on cAMP response element-binding protein (CREB) results in persistent activation of epidermal growth factor receptor (Iwamoto et al., 2007), which leads to tracheal contraction in guinea pigs (Kikuno et al., 2006) and disruption of CREB-mediated transcriptional activity (Endo et al., 2007), respectively. ...
... Taken together, these results suggest that the antibody recognized the naphthalene ring with the dicarbonyl group at the orthoposition, but not at the para-position. Although an epoxide intermediate derived from naphthalene binds to protein (Zheng et al., 1997;Troester et al., 2002) (see Fig. 1), it seems likely that the antibody against 1,2-NQ prepared in our laboratory did not cross-react with the reactive species, because of the absence of a 1,2-dicarbonyl group. ...
Naphthalene undergoes biotransformation by a variety of enzymes to yield 1,2-naphthoquinone (1,2-NQ), a reactive metabolite that binds covalently to proteins. Because this covalent modification is thought to account for naphthalene toxicity, a procedure to detect 1,2-NQ bound to macromolecules is required. In this study, we prepared a polyclonal antibody against 1,2-NQ and examined the specificities of the antibody for various aromatic structures and for the regiochemistry of the quinone functionality. Western blot analysis revealed that the antibody prepared against 1,2-NQ recognized the naphthalene moiety with the ortho-dicarbonyl group, but not with the para-dicarbonyl group; in addition, little cross-reactivity of ortho-quinones with different numbers of aromatic rings (n = 1, 3, 4, 5, 6) was seen. Dot blot and Western blot analyses with the polyclonal antibody enabled quantitative determination of the formation of protein-bound 1,2-NQ during the metabolic activation of naphthalene. The present method can be expected to applicable for the identification of the molecular targets of 1,2-NQ derived from naphthalene in cells and tissues.
... Moreover, naphthalene was known to be metabolized intracellularly into naphthoquinone and naphthalene epoxide that could react with thiol group (SH) of cysteine amino acid residue via nucleophilic substitution and 1,4-Michael addition, respectively. 17 1-Naphthol and naphthoquinone were also reported to produce reactive oxygen species (ROS) under the effect of hepatic microsomal enzymes 18 that recently achieved promising results in cancer treatment. 19 Additionally, naphthoquinone fullled Lipinski's rule of ve to be an orally active drug. ...
Multitarget-directed drugs (hybrid drugs) constitute an efficient avenue for the treatment of multifactorial diseases. In this work, novel naphthalene hybrids with different heterocyclic scaffolds such as nicotinonitrile, pyran, pyranopyrazole, pyrazole, pyrazolopyridine, and azepine were efficiently synthesized via tandem reactions of 3-formyl-4H-benzo[h]chromen-4-one 1 with different nucleophilic reagents. Analysis of these hybrids using PASS online software indicated different predicted biological activities such as anticancer, antimicrobial, antiviral, antiprotozoal, anti-inflammatory, etc. By focusing on antitumor, anti-inflammatory, and antituberculosis activities, many compounds revealed remarkable activities. While 3c, 3e, and 3h were more potent than doxorubicin in the case of HepG-2 cell lines, 3a-e, 3i, 6, 8, 10, 11, and 12b were more potent in the case of MCF-7. Moreover, compounds 3c, 3h, 8, 10, 3d, and 12b manifested superior activity and COX-2 selectivity to the reference anti-inflammatory Celecoxib. Regarding antituberculosis activity, 3c, 3d, and 3i were found to be the most promising with MIC less than 1 mg mL À1. The molecular docking studies showed strong polar and hydrophobic interactions with the novel naphthalene-heterocycle hybrids that were compatible with experimental evaluations to a great extent.
... Naphthalene epoxides and naphthoquinones are the reactive metabolites of naphthalene, responsible for the covalent interaction with cysteine amino acid of cellular proteins and cytotoxicity. Sulfhydryl groups of cysteine residues can react with both naphthalene oxides (epoxide) by SN2 and SN1 reactions and quinone metabolites of naphthalene (1,4and 1,2-naphthoquinone) produced by 1,4-Michael addition reaction [7]. The molecular weight of naphthoquinone is 158.156, ...
... Brachinus fumans (Dean et al. 1990). Ces molécules présentes dans l'environnement sont notamment issues de la dégradation de composés aromatiques tels que la lignine, le benzène ou encore le naphtalène, qui représente l'hydrocarbure aromatique polycyclique le plus abondant dans l'atmosphère (Zheng et al. 1997;Preuss et al. 2003;Lu and Zhu 2007;Ortiz-Bermudez et al. 2007;Kautzman et al. 2010). Ces composés sont très réactifs et les échanges d'électrons entre les formes benzoquinone et hydroquinone au sein de leur cycle redox sont à l'origine de la génération d'espèces oxygénées réactives (Buffinton et al. 1989;O'Brien 1991; Discussion et perspectives Monks et al. 1992;Jarabak and Jarabak 1995). ...
Accès restreint aux membres de l'Université de Lorraine jusqu'au 2015-12-07
... Ingested phenolic compounds become extensively oxidized, which produces ROS (Canada et al., 1990;Barbehenn et al., 2001;Barbehenn et al., 2009). Moreover, quinones, the oxidation products of phenols (Zheng et al., 1997;Barbehenn et al., 2006) may also be toxic and can cause the formation of gut lesions (Thiboldeaux et al., 1998;Perić-Mataruga et al., 2006a) and oxidative stress in the midgut tissues of L. dispar (Pecci, 2011). ...
As a very invasive insect species, Lymantria dispar is adaptable and sensitive to a changing environment. In insects the neuroendocrine system first reacts to stress by production of prothoracicotropic neurohormones (PTTH) that control ecdysteroid synthesis (morphogenetic and stress hormones). In this article, we report changes in the L2' brain neurosecretory neurons that synthesize PTTH in L. dispar larvae after feeding on locust tree leaves (Robinia pseudoacacia), an unsuitable host plant. Groups of larvae (n = 20 per experimental group) were offered this in comparison with oak leaves (Quercus robur), a suitable control diet, for 3 days after molting into the fourth instar. L2' neurons and their nuclei were enlarged and the amount of neurosecretory product in the cytoplasm was increased (15.5%) after consumption of locust tree leaves in comparison to the control. Furthermore, activities of the following antioxidative defense components were estimated: superoxide dismutase (SOD), catalase (CAT), and amount of glutathione in the midgut. Higher SOD activity (13.85 ± 0.9 U/mg prot.) and glutathione amount (0.56 ± 0.06 μMGSH/g tissue) but unchanged CAT activity was found in the midgut of larvae offered locust tree leaves when compared to the control.
... They react with the electron-rich DNA to produce DNA adducts and/or DNA strand breaks, causing DNA mutations and eventually carcinogenesis (Costa et al. 2012). They are also reported to cause enzyme inhibition by epoxide-protein adduct formation (Thomas et al. 1969), and to bind to critical protein targets leading to several toxic effects (Zheng et al. 1997). Recently, the biological functions of several epoxides, most prominently the epoxides of fatty acids, have been reported to mediate several signaling pathways. ...
Epoxide hydrolases (EH) are ubiquitously expressed in all living organisms and in almost all organs and tissues. They are mainly subdivided into microsomal and soluble EH and catalyze the hydration of epoxides, three-membered-cyclic ethers, to their corresponding dihydrodiols. Owning to the high chemical reactivity of xenobiotic epoxides, microsomal EH is considered protective enzyme against mutagenic and carcinogenic initiation. Nevertheless, several endogenously produced epoxides of fatty acids function as important regulatory mediators. By mediating the formation of cytotoxic dihydrodiol fatty acids on the expense of cytoprotective epoxides of fatty acids, soluble EH is considered to have cytotoxic activity. Indeed, the attenuation of microsomal EH, achieved by chemical inhibitors or preexists due to specific genetic polymorphisms, is linked to the aggravation of the toxicity of xenobiotics, as well as the risk of cancer and inflammatory diseases, whereas soluble EH inhibition has been emerged as a promising intervention against several diseases, most importantly cardiovascular, lung and metabolic diseases. However, there is reportedly a significant overlap in substrate selectivity between microsomal and soluble EH. In addition, microsomal and soluble EH were found to have the same catalytic triad and identical molecular mechanism. Consequently, the physiological functions of microsomal and soluble EH are also overlapped. Thus, studying the biological effects of microsomal or soluble EH alterations needs to include the effects on both the metabolism of reactive metabolites, as well as epoxides of fatty acids. This review focuses on the multifaceted role of EH in the metabolism of xenobiotic and endogenous epoxides and the impact of EH modulations.
... Microsomal epoxide hydrolase (mEH) is a key hepatic enzyme involved in the metabolism of numerous xenobiotics, such as polyaromatic hydrocarbons, which undergo cytochromedependent epoxidation (3,4). Additionally, mEH is likely involved in the extrahepatic metabolism of these agents, such as naphthalene in the lungs (5). Generally, the conversion of epoxides to diols results in less mutagenic or carcinogenic compounds. ...
The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to several diseases, such as emphysema, spontaneous abortion, and several forms of cancer. To provide new tools for studying the function of mEH, inhibition of this enzyme was investigated. Inhibition of recombinant rat and human mEH was achieved using primary ureas, amides, and amines. Several of these compounds are more potent than previously published inhibitors. Elaidamide, the most potent inhibitor that is obtained, has a Ki of 70 nM for recombinant rat mEH. This compound interacts with the enzyme forming a noncovalent complex, and blocks substrate turnover through an apparent mix of competitive and noncompetitive inhibition kinetics. Furthermore, in insect cell cultures expressing rat mEH, elaidamide enhances the toxicity effects of epoxide-containing xenobiotics. These inhibitors could be valuable tools for investigating the physiological and toxicological roles of mEH.
... Indeed, recent work with protein disulfide isomerase and actin demonstrate relatively high selectivity for modification of specific cysteines, lysines and histidines by both epoxide and quinone metabolites of naphthalene [33]. The results of the current studies are consistent with previous findings showing epoxide binding to sulfur nucleophiles was minor relative to binding by the 1,2-naphthoquinone in Clara cell incubations with naphthalene [34]. In naphthalene-treated mice, 1,4-NQ adducts were more abundant than NO adducts in all tissue samples studied, with the lung carrying the highest NQ load [35] . ...
Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.
... Naphthalene is metabolized by cytochrome P450 (CYP) isozymes (CYP 1A1, CYP 1A2, CYP 2A1, CYP 2E1 and CYP 2F2) to 1-naphthol, 2-naphthol and naphthalene 1, 2-oxide that are unstable at physiological pH and can react with glutathione to reduce glutathione level and to increase oxidative stress (Jerina et al., 1970;Wilson et al., 1996). Both 1-naphthol and 2-naphthol compounds are oxidized either enzymatically or nonenzymatically to 1,4-naphthoquinone (1,4-NPQ) and 1,2-NPQ, respectively (Chichester et al., 1994;Lin et al., 2009;Wilson et al., 1996;Zheng et al., 1997). These compounds cause DNA damage by binding N3 and N7 position guanine and adenine in DNA and increasing oxidative stress (Bagchi et al., 1998(Bagchi et al., , 2001Saeed et al., 2009). ...
Naphthalene, a bicyclic aromatic hydrocarbon, has toxic effects on animals and humans. Although recent studies stressed on the genotoxic and cytotoxic effects of naphthalene and its metabolites on eukaryotic cells, there is a big controversy among the results of these studies. The aim of this study is to investigate the effects of naphthalene and its metabolites on the cytotoxicity and genotoxicity in the human lymphocytes in the culture. The genotoxic and cytotoxic effects of naphthalene and its metabolites, 1-naphthol and 2-naphthol, were studied using cytotoxicity test (lactate dehydrogenase and cell proliferation (WST-1) assays) and DNA fragmentation assay (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay). Naphthalene and its metabolites had no significant cytotoxic effect on treated samples when compared with untreated ones. This result was also confirmed by WST-1 assay. In the TUNEL assay, DNA fragmentation was induced significantly by all concentrations of naphthalene and 2-naphthol and 50 and 100 µM concentrations of 1-naphthol (p < 0.05 or 0.001). In the DNA fragmentation, the most effective dose of 2-naphthol (63%) was 100 µM, when compared with negative control group (13%). These results suggest that naphthalene and its metabolites, 1-naphthol and 2-naphthol, may cause DNA damage on human lymphocytes.
... In those isolated cells, incubation with 0.5 mM 1,2dihydrodioxy-1,2-dihydronaphthalene (henceforth referred to as dihydrodiol), 1-naphthol, or 1,2-naphthoquinone decreased cell viability approximately as effectively as 0.5 mM naphthalene, whereas incubation with 0.5 M naphthalene oxide or 1,4-naphthoquinone significantly decreased viability more than the same concentration of parent compound; moreover, naphthalene-oxide-induced cytotoxicity was not blocked with piperonyl butoxide (Chichester et al., 1993). In isolated mouse-lung Clara cells exposed to naphthalene, 1,2naphthoquinone was one of two detected types of covalent, naphthalene-related protein adduct (Zheng et al., 1997). Incubation of isolated murine hepatocytes with the (1S,2R)naphthalene-oxide enantiomer-which is converted much more slowly to the dihydrodiol than the (1R,2S)-epoxide-resulted in nearly complete loss of cell viability, whereas incubation with the (1R,2S) enantiomer with a shorter half life had almost no effect on cell viability (Buonarati et al., 1989). ...
This report provides a summary of deliberations conducted under the charge for members of Module C Panel participating in the Naphthalene State-of-the-Science Symposium (NS(3)), Monterey, CA, October 9-12, 2006. The panel was charged with reviewing the current state of knowledge and uncertainty about naphthalene metabolism in relation to anatomy, physiology and cytotoxicity in tissues observed to have elevated tumor incidence in these rodent bioassays. Major conclusions reached concerning scientific claims of high confidence were that: (1) rat nasal tumor occurrence was greatly enhanced, if not enabled, by adjacent, histologically related focal cellular proliferation; (2) elevated incidence of mouse lung tumors occurred at a concentration (30 ppm) cytotoxic to the same lung region at which tumors occurred, but not at a lower and less cytotoxic concentration (tumorigenesis NOAEL=10 ppm); (3) naphthalene cytotoxicity requires metabolic activation (unmetabolized naphthalene is not a proximate cause of observed toxicity or tumors); (4) there are clear regional and species differences in naphthalene bioactivation; and (5) target tissue anatomy and physiology is sufficiently well understood for rodents, non-human primates and humans to parameterize species-specific physiologically based pharmacokinetic (PBPK) models for nasal and lung effects. Critical areas of uncertainty requiring resolution to enable improved human cancer risk assessment were considered to be that: (1) cytotoxic naphthalene metabolites, their modes of cytotoxic action, and detailed low-dose dose-response need to be clarified, including in primate and human tissues, and neonatal tissues; (2) mouse, rat, and monkey inhalation studies are needed to better define in vivo naphthalene uptake and metabolism in the upper respiratory tract; (3) in vivo validation studies are needed for a PBPK model for monkeys exposed to naphthalene by inhalation, coupled to cytotoxicity studies referred to above; and (4) in vivo studies are needed to validate a human PBPK model for naphthalene. To address these uncertainties, the Panel proposed specific research studies that should be feasible to complete relatively promptly. Concerning residual uncertainty far less easy to resolve, the Panel concluded that environmental, non-cytotoxic exposure levels of naphthalene do not induce tumors at rates that can be predicted meaningfully by simple linear extrapolation from those observed in rodents chronically exposed to far greater, cytotoxic naphthalene concentrations.
... It is a key hepatic enzyme involved in the metabolism of numerous xenobiotics such as the epoxides of 1,3-butadiene, styrene, naphthalene, benzo(a)pyrene, phenantoin, and carbamazepine [3][4][5][6][7]. The mEH is also involved in the extrahepatic metabolism of these agents [8,9]. Whereas for most compounds mEH action is a detoxification process [3][4][5], in some cases (e.g., for benzo(a)pyrene 4,5-oxide) diol formation can lead to the stabilization of a secondary epoxide, thereby increasing the mutagenicities and carcinogenicities of the product [10,11]. ...
The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.
... Our initial analyses were directed at two groups of key organic compounds, the PAHs and representative quinones, 1,2-and 1,4-naphthoquinone (1,2-and 1,4-NQ), 9-10-phenanthraquinone (PQ) and 9,10-anthraquinone (AQ). Quinones are directly formed by combustion of gasoline and diesel fuels ( Jakober et al., 2007), and also converted from PAHs either by photochemistry (Eiguren- Fernandez, 2008b) or cellular metabolism ( Penning et al., 1999;Zheng et al., 1997). The quinone assay used here estimates the concentration of four representative quinones. ...
... Dissociation of singly and doubly protonated peptide precursor ions afforded, along with formation of sequence fragments corresponding to the loss of S-(2,5-diidroxyphenyl)cysteine (211u), also non-sequence fragments due to loss of the adducted xenobiotic as neutral hydroquinone-2-thiol (142u) (Mason & Liebler, 2000). The 1,4-quinone metabolite of naphtalene binds to Clara cell proteins, as demonstrated by measurement by GC-MS of the corresponding degradation product 3,4-dimethoxy(methylthio)naphthalene obtained by alkaline permethylation (Zheng et al., 1997). Hemoglobin and albumin adducts of 1,2-naphthoquinone and 1,4-naphthoquinone were also detected after administration of naphthalene to F344 rats (Waidyanatha et al., 2002Waidyanatha et al., , 2004). ...
Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein-electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors.
Accumulated epidemiological and animal studies have suggested that prolonged exposure to ambient particulate matter (PM) is associated with an increased risk of cardiovascular disease and pulmonary dysfunction. While diesel exhaust particles (DEP) contain large variety of compounds, polycyclic aromatic hydrocarbons (PAHs) are a dominant component contaminated in DEP. This article reviews effects of two PAH quinones, 9,10-phenanthraquinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), on vascular and respiratory systems.
Electrophilic quinones are produced during the combustion of gasoline in the atmosphere. Although these reactive species covalently bind to protein-based nucleophiles in cells, resulting in the formation of protein adducts involved in the modulation of redox signaling pathways and cytotoxicity, the extracellular regulation of quinones is not understood. In this study, incubation of 1,2-naphthoquinone (1,2-NQ) with the low-molecular-weight fraction of mouse plasma resulted in the consumption of cysteine (CysSH) in the plasma in a concentration-dependent manner. Covalent modification of albumin was markedly repressed by the addition of either the low-molecular-weight fraction of mouse plasma or CysSH, suggesting that CysSH protects by forming a conjugate with 1,2-NQ. Similar phenomena also occurred for other atmospheric quinones 1,4-NQ and 1,4-benzoquinone (1,4-BQ). The addition of cystine to a culture medium without amino acids enhanced the release of CysSH from A431 cells and blocked 1,2-NQ-mediated arylation of intracellular proteins, suggesting that 1,2-NQ interacts with extracellular CysSH. Liquid chromatography-tandem mass spectrometry analysis revealed that 1,2-NQ and 1,4-BQ undergoes nucleophilic attack by CysSH, yielding a 1,2-NQH2-SCys adduct and 1,4-BQH2-SCys adduct, respectively. Unlike 1,2-NQ and 1,4-BQ, the authentic 1,2-NQH2-SCys adduct and 1,4-BQH2-SCys adduct had little effect on the covalent modification of cellular proteins and viability of A431 cells. These results suggest that electrophilic quinones are readily trapped by CysSH released from A431 cells, forming less-toxic CysSH adducts and thereby repressing covalent modification of cellular proteins. These findings provide evidence for the existence of a "phase zero" reaction of electrophiles prior to their uptake by cells.
Polycyclic aromatic phenols (PAPs or hydroxy-PAHs) are conveniently converted into their corresponding ortho-quinones using commercially available stabilized iodoxybenzoic acid (SIBX). SIBX provides a safer and commercially available alternative to IBX and displays the same selectivity with comparable or better yields in the oxidative dearomatization of phenols to ortho-quinone, including examples where formation of para-quinones is feasible. This ortho-selectivity from all positions of a hydroxy-group allowed for simple synthesis of the prerequisite hydroxy-PAHs by either photochemical cyclization of stilbenes or Pt-catalyzed cycloisomerization. The later synthesis involved a four-step sequence where suitably substituted biphenyls were prepared by Suzuki-Miyaura cross-coupling, followed by the Corey–Fuchs protocol and cycloisomerization by a catalytic amount of PtCl2. 2- and 4-methylphenanthene were also prepared for the first time using this method.
Quinones and quinone imines are highly reactive metabolites (RMs) able to induce dangerous effects in vivo. They are responsible for all kinds of toxicity: e.g. cytotoxicity, immunotoxicity and carcinogenesis. Furthermore, hepatotoxicity of chemicals/drugs in particular can be induced by quinone and quinone imine metabolites. According to their reactivity quinones and quinone imines react as Michael's acceptors with cell proteins and/or DNA and, in this way, cause damage to the cells. Quinones and quinone imines also have high redox potential and, due to their semiquinone radicals, are capable of redox cycling and forming reactive oxygen species (ROS). However, the presence of quinones and quinone imines structures in compounds is not always responsible for a toxic effect. The main question, therefore, is what are the main factors responsible for the toxicity of the chemicals and drugs that form RMs. For this reason, the presence of structural alerts and evidence for the formation of reactive quinones and quinone imines metabolites and their mechanisms of toxicity through cellular effects are discussed in this review, together with examples.
Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H2O2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H2O2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air.
After environmental and occupational exposure to naphthalene, 1,2-dihydroxynaphthalene (1,2-DHN) was shown to be one major metabolite in human naphthalene metabolism. However, the instability of free 1,2-DHN complicates the reliable determination of this promising biomarker in urine. To solve this stability problem, glucuronide conjugates of 1,2-DHN and the corresponding isotopically labelled D6-1,2-dihydroxynaphthalene (D6-1,2-DHN) were synthesised and applied as reference material and internal standard in a gas chromatographic-tandem mass spectrometric (GC-MS/MS) method. The determination of 1- and 2-naphthol (1-MHN, 2-MHN) was included in the procedure to enable a comprehensive assessment of naphthalene metabolism and exposure. The results of the validation showed a high reliability and sensitivity of the method. The detection limits range from 0.05 to 0.16 μg/L. Precision and repeatability were determined to range from 1.4 to 6.6% for all parameters. The simultaneous determination of 1- and 2-MHN as additional parameters besides 1,2-DHN enables the application of the method for further metabolism and kinetic studies on naphthalene. The use of glucuronide-derivative reference substances and the application of structurally matched isotopic-labelled internal standards for each substance guarantee a reliable quantification of the main naphthalene metabolites 1,2-DHN and 1- and 2-MHN. Open image in new windowGraphical abstractReliable quantification of 1,2-dihydroxynaphthalene in urine using a conjugated reference compound for calibration
Included among the many environmental electrophiles aromatic hydrocarbon quinones formed during combustion of gasoline, crotonaldehyde in tobacco smoke, methylmercury accumulated in fish, cadmium contaminated in rice, and acrylamide in baked foods are common examples. These electrophiles can modify nucleophilic functions such as cysteine residues in proteins forming adducts and in the process activate cellular redox signal transduction pathways such as kinases and transcription factors. However, higher concentrations of electrophiles disrupt such signaling by nonselective covalent modification of cellular proteins. Persulfide/polysulfides produced by various enzymes appear to capture of environmental electrophiles because formation of their sulfur adducts without electrophilicity. We therefore speculate that persulfide/polysulfides are candidates for regulation of redox signal transduction pathways (e.g., cell survival, cell proliferation, and adaptive response) and toxicity during exposure to environmental electrophiles.
Benzbromarone (BBR) is a therapeutically useful uricosuric agent but can also cause acute liver injury. The hepatotoxicity of BBR is suggested to be associated with its metabolic activation. Our recent metabolic study demonstrated that BBR was metabolized to epoxide intermediate(s) by cytochromes P450 3A and the intermediate(s) was reactive to N-acetylcysteine. The objectives of the present study were to determine the chemical identity of the interaction of protein with the epoxide intermediate(s) of BBR and to define the association of the protein modification with hepatotoxicity induced by BBR. Microsomal incubation study showed that the reactive intermediate(s) covalently modified microsomal protein at cysteine residues. Such adduction was also observed in hepatic protein obtained from liver of mice given BBR. The protein covalent binding occurred in time- and dose-dependent manners. Pretreatment with ketoconazole attenuated BBR-induced protein modification and hepatotoxicity, while pretreatment with dexamethasone or buthionine sulfoximine potentiated the protein adduction and hepatotoxicity induced by BBR. A good correlation was observed between BBR-induced hepatotoxicity and the epoxide-derived hepatic protein modification in mice. The present study provided in-depth mechanistic insight into BBR-induced hepatotoxicity.
Benzbromarone (BBR) is a benzofuran derivative that has been a quite useful drug for the treatment of gout. However, it was withdrawn from European markets in 2003, due to reported serious incidents of drug-induced liver injury. BBR-induced hepatotoxicity has been suggested to be associated with the formation of a quinone intermediate. The present study reported epoxide-derived intermediate(s) of BBR. An N-acetyl cysteine (NAC) conjugate derived from epoxide metabolite(s) was detected in both microsomal incubations of BBR and urine samples of mice treated with BBR. The NAC conjugate was identified as 6-NAC BBR. We chemically synthesized 6-NAC BBR. Ketoconazole suppressed the bioactivation of BBR to the epoxide intermediate(s), and CYP3A subfamily was the primary enzyme responsible for the formation of the epoxide(s). The present study provided new information on metabolic activation of BBR.
Polycyclic aromatic hydrocarbons (PAHs) are moderately reactive, but undergo photochemical degradation in the atmosphere, and are widely used as chemical raw materials. Aromatic hydrocarbons cause local irritation and changes in endothelial cell permeability and are absorbed rapidly. Accumulation of aromatic hydrocarbons in marine animals occurs to a greater extent and retention is longer compared to alkanes. Toxicity of polynuclear aromatics has been reported comprehensively. It has been reported that exposure to a variety of complex mixtures containing these chemicals, such as soot, coal tar and pitch, mineral oils, coal gasification residues, and cigarette smoke has historically been associated with induction of cancer. Naphthalene causes cataracts in the eyes of experimental animals. Its vapors may cause severe systemic injury. Alkylbenzenes are readily aspirated and produce instant death via cardiac arrest and respiratory paralysis. In general, the acute toxicity of alkylbenzenes is higher for toluene than that for benzene and decreases further with increasing chain length of the substituent, except for highly branched C8 to C18 derivatives. Polycyclic aromatic hydrocarbons are metabolized through epoxides and hydroxides and are excreted as conjugates.
Keywords:
aryl hydrocarbon hydroxylase;
Alkyl benzene;
anthracene;
heterocyclic;
polyphenol;
naphthalene
The Separation of Interference Fringes of the Main Diffraction Maxima (SIFMDM) and the second maximum for Cross Segmented Wedge Array (CSWA) focus system were derived, and the change rules of the Small-scale Irradiation Non-uniformity (SSINU) were confirmed by numerical calculations based on the generalized Huygens-Fresnel diffraction integral theory. The two methods of reducing the SSINU, the Deviation of Wedge Angles (DWA) and the Off-focal Laser Radiation on the Target (OFLRT), were proposed. Theoretical study and experimental results show they agree quite well. By both the DWA and the OFLRT, the SIFMDM can be reduced by 6 times and the SSINU is also improved obviously, when the DWAS and off-focal variables are optimized to δpj = 20%, ΔZ = 2 mm.
A novel lens arrays optical system with continuously variable focus width from several microns to several millimeters for providing uniform irradiation is proposed, which is composed of two lens arrays and an aspherical lens, and the system converts a circular laser beam into a flat-top square focused spot, which realizes the transformation of beam-shape and the uniform distribution of the spatial intensity at the same time. Based on the adaxial matrix optics and scalar quantity diffraction integral theory, the principle of this system is analyzed, and optimum design of the system parameters and numerical calculations for this system are presented in detail. The simulated results show that the nonuniformity along the x and y direction are η x = 4.6%, η y = 5.3% respectively, the energy efficiency can be up to 95.3%, and by moving the target slightly backward from the focal plane, off-focus length Δz = 2mm is obained, which nearly satisfies the need. The theoretical analysis agrees with the results of the emulational experiment with Zemax optical design software.
Naphthalene shows carcinogenic properties in animal experiments. As the substance is ubiquitary present in the environment and has a possibly high exposure at industrial workplaces, the determination of naphthalene metabolites in humans is of environmental-medical as well as occupational-medical importance. Here, biomarkers of 1,2- and 1,4-naphthoquinone, as possibly carcinogenic metabolites in the naphthalene metabolism, are of outstanding significance. We developed and validated a liquid chromatography-tandem mass-spectrometric (LC-MS/MS) method for the simultaneous determination of the naphthoquinone mercapturic acids of 1,2- and 1,4-naphthoquinone in human urine samples as a sum of naphthoquinone- and dihydroxynaphthalene-mercapturic acid. Except for enzymatic hydrolysis and acidification, no further sample preparation is necessary. For sample clean-up, a column switching procedure is applied. The mercapturic acids are extracted from the urinary matrix on a restricted access material (RAM RP 18) and separated on a reversed phase column (Synergi Polar RP C18). The metabolites were quantified by tandem mass spectrometry using labelled D5-1,4-NQMA as internal standard. The limits of detection are 3μg/l for 1,2-NQMA and 1μg/l for 1,4-NQMA. Intraday- and interday precision for pooled urine (spiked with 10μg/l and 30μg/l of the analytes) ranges from 5.9 to 15.1% for 1,2-NQMA and from 2.0 to 10.8% for 1,4-NQMA. The developed method is suited for the sensitive and specific determination of the mercapturic acids of naphthoquinones in human urine. A good precision and low limits of detection were achieved. Application of those new biomarkers in biomonitoring studies may give deeper insights into the mechanisms of the human naphthalene metabolism.
Epoxides are organic three-membered cyclic oxygen compounds that derive from oxidative metabolism of endogenous metabolites (e.g., intermediate in cholesterol biosynthesis), as well as xenobiotic compounds (e.g., metabolite of benzpyrene), via chemical and enzymatic oxidation processes. The resultant unstable, chemically reactive epoxides are known to be mutagenic and carcinogenic initiators. Therefore, it is of vital importance to regulate levels of these reactive species. Epoxide hydrolase, also known as Arene-oxyde hydratase, catalyzes the hydrolysis of arene and aliphatic epoxides to less reactive and more water-soluble dihydrodiols by the trans addition of water. The following general equation characterizes these enzymes: H 2O + epoxide = glycol. There are two epoxide hydrolase subtypes: The microsomal, membrane bound (mEH, EPHX1) and the soluble, cytosolic form (sEH, EPHX2).
This chapter discusses the uses, physical and chemical properties, mammalian toxicology, sources of exposure, industrial hygiene and medical management of naphthalene. Naphthalene is commonly produced from the distillation and fractionation of coal tar. Moth balls and other moth repellants, and some solid block deodorizers used for toilets and diaper pails, are made of crystalline naphthalene. Dermal contact with naphthalene may cause skin irritation and an allergic skin reaction. Contact with the eyes may cause conjunctivitis, corneal injury, diminished visual acuity, and cataracts. The mechanisms of acute naphthalene toxicity are known for hemolytic anemia and cataract formation. The highest occupational exposure to naphthalene occurs in wood treatment (creosote), coke and coal tar production, mothball production, and jet fuel industries. In the case of accidental inhalation exposure, the subject should first be removed to an area with fresh air and good ventilation and then referred for medical attention.
Inhalation of naphthalene causes olfactory epithelial nasal tumors in rats (but not in mice) and benign lung adenomas in mice (but not in rats). The limited available human data have not identified an association between naphthalene exposure and increased respiratory cancer risk. Assessing naphthalene's carcinogenicity in humans, therefore, depends entirely on experimental evidence from rodents. We evaluated the respiratory carcinogenicity of naphthalene in rodents, and its potential relevance to humans, using our Hypothesis-Based Weight-of-Evidence (HBWoE) approach. We systematically and comparatively reviewed data relevant to key elements in the hypothesized modes of action (MoA) to determine which is best supported by the available data, allowing all of the data from each realm of investigation to inform interpretation of one another. Our analysis supports a mechanism that involves initial metabolism of naphthalene to the epoxide, followed by GSH depletion, cytotoxicity, chronic inflammation, regenerative hyperplasia, and tumor formation, with possible weak genotoxicity from downstream metabolites occurring only at high cytotoxic doses, strongly supporting a non-mutagenic threshold MoA in the rat nose. We also conducted a dose-response analysis, based on the likely MoA, which suggests that the rat nasal MoA is not relevant in human respiratory tissues at typical environmental exposures. Our analysis illustrates how a thorough WoE evaluation can be used to support a MoA, even when a mechanism of action cannot be fully elucidated. A non-mutagenic threshold MoA for naphthalene-induced rat nasal tumors should be considered as a basis to determine human relevance and to guide regulatory and risk-management decisions.
Introduction: Honey bees collect floral nectar and pollens, for feeding, which rich with allelochemicals and phenols. The oxidation of these materials produce reactive oxygen species (ROS), among them the hydrogen peroxide (H 2 O 2) and superoxide. The honey bee antioxidant enzymes are of particular interest in detoxification of these ROS. Superoxide dismutases (SODs) are the first line of defense against oxygen free radicals. In concert with catalase; SODs have strong antioxidant properties. Since catalase is inefficient at removing H 2 O 2 because of its high K m the ascorbate peroxidase (APOX) serves better in H 2 O 2 detoxification. Results: The antioxidant enzymes under investigation showed highly significant variation during the whole duration of the experiment. However, a combined effect of months, race (Hybrid and Carniolan) and type (foraging and nursing) showed similar activity in SOD and CAT. Conclusion: The correlations between SOD, CAT and APOX, indicating that there must be a specific manner strategy for managing peroxides at safe levels. The increase or decreases of the antioxidants are according to the contents and levels of peroxides.
Accumulated epidemiological and animal studies have suggested that prolonged exposure to ambient particulate matter (PM) is associated with an increased risk of cardiovascular disease and pulmonary dysfunction. While diesel exhaust particles (DEP) contain large variety of compounds, polycyclic aromatic hydrocarbons (PAHs) are a dominant component contaminated in DEP. This article reviews effects of two PAH quinones, 9,10-phenanthraquinone (9,10-PQ) and l,2-naphthoquinone (l,2-NQ), on vascular and respiratory systems.
The transcriptional regulator QsrR is converted into a genetically encoded fluorescent probe capable of ratiometric monitoring of quinones in living cells with high sensitivity and selectivity.
The article contains sections titled:
Aromatic hydrocarbons are the class of chemicals that include multi‐ring aromatic compounds. Smaller aromatic hydrocarbons, one to two rings, are of considerable economic importance as industrial raw materials, solvents, and components of innumerable commercial and consumer products. However, aromatics differ vastly in chemical, physical, and biological characteristics from the aliphatic and alicyclic hydrocarbons. In addition, aromatics are more toxic to humans and other mammals. Of prime importance are the carcinogenicity of styrene and the polycyclic aromatic hydrocarbons.
Chemically, aromatic hydrocarbons can be divided into three groups: (a) alkyl‐, aryl‐, and alicyclic‐substituted benzene derivatives, (b) di‐ and polyphenyls, and (c) polycyclic compounds composed of two or more fused benzene ring systems. The basic chemical entity is the benzene nucleus, which occurs alone, substituted, joined, or fused.
Aromatics are moderately reactive and undergo photochemical degradation in the atmosphere. Aromatic compounds occur in liquid, vapor, or solid form. The lower molecular weight derivatives possess higher vapor pressures, volatility, absorbability, and solubility in aqueous media than the comparable aliphatic or alicyclic compounds. These properties contribute to their biological activities. They are characterized also by miscibility or conversion to compounds soluble in aqueous body fluids, high lipid solubility, and donor–acceptor and polar interaction. Because of their low surface tension and viscosity, aromatics may be aspirated into the lungs during ingestion, where they can cause chemical pneumonitis.
Aromatics are primary skin irritants, and repeated or prolonged skin contact may cause dermatitis, dehydrating, and defatting of the skin. Eye contact with aromatic liquids may cause lacrimation, irritation, severe burns from prolonged contact. Naphthalene causes cataracts in the eyes of experimental animals. Its vapors are respiratory and mucous membrane irritants and may cause severe systemic injury. Direct aerosol deposition or contact from ingestion and subsequent aspiration can cause severe pulmonary edema, pneumonitis, and hemorrhage. Alkylbenzenes that have C 1 to C 4 side chains are readily aspirated and can produce instant death via cardiac arrest and respiratory paralysis. For example, in hexylbenzene exposure, death occured in 18 min, during which extensive pulmonary edema occured, resulting in a considerable increase in lung weight. The higher alkylbenzenes showed few or no effects. The unique effects of benzene on bone marrow and blood‐forming mechanisms are of major importance. In general, the acute toxicity of alkylbenzenes is higher for toluene than for benzene and decreases further with increasing chain length of the substituent, except for highly branched C 8 to C 18 derivatives. The toxicity increases again for vinyl derivatives. Pharmacologically, the alkylbenzenes are CNS depressants, since they exhibit a particular affinity to nerve tissues.
Aromatic hydrocarbons cause local irritation and changes in endothelial cell permeability and are absorbed rapidly. Secondary effects have been observed in the liver, kidney, spleen, bladder, thymus, brain, and spinal cord in animals. Aromatic hydrocarbons, even from a single dose, exhibit a special affinity to nerve tissue. Animals dosed with alkylbenzenes exhibit signs of CNS depression, sluggishness, stupor, anesthesia, and coma. This is in sharp contrast with benzene, which is a neuroconvulsant and produces tremors and convulsions. The CNS depressant potency of the alkylbenzenes depends on branching or side‐chain length. It diminishes with increasing numbers of substituents or side‐chain carbon number up to dodecylbenzene, which has practically no CNS depressant activity.
Aromatic hydrocarbons accumulate in marine animals to a greater extent and are retained longer than alkanes. In all species tested, the accumulation of aromatic hydrocarbons depended primarily on the octanol/water partition coefficient. Once absorbed, higher molecular weight hydrocarbons are released more slowly.
Polycyclic aromatic hydrocarbons are mainly solid materials that are soluble in fats, oils, and organic solvents. The mutagenic or carcinogenic properties of PAHs have been linked to physicochemical properties, such as electronegativity or K‐ and L‐region reactivity, electrophilic potency, dipole moment, intramolecular and subcellular binding, hydrophobicity, and others. However, these characteristics alone are inadequate for specific predictions.
Polynuclear aromatics are practically nontoxic for acute ingestion and acute dermal application.
Enzyme systems, such as aryl hydrocarbon hydroxylase (AHH), are present in almost all human and animal cell tissues and are inducible by noncarcinogenic and potentially carcinogenic hydrocarbons. The stability of cytochrome P450 epoxidase may depend on immunologic competence, as does the epoxide hydrase. BaP is both teratogenic and mutagenic in rodents.
Four‐ and five‐ring PAHs are carcinogenic. They include the benz(a)anthracenes, benzofluoranthracenes, benzo(a)pyrenes, chrysenes, and dibenz(a,h)anthracene. The OSHA and ACGIH ceiling for coal tar pitch volatiles that contain one or more PAHs is 0.2 ppm with a cancer notation.
Sampling techniques include collecting air particles using an absorbent glass sampler, desorption with pentane, and quantification using spectral analysis. Collection on acrylonitrile‐PVC filters is also recommended. Analytic quantification is also achieved by using gas chromatography high‐resolution mass spectrometry or chemiluminescence. Methods for cleanup from waste water are also available.
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants which may cause cancer and require metabolic activation to exert their carcinogenic effects. As a step toward identifying the spectrum of PAH o-quinone-amino acid adducts that may form in biological systems, several naphthalene-1,2-dione-amino acid adducts were synthesized. Each adduct was formed as a 1,4-Michael addition product and spectral data corroborate that these adducts were either o-quinones or catechols. PAH o-quinone adducts can be used as standards to identify their presence in vitro and in vivo.
Quinones are a group of highly reactive organic chemical species that interact with biological systems to promote inflammatory, anti-inflammatory, and anticancer actions and to induce toxicities. This review describes the chemistry, biochemistry, and cellular effects of 1,2- and 1,4-naphthoquinones and their derivatives. The naphthoquinones are of particular interest because of their prevalence as natural products and as environmental chemicals, present in the atmosphere as products of fuel and tobacco combustion. 1,2- and 1,4-naphthoquinones are also toxic metabolites of naphthalene, the major polynuclear aromatic hydrocarbon present in ambient air. Quinones exert their actions through two reactions: as prooxidants, reducing oxygen to reactive oxygen species; and as electrophiles, forming covalent bonds with tissue nucleophiles. The targets for these reactions include regulatory proteins such as protein tyrosine phosphatases; Kelch-like ECH-associated protein 1, the regulatory protein for NF-E2-related factor 2; and the glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase. Through their actions on regulatory proteins, quinones affect various cell signaling pathways that promote and protect against inflammatory responses and cell damage. These actions vary with the specific quinone and its concentration. Effects of exposure to naphthoquinones as environmental chemicals can vary with the physical state, i.e., whether the quinone is particle bound or is in the vapor state. The exacerbation of pulmonary diseases by air pollutants can, in part, be attributed to quinone action.
The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numerous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been extensively studied and numerous strategies have been employed to develop inhibitors against these enzymes. Herein, we report on the optimization of our novel small-molecule inhibitor SKI-I (N'-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) and the identification of a SphK1-specific analog, SKI-178, that is active in vitro and in vivo. This SphK1 specific small-molecule, non-lipid like, inhibitor will be of use to elucidate the roles of SphK1 and SphK2 in the development/progression of hyperproliferative and/or inflammatory diseases.
Human health risk assessment consists of bringing to bear a large body of in vitro, animal, and epidemiologic studies on the question of whether environmental exposures to a substance are a potential risk to humans. The body of scientific information is typically less than definitive and often contains apparent contradictions. Often various possible conclusions about potential human risks may be drawn from the data and these may vary from very strong to tenuous. The task, therefore, is to communicate the uncertainties in the inferences from the data effectively, giving proper consideration to contrary data and alternative scientifically plausible interpretations. We propose an approach, Hypothesis-Based Weight of Evidence (HBWoE), to organize, evaluate, and communicate the large body of available relevant data on a given chemical, using naphthalene as an example. The goal for our use of the term "weight of evidence" (WoE) is broad in that we express the relative degrees of credence that should be placed in alternative possible interpretations of the naphthalene data and hypothesized carcinogenic modes of action (MoAs), expressed in a way that shows how such credence is tied to specific scientific interpretations, considering consistencies, inconsistencies, and contradictions within the data set.
Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, beta-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.
Naphthalene is metabolized by several cytochrome P-450 (CYP) monooxygenases to 1,2-epoxynaphthalene. However, the subsequent interactions of the epoxide with macromolecules in the cells, and the significance of these interactions to cellular injury, are not well characterized. Additionally, CYP1A1, which can metabolize naphthalene to 1,2-epoxynaphthalene, may be induced by a number of xenobiotics. Yet, the in situ interaction between naphthalene and CYP1A1 alone, without the influence of other xenobiotic metabolizing enzymes, has not been examined. Using a model eukaryotic expression system capable of over-expressing recombinant CYP1A1, we found that naphthalene was toxic to cells expressing CYP1A1 in a dose- (LC50: 0.3 mM) and time-dependent (LT50: 12 h) manner. Naphthalene treatment of CYP1A1-expressing cells resulted in a 47% decrease in cellular glutathione (GSH) levels. Pretreatment with ethyl ester GSH, a GSH analog, protected CYP1A1-expressing cells such that viability was 30% greater than for cells treated with naphthalene alone. Cytotoxicity was strongly correlated ( r2 : 0.96) with covalent binding of cellular proteins. Alkaline permethylation techniques showed that cysteinyl-SH groups of cellular proteins are a nucleophilic target of the epoxide metabolite. These results suggest that, in the absence of other pathways, naphthalene is modified by CYP1A1 to 1,2-epoxynaphthalene, which subsequently binds cellular sulfhydryl groups on proteins and GSH.
En general los procesos de biotransformación de fármacos originan metabolitos excretables por la orina. Nitrofurantoína y Nifurtimox son compuestos lipofílicos, sin embargo no existen antecedentes acerca de la eliminación de metabolitos de estos fármacos en orina. Su administración induce diversos efectos adversos asociados a la nitrorreducción que ellos sufrirían in vivo, reacción que genera un nitro anión radical (NO2.-) intermediario que sufre reciclaje redox con O2, generando ROS. Naftaleno es un xenobiótico metabolizado por oxidación a través del sistema citocromo P450; además, induce estrés oxidativo por esta vía metabólica. En este trabajo realizamos un estudio comparativo entre el metabolismo de naftaleno y, Nitrofurantoína y Nifurtimox catalizado por el sistema citocromo P450. Para ello utilizamos una preparación enriquecida en retículo endoplásmico hepático de rata (microsomas). La incubación de los microsomas con ya sea naftaleno, Nitrofurantoína o Nifurtimox y NADPH indujo lipoperoxidación microsómica, fenómeno que fue inhibido por GSH y un extracto hidroalcohólico de Buddleja globosa (matico) de una forma concentración-respuesta. La lipoperoxidación microsómica inducida por los 3 xenobióticos ensayados tuvo un comportamiento bimodal y fue dependiente de la concentración de los xenobióticos; naftaleno aumentó la lipoperoxidación y, Nitrofurantoína y Nifurtimox, la disminuyeron. Así, la primera pendiente positiva obtenida en presencia de naftaleno fue 2 veces mayor que la segunda; asimismo, la pendiente negativa obtenida a concentraciones µM de ya sea Nitrofurantoína o Nifurtimox fue 20 y 10 veces mayor que la obtenida a concentraciones mM, respectivamente. Además, los 3 xenobióticos: a) disminuyeron el contenido de tioles microsómicos en condiciones de biotransformación, b) se unieron a la monooxigenasa citocromo P450 de una forma concentración-respuesta, c) inhibieron la O-desmetilación de p-nitroanisol, reacción catalizada por el sistema citocromo P450, fenómeno que fue prevenido parcialmente por GSH y DTT. Nuestros resultados muestran que Nitrofurantoína y Nifurtimox pueden ser nitrorreducidos y además, oxidados por el sistema citocromo P450; sin embargo, se requieren nuevos experimentos que permitan confirmar este postulado. Por otra parte, ya que el estrés oxidativo inducido por la nitrorreducción fue inhibido por antioxidantes, la administración de una terapia asociada podría ayudar a disminuir los efectos adversos descritos para estos fármacos.
Epoxide hydrolase plays an important role in the detoxification of genotoxic compounds and in the control of physiological signaling molecules. Altered levels of epoxide hydrolase activity are associated with many diseases, such as emphysema, lung cancer, ovarian cancer, and laryngeal carcinoma. We designed and synthesized a resorufin-based fluorogenic probe, 7-(2-(oxiran-2-yl)ethoxy) resorufin, which was hydrolyzed by microsomal epoxide hydrolase to form the corresponding diol, which upon further treatment with sodium periodate released the strongly fluorescent resorufin. The probe exhibits good biological compatibility and photophysical properties, such as long wavelength excitation (571 nm) and emission (585 nm) and a wide working pH range (from 6.0 to 10.0), and thus facilitates the determination of the activity of microsomal epoxide hydrolase.
A library of diastereomerically pure epoxysterols, prepared by combining chemical and enzymatic methodologies, was evaluated for cytotoxicity toward human cancer and noncancer cell lines. Unsaturated steroids were oxidized by magnesium bis(monoperoxyphthalate) hexahydrate in acetonitrile, and the resulting epimeric epoxides were enzymatically separated using Novozym 435 or lipase AY. Some of the synthesized epoxysterols have potent cytotoxicity and higher activity on cancer cell lines HT29 and LAMA-84.
Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with naphthalene. To further identify the target proteins of the naphthoquinone, we raised polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 microM at 37 degrees C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride) microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 microM 1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was observed in the lane loaded with the protein sample incubated with 1.0 microM 1,2-naphthoquinone, and many chemiluminescent bands at a wide range of molecular weights were observed in the lane loaded with the protein sample incubated with 10 microM quinone. As expected, no chemiluminescent bands were detected on the membrane blotted with the proteins exposed to vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone cysteine adducts and developed immunostaining to detect protein modification by 1,2-naphthoquinone.
Nonciliated bronchiolar epithelial (Clara) cells are selectively damaged by intraperitoneal administration of naphthalene. We examined these changes using light microscopy, transmission electron microscopy, and scanning electron microscopy. Naphthalene administration causes the Clara cells to expand and exfoliated shortly thereafter. Following exfoliation the remaining ciliated cells show morphologic abnormalities, including cilia loss and ballooning of remaining cilia. Upon regeneration of the Clara cells the ciliated cells gradually return to their normal appearance. One possible explanation for these findings is that the Clara cell secretions directly affect the physiologic state of the surrounding ciliated cells.
Injection of a single dose of naphthalene into C57BL/6J mice (225 mg/kg, ip) produced a significant (30–70%) and prolonged (8–15 days) impairment in pulmonary microsomal monooxygenase activities without altering these activities in liver microsomes. The time course of naphthalene-induced morphologic damage to bronchiolar epithelium paralleled compromises in pulmonary monooxygenase activity. No concomitant alterations in hepatic morphology were observed. Five microsomal enzymes were studied: benzphetamine N-demethylase, aryl hydrocarbon hydroxylase, NADPH cytochrome c reductase, 7-ethoxyresorufin O-deethylase (a cytochrome P-448-dependent enzyme), and styrene epoxide hydrolase (a cytochrome P-450-independent enzyme). In general, the time course of the inhibition of these pulmonary enzymes was similar but the magnitude of the inhibition varied somewhat. Maximum inhibition of enzyme activity occurred about 3 days after naphthalene administration; 7-ethoxyresorufin O-deethylase activity was reduced to about 30% of control values whereas benzphetamine N-demethylase declined to about 70% of control. The remaining enzymes clustered midway between these extremes at about 50% of control values. Inhibited activities remained at relatively constant levels between Days 3 and 8 and by Day 15, there was a clear trend returning toward controls. Despite this trend, three of the six pulmonary enzyme activities examined remained significantly below control levels 15 days after a single dose of the hydrocarbon. Histologically, the pulmonary nonciliated bronchiolar epithelial (Clara) cell was the primary target of naphthalene toxicity. At early time points and at low magnifications, it appeared as if the entire bronchiolar epithelium was undergoing necrosis and sloughing into the lumen. However, higher magnifications revealed residual ciliated epithelium. The distribution of Clara cell damage appeared to vary considerably. One could find bronchioles that appeared completely denuded of epithelium and others in the same section whose Clara cells had been spared or, alternatively, were in the process of regeneration. The results are discussed in relation to recent work which has shown selective covalent binding of naphthalene to pulmonary Clara cells.
Alkaline permethylation and GC/MS analysis of urinary mercapturic acids from rats given bromobenzene yielded several quinone-derived bromodimethoxythioanisole isomers as expected. Unexpectedly, seven bromomonomethoxythioanisole isomers were also observed, suggesting the presence of bromomonohydroxyphenyl mercapturic acids in the urine. Alkaline permethylation of synthetic 4- and 5-bromo-2-hydroxyphenyl mercapturic acid gave 4- and 5-bromo-2-methoxythioanisole, respectively, which were also observed after alkaline permethylation of urine from bromobenzene-treated rats, as was 2-bromo-4-methoxythioanisole. To explore the biosynthetic origin of the bromonohydroxyphenyl mercapturic acids, rats were separately dosed intraperitoneally with synthetic racemic 2-, 3-, or 4-bromophenyl mercapturic acid, or biosynthetic L-(-)-4-bromophenyl mercapturic acid, or a biosynthetic mixture of the 3,4- and 4,3-premercapturic acids from bromobenzene, and their urine (0-24 hr) analyzed by alkaline permethylation and GC/MS. The administered mercapturic acids and premercapturic acids were partly excreted unchanged (60-80% and 24%, respectively), but both gave rise to bromomonohydroxyphenyl mercapturic acids (0.1-5.2% of dose). Results indicated that the latter could be formed by 1) dehydrogenation of premercapturic acids and 2) hydroxylation of mercapturic acids (or their cysteine equivalents).
The purpose of this study was to define, in quantitative terms, epithelial alterations produced by the cytochrome P-450-activated Clara cell cytotoxicant, naphthalene, in lobar bronchi and terminal bronchioles of three species with differing sensitivity: mouse, rat, and hamster.
Adult mice, hamsters, and rats were treated intraperitoneally with a single dose of naphthalene ranging from 0 mg/kg up to the approximate LD50. The animals were killed 24 hours postinjection and the changes in airway epithelium characterized by light microscopic morphometry.
In mouse, bronchiolar epithelial thickness was significantly elevated by low, but not high, doses; ciliated cell number increased and Clara cell number decreased in a dose-dependent fashion. Vacuolated Clara cell number increased in all treated mice. In rat and hamster, bronchiolar epithelial thickness or cell number did not change. In mice, bronchial epithelial thickness was unchanged except at high doses, but both ciliated and Clara cell number was decreased. In bronchi of rats, epithelial thickness and numbers of nonciliated, ciliated, and basal cells were unchanged. In bronchi of hamsters, both ciliated and nonciliated cell number were decreased.
(a) In mice, naphthalene-induced acute bronchiolar toxicity involves not only Clara cells, but also affects the purported nontarget cell type (ciliated cells). (b) In rats and hamsters, bronchiolar epithelium is insensitive to naphthalene injury. (c) In mice, injury to bronchi occurs at higher doses than in bronchioles and involves both ciliated and nonciliated cells. (d) In rats, bronchi are insensitive. (e) In hamsters, bronchi are more sensitive than bronchioles. This study emphasizes the variability of response by species, airway and epithelial cell type to cytochrome P-450-mediated pulmonary toxicants and the need for precise quantitative methods of defining both cytotoxic and metabolic events.
The purpose of this study was to define the sites of cytotoxicity within the respiratory tract (nasal cavity and tracheobronchial airway tree) resulting from administration of naphthalene, an organic chemical whose cytotoxic properties require metabolic activation via the cytochrome P-450 monooxygenase system. Three species were compared: mouse, hamster and rat. Naphthalene was administered in corn oil at these doses: mouse (0-400 mg/kg), hamster (0-800 mg/kg) and rat (0-1600 mg/kg), and the animals were sacrificed 24 hr later. In mice, naphthalene produced Clara cell cytotoxicity at 50 mg/kg. The primary alteration was swelling and vacuolation of Clara cells in terminal bronchioles. At 100 mg/kg, the number of terminal bronchioles with vacuolated Clara cells and the number of Clara cells within terminal bronchioles which showed vacuolation increased. At 200 and 300 mg/kg, almost all of the nonciliated cells lining terminal bronchioles in mice were exfoliated and necrotic. In contrast, there was no apparent effect on Clara cells or ciliated cells of terminal bronchioles in rats treated with up to 1600 mg/kg. At 800 mg/kg, minor alterations in Clara cells in some terminal bronchioles were observed in hamsters. At 300 mg/kg, lobar bronchus and trachea showed swelling 'and vacuolation of nonciliated cells in mice, but no detectable change at 200 mg/kg or below. The trachea and lobar bronchus were unaffected in rats, but showed cytotoxic changes in hamsters. In the nasal cavity of mice, cytotoxicity was observed only in the olfactory epithelium and only in animals treated with 400 mg/kg. There were minimal alterations in the respiratory epithelium. The only epithelial population showing cytotoxicity in the rat was the olfactory epithelium. Complete necrosis was observed at 200 mg/kg and higher. In the hamster there was no discernible alteration in the olfactory epithelium at 100 and 200 mg/kg. At 400 mg/kg, the olfactory epithelium was necrotic. Naphthalene injury to the tracheobronchial epithelium of the mouse is: 1) Clara cell specific; 2) dose-related in the terminal bronchiole; and 3) involves more proximal airways in a dose-dependent fashion. The tracheobronchial epithelium of the rat is refractory to Clara cell injury even at the LD50, but proximal airways are more susceptible than distal airways in the hamster. The nasal cavity shows specific injury in one zone (olfactory epithelium) in a dose and species specific manner. The susceptibility to naphthalene-induced injury in the olfactory epithelium does not correlate with the susceptibility of Clara cells in more distal portions of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)
Bromobenzene (BB) hepatotoxicity is widely attributed to the alkylation of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. This laboratory recently reported the first conclusive evidence that BB epoxides actually do alkylate proteins; i.e., acid hydrolysates of hepatic proteins from phenobarbital-(PB-) induced BB-treated rats contain S-(o-, S-(m-, and S-(p-bromophenyl)cysteine [Weller, P.E., and Hanzlik, R.P. (1991) Chem. Res. Toxicol. 4, 17-20]. However, these three compounds account for less than 0.5% of total protein covalent binding. Bromoquinone metabolites of BB are also suspected of alkylating proteins. To search for such adducts to protein cysteinyl or methionyl residues, we heated hepatic proteins from PB-induced BB-treated rats with a two-phase mixture of 16 N KOH and CH3I ("alkaline permethylation"). Under these conditions S-alkylated residues are cleaved via elimination and the phenoxide and thiophenoxide groups on the fragments are methylated. Product analysis by 14C HPLC and GC/MS revealed o-, m-, and p-bromothioanisoles in amounts comparable to the content of S-(bromophenyl)cysteines found by acid hydrolysis (para much greater than meta, ortho). This, too, clearly implicates protein-SH alkylation by BB-2,3- and 3,4-oxides. In addition, 2,3-dimethoxy-5-bromothioanisole and another unidentified isomer were observed. However, by far the major adduct (5-6% of total covalent binding) was 2,5-dimethoxythioanisole (i.e., a debrominated adduct). When BB-d5 was administered, the latter contained mostly 3 deuterium atoms/mol. These latter results clearly show that alkylation of protein sulfur nucleophiles in vivo by quinone metabolites is 10-15 times more extensive than their alkylation by BB epoxides. After BB-d5 was administered, the bromothioanisoles and dimethoxybromothioanisoles contained 4 and 2 deuterium atoms/mol, respectively. A weighted average calculation of deuterium retention across the six major sulfur adducts agreed well with 3H/14C retention ratios determined earlier for total liver protein covalent binding of dual-labeled [3H/14C]BB, indicating that the overall pattern of BB metabolite binding to all protein nucleophiles may closely parallel that seen here specifically for protein sulfhydryl groups. The identification of a variety of specific BB-derived adducts to protein now affords the opportunity to investigate their relative contributions to the toxicity of bromobenzene.
The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Nonciliated bronchiolar cells (Clara cells) are thought to provide important respiratory secretions in the small airways and to have other metabolic functions. In mice, nonciliated bronchiolar cells have been the subject of special investigation because tumors of these cells can be specifically induced by chemical carcinogens. A method for isolating nonciliated bronchiolar cells from mice has been reported. However, we have developed a method to isolate these cells from the lungs of BDF1 mice with approximately 80% purity. Cells were identified by electron microscopy, nitroblue tetrazolium dye reduction in the presence of NADPH, immunocytochemical staining of cytochrome P450 isozymes, and mitochondrial staining with rhodamine 123. The isolated cells were examined in culture for synthesis and secretion of proteins and phospholipids. Protein synthesis and secretion were examined in cells labeled with [34S]methionine for 16 h. Fresh medium was added to washed cells and the cells were incubated for an additional 3h. The secreted proteins were precipitated with 10% trichloroacetic acid. Molecular weights of the most prominent radiolabeled secreted proteins were 6, 36, 43, and 45 kDa. Phospholipid synthesis and secretion were examined in cells labeled with [14C]acetate and 32P. Less than 1% of the radioactive lipids was found in the medium, and secretion of lipid was not stimulated by terbutaline or tetradecanoylphorbol acetate compounds, which stimulate phospholipid secretion by type II cells. These data support the hypothesis that nonciliated bronchiolar cells synthesize and secrete proteins but do not secrete phospholipids in any appreciable amount.
Experimental bronchiolar necrosis was elicited in mice and rats by a single intraperitoneal dose of bromobenzene or other aromatic hydrocarbon. The lesion was associated with the binding of a bromobenzene metabolite to bronchiolar epithelial cells. Pulmonary microsomal enzymes converted bromobenzene to a metabolite that became bound to microsomal proteins in vitro. These studies indicated that so called drug metabolizing enzymes in the lung might be important determinants in the pathogenesis of bromobenzene induced bronchiolar necrosis. It is possible that the activities of similar pulmonary enzymes could play a significant role in the pathogenesis of other pulmonary lesions related to foreign compounds.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Previous studies have shown that cytochrome P-450-mediated metabolism of naphthalene results in dose-dependent bronchiolar necrosis in mice and in the formation of reactive metabolites which deplete reduced glutathione and become bound covalently to tissue macromolecules. The finding that pulmonary glutathione levels were nearly totally depleted after large doses of naphthalene suggested that hepatic formation of reactive metabolites may contribute substantially to glutathione depletion and covalent binding in extrahepatic tissues. This possibility has been supported by several new lines of evidence: 1) similar levels of covalent binding were observed in lung, liver and kidney in vivo, yet the rate of kidney microsomal metabolic activation of naphthalene was much lower than in liver or lung; 2) phenobarbital pretreatment markedly increased in vivo covalent binding in lung, liver and kidney and increased hepatic but decreased pulmonary microsomal covalent binding; 3) 3-methylcholanthrene pretreatment resulted in slightly increased levels of covalent binding in lung, liver and kidney yet decreased pulmonary microsomal covalent binding; 4) administration of p-xylene, at doses which selectively decreased pulmonary microsomal metabolism of biphenyl (4-hydroxylation) and naphthalene (to reactive metabolites), decreased in vivo covalent binding in liver and kidney to the same extent as lung after [14C]naphthalene; and 5) pretreatment with buthionine sulfoximine preferentially depleted hepatic and renal but not pulmonary glutathione levels and markedly increased covalent binding in all three tissues. The severity of naphthalene-induced bronchiolar damage was unaffected by pretreatment with phenobarbital, 3-methylcholanthrene or p-xylene but was increased by prior administration of buthionine sulfoximine. These studies suggest that a portion of the reactive metabolites which deplete glutathione and bind covalently in extrahepatic tissues originate in the liver. Whether these circulating metabolites play a role in naphthalene-induced pulmonary bronchiolar damage is not clear.
Intraperitoneal administration of the volatile hydrocarbon, naphthalene, resulted in severe bronchiolar epithelial cell necrosis in mice, while hepatic or renal necrosis was not observed. Pulmonary damage and mortality by naphthalene were increased by prior treatment with diethyl maleate and decreased by prior treatment with piperonyl butoxide (1600 mg/kg). SKF 525A pretreatment had no effect on naphthalene-induced pulmonary damage. Administration of [14C]naphthalene resulted in the covalent binding of radiolabel to tissue macromolecules. Highest levels of binding occurred in lung, liver and kidney. Levels of covalent binding reached a maximum 2--4 h after treatment and corresponded to rapid glutathione depletion in lung and liver. Covalent binding was dose-dependent and showed a threshold between 200 and 400 mg/kg which coincided with almost total depletion of tissue glutathione levels. Covalent binding of reactive metabolites was increased 3--4-fold by prior treatment with diethyl maleate, and was decreased 3--4-fold by pretreatment with piperonyl butoxide. These studies support the view that naphthalene-induced pulmonary damage is mediated by the cytochrome P-450-dependent metabolism of naphthalene and that glutathione plays an important role in the detoxification of the lung damaging metabolite(s).
Naphthalene produces selective necrosis of Clara cells in the mouse but not in the rat. The pulmonary toxicity depends on cytochrome P450-mediated metabolism; however, the selective pulmonary toxicity of naphthalene in the mouse does not correspond to tissue-selective covalent binding of reactive naphthalene metabolites in vivo. These studies compare reactive metabolite binding in target and nontarget cells and in various subcompartments of mouse lung and characterize, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins to which arylating metabolites are bound. Reactive metabolite binding was substantially higher in incubations of [3H]-naphthalene with distal bronchioles and isolated Clara cells than with explants of trachea or bronchus from the mouse. Likewise, binding was substantially higher in incubations of murine Clara cells than in identical incubations with mouse hepatocytes (nontarget cells) or rat trachea cells (nonsusceptible species). These data show a good correlation between cellular susceptibility to toxicity and the amount of reactive metabolite bound in vitro. Concentrations of adduct were highest in the medium and the nuclear/cell debris fraction (1000 x g pellet) of isolated Clara cells incubated with naphthalene; very small amounts of adduct were noted in pellets isolated at 20,000 or at 100,000 x g (mitochondrial and microsomal fractions) or in cytosol. These observations were consistent with the finding that adduct concentrations in bronchoalveolar lavage were substantially higher than in the lung at low doses of naphthalene and suggest that monitoring adducts in lavage may serve as a useful biomarker of exposure and effect.(ABSTRACT TRUNCATED AT 250 WORDS)
The measurement of metabolites constitutes a useful tool for detection of exposure and in pharmacokinetic studies. Epoxidation with subsequent glutathione conjugation and mercapturic acid formation is an important deactivation pathway for naphthalene, a toxin which presumably causes lung disease. The mercapturic acid conjugates of naphthalene [NaphMA (1), N-acetyl-S-(1,2-dihydro-1-hydroxy-2-naphthyl)cysteine (1a), and N-acetyl-S-(1,2-dihydro-2-hydroxy-1-naphthyl)cysteine (1b)], its most important urinary metabolites, and other structurally related derivatives, such as N-acetyl-S-(1,2,3,4-tetrahydro-2-hydroxy-1-naphthyl) cysteine (2), N-acetyl-S-(3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl)cysteine (3), and N-acetyl-S-(2-hydroxy-1-phenylethyl)cysteine (4a) and N-acetyl-S-(2-hydroxy-2-phenylethyl)cysteine (4b) as an isomeric mixture, were synthesized to develop an ELISA (enzyme-linked immunosorbent assay) for the specific detection of NaphMA (1). Compound 1, as an isomeric mixture, was used to raise antibodies by immunizing six rabbits with the corresponding KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) derivatives (1KLH and 1BSA). The remaining compounds were covalently attached to BSA, conalbumin, and ovalbumin to be used as coating antigens. The best assay was obtained in a homologous system combining serum Ab2357 (1KLH) and 1BSA as coating antigen. The immunoassay has an I50 of 4-6 ng/mL and a detection limit of 1-2 ng/mL. Because of the known instability of the mercapturic acid conjugate of naphthalene 1, leading to the fully aromatic compound 20, a system involving HPLC is described to check the stability of the NaphMA stock solutions used in the assay. Cross-reactivity studies show high specificity toward the NaphMA. Other related compounds as well as the dehydrated derivative 20 are not recognized by the antibody in this ELISA system.
We recently reported on the identification of metabolites of the hepatotoxin bromobenzene covalently bound to rat liver protein sulfur nucleophiles (D. E. Slaughter and R. P. Hanzlik, Chem. Res. Toxicol. 4, 349-359 (1991). Central to that study was our development of a method called alkaline permethylation which converts protein-S adducts of xenobiotic electrophiles to stable extractable thioanisole derivatives. We report here on substantial improvements to our original alkaline permethylation method which should greatly expand its potential utility. Specifically, we have developed significantly milder reaction conditions, eliminated side reactions, improved the amount of and consistency of thioanisole yields from various mercapturic acid model compounds, and increased the overall sensitivity of the method at least 50-fold. Using the procedure described herein it is routinely possible to generate, detect, and identify by GC/MS as little as 2 pmol of a thioanisole derivative. This method is potentially quite general and should prove useful for studies in the toxicology of reactive metabolites, for industrial hygiene and biomonitoring, and for agrichemical residue analysis.
Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with naphthalene. To further identify the target proteins of the naphthoquinone, we raised polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 microM at 37 degrees C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride) microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 microM 1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was observed in the lane loaded with the protein sample incubated with 1.0 microM 1,2-naphthoquinone, and many chemiluminescent bands at a wide range of molecular weights were observed in the lane loaded with the protein sample incubated with 10 microM quinone. As expected, no chemiluminescent bands were detected on the membrane blotted with the proteins exposed to vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone cysteine adducts and developed immunostaining to detect protein modification by 1,2-naphthoquinone.
- L F Fieser
Fieser, L. F. (1943) 1,2-Naphthoquinone. Org. Synth. 2, 430-433.