9,10-Phenanthrenequinone induces DNA deletions and forward mutations via oxidative mechanisms in the yeast Saccharomyces cerevisiae.

The estimated cancer risk from diesel exhaust particles (DEP) in the air is approximately 70% of the cancer risk from all air pollutants. DEP is comprised of a complex mixture of chemicals whose carcinogenic potential has not been adequately assessed. The polycyclic aromatic hydrocarbon quinone 9,10-phenanthrenequinone (9,10 PQ) is a major component of DEP and a suspect genotoxic agent for DEP induced DNA damage. 9,10 PQ undergoes redox cycling to produce reactive oxygen species that can lead to oxidative DNA damage. We used two systems in the yeast Saccharomyces cerevisiae to examine possible differential genotoxicity of 9,10 PQ. The DEL assay measures intra-chromosomal homologous recombination leading to DNA deletions and the CAN assay measures forward mutations leading to canavanine resistance. Cells were exposed to 9,10 PQ aerobically and anaerobically followed by DNA damage assessment. The results indicate that 9,10 PQ induces DNA deletions and point mutations in the presence of oxygen while exhibiting negligible effects anaerobically. In contrast to the cytotoxicity observed aerobically, the anaerobic effects of 9,10 PQ seem to be cytostatic in nature, reducing growth without affecting cell viability. Thus, 9,10 PQ requires oxygen for genotoxicity while different toxicities exhibited aerobically and anaerobically suggest multiple mechanisms of action.     

Reductive activation of terpenylnaphthoquinones

Four terpenylnaphthoquinones were found to enhance the rate of superoxide production in the presence of ascorbate as detected from the superoxide dismutase (SOD)-inhibitable initial oxygen consumption rates. Initial rates of oxygen consumption in the presence of ascorbate plus quinone increase with an increase in the half-wave reduction potentials of the quinones. These quinones also enhance the rate of Cyt(III)c reduction by xanthine/xanthine oxidase (X/XO) in both air- and nitrogen-saturated aqueous solutions at pH 7.4. Maximum rates of Cyt(III)c reduction in nitrogen and oxygen-saturated solutions (V(max)), in the presence of X/XO, increase with an increase in the half-wave reduction potentials of the quinones. SOD inhibits Cyt(III)c reduction rates in the presence of these quinones and X/XO in a manner which is also dependent on the quinone half-wave redox potential. The relative antineoplastic activity of two of these quinones follows the order in rates of oxygen consumption or Cyt(III)c reduction. This is consistent with an antineoplastic action of these quinones through the mechanism of redox cycling or possible interference or inhibition of mitochondrial respiration.     

Contribution of reductase activity to quinone toxicity in three kinds of hepatic cells

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.     

Role of quinones in toxicology

Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).     

Induction of phenotypes resembling CuZn-superoxide dismutase deletion in wild-type yeast cells: an in vivo assay for the role of superoxide in the toxicity of redox-cycling compounds

Yeast (Saccharomyces cerevisiae) lacking the enzyme CuZn-superoxide dismutase (sod1delta) display a large number of dioxygen sensitive phenotypes, such as amino acid auxotrophies, sensitivity to elevated temperatures, and sensitivity to 100% dioxygen, which are attributed to superoxide stress. Such cells are exquisitely sensitive to small amounts of the herbicide paraquat (methyl viologen), which is known to produce high fluxes of superoxide in vivo via a redox-cycling mechanism. We report that dioxygen sensitive phenotypes similar to those seen in sod1delta cells can be induced in wild-type cells by treatment with moderate concentrations of paraquat or diquat, another bipyridyl herbicide, providing strong evidence that the mechanism of toxicity for both of these compounds is attributable to superoxide stress. Certain redox-cycling quinone compounds (e.g., menadione and plumbagin) are also far more toxic toward sod1delta than to wild type. However, treatment of wild-type yeast with menadione or plumbagin did not induce sod1delta-like phenotypes, although toxicity was evident. Thus, their toxicity in wild type cells is predominantly, but not exclusively, due to mechanisms unrelated to superoxide production. Further evidence for a different basis of toxicity toward wild-type yeast in these two classes of redox-cycling compounds includes the observations that (i) growth in low oxygen alleviated the effects of paraquat and diquat but not those of menadione or plumbagin and (ii) activity of the superoxide sensitive enzyme aconitase is affected by very low concentrations of paraquat but only by higher, growth inhibitory concentrations of menadione. These results provide the basis for an easy qualitative assay of the contribution of redox-cycling to the toxicity of a test compound. Using this method, we analyzed the Parkinsonism-inducing compound 1-methyl-4-phenylpyridinium and found that redox cycling and superoxide toxicity are not the predominant factor in its toxic mechanism.     

Nitric oxide mitigates apoptosis in human endothelial cells induced by 9,10-phenanthrenequinone: Role of proteasomal function

It has been widely recognized that nitric oxide (NO) suppresses oxidative damage of endothelial cell, but little is known about its pathophysiological role in apoptotic induction by 9,10-phenanthrenequinone (9,10-PQ), a major quinone component in diesel exhaust particles. Here, we have investigated the change in NO level in human aortic endothelial cells and the effect of NO in each step of apoptotic signaling initiated by 9,10-PQ. Treatment with 9,10-PQ evoked a bell-shaped production of NO, which was presumably due to increase in an active form of endothelial NO synthase. Pretreatment with exogenous NO decreased the susceptibility of the cells to 9,10-PQ, and retrieved from apoptotic signaling (reactive oxygen species generation, glutathione depletion and caspase activation) induced during exposure to high concentrations of 9,10-PQ. In addition, inhibition of endogenous NO production augmented the toxicity of 9,10-PQ. Interestingly, the 9,10-PQ treatment resulted in marked decreases in the proteasomal activities, which were partially abrogated by NO and a cell-permeable cGMP analog. These results indicate that proteasomal dysfunction by oxidative stress participates in the 9,10-PQ-induced apoptotic signaling and is ameliorated by NO via a cGMP-dependent pathway, thereby suggesting the protective role of NO in vascular damage caused by 9,10-PQ.     

Application of quantitative structure-toxicity relationships for the comparison of the cytotoxicity of 14 p-benzoquinone congeners in primary cultured rat hepatocytes versus PC12 cells.

Quinones are believed to induce their toxicity by two main mechanisms: oxygen activation by redox cycling and alkylation of essential macromolecules. The physicochemical parameters that underlie this activity have not been elucidated, although redox potential is believed to play a significant role. In this study, we have evaluated the cytotoxicity, formation of reactive oxygen species (ROS), and the glutathione (GSH) depleting ability of 14 p-benzoquinone congeners in primary rat hepatocyte and PC12 cell cultures. All experiments were performed under identical conditions (37 degrees C, 5% CO2/air) in 96-well plates. The most cytotoxic quinone was found to be tetrachloro-p-benzoquinone (chloranil), and the least toxic was duroquinone or 2,6-di-tert-butyl-p-benzoquinone. The cytotoxic order varied between the cell types, and in particular, the di-substituted methoxy or methyl p-benzoquinones were particularly more cytotoxic towards PC12 cells. We have derived one- and two-parameter quantitative structure-toxicity relationships (QSTRs) which revealed that the most cytotoxic quinones had the highest electron affinity and the smallest volume. Cytotoxicity did not correlate with the lipophilicity of the quinone. Furthermore, we found that p-benzoquinone cytotoxicity correlated well with hepatocyte ROS formation and GSH depletion, whereas in PC12 cells, cytotoxicity did not correlate with ROS formation and somewhat correlated with GSH depletion. Hepatocytes had far greater hydrogen peroxide detoxifying capacity than PC12 cells, but PC12 cells contained more GSH/mg protein. Thus, p-benzoquinone-induced ROS formation was greater towards PC12 cells than with hepatocytes. To our knowledge, this is the first QSTR derived for p-benzoquinone cytotoxicity in these cell types and could form the basis for distinguishing certain cell-specific cytotoxic mechanisms.     

Bioreductive activation of quinones: A mixed blessing

Quinones can be metabolized by various routes: substitution or reductive addition with nucleophilic compounds (mainly glutathione and protein thiol groups), one-electron reduction (mainly by NADPH: cytochrome P-450 reductase) and two-electron reduction (by D,T-diaphorase). During reduction semiquinone radicals and hydroquinones are formed, which can transfer electrons to molecular oxygen, resulting in the formation of reactive oxygen intermediates and back-formation of the parent quinone (redox cycling). Reaction of semiquinones and reactive oxygen intermediates with DNA and other macromolecules can lead to acute cytotoxicity and/or to mutagenicity and carcinogenicity. The enhanced DNA-alkylating properties of certain hydroquinones are exploited in the bioreductive alkylating quinones. Acute cytotoxicity of quinones appears to be related to glutathione depletion and to interaction with mitochondria and subsequent disturbance of cellular energy homoeostasis and calcium homoeostasis. These effects can to a certain extent be predicted from the electron-withdrawing and electron-donating effects of the substituents on the quinone nucleus of the molecule. Prediction of cytostatic potential remains much more complicated, because reduction of the quinones and the reactivity of the reduction products with DNA are modulated by the prevailing oxygen tension and by the prevalence of reducing enzymes in tumour cells.This article is based on a lecture given at the 16th LOF Symposium, 27 October 1989, Utrecht, the Netherlands.     

Redox-active quinones and ascorbate: an innovative cancer therapy that exploits the vulnerability of cancer cells to oxidative stress

Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.     

Xanthine oxidase-catalyzed reduction of estrogen quinones to semiquinones and hydroquinones.

Metabolic redox cycling between the stilbene estrogen diethylstilbestrol (DES) and diethylstilbestrol-4',4"-quinone (DES Q) has been demonstrated previously. The xanthine and xanthine oxidase-catalyzed reduction of estrogen quinone has been studied in this work to understand the role of metabolic redox cycling in estrogen metabolism. Xanthine and xanthine oxidase catalyzed the reduction of DES Q to 44% Z-DES and 9% E-DES. This reaction was inhibited by the addition of superoxide dismutase or by a lack of oxygen (under anaerobic conditions). DES Q was also reduced in a non-enzymatic reaction by superoxide radicals generated by potassium superoxide and crown ether. The reaction between the O2-. and DES Q was also investigated by an electron spin resonance spin-trapping technique. The superoxide anion generated in an oxygen-saturated xanthine and xanthine oxidase system was detected as 5,5-dimethyl-1-pyrroline-1-oxide-superoxide adduct. The addition of DES Q or 2,3-estradiol quinone totally inhibited the formation of this adduct. The reduction of DES Q by superoxide radicals was taken as evidence that this reaction was one possible mechanism of xanthine and xanthine oxidase-mediated reduction. In addition, reduction of DES Q by direct electron transfer to quinone by the enzyme may also occur. The intermediate formation of semiquinone free radicals in the reduction is implied by the nature of the single electron transfer reactions and, in addition, has been demonstrated for the catechol estrogen by electron spin resonance measurements. It is concluded that the reduction of estrogen quinones to their hydroquinones by xanthine oxidase occurs by both one electron transfer to the quinone and by formation of superoxide which then reduces the quinone.     

Generation of photoemissive species by mitomycin C redox cycling in rat liver microsomes

Generation of reactive oxygen species during redox cycling is thought to be involved in the chemotherapeutic action of quinone anticancer drugs. A clinically used agent which contains a quinone moiety is mitomycin C (Mit C). With isolated rat liver microsomes we detected photoemissive species during Mit C-induced redox cycling. After addition of reduced glutathione (GSH) a large increase in Mit C-induced chemiluminescence was observed. The increase of photoemission in deuterium oxide as well as greater than 90% of intensity at wavelengths greater than 610 nm suggest that singlet oxygen is a photoemissive species generated by this system. Glutathione disulfide (GSSG) accumulates during the reaction. We propose that superoxide anion radicals formed during redox cycling of Mit C react with GSH. Generation of glutathionyl radicals followed by oxygen addition then leads to the formation of photoemissive species and GSSG.     

9,10-Phenanthrenequinone Induces Monocytic Differentiation of U937 Cells through Regulating Expression of Aldo-Keto Reductase 1C3

Persistent inhalation of diesel exhaust particles results in damaged lung cells through formation of reactive oxygen species (ROS), but the details of the toxicity mechanism against monocytes are poorly understood. In this study, we used human promyelomonocytic U937 cells as surrogates of monocytes and investigated the toxicity mechanism initiated by exposure to 9,10-phenanthrenequinone (9,10-PQ), a major quinone component in diesel exhaust particles. A 24-h incubation with 9,10-PQ provoked apoptotic cell death, which was due to signaling through the enhanced ROS generation and concomitant caspase activation. Flow cytometric analyses of U937 cells after long-term exposure to 9,10-PQ revealed induction of differentiation that was evidenced by increasing expression of CD11b/CD18, a cell-surface marker for monocytic differentiation into macrophages. The 9,10-PQ-induced differentiation was significantly abolished by ROS inhibitors, suggesting that ROS generation contributes to cell differentiation. The 9,10-PQ treatment increased the expression of aldo-keto reductase (AKR) 1C3, which reached a peak at 1 to 2?d post-treatment and then declined. The bell-shaped curve of the AKR1C3 expression by 9,10-PQ resembled that caused by phorbol 12-myristate 13-acetate, a differentiation inducer. Additionally, the concomitant treatment with tolfenamic acid, a selective AKR1C3 inhibitor, sensitized the differentiation induced by 9,10-PQ. These results suggest that ROS formation during 9,10-PQ treatment acutely leads to apoptosis of U937 cells and the initiation of monocytic differentiation, which proceeds after the provisional overexpression of AKR1C3.     

Quinones as mutagens, carcinogens, and anticancer agents: introduction and overview

Quinones are widespread in our environment, occurring both naturally and as pollutants. Human exposure to them is therefore extensive. Quinones also form an important class of toxic metabolites generated as a result of the metabolism of phenols and related compounds, including phenol itself, 1-naphthol, and diethylstilbestrol. The mechanisms by which quinones exert their toxic effects are complex, but two processes appear to be centrally involved: the direct arylation of sulfhydryls, and the generation of active oxygen species via redox cycling. Certain quinones have been shown to be mutagenic via the formation of active oxygen species and others via their conversion to DNA-binding semiquinone free radicals. Paradoxically, quinones are not only mutagenic and therefore potentially carcinogenic, they are also effective anticancer agents. Classic examples are Adriamycin (doxorubicin hydrochloride) and mitomycin C, but other less complex quinones also show effective antitumor activity. The design of novel quinones that are more selective in their toxicity to human tumor cells and whose mechanism of action is understood seems a promising approach in cancer treatment, especially if host toxicity can be prevented via the use of chemoprotective agents.     

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-l-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling.     

A rapid and sensitive micro-assay to determine the capacity of quinones to undergo redox cycling

Using a series of aziridinyl-benzoquinones it is shown that the conversion of oxyhemoglobin to methemoglobin in sheep erythrocytes is correlated with the capacity of each quinone to undergo redox cycling. Based on these findings a semiquantitative assay is developed for the rapid screening of redox cycling quinones.     

The toxicity of menadione and mitozantrone in human liver-derived Hep G2 hepatoma cells

The cytotoxic properties of quinone drugs such as menadione and adriamycin are thought to be mediated through one-electron reduction to semiquinone free radicals. Redox cycling of the semiquinones results in the generation of reactive oxygen species and in oxidative damage. In this study the toxicity of mitozantrone, a novel quinone anticancer drug, was compared with that of menadione in human Hep G2 hepatoma cells. Mitozantrone toxicity in these cells was not mediated by the one-electron reduction pathway. In support of this, inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells from oxidative damage, did not affect the response of the Hep G2 cells to mitozantrone, whereas it exacerbated menadione toxicity. In addition, the toxicity of menadione was preceded by depletion of reduced glutathione which was probably due to oxidation of the glutathione. Mitozantrone did not cause glutathione depletion prior to cell death. DT-diaphorase activity and intracellular glutathione were found to protect the cells from the toxicity of both quinones. Inhibition of epoxide hydrolase potentiated mitozantrone toxicity but did not affect that of menadione. Our experiments indicate that mitozantrone toxicity may involve activation to an epoxide intermediate. Both quinone drugs inhibited cytochrome P-450-dependent mixed-function oxidase activity, although menadione was more potent in this respect.     

Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites.

PCBs are industrial chemicals that continue to contaminate our environment. They cause various toxic effects in animals and in exposed human populations. The mechanisms of toxicity, however, are not completely understood. PCBs are metabolized by cytochromes P450 to mono- and dihydroxylated compounds. Dihydroxy-PCBs can potentially be oxidized to the corresponding quinones. We hypothesized that reactive oxygen species (ROS) are produced by redox reactions of PCB metabolites. We tested several synthetic dihydroxy- and quinoid-PCBs with 1-3 chlorines for their potential to produce ROS in vitro and in HL-60 human leukemia cells, and DNA strand breaks in vitro. All dihydroxy-PCBs tested produced superoxide. The quinones generated superoxide only in the presence of GSH, probably during the autoxidation of the glutathione conjugates. We observed increased superoxide production with decreasing halogenation. Incubation of dihydroxy-PCBs or PCB quinones + GSH with plasmid DNA resulted in DNA strand break induction in the presence of Cu(II). Tests with various ROS scavengers indicated that hydroxyl radicals and singlet oxygen are likely involved in this strand break induction. Finally, dihydroxy- and quinoid PCBs also produced ROS in HL-60 cells in a dose- and time-dependent manner. We conclude that dihydroxylated PCBs, and PCB quinones after reaction with GSH, produce superoxide and other ROS both in vitro and in HL-60 cells, and oxidative DNA damage in the form of DNA strand breaks in vitro. The reactions seen in vitro and in cells may well be a predictor of the toxicity of PCBs in animals.     

Specificity of human aldo-keto reductases, NAD(P)H:quinone oxidoreductase, and carbonyl reductases to redox-cycle polycyclic aromatic hydrocarbon diones and 4-hydroxyequilenin-o-quinone

Polycyclic aromatic hydrocarbons (PAHs) are suspect human lung carcinogens and can be metabolically activated to remote quinones, for example, benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases, and to non-K region o-quinones, for example B[a]P-7,8-dione, by the action of aldo keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS), and produce the mutagenic lesion 8-oxo-dGuo and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze the reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene, and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione, and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion, and hydrogen peroxide formation established that ROS were produced as a result of the redox cycling. When compared with human recombinant NAD(P)H:quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and last CBR1 and CBR3. In A549 cells, two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol, suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.     

Effect of copper on the cytotoxicity of phenanthrene and 9,10-phenanthrenequinone to the human placental cell line, JEG-3

The trophoblast cell line, JEG-3, was used to study the cytotoxicity of phenanthrene, 9,10-phenanthrenequinone (PHEQ), anthracene, and 9,10-anthracenedione alone and with copper. The endpoints were the capacity of cultures to reduce alamar Blue (AB), a measure of energy metabolism, and to convert carboxyfluorescein diacetate acetoxymethyl ester (CFDA AM) to carboxyfluorescein, an indication of membrane integrity. Only PHEQ elicited a cytotoxic response. PHEQ caused a concentration-dependent decline in AB but not in CFDA AM readings, suggesting an impairment to energy metabolism. In the presence of copper, PHEQ concentration–response curves were shifted to the left for AB and were obtained with CFDA AM. The Cu/PHEQ synergy is attributed to an increase in redox cycling and production of reactive oxygen species (ROS), which overwhelm antioxidant defenses, damaging energy metabolism first and then membrane integrity. The impermeable copper chelator, bathocuproine, reduced the PHEQ/copper interaction, but the permeable chelator, neocuproine, and copper together were cytotoxic.     

Mechanisms of superoxide radical-mediated toxicity

Free radicals generated from metabolism of foreign compounds can have extremely detrimental consequences on cell function and survival. Due to their high reactivity, free radicals may potentially perturb a wide spectrum of important cellular macromolecules such as nucleic acids, proteins, lipids and polysaccharides. Recently, the toxicity of several xenobiotics has been suggested to be mediated by formation of free radicals derived from reduction of molecular oxygen, forming superoxide anion (O(2)) and hydroxyl radical (OH .). For example, the pulmonary toxicity of the bipyridylium herbicide paraquat has been attributed to an enzymatically catalyzed one-electron redox cycling of the parent molecule, resulting in generation of O(2). Examples of other compounds that are subject to redox cycling with associated O(2) formation are those agents containing quinone or aromatic nitro structural elements. An important aspect of free-radical-mediated toxicity is that it is moderated by several cellular defense mechanisms including superoxide dismutase, catalase, glutathione peroxidase, vitamin E and reduced glutathione. Thus, toxicity mediated by free radical generation may not occur unless defense mechanisms are overwhelmed by radical production.