Sulfhydryl binding and topoisomerase inhibition by PCB metabolites

Polychlorinated biphenyls (PCBs) are highly persistent contaminants in our environment. Their persistence is due to a general resistance to metabolic attack. Lower halogenated PCBs, however, are metabolized to mono- and dihydroxy compounds, and the latter may be further oxidized to quinones with the formation of reactive oxygen species (ROS). We have shown that PCB metabolism generates ROS in vitro and in cells in culture and this leads to oxidative DNA damage, like DNA strand breaks and 8-oxo-dG formation. In the present study, we have evaluated the reactivity of PCB metabolites with other nucleophiles, like glutathione (GSH), by assessing (1) quantitative GSH binding in vitro, (2) GSH and thiol (sulfhydryl) depletion in HL-60 cells, (3) the associated cytotoxicity, and (4) the inhibition of topoisomerase II activity in vitro. PCB quinones were found to bind GSH in vitro at a ratio of 1:1.5 and to deplete GSH in HL-60 cells as measured by both spectrophotometric and spectrofluorometric methods. By flow cytometry analysis, we confirmed that there was intracellular GSH depletion in HL-60 cells by PCB quinones and this is associated with cytotoxicity. On the other hand, the PCB hydroquinone metabolites did not bind GSH or other thiols within 1 h of exposure. However, by spectral analyses we found that the PCB hydroquinones could be oxidized enzymatically to the quinones, which could then bind GSH. The resulting hydroquinone-glutathione addition product(s) could undergo a second and third cycle of oxidation and GSH addition with the formation of di- and tri-GSH-PCB adducts. The effect of the PCB metabolites was also tested on a sulfhydryl-containing enzyme, topoisomerase II. PCB quinones inhibited topoisomerase II activity while the PCB hydroquinone metabolites did not. Hence, the oxidation of PCB hydroquinone metabolites to quinones in cells followed by the binding of quinones to GSH and to protein sulfhydryl groups and the resulting oxidative stress may be important aspects of the toxicity of these compounds.     

Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells

Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-l-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation.     

The role of reactive oxygen species in microcystin-LR-induced DNA damage

Microcystins are cyclic heptapeptides produced by different freshwater cyanobacterial species such as Microcystis aeruginosa. They have been shown to induce DNA damage in vitro and in vivo, however, the mechanisms of their genotoxic activity remain unclear. With the comet assay we demonstrate that, in human hepatoma HepG2 cells, microcystin-LR (MCLR) induced DNA strand breaks which were transiently present and probably produced during the cellular repair of MCLR-induced DNA damage. Digestion of DNA from MCLR-treated HepG2 cells with purified formamidopyrimidine-DNA glycosylase (Fpg), which recognizes specific oxidized purines, displayed a greater extent of DNA strand breaks than non-digested DNA, providing evidence that MCLR induced oxidation of purines. The number of DNA strand breaks detected after digestion with Fpg increased with time of exposure of the cells to MCLR, indicating that oxidized purines were not repaired. Using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluoroprobe we showed that MCLR, at non-cytotoxic concentrations, induced a time and dose dependent increase of intracellular reactive oxygen species (ROS) formation in HepG2 cells. The role of ROS in MCLR-induced DNA damage was further confirmed by exposing the cells to MCLR in the presence of different ROS scavengers. The formation of DNA strand breaks and oxidized purines was completely prevented by a superoxide dismutase mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), an iron chelator, deferoxamine (DFO), a precursor of glutathione (GSH) and intracellular ROS scavenger, N-acetyl-L-cysteine (NAC), and partly by hydroxyl radical scavengers dimethylsulphoxide (DMSO) and 1,3-dimethyl-2-thiourea (DMTU). The results provide evidence that the genotoxicity of MCLR is mediated by ROS.     

Protective role of estrogen receptor-alpha on lower chlorinated PCB congener-induced DNA damage and repair in human tumoral breast cells

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. Much of the research has focused on the carcinogenic potential of higher chlorinated PCBs, but accumulative evidence has shown that lower chlorinated PCB congeners have initiating and promoting activities. The goal of this study was to examine the potential of lower chlorinated PCBs, including 2,2′,5,5′-tetrachlorobiphenyl (PCB52) and 3,3′,4,4′-tetrachlorobiphenyl (PCB77), to induce DNA damage and apoptosis in human MDA-MB-231 (MDA) and MCF-7 breast cancer cells. Results confirmed that treatment of cells with PCB52 and PCB77 resulted in oxidative stress and caspase-dependent apoptosis in both MDA and MCF-7 cells. We noticed that at non-cytotoxic concentrations PCB52 and PCB77-induced decreases in intracellular NAD(P)H in MDA cells but not in MCF-7 cells. Further investigation confirmed that decreases in intracellular NAD(P)H in PCB-treated MDA cells are primarily due to reduction in intracellular NAD⁺ pool mediated by poly(ADP-ribose)polymerase-1 activation through formation of DNA strand breaks. Antagonism was observed between PCB52 and PCB77 for the effect on induction of DNA strand breaks in MDA cells. Overall, this evidence demonstrates that at non-cytotoxic concentrations, lower chlorinated PCB congeners are capable of inducing oxidative DNA lesions in ERα(−)/MDA cells but not in ERα(+)/MCF-7 cells and that functional ERα plays a protective role in modulating the PCB-induced DNA damage in human breast cancer cells.     

Cytotoxic and DNA-damaging effects of diterpenoid quinones from the roots of Salvia officinalis L. on colonic and hepatic human cells cultured in vitro

Three diterpenoid quinones (royleanone- SAR 3, horminone- SAR 26, and acetyl horminone- SAR 43) isolated from the roots of Salvia officinalis L. were tested for their cytotoxic and DNA-damaging activity in human colon carcinoma cells Caco-2 and human hepatoma cells HepG2 cultured in vitro. Cytotoxicity was measured by the trypan blue exclusion technique and induction of apoptosis was evaluated by flow immunofluorocytometry after 30-300 min. exposure of HepG2 and Caco-2 cells to diterpenoid quinones and following 24 hr post-incubation in the culture medium. Induction of DNA breaks was measured after 60 min. exposure of cells to different concentrations of the compounds studied by the alkaline elution of DNA and by the Comet assay. Though all the quinones tested decreased the viability of the cells studied proportionally to the concentration and to the time of treatment (cytotoxicity= 30-60%), the increased level of apoptotic nuclei comparable to the level of apoptotic nuclei induced by a topoisomerase I inhibitor was proved only in HepG2 cells treated with 1x10(-4) mol/l SAR 26 or SAR 43. Either no or marginal increase of the level of apoptotic nuclei was observed in SAR 3-treated HepG2 cells and in SAR 3-, SAR 26- or SAR 43-treated Caco-2 cells. All compounds tested induced creation of DNA strand breaks in both cell types at concentrations >1x10(-7)-1x10(-6) mol/l. The occurrence of DNA strand breaks at different pH values as well as the kinetics of DNA breaks rejoining were evaluated only in colonic cells Caco-2. The Comet assay processed in parallel at pH 13.0 and pH 12.1 showed that strand breaks detected in SARs-treated colonic Caco-2 cells originated from alkali-labile sites, as induced DNA lesions were converted to DNA strand breaks only under strong alkaline conditions. The kinetics of DNA rejoining revealed that SARs-induced DNA breaks were repaired very slowly.     

DNA oxidative damage during differentiation of HL-60 human promyelocytic leukemia cells.

DNA oxidative damage was measured in human promyelocytic leukemia HL-60 cells, in the same cells committed to granulocytic differentiation with dimethyl sulfoxide (DMSO) or all-trans-retinoic acid (RA) and in mature human peripheral granulocytes (HPG). DNA damage was evaluated as single strand breaks and 8-OHdG adducts, measured by single cell electrophoresis or by monoclonal antibodies, respectively. The basal levels of either marker of DNA damage were higher in undifferentiated HL-60 cells than in HPG and DMSO- or RA-differentiated cells. Treatment with H(2)O(2) increased 8-OHdG formation in all cells, but the levels of DNA damage remained higher in undifferentiated cells as compared to the differentiated ones. Three lines of evidence suggested that the higher levels of DNA damage observed in undifferentiated cells were at least in part attributable to a reduced detoxification of reactive oxygen species (ROS). First, undifferentiated cells were shown to accumulate higher levels of dichlorodihydrofluorescein-detectable ROS than HPG and DMSO- or RA-differentiated cells. Second, undifferentiated HL-60 cells were characterized by reduced levels of GSH and lower GSH/GSSG ratios as compared to the differentiated cells. Third, pretreatment of undifferentiated HL-60 cells with antioxidants such as alpha-tocopherol or beta-carotene suppressed the elevation of ROS and the formation of 8-OHdG induced by H(2)O(2). Further evidence for the importance of the oxidant/antioxidant balance was obtained by modulating the iron-catalyzed decomposition of H(2)O(2) to hydroxyl radicals in undifferentiated HL-60 cells. In fact, pretreatment with FeSO(4) increased the formation of 8-OHdG induced by H(2)O(2), whereas pretreatment with the iron chelator deferoxamine produced the opposite effect. These results illustrate correlations between the oxidant/antioxidant balance and DNA damage and suggest that the capability of a cell population to withstand oxidative stress and DNA damage may depend on its degree of differentiation.     

Polychlorinated biphenyls activate caspase-3-like death protease in vitro but not in vivo.

We prove here that serum albumin inhibits apoptosis induced by polychlorinated biphenyls (PCBs), confirming that serum albumin binds to PCB, and that the albumin-PCB complexes inhibit apoptosis in HL-60 cells. We found that PCB (50 microM) increased the activity of caspase-3-like protease when HL-60 cells, as well as splenocytes, were cultured in "serum-free medium." Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited apoptosis in cells cultured in the serum-free medium containing 50 microM PCB. To elucidate whether or not PCBs induce apoptosis in vivo, we examined apoptosis of splenocytes by administering PCB to ICR mice (100, 500, 1000 mg x kg(-1) x d(-1)) for 5 d and characterizing splenocytes. Interestingly, splenocytes treated with PCB did not show any changes characteristic of apoptosis. These results demonstrate that PCB activates the caspase-3-like death protease in vitro in serum-free medium, but does not induce apoptosis of splenocytes in vivo, suggesting that blood serum may mask the apoptosis induced by PCB.     

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity—HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.     

Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks

It was found that reactive oxygen species (ROS) were generated in the interactions of nitric oxide (NO) with glutathione (GSH) or cysteine (CySH) under aerobic conditions. When supercoiled DNA was incubated with a mixture of NO/GSH, NO/CySH, NOC-7 (a NO donor)/GSH or NOC-7/CySH under aerobic conditions, DNA single-strand breaks were observed on agarose gel electrophoresis. The strand breaks were inhibited by common ROS scavengers: superoxide dismutase+catalase, the spin trapping agent 5,5-dimethyl-1-pyrroline-N oxide (DMPO), ethanol, and EDTA. The strand breaks were also caused by incubation with a mixture of S-nitrosoglutathione (GSNO) with GSH or CySH, which was inhibited by ROS scavengers. In the reaction of NO/GSH, GSNO rapidly formed and then gradually decreased, and in the reaction of GSNO/GSH, GSNO was gradually decreased. The decrease inf GSNO was accelerated in the presence of superoxide+catalase. Hydroxyl radical was detected in the mixtures of NO with GSH or CySH under aerobic conditions, and thiyl radicals were detected in the mixtures of GSNO with GSH or CySH under anaerobic conditions as examined in electron spin resonance studies using DMPO as a spin trap. The results indicate that the interaction of NO with thiols in the presence of O2 generates ROS that caused DNA single-strand breaks.     

Induction of cell damage by menadione and benzo(a)pyrene-3,6-quinone in cultures of adult rat hepatocytes and human fibroblasts

The cytotoxicity of menadione (2-methyl-1,4-naphthoquinone) and benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was tested in cultures of adult rat hepatocytes and human fibroblasts. Menadione induced DNA strand breaks, cell membrane damage and depletion of reduced glutathione (GSH) in both hepatocytes and fibroblasts. In fibroblasts, effects on both DNA and membrane integrity were potentiated by the presence of dicoumarol, a specific inhibitor of the 2-electron reduction of quinones by DT-diaphorase, whereas in hepatocytes only the cell membrane damage was sensitive to dicoumarol. Results indicate that menadione toxicity is mediated via 1-electron reduction, although in hepatocytes different reactive species may be responsible for damage to DNA and to the membrane. BP-3,6-Q induced DNA strand breaks in fibroblasts at concentrations as low as 1 microM. The extent of DNA damage was insensitive to dicoumarol. Even after GSH depletion and inhibition of glucuronidation and sulphate conjugation, BP-3,6-Q caused no DNA damage in hepatocytes. In contrast to menadione, BP-3,6-Q did not induce cell membrane leakage or decrease in GSH levels in either hepatocytes or fibroblasts. These studies show the complexity of the metabolic pathways involved, in terms of activation and detoxification processes, in the toxicity of quinones.     

Oxidative stress and cell-cycle change induced by coexposed PCB126 and benzo(a) pyrene to human hepatoma (HepG2) cells

Benzo(a)pyrene (BaP) never exists in the environment as a single compound but always coexists with other chemicals. These chemicals may affect the toxicity of BaP. Our previous study confirmed that polychlorinated biphenyls (PCBs), which were recently found coexisting with BaP in various environmental media, dramatically enhanced the genotoxicity of BaP. But the known mechanisms associated with this phenomenon are limited. Because BaP's genotoxicity is highly associated with its ability to induce the oxidative stress, we propose that the coexistence of PCBs may enhance BaP's genotoxicity by affecting BaP-induced oxidative stress. In this study, the HepG2 cells were treated with either BaP (50 μM), 3,3',4,4',5-pentachlorobiphenyl (PCB126) (0.01, 0.1, 1, and 10 nM), or pretreated with PCB126 followed by a coexposure to BaP and PCB126. We found that the exposure to BaP alone effectively increased the level of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and the percentage of cells in G0/G1 phase, but decreased the percentage of S-phase cells. Compared to BaP alone, coexposure to both BaP and PCB126 effectively enhanced the levels of ROS and MDA as well as the percentage of cells in S phase, but decreased the levels of GSH and percentage of cells in G0/G1 phase. Our findings suggest that increasing oxidative stress and impairing the normal cell-cycle control may be mechanisms by which PCB126 enhances the genotoxity of BaP exposure.     

Apoptosis-inducing active components from Corbicula fluminea through activation of caspase-2 and production of reactive oxygen species in human leukemia HL-60 cells.

The anti-cancer effects and possible mechanisms of the freshwater clam (Corbicula fluminea Muller) and its active compounds (FME) on cell viability in human leukemia HL-60 cells were investigated. This study demonstrated that FME was able to inhibit cell proliferation in a concentration- and time-dependent manner. Treatment with FME caused induction of caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 activity in a time-dependent manner, but not affect caspase-1 activity; it induced the proteolysis of DNA fragmentation factor (DFF-45) and poly(ADP-ribose) polymerase (PARP). Induction of cell death by FME was completely prevented by a pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and a caspase-2 inhibitor, Z-Val-Asp-Val-Ala-Asp-FMK (Z-VDVAD-FMK). Furthermore, treatment with FME caused a rapid loss of mitochondrial transmembrane potential, stimulation of generation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and GSH depletion. Anti-oxidants such as N-acetylcysteine, catalase, superoxide dismutase, allopurinol, and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibited FME-induced cell death. In addition, the results showed that FME-induced apoptosis was accompanied by up-regulation of Bax and Bad, and down-regulation of Bcl-2 and Bcl-XL. Taken together, induction of apoptosis on HL-60 cells by FME was mainly associated with ROS production, GSH depletion, mitochondrial dysfunction, and caspase activation.     

Effects of benzene on DNA strand breaks in vivo versus benzene metabolite-induced DNA strand breaks in vitro in mouse bone marrow cells

Previously, we identified p-benzoquinone (BQ) and 1,2,4-benzenetriol (BT) as toxic metabolites of benzene on the basis of their inhibitory effect on DNA synthesis. In the present study, the capability of benzene and the two metabolites to induce DNA strand breaks was investigated in either the in vivo or the in vitro system by comparing the DNA elution rate on a fine membrane filter at alkaline pH. In the in vitro system were bone marrow cells were reacted with test chemicals for 60 min, both BQ and BT induced a dose-related increase in alkali-labile DNA single-strand breaks (SSBs) of bone marrow cells. However, when glutathione (350 micrograms/ml) was added to the same reaction system, the DNA damaging effect of BQ (24 microM) and BT (24 microM) was blocked by 100 and 53%, respectively. Catalase (130 units/ml) completely blocked the DNA damaging effect of BT, while no protection was afforded with BQ. Consistent with these observations, no induction of alkali-labile DNA SSBs was observed in the in vivo system by an anesthetic dose of benzene (1760 mg/kg, ip or po) at 1, 24, and 36 hr postadministration in both male and female ICR mice. These results suggest that benzene exposure would not induce direct DNA strand breaks in vivo under realistic work-related or accidental exposure conditions and also indicate that caution should be exercised in the interpretation of in vitro data for whole-body toxicity evaluation.     

Effects of 1, 6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantane (DPD), a reactive oxygen species and apoptosis inducing agent, on human leukemia cells in vitro and in vivo

1, 6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantine (DPD), a new cytostatic and differentiation inducing agent, was found to inhibit the growth of several cancer cell lines in the National Cancer Institute (NCI) Anticancer Drug Screen system. Previously, we demonstrated that DPD inhibited the growth of human colon cancer cell lines both in vitro and in vivo. In this study, we examined the anticancer effects of DPD on two human leukemia cells lines. DPD exerted growth inhibitory activities in vitro against two human leukemia cell lines, the promyeloid line HL-60 and the lymphoblastic line Molt-3. The in vivo effect of tumor growth suppression by DPD was also observed in mouse xenografts. No acute toxicity was observed after an intra-peritoneal challenge of DPD in "severe combined immune-deficiency" (SCID) mice twice a week. The in vitro study showed HL-60 was more sensitive to DPD than Molt-3 through induction of G(0)/G(1) cell-cycle arrest with the appearance of a hypodiploid DNA fraction. The increased superoxide (O(2)(-)), dissipation of the mitochondrial membrane potential, activation of caspase 3, and increase in annexin V binding were evident before apoptosis in DPD-treated cells. The superoxide dismutase 1 (SOD1) mRNA expression was also decreased in DPD-treated HL-60 and Molt-3 cells. Thus, it appeared that inhibition of SOD might be the major cause for the production of cellular superoxide with concomitant decrease of H(2)O(2) in DPD-treated cells. Addition of antioxidant can reduce DPD-induced mitochondrial damage, caspase activation, and annexin V binding in HL-60 cells. The results suggest that the cellular generation of O(2)(-) plays a role in initiating and coordinating DPD-mediated growth arrest and apoptosis of HL-60 cells. Importantly, addition of arsenic trioxide, a compound capable of reactive oxygen species (ROS) generation, significantly enhanced the in vitro activity of DPD. These results suggest that DPD appears to be a potential new modality in human leukemia therapy.     

An in vitro approach to assess the toxicity of certain food contaminants: methylmercury and polychlorinated biphenyls

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants and food contaminants, and known developmental neurotoxicants. Aim of this study was to evaluate the effects of MeHg, PCB 126 and PCB 153 in a battery of in vitro cell systems. A total of 17 cell types were utilized, including nervous system (neuronal and astroglial) and non-nervous system cells. End-points measured included MTT reduction, Trypan blue exclusion and (3)H-thymidine incorporation into DNA. Results indicate that this approach would identify these three compounds as neurotoxicants, and would also point out to the thyroid (for PCB 126 and MeHg) and the prostate (for both PCBs) as important additional targets. Tests of binary combinations of MeHg and PCBs indicated no interaction and an additive response, in agreement with other recent reports. Cerebellar granule neurons from mice with genetically determined low glutathione levels were more sensitive than wild-type neurons to the toxicity of all three compounds, supporting a role for oxidative stress in their neurotoxicity. These findings provide initial evidence that a relatively rapid in vitro screening approach can be developed, that would provide initial information useful for assessing neurotoxicity, as well as indication on potential other targets of biological action or toxicity.     

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).     

Polychlorinated biphenyls: correlation between in vivo and in vitro quantitative structure-activity relationships (QSARs)

The in vivo quantitative structure-activity relationships (QSARs) for several polychlorinated biphenyls (PCBs) were determined in the immature male Wistar rat. The ED25 and ED50 values for hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction as well as for body weight loss and for thymic atrophy were determined for nine PCB congeners and 4'-bromo-2,3,4,5-tetrachlorobiphenyl. The most active compounds were the coplanar PCB congeners, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl; for example, their ED50 values for body weight loss were 3.25 and 15.1 mumol/kg, respectively. The in vivo toxicity of the coplanar PCB, 3,3',4,4'-tetrachlorobiphenyl, was significantly lower (ED50 for body weight loss = 730 mumol/kg) than the values observed for the more highly chlorinated homologs, and this was consistent with the more rapid metabolism of the lower chlorinated congener. The dose-response biologic and toxic effects of several mono-ortho-chloro-substituted analogs of the coplanar PCBs, including 2,3,4,4'5-, 2,3,3',4,4'-, 2',3,4,4',5- and 2,3',4,4',5-penta-, 2,3,3',4,4',5- and 2,3,3',4,4',5'-hexachlorobiphenyl were also determined, and members of this group of compounds were all less toxic than 3,3',4,4',5-penta and 3,3',4,4',5,5'-hexachlorobiphenyl. There was a good rank order correlation between the in vivo QSAR data and the in vitro QSARs for PCBs that were developed from their relative receptor binding affinities and potencies as inducers of AHH and EROD in rat hepatoma H-4-II E cells in culture. These results are consistent with the proposed receptor-mediated mechanism of action for PCBs. In addition, for this series of halogenated biphenyls there was a linear correlation between their in vivo toxicity in rats and their in vitro monooxygenase enzyme induction results. Assuming that the in vivo toxic responses in the rat are representative toxic responses to PCBs, then these results support the predictive utility of the in vitro bioassay with rat hepatoma H-4-II E cells as a short-term test system for the potential toxicity of this class of halogenated aryl hydrocarbons.     

An examination of quinone toxicity using the yeast Saccharomyces cerevisiae model system

The toxicity of quinones is generally thought to occur by two mechanisms: the formation of covalent bonds with biological molecules by Michael addition chemistry and the catalytic reduction of oxygen to superoxide and other reactive oxygen species (ROS) (redox cycling). In an effort to distinguish between these general mechanisms of toxicity, we have examined the toxicity of five quinones to yeast cells as measured by their ability to reduce growth rate. Yeast cells can grow in the presence and absence of oxygen and this feature was used to evaluate the role of redox cycling in the toxicity of each quinone. Furthermore, yeast mutants deficient in superoxide dismutase (SOD) activity were used to assess the role of this antioxidant enzyme in protecting cells against quinone-induced reactive oxygen toxicity. The effects of different quinones under different conditions of exposure were compared using IC50 values (the concentration of quinone required to inhibit growth rate by 50%). For the most part, the results are consistent with the chemical properties of each quinone with the exception of 9,10-phenanthrenequinone (9,10-PQ). This quinone, which is not an electrophile, exhibited an unexpected toxicity under anaerobic conditions. Further examination revealed a potent induction of cell viability loss which poorly correlated with decreases in the GSH/2GSSG ratio but highly correlated (r2 > 0.7) with inhibition of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), suggesting disruption of glycolysis by this quinone. Together, these observations suggest an unexpected oxygen-independent mechanism in the toxicity of 9,10-phenanthrenequinone.     

Does metformin prevent short-term oxidant-induced dna damage? In vitro study on lymphocytes from aged subjects

Metformin(1-(diaminomethylidene)-3,3-dimethyl-guanidine), an anti-hyperglycemic agent, also has antioxidant effects. Although the origin is not clearly understood, the antioxidant activity of metformin might result from a direct effect on reactive oxygen species (ROS) or could have an indirect action on the superoxide anions produced by hyperglycemia. The ability of metformin to modulate DNA damage produced by oxidative stress is not known. For this reason, we examined the short term effect of metformin (50 microM, 2 h) on the DNA damage of cumene hydroperoxide (CumOOH)-induced lymphocytes from aged and young control groups (n = 10 each). In this study, DNA damage elicited by CumOOH (1 mM) was detected with the Comet Assay and the ELISA technique. Our results showed a significant increase in apoptotic DNA fragmentation and DNA strand breaks (Comet assay tail factor %) that was detected before and after CumOOH induction in lymphocytes of healthy elderly subjects when compared with healthy young control. Metformin significantly decreased CumOOH-induced apoptotic DNA fragmentation and DNA strand breaks in lymphocytes from aged subjects, although it did not produce a long-term effect. The in vitro results indicate that the short-term effect of metformin can protect against prooxidant stimulus-induced DNA damage in lymphocytes from elderly subjects.     

Reduced DNA double strand breaks in chlorambucil resistant cells are related to high DNA-PKcs activity and low oxidative stress

Modulation of DNA repair represents a strategy to overcome acquired drug resistance of cells to genotoxic chemotherapeutic agents, including nitrogen mustards (NM). These agents induce DNA inter-strand cross-links, which in turn produce double strand breaks (dsbs). These breaks are primarily repaired via the nonhomologous end-joining (NHEJ) pathway. A DNA-dependent protein kinase (DNA-PK) complex plays an important role in NHEJ, and its increased level/activity is associated with acquired drug resistance of human tumors. We show in this report that the DNA-PK complex has comparable levels and kinase activity of DNA-PK catalytic subunit (DNA-PKcs) in a nearly isogenic pair of drug-sensitive (A2780) and resistant (A2780/100) cells; however, treatment with chlorambucil (Cbl), a NM-type of drug, induced differential effects in these cells. The kinase activity of DNA-PKcs was increased up to 2h after Cbl treatment in both cell types; however, it subsequently decreased only in sensitive cells, which is consistent with increased levels of DNA dsbs. The decreased kinase activity of DNA-PKcs was not due to a change in its amount or the levels of Ku70 and Ku86, their subcellular distribution, cell cycle progression or caspase-mediated degradation of DNA-PK. In addition to DNA cross-links, Cbl treatment of cells causes a 2.2-fold increase in the level of reactive oxygen species (ROS) in both cell types. However, the ROS in A2780/100 cells were reduced to the basal level after 3-4h, while sensitive cells continued to produce ROS and undergo apoptosis. Pre-treatment of A2780 cells with the glutathione (GSH) precursor, N-acetyl-L-cysteine prevented Cbl-induced increase in ROS, augmented the kinase activity of DNA-PKcs, decreased the levels of DNA dsbs and increased cell survival. Depletion in GSH from A2780/100 cells by L-buthionine sulfoximine (BSO) resulted in sustained production of ROS, lowered DNA-PKcs kinase activity, enhanced levels of DNA dsbs, and increased cell killing by Cbl. We propose that oxidative stress decreases repair of DNA dsbs via lowering kinase activity of DNA-PKcs and that induction of ROS could be the basis for adjuvant therapies for sensitizing tumor cells to nitrogen mustards and other DNA cross-linking drugs.