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.     

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.     

Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytes

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that exhibit various toxic effects in animals and exposed human populations. The molecular mechanisms of PCB toxicity have been attributed to the toxicological properties of its metabolites, such as hydroquinones, formed by cytochrome‐P‐450 oxidation. The effects of PCB hydroquinone metabolites towards freshly isolated rat hepatocytes were investigated. Hydroquinones can be oxidized to semiquinones and/or quinone metabolites. These metabolites can conjugate glutathione or can oxidize glutathione as a result of redox cycling. This depletes hepatocyte glutathione, which can inhibit cellular defence mechanisms, causing cell death and an increased susceptibility to oxidative stress. However in the following, glutathione‐depleted hepatocytes became more resistant to the hydroquinone metabolites of PCBs. This suggested that their glutathione conjugates were toxic and that there was a third type of quinone toxicity mechanism which involved a hydrogen peroxide‐accelerated autoxidation of the hydroquinones to form toxic electrophilic quinone and semiquinone–glutathione conjugates. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells

Benzene (BZ) is a class I carcinogen and its oxidation to reactive intermediates is a prerequisite of hematoxicity and myelotoxicity. The generated metabolites include hydroquinone, which is further oxidized to the highly reactive 1,4-benzoquinone (BQ) in bone marrow. Therefore, we explored the mechanisms underlying BQ-induced HL-60 cell proliferation by studying the role of BQ-induced reactive oxygen species (ROS) in the activation of the ERK-MAPK signaling pathway. BQ treatment (0.01-30 microM) showed that doses below 10 microM did not significantly reduce viability. ROS production after 3 microM BQ treatment increased threefold; however, catalase addition reduced ROS generation to basal levels. FACS analysis showed that BQ induced a fivefold increase in the proportion of cells in S-phase. We also observed a high proportion of Bromodeoxyuridine (BrdU) stained cells, indicating a higher DNA synthesis rate. BQ also produced rapid and prolonged phosphorylation of ERK1/2 proteins. Simultaneous treatment with catalase or PD98059, a potent MEK protein inhibitor, reduced cell recruitment into the S-phase and also abolished the ERK1/2 protein phosphorylation induced by BQ, suggesting that MEK/ERK is an important pathway involved in BQ-induced ROS mediated proliferation. The prolonged activation of ERK1/2 contributes to explain the increased S-phase cell recruitment and to understand the leukemogenic processes associated with exposure to benzene metabolites. Thus, the possible mechanism by which BQ induce HL-60 cells to enter the cell cycle and proliferate is linked to ROS production and its growth promoting effects by specific activation of regulating genes known to be activated by redox mechanisms.     

2-(4'-CHLOROPHENYL)-1,4-BENZOQUINONE INCREASES THE FREQUENCY OF MICRONUCLEI AND SHORTENS TELOMERES

The toxicity of polychlorinated biphenyls (PCBs) has been attributed widely to receptor-mediated effects, buttressed by the popularity of the Toxic Equivalency Factor. We propose that a crucial toxic mechanism of lower-chlorinated PCBs is their enzymatic biotransformation to electrophiles, including quinoid metabolites, that bind intracellular sulfhydryl groups, such as those found in microtubulin and enzymes like telomerase. To test this hypothesis, we have examined micronuclei induction, cell cycle, and telomere shortening in cells in culture. Our findings show a large increase in micronuclei frequency and cell cycle perturbation in V79 cells, and a marked decrease in telomere length in HaCaT cells exposed to 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3pQ).     

Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.     

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo IIalpha activity through the accumulation of Topo II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC50 of 0.9 microM, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC50 of 9.6 microM, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 microM. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC50 about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design.     

Modulation of the toxicity and macromolecular binding of benzene metabolites by NAD(P)H:Quinone oxidoreductase in transfected HL-60 cells.

Benzene is oxidized in the liver to produce a series of hydroxylated metabolites, including hydroquinone and 1,2,4-benzenetriol. These metabolites are activated to toxic and genotoxic species in the bone marrow via oxidation by myeloperoxidase (MPO). NAD(P)H:quinone oxidoreductase (NQO1) is an enzyme capable of reducing the oxidized quinone metabolites and thereby potentially reducing their toxicities. We introduced the NQO1 gene into the HL-60 cell line to create a high MPO-, high NQO1-expressing cell line, and tested its response in assays of benzene metabolite toxicity. NQO1 expression reduced a class of hydroquinone- and benzenetriol-induced DNA adducts by 79-86%. The cytotoxicity and apoptosis caused by hydroquinone were modestly reduced, while protein binding was unchanged and the rate of glutathione depletion increased. NQO1's activity in reducing a class of benzene metabolite-induced DNA adducts may be related to its known activities in maintaining membrane-bound endogenous antioxidants in reduced form. Alternatively, NQO1 activity may prevent the formation of adducts which result from polymerized products of the quinones. In either case, this protection by NQO1 may be an important mechanism in the observation that a lack of NQO1 activity affords an increased risk of benzene poisoning in exposed individuals [Rothman, N., et al. (1997) Cancer Res. 57, 2839-2842].     

The putative benzene metabolite 2,3, 5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells

In this study, we show that 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a putative metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Prior to the onset of apoptosis, TGHQ depletes intracellular glutathione (GSH) in a reactive oxygen species (ROS)-independent manner. Neutral, Mg(2+)-dependent sphingomyelinases, which are normally inhibited by GSH, are subsequently activated, as evidenced by increases in intracellular ceramide and depletion of sphingomyelin. As ceramide levels rise, effector caspase (DEVDase) activity steadily increases. Interestingly, while catalase has no effect on TGHQ-mediated depletion of GSH, this hydrogen peroxide (H(2)O(2)) scavenger does inhibit DEVDase activity and apoptosis, provided the enzyme is added to HL-60 cells before an increase in ceramide can be observed. Since ceramide analogues inhibit the mitochondrial respiratory chain, these data imply that ceramide-mediated generation of H(2)O(2) is necessary for the activation of effector caspases-3 and/or -7, and apoptosis. In summary, these studies indicate that TGHQ, and perhaps many quinol-based toxicants and chemotherapeutics, may induce apoptosis in hematopoietic cells by depleting GSH and inducing the proapoptotic ceramide-signaling pathway.     

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.     

Tocopherol metabolism using thermochemolysis: chemical and biological properties of gamma-tocopherol, gamma-carboxyethyl-hydroxychroman, and their quinones

Identification and quantitative estimation of quinone metabolites of gamma-tocopherol (gamma-T) and its derivative gamma-carboxyethyl hydroxychroman (gamma-CEHC) are complicated by their functions as arylating electrophiles. We hypothesize that their biological properties are expressed through arylating quinone electrophile addition (Michael reaction) with thiol nucleophiles in cells and tissues. Glutathione (GSH) reacted with gamma-tocopheryl quinone (gamma-TQ) to form the hydroquinone adduct, which was identified by electrospray time-of-flight MS (ESI-TOF-MS). Tetramethylammonium hydroxide (TMAH) thermochemolysis reduced and methylated quinones and cleaved and methylated thioether adducts. These relatively nonpolar derivatives were readily separated by GC and identified by MS fragmentation patterns. gamma-CEHC was synthesized and oxidized to a product identified as the quinone lactone (gamma-CEHC-QL). TMAH methylated both gamma-CEHC-QL and its GSH adduct without opening the lactone ring, and these products were separated by GC and identified by MS fragmentation patterns. gamma-CEHC-QL reacted with both the cysteinyl enzyme papain and fetal bovine serum, and TMAH thermochemolysis showed that each product mixture contained unreacted precursor and thioether adduct. Cytotoxicities of phenolic precursors, gamma-T and gamma-CEHC, and their quinones, gamma-TQ and gamma-CEHC-QL, respectively, were compared in COS1, NT2, 3T3, and N2a cell lines. Phenolic precursor gamma-T had a small effect only with NT2 and 3T3 cells while gamma-CEHC had no effect in any cell line. Arylating quinones were highly cytotoxic in all cell lines with gamma-TQ showing a significantly greater cytotoxicity than gamma-CEHC-QL. These data are consistent with our arylating electrophile hypothesis as an explanation for some biological activities of Ts through their quinone metabolites.     

Effects of benzene metabolites on DNA cleavage mediated by human topoisomerase II alpha: 1,4-hydroquinone is a topoisomerase II poison

Although benzene induces leukemias in humans, the compound is not believed to generate chromosomal damage directly. Rather, benzene is thought to act through a series of phenolic- and quinone-based metabolites, especially 1,4-benzoquinone. A recent study found that 1,4-benzoquinone is a potent topoisomerase II poison in vitro and in cultured human cells [Lindsey et al. (2004) Biochemistry 43, 7363-7374]. Because benzene is metabolized to multiple compounds in addition to 1,4-benzoquinone, we determined the effects of several phenolic metabolites, including catechol, 1,2,4-benzenetriol, 1,4-hydroquinone, 2,2'-biphenol, and 4,4'-biphenol, on the DNA cleavage activity of human topoisomerase II alpha. Only 1,4-hydroquinone generated substantial levels of topoisomerase II-mediated DNA scission. DNA cleavage with this compound approached levels observed with 1,4-benzoquinone (approximately 5- vs 8-fold) but required a considerably higher concentration (approximately 250 vs 25 microM). 1,4-Hydroquinone is a precursor to 1,4-benzoquinone in the body and can be activated to the quinone by redox cycling. It is not known whether the effects of 1,4-hydroquinone on human topoisomerase II alpha reflect a lower reactivity of the hydroquinone or a low level of activation to the quinone. The high concentration of 1,4-hydroquinone required to increase enzyme-mediated DNA cleavage is consistent with either explanation. 1,4-Hydroquinone displayed attributes against topoisomerase II alpha, including DNA cleavage specificity, that were similar to those of 1,4-benzoquinone. However, 1,4-hydroquinone consistently inhibited DNA ligation to a greater extent than 1,4-benzoquinone. This latter result implies that the hydroquinone may display (at least in part) independent activity against topoisomerase II alpha. The present findings are consistent with the hypothesis that topoisomerase II alpha plays a role in the initiation of specific types of leukemia that are induced by benzene and its metabolites.     

Effects of 2,2',4,4'-tetrachlorobiphenyl on granulocytic HL-60 cell function and expression of cyclooxygenase-2.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that affect a number of cellular systems, including neutrophils. It has been demonstrated that noncoplanar PCBs (i.e., ortho- substituted PCBs) alter function of primary rat neutrophils. The objectives of these experiments were to determine if responses in a human, neutrophil-like cell line exposed to PCBs were similar to those reported for rat neutrophils and to explore further PCB-mediated alterations in neutrophil function. The human promyelocytic leukemia cell line (HL-60) was differentiated with DMSO to a neutrophil-like phenotype. Treatment of differentiated HL-60 cells with 2,2',4,4'-tetrachlorobiphenyl, a noncoplanar, ortho-substituted PCB congener, caused an increase in f-Met-Leu-Phe-induced degranulation, as measured by release of myeloperoxidase (MPO). Treatment with the coplanar, non-ortho-substituted congener 3,3',4,4'-tetrachlorobiphenyl had no effect on MPO release. 2,2',4,4'-Tetrachlorobiphenyl caused a time- and dose-dependent release of [3H]-arachidonic acid (3H-AA). A significant increase in 3H-AA release was observed after 60 min of exposure, and concentrations of 10 microM or larger increased 3H-AA release. In contrast, 3,3',4,4'-tetrachlorobiphenyl had no effect on 3H-AA release. The effect of PCBs on mRNA levels for cyclooxygenase-2 (COX-2) was examined using semiquantitative RT-PCR. COX-2 mRNA was significantly elevated in response to 2,2',4,4'-tetrachlorobiphenyl in a concentration-dependent manner. COX-2 expression was maximal by 30 min of exposure to 2,2',4,4'-tetrachlorobiphenyl. COX-2 protein and activity were also increased after exposure to 2,2',4,4'-tetrachlorobiphenyl; COX-1 protein and activity were unaffected. 3,3',4,4'-Tetrachlorobiphenyl did not increase COX-2 mRNA levels. These results demonstrate that a noncoplanar PCB alters the functional status of granulocytic HL-60 cells, causing enhanced degranulation and upregulation of COX-2, whereas a coplanar PCB lacks this activity. These data suggest that noncoplanar PCBs alter HL-60 cell function and COX-2 expression via an Ah-receptor-independent mechanism.     

Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely used chemotherapeutic drugs, but its effectiveness is limited by tumor cell resistance and the severe side effects it causes. One strategy for overcoming this problem is the concomitant use of natural dietary compounds as therapeutic agents. Benzyl isothiocyanate (BITC) is a promising chemopreventive agent found in cruciferous vegetables and papaya fruits. The aim of this study was to investigate the effects of BITC on cisplatin-induced cytotoxicity in human promyelocytic leukemia cells and normal human lymphocytes. The combined treatment of HL-60 cells with BITC followed by cisplatin (BITC/cisplatin) caused a significant decrease in cell viability. BITC also increased apoptotic cell death compared to cisplatin treatment alone. In normal human lymphocytes, BITC did not enhance the cytotoxic effects of cisplatin. Cellular exposure to BITC/cisplatin increased reactive oxygen species (ROS) generation but decreased the total glutathione (GSH) level in HL-60 cells. Pretreatment of HL-60 cells with N-acetylcysteine or glutathione monoethyl ester effectively decreased BITC/cisplatin-induced cell death. The addition of the extracellular signal-regulated kinase (ERK) inhibitor PD98059 abolished BITC/cisplatin-induced apoptosis. Taken together, our results suggest that BITC enhances cisplatin-induced cytotoxicity through the generation of ROS, depletion of GSH, and ERK signaling in HL-60 cells.     

DNA topoisomerase II inhibition by peroxisomicine A(1) and its radical metabolite induces apoptotic cell death of HL-60 and HL-60/MX2 human leukemia cells

Peroxisomicine A(1) (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an antitumor drug. To date, the molecular basis for its diverse biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with DNA and its effects on human DNA topoisomerases. Practically no interaction with DNA was detected. Peroxisomicine was found to inhibit topoisomerase II but not topoisomerase I. DNA relaxation and decatenation assays indicated that the drug interferes with the catalytic activity of topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase poisons such as etoposide. Two human leukemia cell lines sensitive or resistant to mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/MX2 cells (with reduced topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with T-514 stimulated the cleavage of the poly(ADP-ribose) polymerase by intracellular proteases such as caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant leukemia cell lines than T-514. Collectively, the results suggest that topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of topoisomerase II-targeted anticancer agents.     

Doxazolidine induction of apoptosis by a topoisomerase II independent mechanism

The mechanism of doxorubicin is compared with that of doxazolidine, a doxorubicin-formaldehyde conjugate. The IC(50) for growth inhibition of 67 human cancer cell lines, but not cardiomyocytes, is 32-fold lower with doxazolidine than with doxorubicin. Growth inhibition by doxazolidine correlates better with growth inhibition by DNA cross-linking agents than with growth inhibition by doxorubicin. Doxorubicin induces G2/M arrest in HCT-116 colon cancer cells and HL-60 leukemia cells through a well-documented topoisomerase II dependent mechanism. Doxazolidine fails to induce a G2/M arrest in HCT-116 cells but induces apoptosis 4-fold better than doxorubicin. The IC(50) for doxazolidine growth inhibition of HL-60/MX2 cells, a topoisomerase II deficient derivative of HL-60 cells, is 1420-fold lower than the IC(50) for doxorubicin, and doxazolidine induces apoptosis 15-fold better. Further, doxazolidine has little effect in a topoisomerase II activity assay. These data indicate that doxorubicin and doxazolidine induce apoptosis via different mechanisms and doxazolidine cytotoxicity is topoisomerase II independent.     

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.     

Changes in antioxidant defense systems by 2,2',5,5'-tetrachlorobiphenyl exposure in neuronal SK-N-MC cells

Polychlorinated biphenyls (PCBs) are known to alter the mammalian antioxidant defense system. To determine whether similar detoxification processes are activated in human neuronal cells, we investigated activities of antioxidant enzymes and the glutathione status (i.e., the levels of reduced and oxidized glutathione, GSH and GSSG) in human neuronal SK-N-MC cells exposed to 2,2',5,5'-tetrachlorobiphenyl (PCB 52). Upon PCB 52 treatment, time- and concentration-dependent inhibitions of cell viability were observed. PCB 52 did not affect GSH contents upon increasing the concentration up to 15 microg/ml, but significant depletions in GSH were observed at the concentrations of 20 and 25 microg/ml. PCB 52 exposure increased GSSG levels in the SK-N-MC cells, while GSH levels were decreased, and these changes naturally modified the GSSG/GSH ratios. Cytosolic glutathione S-transferase (GST) activity with 1-chloro-2,4-dinitrobenzene as substrate was enhanced by two-fold in neuronal cells after exposure to PCB 52 versus controls. In contrast, neuronal cells showed a sustained decrease in glutathione peroxidase activity with increasing concentrations of PCB 52, and a sustained decrease in Cu/Zn-superoxide dismutase (SOD) activity with increasing concentrations of PCB 52. Catalase activity was increased until 12 h after exposure to PCB 52, but was decreased 24 h after exposure. Overall, these results imply a major effect of PCB 52 on GSH status and upon the activities of antioxidant enzymes in human neuronal SK-N-MC cells, and upon the overall process of detoxification in human neuronal cells.     

Genotoxic effects of polychlorinated biphenyls (PCB 153, 138, 101, 118) in a fish cell line (RTG-2)

Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. The primary aim of this study was to investigate the genotoxic effects of some PCBs (PCB153 (2,2′,4,4′,5,5′-hexachlorobiphenyl) and 138 (2,2′,3,4,4′,5′-hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118 (2,3′,4,4′,5-) and 101 (2,2′,4′,5,5′-), the former an ortho-substituted, low-affinity dioxin-like compound and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial step for the identification of genotoxic effects.The alkaline comet assay and the micronucleus test show clear genotoxic damage after short and longer exposure (2 and 24 h) to maximum soluble, non-cytotoxic doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release, reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity. Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress.