Chemico-Biological Interactions 167 (2007) 184192

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes
Hojjat Sadeghi-Aliabadi a , Katie Chan c , Hans-Joachim Lehmler b , Larry W. Robertson b , Peter J. OBrien c,
c

Department of Medicinal Chemistry, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran b Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA 52242, USA Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ont., Canada M5S 2S2 Received 29 November 2006; received in revised form 13 February 2007; accepted 15 February 2007 Available online 23 February 2007

Abstract Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC50 (2 h) was 3 ,4 -diCl-2,3-diOH-biphenyl > PHQ > 4 -Cl-2,5-diOH-biphenyl, 4 -Cl-2,3-diOH-biphenyl > 2 ,5 -diCl-3,4-diOH-biphenyl > 2 ,3 -diCl-3,4-diOH-biphenyl > 3 ,4 -diCl3,4-diOH-biphenyl > 4 Cl-3,4-diOH-biphenyl > 4 -Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2 ,5 > 2 ,3 > 3 ,4 . Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3 ,4 -diCl-3,4-diOH-biphenyl, 3 ,4 -diCl-2,3-diOH-biphenyl and 4 -Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites. 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: PCBs; Polychlorinated biphenyls; Hepatocytes; 4-Chlorobiphenyl; Dihyroxylated metabolites; Cytotoxicity; Glutathione; Oxidative stress; Catechols

Abbreviations: AG, aminoguanidine; BQ, benzoquinone; 4-ClBP, 4-chloro-biphenyl; COMT, catechol-O-methyl transferase; GSH, glutathione; HQ, hydroquinone; PCBs, polychlorinated biphenyls; ROS, reactive oxygen species; SULT, cytosolic sulfotransferase; UGT, uridine diphosphate glucuronosyltransferases Corresponding author. Tel.: +1 416 978 2716; fax: +1 416 978 8511. E-mail address: peter.obrien@utoronto.ca (P.J. OBrien). 0009-2797/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2007.02.011

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1. Introduction PCBs were widely used in a variety of industrial and consumer products for several decades before their production was banned in the 1970s. As a result of their extensive use and their chemical stability, PCBs are still ubiquitous environmental contaminants that are frequently found as complex mixtures of isomers and congeners in air, water, soil, dust and on surfaces in homes and in factories [1]. Initial concerns regarding PCBs focused on their accumulation in the tissues of sh, birds, terrestrial and marine mammals and humans. These compounds are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. Recently, attention has been directed to the biological activity of the phenolic and catecholic metabolites of PCB [24]. Ten individual dichlorobiphenyls were shown to be metabolized to phenol dihydrodiol metabolites and unstable metabolites of intermediate polarity by microsomes (+NADPH) from rats treated with phenobarbital or 3-methylcholanthrene (to induce different P450s) [5]. Later induced rat microsomes were also shown to metabolize 4-ClBP to three phenolic compounds 4 Cl-2-OH-BP, 4 -Cl-3-OH-BP and 4 -Cl-4-OH-BP, as well as three dihydroxy metabolites, 4 -Cl-2,3-diOHBP, 4 -Cl-2,5-diOH-BP and 4 -Cl-3,4-diOH-BP, the three one-ring precursors of ortho- and para-quinones [6]. PCB catechols and hydroquinones are probably formed either by the further oxidation of phenolic PCB metabolites or by the hydrolysis of the epoxide intermediates catalyzed by epoxide hydrolase, to form dihydrodiols that may undergo further oxidation catalyzed by dihydrodiol dehydrogenase to form catechols [7]. PCB catechols can be further oxidized to semiquinones and quinones [8]. Generally catecholic metabolites of xenobiotics undergo detoxication by conjugation in which the enzymes involved in this process usually include UGTs, COMT or sulfotransferases [2,9]. The present study was conducted to determine the cytotoxicity of some catecholic PCB metabolites (Table 1) towards normal hepatocytes, COMT inhibited hepatocytes and UGT inhibited hepatocytes. We also investigated the effects of these PCB metabolites on the mitochondrial membrane potential and hepatocyte GSH levels. The results of these studies provide further information on phase II metabolism of PCB congener metabolites and also investigates whether the chlorine substitution pattern of hydroxylated PCBs is a factor affecting their rate of cytotoxicity and metabolism.

2. Experimental 2.1. Chemicals Trypan blue, iodoacetic acid, GSH, rhodamine 123, meta-phosphoric acid (MPA), dimethyl sulfoxide (DMSO) and borneol were obtained from SigmaAldrich Co. Canada Ltd. (Oakville, Ont., Canada). Collagenase (from Clostridium histoliticum), bovine serum albumin (BSA), 1-uoro2,4-dinitrobenzene (FDNB) and N-(2-hydroxyethyl) piperazine-N -(2-ethanesulfonic acid) (HEPES) were purchased from Boehringer-ingelheim (Montreal, Que., Canada). Sodium pentobarbital solution was obtained from MTC Pharmaceuticals (Cambridge, Ont., Canada). Entacapone was purchased from Novartis Pharmaceutical (East Hanover, NJ, USA). All other chemicals were purchased from a local supplier and were of the highest commercial grade available. The stock solutions of PCBs were prepared in DMSO. 2.2. Test compounds All six PCB catechol metabolites were synthesized as described previously [10]. 4 -Cl-2,5-diOH-BP was synthesized via the corresponding benzoquinone [11,12] by reduction with sodium dithionite [6]. The structure and abbreviated nomenclature of PCB metabolites used in this study are shown in Table 1. Stock solutions of metabolites were prepared as 100 mM solutions in DMSO. 2.3. Animals Male SpragueDawley rats (200250 g), fed a standard chow diet and tap water ad libitum, were used for hepatocyte preparation in all experiments. 2.4. Isolation and incubation of hepatocytes Freshly isolated rat hepatocytes were prepared from a single rat by collagenase perfusion of the liver as described by Moldeus et al. [13]. Approximately 85% of freshly isolated hepatocytes excluded trypan blue. Cells were suspended at a density of 106 cells/ml and were preincubated in KrebsHenseleit bicarbonate buffer (pH 7.4) supplemented with 12.5 mM HEPES for 30 min under an atmosphere of 10% O2 :5% CO2 :85% N2 at 37 C in continuously rotating 50 ml round bottom asks before the addition of chemicals.

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Table 1 CatecholPCB metabolites used in this study, their chemical name, structure and LC50 in normal hepatocytes Chemical names Structures Cytotoxicity LC50 ( M) normal cells

4 -Cl-3,4-diOH-biphenyl

250 25

4 -Cl-2,3-diOH-biphenyl

150 14

3 ,4 -diCl-3,4-diOH-biphenyl

200 17

2 ,3 -diCl-3,4-diOH-biphenyl

180 16

2 ,5 -diCl-3,4-diOH-biphenyl

175 12

3 ,4 -diCl-2,3-diOH-biphenyl

100 10

4-Chlorobiphenyl

400 24

4 -Cl-2,5-diOH-biphenyl

150 8

Phenyl hydroquinone

130 8

Cytotoxicity of catechol PCB metabolites in normal hepatocytes was determined at four time points (30, 60, 120 and 180 min) by % trypan blue uptake; only 2 h data are presented. Mean S.E.M. for three separate experiments are given. Reaction conditions are described under Section 2.

2.5. Preparation of UGT or COMT inhibited hepatocytes For inhibition experiments, inhibitors of metabolism, namely ()-borneol (inhibitor of glucuronidation, nal concentration 500 M) and entacapone (COMT

inhibitor, nal concentration 150 M) were added to hepatocyte incubations [14,15] and a further 5 min period was allowed before the addition of chemicals at a concentration of 125 M each. No loss of hepatocyte viability occurred even at twice the inhibitor concentration used. The cytotoxicity of metabolites to these UGT

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or COMT inhibited hepatocytes was assessed at indicated time points (30, 60, 120 and 180 min) following the addition of catecholic metabolites to the hepatocytes. Stock solutions of ()-borneol and entacapone in absolute ethanol were prepared as 100 and 20 mM solutions, respectively. 2.6. Cell viability The viability of isolated hepatocytes was assessed from the intactness of the plasma membrane as determined by the trypan blue (0.1%, w/v) exclusion assay. Aliquots of the hepatocyte incubate were taken at different time points during the 3 h incubation period. At least 80% of the control cells were still viable after 3 h. 2.7. HPLC analysis of hepatocytes GSH The total amount of GSH in the isolated hepatocyte suspension was measured by HPLC analysis of deproteinized samples (25% MPA) after derivatization with iodoacetic acid and 1-uoro-2,4-dinitrobenzene [16], using a Waters HPLC system (Model 510 pumps, WISP710B auto injector, and model 410 UV/VIS detector) equipped with a Bondapak NH2 (10 m) 3.9 mm 300 mm column and a model 660 solvent programmer. GSH was used as an external standard. The procedure is based upon the initial formation of Scarboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to 2,4-dinitrophenyl derivatives by reaction with 1uoro-2,4-dinitrobenzene (FDNB). Determination of nanomole levels of GSH is possible with this method. Briey, 0.8 ml of the treated hepatocyte suspension was incubated with 0.2 ml of 25% metaphosphoric acid for 30 min at room temperature and then centrifuged at 1000 rpm for 5 min. 0.5 ml of the supernatant and 0.05 ml of freshly prepared iodoacetic acid (15 mg/ml) were mixed with approximately 300 mg of sodium bicarbonate. The mixture was sealed and left up to 1 h or overnight in the dark at room temperature. FDNB solution (1.5%, v/v in ethanol) was added to the samples and were left in the dark for 4 h at 4 C and then analyzed by HPLC as described previously by Reed et al. [16]. Briey a gradient mobile phase comprised of two solvent systems, solvent A: methanol/water 4:1 and solvent B: methanol/acetate buffer 4:1, was used to elute the sample. Acetate buffer was prepared by addition of sodium acetate trihydrate (272 g) and glacial acetic acid (378 ml) to 122 ml Millipore water (128 ml). The HPLC gradient was programmed at a ow rate of 1.0 ml/min and started

at 10% and programmed to 90% solvent B over a 30 min period. 2.8. Mitochondrial membrane potential assay The uptake of the cationic uorescent dye, rhodamine 123, has been used for the estimation of mitochondrial membrane potential as mentioned by Andersson and co-worker [17]. Briey, 500 l aliquots of the cell suspension were separated from the incubation medium by centrifugation at 700 rpm for 30 s. The cell pellet was resuspended in 2 ml of fresh incubation medium containing 1.5 M rhodamine 123 and incubated at 37 C in a thermostatic bath for 10 min with gentle shaking. Hepatocytes were then separated by centrifugation and the amount of rhodamine 123 remaining in the incubation medium was measured uorimetrically using a Shimadzu RF5000U uorescence spectrophotometer set at 490 nm excitation and 520 nm emission wavelengths. The capacity of mitochondria to take up the rhodamine 123 dye was calculated as the difference (between control and cells treated with PCB metabolites) in rhodamine 123 uorescence. 2.9. Statistical analysis Results were analyzed by one-way analysis of variance and Dunnetts post hoc method, using Sigmastat Version 2.0. Results are expressed as the means S.E.M. p-Values less than 0.05 were considered statistically signicant and n = 3. 3. Results 3.1. Cytotoxicity of catecholic PCB metabolites towards hepatocytes The cytotoxicity of phenyl hydroquinone (PHQ) or 4-ClBP and its chlorinated catecholic metabolites in rat hepatocytes was measured by the trypan blue exclusion test [18], as the percentage of cells that take up trypan blue. Cytotoxicity was expressed as the concentration that caused 50% cell death at 2 h (LC50 ) following the addition of metabolites to hepatocytes as shown in Table 1. 4-ClBP was used as parent compound to these metabolites and PHQ was added to this study as a representative of non-chlorinated dihydroxy metabolites. The order of hepatocyte toxicity of compounds tested was 3 ,4 -diCl-2,3-diOH-biphenyl > PHQ > 4 -Cl-2,5diOH-biphenyl, 4 -Cl-2,3-diOH-biphenyl > 2 ,5 -diCl3,4-diOH-biphenyl > 2 ,3 -diCl-3,4-diOH-biphenyl > 3 , 4 -diCl-3,4-diOH-biphenyl > 4 -Cl-3,4-diOH-biphenyl

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> 4-ClBP with LC50 values varying from 100 to 400 M. 3.2. The cytotoxicity of catechols towards UGT or COMT inhibited hepatocytes As shown in Table 2, borneol (500 M), a UGT inhibitor, markedly increased the cytotoxicity of all catecholic PCB metabolites suggesting that all catechol metabolites were readily glucuronidated. Entacapone (150 M), a COMT inhibitor, whilst markedly increasing 3 ,4 -diCl-3,4-diOH-biphenyl, 3 ,4 -diCl-2,3-diOHbiphenyl and 4 -Cl-2,3-diOH-biphenyl cytotoxicity but did not increase 2 ,3 -diCl-3,4-diOH-biphenyl or 2 ,5 diCl-3,4-diOH-biphenyl cytotoxicity suggesting that 2 -Cl prevented the methylation of the catechol.

3.3. Hepatocyte GSH depletion by catecholPCB metabolites GSH depletion induced by PCB metabolites after 30 min of incubation were determined by HPLC analysis as described by Reed et al. [16]. The order of effectiveness for catalyzing hepatocyte GSH depletion and oxidation after 30 min incubation was PHQ, 3 ,4 -diCl-2,3-diOH-biphenyl > 4 -Cl-3,4-diOHbiphenyl > 2 ,3 -diCl-3,4-diOH-biphenyl > 2 ,5 -diCl3,4-diOH-biphenyl > 4 -Cl-2,3-diOH-biphenyl > 3 ,4 diCl-3,4-diOH-biphenyl as shown in Table 3. The results showed that all PCB metabolites used in this study decreased GSH levels significantly except for 3 ,4 -diCl-3,4-diOH-biphenyl (p < 0.05).

Table 2 Increasing PCB catecholic metabolite cytotoxicity towards isolated hepatocytes with UGT or COMT inhibitors Catecholic PCB metabolite % cytotoxicity at 2 h (trypan blue uptake) No inhibitor None 4 -Cl-3,4-diOH 3 ,4 -Cl2 -3,4-diOH 2 ,3 -Cl2 -3,4-diOH 4 -Cl-2,3-diOH 2 ,5 -Cl2 -3,4-diOH 3 ,4 -Cl2 -2,3-diOH
* **

UGT inhibition* 20 2 100 100 94 8 97 6 93 8 98 6

COMT inhibition** 23 36 83 58 67 49 58 2 3 7 6 7 4 7

19 38 52 53 44 41 38

2 3 6 6 5 5 4

()-Borneol, inhibitior of UGT catalyzed glucuronidation, was added to hepatocytes at a nal concentration of 500 M. Entacapone, inhibitor of COMT catalyzed methylation of catechols, was added at a nal concentration of 150 M. CatecholPCB metabolites were added to hepatocytes at 125 M. Results for % cytotoxicity at 2 h were taken as mean S.E.M. from three separate experiments. Statistical signicance compared to no inhibitor treated cells (p < 0.05).

Table 3 Total GSH of hepatocyte suspension and hepatocyte mitochondrial membrane potential Compounds tested Concentration ( M) GSHa ( M/106 cells) 30 min Control PHQ 4 -Cl-3,4-diOH-biphenyl 3 ,4 -diCl-3,4-diOH-biphenyl 2 ,3 -diCl-3,4-diOH-biphenyl 4 -Cl-2,3-diOH-biphenyl 2 ,5 -diCl-3,4-diOH-biphenyl 3 ,4 -diCl-2,3-diOH-biphenyl 130 250 250 200 150 175 100 65 39 45 66 47 52 50 38 5 5 4 6* 5 5 5 4 GSHa ( M/106 cells) 90 min 57 17 33 53 37 38 34 16 6 2 3 5* 3 4 3 2 % mitochondrial membrane potentialb 30 min 100 47 5 69 8 70 3 56 7 80 7 72 8 55 5 60 min 100 18 2 57 6 82 7 54 6 78 4 77 3 22 3

Mean S.E.M. for three separate experiments are given. a,b Determined as indicated in Section 2. Hepatocytes, 106 cells/ml, were preincubated for 30 min and then PCB metabolites were added in mentioned concentrations; samples were taken at 30 and 90 min after incubation. Control hepatocytes were incubated in KrebsHenseleit buffer, pH 7.4, containing HEPES (12.5 mM) at 37 C. Other reaction conditions are described under Section 2. For structure of compounds tested see Table 1. * Statistically not signicant as compared to control (p < 0.05).

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3.4. Mitochondrial membrane potential collapse by catecholPCB metabolites Table 3 shows the effect of 4-ClBP metabolites on the mitochondrial membrane potential of hepatocytes. 4 -Cl-2,5-diOH-biphenyl and PHQ are hydroquinone metabolites, 4 -Cl-4-OH-biphenyl is a phenolic metabolite and all tested compounds were catecholic metabolites of 4-ClBP. As shown in Table 3, the mitochondrial membrane potential partly collapsed before cytotoxicity occurred with the following order of effectiveness: PHQ > 3 4 -diCl-2,3-diOHbiphenyl > 2 ,3 -diCl-3,4-diOH-biphenyl > 4 -Cl-3,4diOH-biphenyl > 2 ,5 -diCl-3,4-diOH-biphenyl > 3 ,4 diCl-3,4-diOH-biphenyl > 4 -Cl-2,3-diOH-biphenyl. 4. Discussion The use of accelerated cytotoxic mechanism screening (ACMS) techniques has been used here to study the acute cytotoxicity of various catecholic PCB metabolites. The ACMS technique assumes that drug metabolic pathways at cytotoxic drug concentrations in vitro in 2 h give a good correlation for that which occurs in vivo [19]. This is supported by our recent ndings that there is a good correlation for in vivo induced hepatotoxicity and in vitro hepatocyte cytotoxicity using the ACMS technique for halobenzene compounds [20]. The ACMS methods used here are useful for understanding the role of different metabolic pathways in determining acute toxicity but may not be relevant for understanding low dose chronic exposure. The position of hydroxyl groups and chlorine atom on the PCB catechols may be critical for the cytotoxicity of these compounds. In the present study 3 ,4 -diCl-2,3diOH-biphenyl was found to be two-fold more cytotoxic to isolated rat hepatocytes than 3 ,4 -diCl-3,4-diOHbiphenyl. The only difference in their structure was the positions of hydroxyl groups, which was 2,3 in 3 ,4 -diCl-2,3-diOH-biphenyl and 3,4 in 3 ,4 -diCl-3,4diOH-biphenyl. The large increase in PCB catechol cytotoxicity towards UGT inhibited hepatocytes also suggests that glucuronidation was a major metabolic detoxication pathway for all PCB catechols. Previously it was shown that borneol completely inhibited hepatic glucuronidation of phenylpropionic acid in vivo or in vitro for 2 h [21]. Borneol has been shown to be the best UGT2b12 substrate, as was 3-hydroxy and 2-hydroxy biphenyls [22]. This could suggest that the PCB metabolites studied are also substrates as they have similar chemical structures to the latter. PCB phenolic metabolites, e.g. 4-

OH-3,5 dichlorobiphenyl were also found to be readily glucuronidated [2]. Other UGTs may also contribute as UGT 1A6 catalyzed the glucuronidation of tetrachlorocatechol [9]. Methylation of the PCB catechols catalyzed by COMT also detoxied 3 ,4 -diCl-3,4-diOH-biphenyl, 3 ,4 -diCl-2,3-diOH-biphenyl and 4 -Cl-2,3-diOHbiphenyl, as their cytotoxicity was increased towards COMT inhibited hepatocytes. However 2 3 -diCl3,4-diOH-biphenyl or 2 ,5 -diCl-3,4-diOH-biphenyl cytotoxictiy was not increased suggesting that 2 -Cl substitution prevented methylation. Previously it was shown that 2 ,4 ,6 -triCl-3,4-diOH-biphenyl methylation catalyzed by a hepatic S9 fraction was faster than 2 ,5 -diCl-3,4-diOH-biphenyl [23]. Semiquinone/phenoxyl radicals and quinone species contributed to the cytotoxicity of PCB catecholic metabolites. A more detailed mechanism of this oxidation proposed by Oikawa and co-workers [24] is illustrated in Fig. 1. GSH is a ubiquitous intracellular tripeptide involved in many physiological and protective functions such as conjugation of xenobiotics, thus facilitating their excretion, detoxication of metal ions, scavenging and removal of ROS and the maintenance of protein sulfhydryls [2527]. According to our previous results, quinones can be cytotoxic by two mechanisms: (i) by binding to GSH and cellular macromolecules, causing irreversible changes resulting in cell death; and (ii) by the generation of ROS through redox cycling [28]. Toxicity and depletion of cellular GSH by naphthoquinones was shown to be caused by both direct arylation of GSH and redox cycling [29]. Srinivasan et al. showed HL-60 cells were much more susceptible and formed ROS much more effectively with 4-chlorophenyl benzoquinone than with 4-chlorophenylhydroquinone. They attributed the ROS formation to autoxidation and redox cycling of the GSH conjugate formed when 4chlorophenylhydroquinone reacted with GSH. Thus, the toxicity of PCB quinones could be partly due to GSH depletion with subsequent disturbance of calcium and cellular energy homeostasis [30] and/or inhibition of many other essential proteins, like topoisomerase II [31]. Hepatocyte cell death catalyzed by catecholic PCB metabolites may also be attributed to mitochondrial toxicity as the mitochondrial membrane potential collapsed before cytotoxicity ensued (Table 3). The effectiveness of the catecholic PCB metabolites at inducing mitochondrial toxicity mostly correlated with their effectiveness at depleting hepatocyte GSH. When chlorinated catechols were added to the isolated mitochondria, there was effective uncoupling of

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Fig. 1. Proposed pathway for PCB metabolite metabolism in isolated hepatocytes: GPx, glutathione peroxidase; COMT, catechol-Omethyltransferase; UGT, UDP-glucuronosyltransferase.

mitochondrial oxidative phosphorylation and increased mitochondrial respiration [32], whereas tetrachlorobiphenylols inhibited mitochondrial state 3 respiration [33]. 4-Hydroxybiphenyl also uncoupled hepatocyte mitochondrial respiration and increased state 4 respiration, whereas 2-hydroxybiphenyl or its metabolite

phenylhydroquinone inhibited mitochondrial oxidative phosphorylation and state 3 respiration in rat hepatocytes [34]. PCB phenolic and hydroquinone metabolites were much more effective than catecholic metabolites at generating a high level of ROS (data not shown). In a

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separate study, Oakley et al. [35] reported that several catecholic and hydroquinone PCB metabolites used in this paper were oxidized [using peroxidase or Cu(II) as catalysts] to reactive intermediates that produced DNA oxidative damage and that the hydroquinone PCB metabolites were ve to seven times more effective than that induced by catecholic metabolites. Thus, these studies have shown that the cytotoxicity of PCB catecholic metabolites can be attributed to GSH depletion, mitochondrial toxicity and oxidative stress likely caused by reactive phenoxyl/semiquinone radicals and/or quinone species formed by oxidative metabolism. Acknowledgements Authors thank Dr. Jonathan W. Martin, Tom S. Chan and Arno Siraki (Faculty of Pharmacy, University of Toronto) for their assistance during this project. This project was supported mainly by the National Science and Engineering Council of Canada (PJOB), Isfahan University of Medical Sciences, Iran (HS) and in part by grant numbers P42 ES 013661 and ES 012475 from the National Institute of Environmental Health Sciences (HJL and LWR). Its contents are solely the responsibility of the authors and do not necessarily represent the ofcial views of the NIEHS. References
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