Application of Quantitative Structure-Toxicity Relationships for the Comparison of the Cytotoxicity of 14 p-Benzoquinone Congeners in Primary Cultured Rat Hepatocytes Versus PC12 Cells

Arno G. Siraki, Tom S. Chan and Peter J. O'Brien1

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada M5S 2S2

Received April 1, 2004; accepted June 1, 2004


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

Key Words: quantitative structure-toxicity relationships; quinone; primary rat hepatocytes; PC12 cell cultures; ROS formation; GSH depletion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzoquinones induce a broad spectrum of effects that range from being vitally important for homeostasis to extremely toxic or carcinogenic. For example, vitamin K (phylloquinone) is an essential dietary blood clotting factor and ubiquinone (coenzyme Q10), an essential electron carrier used for mitochondrial respiration is synthesized in the body from phenylalanine or tyrosine precursors. However, most quinones are generally cytotoxic and or genotoxic compounds. They can be formed exogenously from benzene or polycyclic aromatic hydrocarbons, or endogenously from estrogens and catecholamines. Consequently, certain disease states are proposed to be mediated by endogenous quinones, such as breast cancer or Parkinson's disease by estrogen o-quinone (Nutter et al., 1994Go), or by dopamine-o-quinones (Graumann et al., 2002Go), respectively. Quinones have also been implicated in acute nephrotoxicity, immunotoxicity, and carcinogenesis (Bolton et al., 2000Go).

The molecular mechanisms of quinone cytotoxicity have been extensively reviewed (Bolton et al., 2000Go; Monks and Jones, 2002Go; O'Brien, 1991Go) and are comprised of two main mechanisms: (1) covalent binding to macromolecules (protein, DNA) via Michael addition, and (2) formation of reactive oxygen species (ROS), resulting in oxidative stress that can oxidize lipid, protein, or DNA. Covalent binding is preceded by glutathione (GSH) depletion either by conjugation, or via oxidation of GSH to GSH disulfide. It was shown in isolated rat hepatocytes that benzoquinones which alkylated GSH were more cytotoxic than benzoquinones that only oxidized GSH (Rossi et al., 1986Go). Although these are deleterious cellular events, if they are targeted to malignant tumors they can then become useful therapeutically as anticancer agents, e.g., doxorubicin, mitomycin C, etc. (Sinha and Mimnaugh, 1990Go). Diaziquone (AZQ), for example, appears to utilize a combination of covalent binding and oxygen activation in its chemotherapeutic mechanism (O'Brien et al., 1990Go).

In this study, we have tested the cytotoxicity of 14 para-benzoquinone congeners in vitro towards cultured primary rat hepatocytes and cultured rat PC12 cells in order to compare the cytotoxicity of these compounds between normal versus clonal cells. The p-benzoquinones in this series range from metabolites of environmental contaminants to quinones that are considered to be cytoprotective. Pentachlorophenol is an environmental contaminant used mainly as a wood preservative and has recently been suggested to be responsible for the neurological conditions that were observed in residents living nearby a wood treatment plant (Dahlgren et al., 2003Go). Tetrachloro-1,4-p-benzoquinone (chloranil) is a peroxidase metabolite of pentachlorophenol (Samokyszyn et al., 1995Go). p-Benzoquinone is a benzene metabolite believed to be the ultimate carcinogen in benzene-induced acute myelogenous leukemia (Hutt and Kalf, 1996Go). Interestingly, these compounds induced DNA damage similar to the cytotoxic mechanisms discussed above, i.e., chloranil and p-benzoquinone induced DNA damage by both covalent binding and oxidative stress (Lin et al., 2001Go; Pongracz et al., 1990Go; Winn, 2003Go). Therefore, investigating the cytotoxicity of these compounds may also provide insight into their potential genotoxicity. On the other hand, Coenzyme Q10 (ubiquinone), which is marketed as an anti-aging compound, was shown to reduce UVA mediated oxidative stress (Hoppe et al., 1999Go). We have evaluated a smaller congener of ubiquinone, Coenzyme Q1 (contains one isoprenyl group at C6), which was reported to prevent cumene hydroperoxide induced cytotoxicity towards isolated rat hepatocytes (Chan and O'Brien, 2003Go; Galati and O'Brien, 2003Go).

Consequently, it is desirable to identify and understand the physicochemical parameters that underlie the cytotoxic or cytoprotective effect of benzoquinones. Herein, we have analyzed the cytotoxicity of 14 p-benzoquinone derivatives by one- and two-parameter quantitative structure-activity relationships (QSTRs), and have correlated their cytotoxicity with ROS formation as well as GSH depletion. In a previous study, the derivation of a QSTR was attempted for the inhibition of Tetrahymena pyriformis growth when exposed to quinones for 40 h, but only a subset of five quinones could be correlated to one-electron redox potential. Any combination log P (log KOW), one-electron redox potential, or energy of the lowest unoccupied molecular orbital was otherwise unsuccessful (Schultz et al., 1997Go). In our study, the significant physicochemical parameters found included volume (VOL), electron affinity (EA), Hammett constant ({sigma}), and one-electron redox potential [E(Q/Q•–)], but similar to the previous study, not C log P. We attempted to explain why duroquinone was an outlier (i.e., not fitting the model) and we discuss the usage of physicochemical parameters to explain the cytotoxic mechanisms of benzoquinones.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and equipment. All p-benzoquinones and reagents were obtained from Sigma-Aldrich Co. (Oakville, ON) and were of the best possible grade. 3,6-Diaziridinyl-2,5-bis(carboethoxyamino)-p-benzoquinone (AZQ) was a gift from Dr. A. M. Rauth. Collagenase was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Dulbecco's minimum essential medium (DMEM) and Penicillin-Streptomycin (10,000 U/ml and 10 mg/ml, respectively) was obtained from Invitrogen Corp. (GIBCO, Grand Island, NY). Costar 96-well plates (clear for cytotoxicity, and black for ROS and GSH determination) were obtained from Fisher Scientific Co. (Corning Inc., Corning, NY). The plate reader used for visible spectrum readings was a SpectraMax Plus 384, and for fluorescent readings a SpectraMax Gemini XS was used (Molecular Devices Inc., Sunnyvale, CA). Cells were incubated in a Sanyo CO2 incubator (ESBE Scientific Industries Inc., Toronto, ON) and the laminar flow hood used was a model BioKlone-2–4 (Microzone Corp., Nepean, ON).

Cell culture and cytotoxicity. Freshly isolated rat hepatocytes were obtained from Sprague-Dawley rats (225–250 g) by collagenase perfusion of the liver (Moldeus et al., 1978Go). Immediately after isolation, hepatocytes were then washed once (50 x g, 3 min) with culture medium (DMEM containing 10% fetal bovine serum, 10 µg/ml insulin, 10–7 M dexamethasone, and 0.5% penicillin-streptomycin) and resuspended to a final concentration of 5 x 105 cells/ml. The hepatocytes were then plated in 96-well plates (100 µl/well) and allowed 2–3 h to adhere in a water-jacketed incubator at 37°C, 5% CO2/air. Hepatocytes were used within 4 h to minimize the effect of gene expression changes of Phase I and Phase II metabolizing enzymes (Baker et al., 2001Go). PC12 cells, which were first derived from rat phaeochromocytoma (Greene and Tischler, 1976Go), were grown in 75 ml culture treated flasks to confluence with culture medium (DMEM containing 10% fetal bovine serum, 5% horse serum, and 0.5% penicillin-streptomycin). Similar to hepatocytes, the PC12 cells were placed in 96-well plates (100 µl/well) at a density of 5 x 105 cells/ml and allowed to adhere before performing experiments.

Cell viability. Before the addition of p-benzoquinones to hepatocytes or PC12 cells, the culture medium was removed from each well and replaced with Earl's balanced salt solution (EBSS). This step was performed in order to standardize both hepatocytes and PC12 cells by preventing the proliferation of the latter but maintaining their adherence to the plate. Cytotoxicity was determined by the MTT assay, where the reduction of the latter to its formazan dye measured by its absorbance at 570 nm was proportional to cell viability. A minimum of four different doses of benzoquinones were used to derive a dose-viablity curve for the determination of the p-benzoquinone concentration that formed 50% of MTT-formazan compared to control cells, after a 2 h incubation period (LD50).

Measurement of reactive oxygen species. ROS formation was determined by measuring 2',7'-dichlorofluorescein, the oxidation product of 2',7'-dichlorofluorescin diacetate (DCFH-DA) as described previously (Wang and Joseph, 1999Go). Briefly, the media was removed from the black plates (bottom and walls) and replaced with EBSS containing 10 µM DCFH-DA. The benzoquinones were then added to the wells of the 96-well plate and immediately placed in a fluorescent plate reader preheated to 37°C. Readings were taken every 5 min for 30 min ({lambda}ex = 485 nm, {lambda}em = 538 nm). At least four different doses of benzoquinones were used to determine a dose-ROS formation curve for the derivation of the concentration that produced two-fold more ROS than control cells after a 30 min incubation period (EC200).

Glutathione assay in 96-well plates. Total glutathione (GSH) content was assayed by its reaction with o-phthalaldehyde which forms a fluorescent isoindole derivative using an assay previously described (Senft et al., 2000Go) that we have adapted for use in 96-well black plates. Briefly, after the 2-h incubation of cells with benzoquinones, 10 µl of 62.5% trichloroacetic acid was added to each well. After 5 min, 40 µl of 1 M sodium phosphate (pH 7) was added to each well and the same buffer containing 7.5 mM N-ethylmaleimide was added to a duplicate sample to account for background fluorescence. After 15 min, 100 µl of 0.16 M sodium phosphate solution containing 37.5 mM o-phthalaldehyde was added to each well and the fluorescence was determined ({lambda}ex = 365 nm, {lambda}em = 430 nm) after 30 min of incubation at room temperature in the darkness.

Selection of physicochemical parameters and QSTR derivation. The general cytotoxic mechanism of action of benzoquinones was taken into account when choosing the parameters. C Log P, the calculated logarithm of the partition coefficient, is usually a requirement because, in general, the more hydrophobic a compound is the greater its cytotoxicity. Because of the major role of NADPH:Quinone Oxidoreductase (EC 1.6.99.2) in quinone metabolism, we considered solvent accessible surface area, VOL, and molecular connectivity indices as structural parameters. To parameterize electrophilicity, we evaluated electron affinity (EA) which is the positive value of the energy of the lowest unoccupied molecular orbital, the Hammett substituent constant, {sigma}, (phenol values), and the one-electron reduction potentials [E(Q/Q•–)]. EA and {sigma} were previously shown to be analogous and interchangeable (Mekapati and Hansch, 2002Go). To prevent collinearity (and thus, a high error of regression) between parameters, a correlation matrix (see Table 1) showed that EA, E(Q/Q•–), and {sigma} were inter-correlated. Interestingly, VOL was not correlated with C log P in this data set. Therefore EA, E(Q/Q•–), and {sigma} were not used in the same equation. The use of {sigma} was useful since it is correlated (r2 = 0.93) to the pKa values (data not shown) of the corresponding hydroquinones of the benzoquinones shown in Figure 1. In fact, {sigma} can be used to calculate the pKa value of phenols, anilines, and other organic acids and bases (Perrin et al., 1981Go); and was also used to calculate redox potentials (Candeias et al., 1996Go). Quantitative structure-activity relationship (QSAR) is the more common term for the analysis carried out in this study (also called Hansch analysis). However, since the activity in this case is toxicity, it is appropriate to use the term QSTR. QSTR equations were derived by multiple linear regression analysis using Sigma Stat (V2.03, 1992–1997 SPSS, Inc.). VOL and EA were calculated by MOPAC 2002 using the CAChe Worksystem Pro V4.9 for Macintosh, (2000–2002 Fujitsu, Ltd.) after geometry optimization using PM3 parameters, and C log P values were obtained from ALOGPS 2.1 (http://146.107.217.178/lab/alogps/index.html). Hammett constants ({sigma}) were obtained for the values derived for phenols (Hansch et al., 1995Go). Physicochemical parameters used for correlations or QSTR equations are shown in Table 2.


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TABLE 1 Correlation Matrix of the Parameters Evaluated in This Study

 


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FIG. 1. Chemical structures of the p-benzoquinones used in this study. Each number corresponds to that chemical in Tables 2 and 3, and Figures 2 and 3.

 

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TABLE 2 Physicochemical Parameters of p-Benzoquinone Derivatives

 

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TABLE 3 Cytotoxicity, ROS Formation, and GSH Depletion of p-Benzoquinones in Primary Rat Hepatocytes and PC12 Cells

 


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FIG. 2. Calculated versus experimental LD50 values for hepatocytes treated with BQs. Each data point corresponds to a benzoquinone (shown in Table 1). The line was derived by plotting the actual data to the values calculated from Equation 3 (see Table 4). Duroquinone (13) was an outliers that was not accurately predicted by the equation and did not fit the model.

 


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FIG. 3. Calculated versus experimental LD50 values for PC12 cells treated with p-benzoquinones. Each data point corresponds to a benzoquinone shown in Table 1. The line was derived by plotting the actual data to the values calculated from Equation 5 (see Table 4). White circles are outliers (2,6-dimethoxy-p-benzoquinone (8) and duroquinone (13)) that were not accurately predicted by the equation and did not fit the model.

 
Mitochondrial membrane potential. Mitochondria were prepared from rat liver as described previously (Mullock and Snell, 1987Go). A mitochondrial suspension of 86.2 µg/ml was constantly stirred at 37°C in a 3 ml, four-sided plastic cuvette, in respiration buffer. The uptake and quenching of tetramethylrhodamine by mitochondria was used as a fluorescent indicator of mitochondrial membrane potential ({Delta}{Psi}m). These experiments were performed on a Shimadzu RF-5000U spectrofluorophotometer by recording kinetic scans specific for tetramethylrhodamine ({lambda}ex = 540 nm, {lambda}em = 570 nm). Mitochondria were isolated as described previously (Mullock and Snell, 1987Go). After tetramethylrhodamine was taken up by the mitochondria, pyruvate/malate (1/0.1 M) were added and after a short equilibration period (~2 min), 5 µM of p-benzoquinone was added. In cases where a protective effect was suspected, 0.5 µM of rotenone (a complex I inhibitor), was added to observe the effect on membrane potential.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of Benzoquinones towards Hepatocytes and PC12 Cells
As shown in Table 3, the benzoquinones showed varying degrees of effectiveness in killing hepatocytes and PC12 cells. LD50 values ranged approximately 2 log units (20–900 µM) with chloranil (1) being the most cytotoxic benzoquinone and 2,6-di-tert-butyl-p-benzoquinone being the least cytotoxic in hepatocytes. In PC12 cells, chloranil was also the most cytotoxic and duroquinone was the least cytotoxic. The relative order of benzoquinone cytotoxicity between the two cell typeshowever, was different. 2,6-Dimethoxy-p-benzoquinone as well as 2,6- and 2,5-dimethyl-p-benzoquinone appeared more cytotoxic towards PC12 cells and were relatively less toxic in hepatocytes. However, 2,6-dimethoxy-p-benzoquinone was almost as toxic to PC12 cells as chloranil. The greatest difference in cytotoxicity between the two cell types was observed with CoQ1 (Coenzyme Q1, ubiquinone-5) and AZQ (which were approximately 10-fold more cytotoxic to PC12 cells). Overall, it appeared that the most chlorinated substituted benzoquinone was the most cytotoxic and the most alkyl substituted benzoquinone was the least toxic.

ROS Formation by BQs
ROS formation was detected with most of the p-benzoquinones tested (Table 3). The EC200 values represent the dose needed to produce two-fold greater fluorescence of DCFH-DA than untreated cells. Chloranil was the most effective producer of ROS, closely followed by 2,5-dichloro-p-benzoquinone in both cell types. The least effective producers of ROS were 2,6-dimethoxy-, 2,6- and 2,5-dimethyl-p-benzoquinone. ROS production was not detected at 10-fold the LD50 dose for CoQ0, 2-tert-butyl-p-benzoquinone, CoQ1, AZQ, duroquinone, and 2,6-di-tert-butyl-p-benzoquinone, although CoQ0-induced ROS formation was detected in PC12 cells only. Interestingly, the compounds that did not produce detectable ROS were also the least potent cytotoxins. The exceptions, however, were CoQ0 (hepatocytes) and 2-tert-butyl-benzoquinone, which were potent cytotoxins. We classified quinones as not ROS-producing when ROS was not detected at 10 times the LD50. Since the time point for ROS was measured 90 min before the LD50 (i.e., at 30 min), it was expected that some compounds would require a higher dose to form sufficient amounts of ROS (see compounds 8–10). However, p-benzoquinones for which ROS was not detected, did produce ROS when catalese was inhibited (data not shown) and was associate with increased cytotoxicity. This is consistent with previous observations, where duroquinone and AZQ cytotoxicity was enhanced 10-fold by catalase inhibition (Rossi et al., 1986Go; O'Brien, 1991Go).

GSH Depletion
GSH content, which was detected by conjugation of GSH to o-phthalaldehyde, was determined for each BQ. The data were analyzed to determine the dose of BQ required to decrease the level of GSH to half that of untreated cells after a 2-h incubation period (EC50). For hepatocytes, chloranil (1) was the most effective at GSH depletion, and both duroquinone and 2,6-di-tert-butyl-p-benzoquinone were the least effective. At the highest dose tested for the latter two, GSH was depleted to approximately 80% of control levels for each compound, but we could not determine EC50 values because of solubility limitations. Similarly, for PC12 cells, chloranil was also the most potent GSH consumer and both 2,6-di-tert-butyl-p-benzoquinone and duroquinone did not display any GSH depletion within their solubility range. GSH was depleted to approximately 70% of control levels at the highest dose of duroquinone used (800 µM). Specifically in PC12 cells, 2-tert-butyl- and 2,6-dimethoxy-p-benzoquinone depleted GSH to near 50% of control GSH levels, but did not reduce GSH below this level. None of the p-benzoquinones used in this assay produced interference (i.e., fluorescence at these wavelengths, data not shown).

Also, we analyzed the GSH content of 5 x 105 cells/ml for both cell types and compared this to the protein content of the cells. Hepatocytes contained 20.69 ± 2.72 nmol GSH/5 x 105 cells, and PC12 cells contained 5.48 ± 0.36 nmol GSH/5 x 105 cells. However, hepatocytes contained 30.75 ± 1.80 nmol GSH/mg protein and PC12 cells contained 93.52 ± 4.60 nmol GSH/mg protein. Thus, the GSH content in the hepatocytes used in our experiments was overall greater; however, PC12 cells have a greater amount of GSH/mg protein, even though according to protein content, hepatocytes contained approximately 10-fold more protein. Protein content was determined by the Bradford protein assay (Bradford, 1976Go).

QSTR Equations
To determine the most significant parameter that correlated to cytotoxicity, we first derived one-parameter QSTR equation. In Table 4, we have shown that in hepatocytes, EA was the most significant parameter (p < 0.001). An equation derived for C log P yielded a poor regression (r2 = 0.0336) and was not significant (p = 0.531). After removal of the most significant outlier, duroquinone, the inclusion of VOL further improved the QSTR equation (Equation 3). Outliers were determined as described previously (Moridani et al., 2003Go). The equation numbers in bold indicate the best statistical fit to the data. For both cell types, EA was a required parameter, suggesting that electrophilicity is not specific to cell type. However, a better correlation was derived for hepatocytes, suggesting that EA did not entirely parameterize p-benzoquinone cytotoxicity in PC12 cells. In Figure 2, the experimental values for hepatocytes log LD50 values were plotted against those calculated by Equation 3. The outlier, duroquinone (13), was plotted as predicted by Equation 3 (but was not included in the regression line). Duroquinone cytotoxicity was overestimated by Equations 2–3 (i.e., the calculated LD50 was higher than the actual LD50). Similarly, Figure 3 shows the experimental log LD50 values for PC12 cells plotted against the values calculated from Equation 5 (Table 4). Duroquirone was also an outliner for PC12 cells where the calculated LD50 overestimated the actual value whereas 2,6-dimethoxy-p-benzoquinone was the opposite type of outliner in which the calculated LD50 underestimated the actual value.


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TABLE 4 A Comparison of the QSTRs Derived for Cultured Primary Rat Hepatocytes and Cultured Rat PC12 Cells

 
Correlation of Cytotoxicity to ROS Formation and GSH Depletion
In order to evaluate the involvement of GSH depletion and/or ROS formation in the cytotoxic mechanism of BQs, we correlated these events with cytotoxicity. Some points had to be omitted in this analysis since not every BQ depleted GSH or formed ROS (as shown in Tables 3 and 4). In Table 5, both GSH depletion and ROS formation are shown to significantly correlate with hepatocyte LD50 values. The omission of AZQ produced a better correlation between cytotoxicity and GSH depletion, most likely because the aziridine groups of AZQ rapidly bind to GSH (Gutierrez and Siva, 1995Go) and/or result in GSH oxidation (Silva and O'Brien, 1989Go). For PC12 cells, however, there was no significant correlation of cytotoxicity with ROS formation, and a poor (but statistically significant, p = 0.02) correlation of cytotoxicity to GSH depletion. Furthermore, we attempted to identify the most significant parameter for ROS formation and GSH depletion (Table 6). We found that E(Q/Q•–), EA, and {sigma} correlated well in both cell types, further suggesting that the mechanism of ROS formation may be similar in both cell types. Also, EA and E(Q/Q•–) correlated with GSH depletion, albeit not as well as with ROS formation, and {sigma} was not significant. Therefore, electrophilic parameters were better correlated to ROS formation and that other factors in addition to electrophilicity, are responsible for GSH depletion.


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TABLE 5 Correlation of Cytotoxicity to GSH Depletion and ROS Formation

 

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TABLE 6 Correlation of ROS Formation and GSH Depletion in Hepatocytes and PC12 Cells with EA, {sigma}, and E(Q/Q•–)

 
Effectiveness at H2O2 Detoxification
To account for the poor correlation of ROS to cytotoxicity in PC12 cells, we assayed H2O2 detoxifying capacity (catalase activity) in both cell types. Figure 4 shows representative traces of oxygen production by hepatocytes and PC12 cells after the addition of 100 and 500 µM H2O2. In order to make an accurate comparison, cell concentrations were standardized based on the protein content. Hepatocytes are approximately 10 times larger than PC12 cells and contain approximately 10 times more protein (data not shown). The traces in Figure 4, therefore, represent the O2 produced by 1.5 x 106 PC12 cells/ml and 1.5 x 105 hepatocytes/ml. At both concentrations of H2O2, hepatocytes were much more effective at metabolizing H2O2 than PC12 cells.



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FIG. 4. Metabolism of H2O2 by hepatocytes and PC12 cells. One-hundred and 500 µM H2O2 was added to PC12 cells (dashed lines a and c, respectively) and hepatocytes (solid lines b and d, respectively). Hepatocytes more efficiently metabolized hydrogen peroxide (H2O2) than PC12 cells as determined by the production of O2. Cell concentration was standardized based on protein content, thus 1 x 106 PC12 cells/ml and 1 x 105 hepatocytes/ml were used.

 
Mitochondrial Membrane Potential
In order to investigate the outliers in our model, we investigated their effects on mitochondrial membrane potential ({Delta}{Psi}m). Because quinones are known to induce mitochondrial dysfunction (Henry and Wallace, 1995Go), we hypothesized that the outliers may affect {Delta}{Psi}m differently from the other p-benzoquinones. Chloranil (1) was the most effective compound at disrupting mitochondrial membrane potential, as characterized by the release and detection of tetramethylrhodamine from isolated rat hepatocyte mitochondria (data not shown). The order of effectiveness at causing the most release of tetramethylrhodamine (decreasing {Delta}{Psi}m) was chloranil (1) > 2-tert-butyl-p-benzoquinone (4) > p-benzoquinone (7) > duroquinone (13) > 2,6-di-tert-butyl-p-benzoquinone. The latter was the weakest cytotoxin in hepatocytes, and accordingly, had the least effect on {Delta}{Psi}m. Interestingly, approximately 2 min after the addition of duroquinone, more tetramethylrhodamine was taken up by the mitochondria than before the addition.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have attempted to compare, under identical conditions, the cytotoxic effects that p-benzoquinones impart toward primary cultured rat hepatocytes and PC12 cells with the use of QSTR analysis (Hansch analysis) (Hansch et al., 1995Go). We performed this study in order to determine the usefulness of QSTRs in determining if there are differences in the cytotoxic mechanisms of p-benzoquinones in normal cells compared to tumor cells. From the QSTR equations derived, it was apparent that the EA of p-benzoquinones was a common requirement to model the cytotoxicity to both cell types. Although C log P did not correlate to cytotoxicity in hepatocytes, the inclusion of VOL together with EA improved the correlation. We were unsuccessful in correlating a structural parameter for PC12 cell cytotoxicity. The proposed role of EA and VOL in cytotoxicity is depicted in Scheme 1.



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Scheme 1. Cellular reaction pathways for p-benzoquinones and proposed involvement of their physicochemical parameters in this study. EA and E(Q/Q•–) appear to significantly parameterize one electron reduction pathways that lead to ROS presumably from redox cycling. EA also appears to partially parameterize alkylation pathways, but likely requires another variable. VOL appears to parameterize NQO detoxification, and together with EA may also play a role in essential alkylation reactions that lead to cytotoxicity.

 
The order of cytotoxic effectiveness of p-benzoquinones towards hepatocytes and PC12 cells was not similar. In both cell types, however, chloranil was the most cytotoxic p-benzoquinone tested, and duroquinone or 2,6-di-tert-butyl-p-benzoquinone were the least cytotoxic.

Chlorinated compounds are relatively more hydrophobic, which allows them to partition through membranes and interact with macromolecules (i.e., enzymes), even though C log P did not correlate to cytotoxicity in this study. Furthermore, halogen substitutions are electron withdrawing, rendering the molecule more electrophilic than its parent and more likely to undergo nucleophilic attack. Chloranil is a very effective mechanism-based inhibitor of rat and human glutathione-S-transferases (Dierickx, 1983Go; van Ommen et al., 1988Go). A series of p-benzoquinone analogs demonstrated that the best glutathione-S-transferase inhibitors were the fully substituted halogenated p-benzoquinones and the least effective was duroquinone (tetramethyl-p-benzoquinone) (Vos et al., 1989Go). Although duroquinone is also relatively lipophilic, which allows it to partition into membranes just as effectively, it is far less electrophilic, since the methyl groups are electron donating and cannot be substituted by GSH. Consequently, it had no effect on glutathione-S-transferase activity. Furthermore, this electrophilic characteristic also explains the effectiveness of GSH depletion observed by this set of p-benzoquinones in our study.

Although PC12 cells are much smaller and contain 10-fold less protein per cell than hepatocytes, a relatively high dose of p-benzoquinones was required to deplete their GSH. It is likely that PC12 cells could tolerate p-benzoquinone doses of similar magnitude to hepatocytes because we showed that they contain approximately 3-fold more GSH per mg protein than hepatocytes. In certain cases, a greater dose of p-benzoquinone was required to achieve the EC50 value for GSH depletion than for hepatocytes. However, it is puzzling as to why 2,6-dimethoxy-p-benzoquinone was the second most lethal p-benzoquinone toward PC12 cells. We have previously shown that 2,6-dimethoxy-p-benzoquinone was an effective redox cycling compound (O'Brien, 1991Go), which the PC12 cell may be particularly susceptible to.

Similar to tert-butyl-p-benzoquinone, GSH depletion below 50% of control levels could not be achieved using 2,6-dimethoxy-p-benzoquinone. It is possible that these compounds do not have the structural characteristics to activate microsomal glutathione-S-transferase in PC12 cells just as 2,6-dimethoxy-p-benzoquinone was shown not to activate (glutathione-S-transferase (measured by the rate of enzyme-catalyzed glutathione conjugation to chlorodinitrobenzne) in contrast to 2,6-dimethyl-p-benzoquinone (Svensson et al., 2000Go). CoQ0 was shown to lock in a partially activated state (Svensson et al., 2000Go) suggesting that it may be a partial substrate for this enzyme which could account, in part, for its potent GSH-depleting activity.

ROS formation resulted from the one-electron reduction of the p-benzoquinone that produced a transient semiquinone radical which reacted quickly with oxygen to form superoxide and the parent p-benzoquinone. This reduction was carried out enzymatically by NADPH:cytochrome P450 reductase (Cenas et al., 1994Go), xanthine oxidase (Pawlowska et al., 2003Go) and NADH dehydrogenase (Complex I) (Di Virgilio and Azzone, 1982Go) or nonenzymatically by ascorbic acid (Roginsky et al. 1999Go). The ease of reduction or electrophilicity may be related to the rate of this reaction. We found that EA and E(Q/Q•–) significantly correlated with ROS formation and cytotoxicity, as did {sigma} values albeit not as well. We utilized {sigma} values derived for phenolic substituents since it was shown previously that they could be used interchangeably with the energy of the lowest unoccupied molecular orbital (Mekapati and Hansch, 2002Go) which is approximately the negative value of EA. The chlorinated p-benzoquinone analogs were the most effective ROS producers in both cell types. This may be independent of cellular bioactivation, since ESR evidence has shown that chloranil formed a semi-quinone radical in chelex pretreated phosphate buffer (ph 7.4) (Zhu et al., 2002Go). We have previously shown that if ROS is involved in the cytotoxic mechanism, it will precede cytotoxicity (Siraki et al., 2002Go). All the p-benzoquinones formed ROS at doses lower than their cytotoxic LD50 doses, except for the 2,6-dialkyl(oxy)-substituted compounds (8–10) that produced ROS at doses greater than their LD50 values in both cell types. This may be because they are less electrophilic, and are more difficult to reduce (i.e., have negative redox potentials). Moreover, AZQ duroquinone CoQ1, and 2,6-di-tert-butyl-p-benzoquinone (all have negative redox potentials) did not produce detectable ROS at any dose. However, p-benzoquinones that did not yield detectable ROS did produce ROS when catalase was inhibited. Furthermore, catalase inhibition could also enhance their cytotoxic effectiveness in hepatocytes (data not shown). This is consistent with previous observations where duroquinone and AZQ cytotoxicity was enhanced 10-fold by catalase inhibition (O'Brien, 1991Go). Interestingly, duroquinone, the outlier of both QSTR equations, did not produce detectable ROS. This could be because the reduced hydroquinone can paradoxically act as a powerful antioxidant that scavenges freeradicals. A practical example of this is the use of tert-butylhydroquinone as an antioxidant used to extend theshelf-life of food (Byrd, 2001Go).

An exception in the PC12 cells was finding that ROS was detected from CoQ0. The latter was found to be more cytotoxic to neuroblastoma cells treated with 1 mM ascorbate, suggesting that redox cycling was involved in CoQ0 cytotoxicity (Roginsky et al., 1998aGo). Importantly, 2,6-dimethoxy-p-benzoquinone was a significant outlier in the QSTR equation derived for PC12 cells. It is possible that the relative long half-life of the 2,6-dimethoxy-p-semiquinone redical (-195s), which was proposed to be associated wih cytotoxicity toward Ehlich ascites-bearing mice (Pethlg et al., 1983Go), could explain the enhanced killing of PC12 cells by 2,6-dimethoxy-p-benzoquinone. Similarly, phenoxy radical formation has been correlated with cytotoxicity of L1210 murine leukemia cells (Selassie et al., 1998Go) and isolated hepatocytes (Moridanl et al., 2003Go). Therefore, ROS detection could represent semiquinone radical-induced cytotoxicity.

Furthermore, the doses required to produce ROS and cytotoxicity seemed significantly lower for PC12 cells which prompted us to evaluate the ability of PC12 cells and hepatocytes at metabolizing H2O2 formed from p-benzoquinone redox cycling. Hepatocytes were found to be much more effective at metabolizing H2O2 than PC12 cells. If tumor cell biochemistry is representative of to the PC12 cell, it would appear that induction of oxidative stress may be a more selective mode of preferentially targeting them over normal host cells. On the other hand, CoQ1 and AZQ (anti-cancer agent) were 10-fold more cytotoxic to PC12 cells which was the largest difference between the two cell types, yet ROS formation was not detected. This finding is contrary to previous observations performed in epidermal cell lines (Li et al., 1999Go). This may be a result of the incubation buffer we used (Earl's balanced salt solution) that contained 5 mM glucose, which may have caused a reduction in the fluorescence of dichlorofluorescein after cells were treated with p-benzoquinones (work in progress).

Contrary to one-electron reduction, two-electron reduction by NADPH:Quinone Oxidoreductase (NQO, EC 1.6.99.5) is believed to be cytoprotective towards quinones (except for reductively activated anti-cancer agents). Another group derived a QSAR for NQO showing that the kcat/Km would be greatest for the quinones that had the smallest Van der Waals volume and the highest redox potential (Anusevicius et al., 2002Go). It is interesting that our QSTR for cytotoxicity demonstrated that the most cytotoxic quinones would have the smallest VOL, and the highest EA. This similarity between these two independently derived QSARs highlights the essential role that NQO plays in p-benzoquinone detoxification.

Finally, in order to address why duroquinone did not fit our QSTR equations, we determined its effect on isolated hepatocyte mitochondria. Duroquinone cytotoxicity was likely overestimated because it could minimize rotenone-induced disruption of mitochondrial membrane potential (data not shown) possibly because it is reduced by a rotenone-insensitive site on mitochondria (Di Virgilio and Azzone, 1982Go). Furthermore, approximately 2 min after the addition of duroquinone, the {Delta}{Psi}m was greater than control levels before it was added to mitochondria. Thus, duroquinone may reduce its own cytotoxicity through enhanced mitochondrial function via bypassing electrons to Complex III (Ruzicka and Crane, 1971Go).

In summary, we have utilized QSTR as a tool to determine the physicochemical parameters that best model p-benzoquinone induced cytotoxicity towards primary rat hepatocyte culture and PC12 cells. This approach has shown that EA and E(Q/Q•) appear to be closely related to ROS formation and thus, one-electron p-benzoquinone reduction and redox cycling. Also, EA was the most significant parameter for cytotoxicity in both cell types, but more so with hepatocytes. Although the QSTR for hepatocytes could be improved by including VOL in addition to EA, no such improvement was found for PC12 cells using these parameters. ROS formation and GSH depletion were highly correlated with cytotoxicity in hepatocytes, but did not correlate as well with cytotoxicity in PC12 cells. It is possible that the p-benzoquinones may induce dopamine release and oxidation, since this occurred with mitochondrial toxins such as MPTP and cyanide (Kanthasamy et al., 1991Go; Obata, 2002Go). Such events are difficult to model with QSTRs, but also highlight the biological difference between hepatocytes and PC12 cells.


    ACKNOWLEDGMENTS
 
We thank Dr. Linda Mills (Toronto Western Hospital) for donating the PC12 cells, and Dr. Reina Bendayan (University of Toronto) and her lab for the use of their incubator and laminar flow hood. We also wish to thank Dr. Jack Uetrecht (University of Toronto) and his lab for the use of their 96-well plate readers. We also thank Dr. Chris Yip (University of Toronto) for the use of CAChe software. We also acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for the financial support of A.G.S. and T.S.C. (postgraduate scholarship awardees).


    NOTES
 

1 To whom correspondence should be addressed. Fax: (416) 978-8511. E-mail: peter.obrien{at}utoronto.ca


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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