TY - JOUR
AU - Schmieder, P. K.
AB - Abstract A method to measure protein thiols (PrSH), reduced and oxidized, was adapted to determine PrSH depletion in isolated rainbow trout hepatocytes exposed to arylating agent 1,4-benzoquinone (BQ). Toxicant analysis revealed rapid conversion of BQ to 1,4-hydroquinone (HQ) upon addition to hepatocytes. Hepatocytes exposed to 200 μM BQ+HQ showed 80% decline in glutathione (GSH) (1 h), 30% loss of PrSH (6 h), and no loss of viability (24 h). Recoverable oxidized PrSH was detected only after 24 h (200 μM BQ+HQ). Exposure to 600 μM BQ+HQ caused rapid (10 min) loss of > 90% GSH and > 60% PrSH, with eventual cell death. Half of the PrSH depletion at 6 h observed in hepatocytes exposed to 600 μM BQ+HQ was recoverable by reduction with dithiothreitol. Following the loss of GSH in hepatocytes exposed to 600 μM BQ+HQ, cellular PrSH were susceptible to direct arylation and oxidation. Rainbow trout hepatocytes, which contained 10-fold less GSH than rat cells, had a GSH:PrSH ratio of 1:82 compared with rat ratios of 1:2 to 1:6. The methods reported are useful for further study and discrimination of reactive modes of action needed for prediction of aquatic organism susceptibility to these types of toxicants. glutathione, 1,4-hydroquinone, redox cycling, Oncorhynchus mykiss, monobromobimane The U.S. Environmental Protection Agency (U.S. EPA) uses quantitative structure-activity models (QSARs) extensively to predict the ecologic risk of chemicals to aquatic organisms (Bradbury, 1995). Further advancement of QSARs for the prediction of electrophile/proelectrophile (reactive) toxicity in aquatic organisms is dependent on the ability to discriminate mechanisms of reactivity (Hermens, 1990; Russom et al., 1997), including redox cycling and arylation. Once mechanisms are discriminated, chemicals are assigned to groups defined by mode of action, and quantum chemical descriptors are selected that accurately relate chemical structure to the propensity of a chemical to interact with nucleophilic sites on target biomolecules (Karabunarliev et al., 1996). However, this approach is currently hindered by the lack of ability to discriminate reactive mechanisms. Predicting toxicity of chemicals such as quinones is particularly challenging due to their mixed mechanisms of reactivity. A given quinone may possess the ability to a) redox cycle, wherein the cyclical reduction and reoxidation of the quinone results in generation of reactive oxygen species and depletion of reducing equivalents, and/or b) directly arylate (via Michael addition) nucleophiles (O'Brien, 1991). Thus, the ability to assess the degree to which redox cycling and direct arylation/alkylation contribute to the toxicity of chemicals like quinones is an obligate step for development of mechanistically based QSARs for reactive toxicants. Many studies conducted using mammalian models have attempted to discern the relative importance of redox cycling and arylation in quinone-induced cytotoxicity (Gant, et al., 1988; Henry and Wallace, 1995; Miller et al., 1986; Ross et al., 1986; Toxopeus et al., 1994). Cellular glutathione (GSH), as the first line of defense against chemically induced oxidative stress, has been the focus of many investigations (Doroshow, 1995; Ross et al., 1986). Once the cellular nonprotein thiol defenses are overwhelmed, arylation and/or oxidation of critical sulfhydryl-containing proteins is thought to occur (Bellomo and Orrenius, 1985; Cho et al., 1997; Gant et al., 1988; Pascoe et al., 1987). These reactions may alter the function of protein thiol groups (PrSH), which participate in protein folding, metabolic regulation, transport of reducing equivalents, regulatory pathways, and antioxidant defense (Pumford and Halmes, 1997; Willis and Schleich, 1996). The electrophilic centers of quinones [e.g., 2,3,5, and/or 6 positions of 1,4-benzoquinone, (BQ)] and other electrophilic compounds may react directly with nucleophilic thiol moieties, including those present on many proteins (Fig. 1). Glutathione disulfide (GSSG) produced during redox cycling of quinones and other toxicants may also react with thiol groups on proteins to form mixed protein disulfides (PrSSG, PrSSR) (Bellomo et al., 1987; Lii et al., 1996). Both reactions result in a net loss of cellular-reduced PrSH, thus measuring a decrease in total reduced PrSH may be an indication of toxicity for compounds acting through these mechanisms. Protein thiol depletion has been directly measured in isolated rat hepatocytes exposed to toxicants including quinones, e.g., 1,4-naphthoquinone, 2,3-methyl-1,4-naphthoquinone, 2-methyl-1,4-NQ, 2,3-dimethoxy-1,4-naphthoquinone, 1,4-benzoquinone, 2- and 5-hydroxy-1,4-naphthoquinone, and N-acetyl-p-benzoquinone imine (Albano et al., 1985; Bellomo et al., 1990; d'Arcy Doherty et al., 1987; Toxopeus et al., 1993). These investigators attempted to discriminate between reduction in PrSH due to direct arylation and indirect oxidation by measuring total loss of PrSH and PrSH recoverable upon addition of the reducing agent dithiothreitol (DTT) or other reductive treatment, in rat hepatocytes. The time course and magnitude of loss of PrSH in relation to changes in cellular GSH/GSSG, in combination with measures of cell function and viability, may indicate whether a chemical is primarily undergoing redox cycling or directly alkylating/arylating cellular proteins as a main event, leading to observed toxic responses. The rainbow trout is a widely used model in aquatic toxicology. Although various studies have monitored changes in GSH concentrations upon exposure to toxicants (e.g., Dalich and Larson, 1985; Davies, 1985; Otto and Moon, 1995; Parker et al., 1981; Rabergh and Lipsky, 1997; Singh et al., 1996), the status of PrSH groups, especially upon exposure to reactive toxicants, has not been well characterized. BQ was chosen to serve as a model reactive toxicant to assess cellular protein thiol depletion in trout. The objectives of this study were to a) adapt a method to measure PrSH in isolated rat hepatocytes (Cotgreave and Moldeus, 1986) for the measurement of PrSH in isolated trout hepatocytes; b) determine the time course of PrSH depletion relative to changes in GSH redox status upon exposure of trout hepatocytes to BQ; and c) determine the relative contributions of BQ-mediated arylation and indirect oxidation to observed depletion of intracellular PrSH in isolated rainbow trout hepatocytes, through the measurement of the DTT-recoverable component of PrSH. MATERIALS AND METHODS Reagents. Acetonitrile (ACN) was procured from Burdick and Jackson, Muskegon, MI. Monobromobimane (mBBr; 4-bromomethyl-3,6,7-trimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione) was obtained from Molecular Probes Inc., Eugene, OR. N-[2-hydroxyethyl]piperazine-N`-[2-ethanesulfonic acid]) ½ sodium salt (HEPES) was purchased from Research Organics, Inc., Cleveland, OH. Hydroquinone, 1,4-benzenediol (HQ), was secured from Aldrich Chemical Company, Milwaukee, WI. MEM Amino Acid Solution 50X and MEM Vitamin Solution 100X were purchased from Gibco BRL, Grand Island, NY. Trichloroacetic acid (TCA), perchloric acid (PCA), γ-glutamyl glutamate, GSH, GSSG, DTT, Coomassie Blue, bovine serum albumin (BSA), collagenase type IV from Clostridium histolyticum, Percoll, 3-aminobenzoic acid ethyl ester (MS-222), trypan blue, BQ, diethylenetriaminepentaacetic acid (DEPA), and all other chemicals were purchased from Sigma Chemical Co., St. Louis, MO. Fish. Immature male and female rainbow trout (Oncorhychus mykiss, 250 to 550 g) were used. Fish were obtained from the Seven Pines Fish Hatchery, Lewis, WI, and allowed to acclimate to U.S. EPA holding facilities [Lake Superior water, 2 μm filtered, ultraviolet light (UV) treated, 11°C, pH = 7.7, hardness = 45 mg/l; Silver Cup Fish Food, Nelson and Sons, Murray, UT] for at least 2 weeks prior to use. Hepatocyte isolation and incubation. Hepatocytes were isolated by a two-step collagenase perfusion technique (Mommsen et al., 1991), yielding approximately 4 × 108 cells from two 2.5–5 g livers. Briefly, 48-h fasted trout were anesthetized with MS-222, and their livers were perfused in situ with a modified Hanks Balanced Salt Solution (137.9 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 10 mM HEPES, 4.2 mM NaHCO3, 2.5 mM EDTA; pH 7.8; buffer I) at 11°C for 10 min, followed by perfusion with Hanks Balanced Salt Solution (HBSS; 137.9 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 10 mM HEPES, 0.41 mM Mg SO4, 0.49 mM MgCl2, 1.3 mM CaCl2, 4.2 mM NaHCO3, 2.5 mM EDTA; pH 7.8) and collagenase 125 U/ml (buffer II) for 15 min. Following collagenase digestion, the livers were placed in a 1% BSA, 1X vitamin- and 1X amino acid-enriched HBSS (buffer III) before being disjoined with forceps and passed through a mesh screen (100 μm opening) to remove undigested regions. The resulting cell suspension was mixed with Percoll and centrifuged at 100 × g for 10 min at 11°C to separate viable from nonviable cells. The viable cells were resuspended in incubation medium, HBSS, containing 5.5 mM glucose and 1% BSA (buffer IV). Cell concentrations were determined by counting cells in a Neubauer improved hemacytometer. Cell viability was established by trypan blue dye exclusion, and in all cases viability was > 90%. Hepatocyte exposure to BQ was begun by gently pelleting cells (100 × g, 2 min), resuspending in control (buffer IV) or toxicant solution (BQ in buffer IV) in round-bottom flasks (2 to 4 × 106 cells/ml; 20 to 50 ml/flask), and placing in a orbital shaker (11°C) under an atmosphere of 0.25% CO2:balance air. Cells in control and exposure flasks were monitored for cell number and viability at 0, 1.5, 6, and 24 h; for total and DTT recoverable PrSH at 0, 0.25, 1.5, 6, and 24 h; for intracellular GSH and GSSG concentrations at 0, 0.25, 0.5, 1, 3, 4.5, 7, and 24 h; and for extracellular and intracellular toxicant concentrations at 0.5, 1.5, 3, and 6 h. Protein thiol and viability reported are means from five separate experiments run on different days, each using a homogenous mixture of hepatocytes freshly isolated from at least two trout. Toxicant concentrations are means from four experiments; glutathione concentrations are means from six experiments. Toxicant analysis. Solutions of BQ were prepared by adding known quantities of recrystallized BQ to buffer IV, stirring in the dark, and sonicating. The BQ stock solutions were filtered (0.2 μm), and diluted with appropriate volumes of buffer IV to achieve nominal concentrations of 200 and 600 μM. BQ solutions were cooled to 11°C prior to hepatocyte exposure and held in the dark throughout the experiment. Concentrations of the stock solutions and dilutions were measured prior to mixing with cells. Extracellular and intracellular concentrations of BQ and its reduced form HQ were measured at 0.5, 1.5, 3, and 6 h as follows. Samples (500 μl) were removed from the cell suspension and centrifuged (14,400 × g for 30 s) to pellet cells; the supernatant (extracellular fluid) was subsampled (400 μl) for analysis of the extracellular BQ and HQ concentrations. The remaining solution was aspirated off the pelleted cells and discarded, saving the pellet for measurement of intracellular BQ and HQ concentrations. The 400-μl extracellular sample was mixed with 400 μl ACN. Cell debris was pelleted by centrifugation (14,400 × g for 30 s), and the supernatant was sampled for analysis. Intracellular concentrations were determined by adding 300 μl ACN:deionized water (50:50) to the pellet, resuspending, sonicating to completely disrupt cells, centrifuging (14,400 × g for 30 s), and sampling for HPLC analysis. Analysis of extracted extracellular and intracellular BQ+HQ fractions were performed by isocratic reverse-phase HPLC using a mobile phase of 0.1 M sodium acetate, pH 4.2, 7.7% ACN on a Shandon Hypersil ODS C185U (4.6 mm × 250 mm) column (Alltech, Deerfield, IL) with a guard column (4.6 mm × 10 mm) (Alltech, Deerfield, IL). Different detection systems were used to quantify the higher extracellular concentration of BQ and HQ concentrations (UV detection at 254 nm for BQ and 288 nm for HQ) and the lower intracellular concentrations (electrochemical detection at 50 mV for BQ and 425 mV for HQ) (Kolanczyk et al., 1999), because greater analytical sensitivity was needed to detect the intracellular concentrations. BQ and HQ concentrations were quantified using linear standard curves. Glutathione analysis. Concentrations of GSH and GSSG in cellular suspensions were determined using a modification of a fluorescence detection (FLD) method of Martin and White (1991) combined with a separation technique developed by Reed et al. (1980) using HPLC (Hammermeister et al., 2000). An 800-μl sample from cell suspension flasks was gently added to a tube containing an oil layer (400 μl di-n-butyl phthalate) over an acid layer (250 μl of 10% PCA). This three-layered system was then centrifuged to allow only viable cells to move into the bottom acid layer (Fariss et al., 1985). The viable cells were lysed as they entered the acid layer, releasing the thiols of interest. Reagents and samples were kept on ice to diminish oxidation of GSH throughout the procedure. The PCA layer was subsampled and derivatized with dansyl chloride (Hammermeister et al., 2000). Analytes were determined by HPLC with fluorescence detection. Protein thiol analysis. The determination of intracellular PrSH and DTT-recoverable PrSH was based on a modification of the method of Cotgreave and Moldeus (1986) as follows. Approximately 0.5 × 106 cells in suspension were washed in a 50 mM Tris-HC1 mM EDTA buffer (TE), pH 7.8, to remove residual BSA. Cells were resuspended in TE containing 8 mM mBBr and allowed to derivatize for 5 min in the dark. The reaction was terminated, and proteins were precipitated by addition of 100% TCA. Protein precipitates were washed to remove excess reagents and resuspended in TE + 1% SDS. Sample fluorescence (Ex = 394 nm, Em = 480 nm) was measured using a spectrofluorophotometer (Shimadzu Corp., Kyoto, Japan) and quantified based upon a GSH standard curve. Content of free PrSH (i.e., that portion available to react with mBBr) was reported as nmole GSH equivalents/106 cells. The contribution of oxidation to the total depletion of cellular PrSH was determined by treating samples with 100 mM DTT for 30 min at room temperature in 10% Triton X-100 prior to derivatization with mBBr (20 mM). DTT reduces oxidized protein-mixed disulfides, allowing the freed PrSH groups to react with mBBr (Cotgreave and Moldeus, 1986). Assuming oxidation and arylation are primarily responsible for the depletion of cellular PrSH, reversing the apparent depletion of PrSH due to oxidation with DTT allows the remaining loss to be attributed to arylation. Statistical analyses of PrSH and DTT data were done using paired t-test to analyze differences between low and high treatment concentrations, and PrSH versus DTT responses for a given treatment. A one-sample t-test was used to determine differences between treatment response and control by testing percent change difference from zero. All analyses were done using Minitab for Windows (Minitab, Inc., State College, PA). RESULTS Addition of BQ nominal stock solutions (200 and 600 μM) with suspension medium (containing BSA, 11°C) prior to mixing with isolated hepatocytes resulted in measured total concentrations of BQ plus HQ (BQ+HQ) that were 25% and 50% of nominal, respectively (Table 1). Analysis of exposure solutions also revealed that a large portion of the BQ dose had been converted to HQ, i.e., 86% of the 57-μM total BQ+HQ low concentration solution, and 50% of the 331-μM total BQ+HQ high concentration solution. Upon initiation of exposure to trout hepatocytes, total extracellular BQ+HQ decreased only marginally from previously measured values. However, virtually all measurable extracellular toxicant in the presence of hepatocytes was in the form of HQ (Table 1). Extracellular BQ was detected following exposure to cells, 16% of the total BQ+HQ at 0.5 h, in only one of four replicate experiments (data not shown). By 1.5 h there was no measurable BQ in the extracellular medium in any flask (data not shown), and by 6 h total BQ+HQ decreased to 15 and 217 μM total BQ+HQ. Intracellular concentrations of BQ+HQ were also determined, with no detectable BQ present at any time (Table 1). At 30 min, there was approximately six times greater measurable intracellular BQ+HQ present in cells exposed to the high concentration than in cells exposed the low concentration. By 6 h this discrepancy was even greater, with hepatocytes exposed high concentration BQ having more than 10 times the intracellular concentration than those exposed to low concentration. Exposure solutions are subsequently referred to by the target BQ+HQ concentrations, i.e., 200 and 600 μM BQ, keeping in mind these measured concentrations varied significantly from target and nominal values in concentration and content. This is done for comparability with previous studies using unmeasured concentration. Viability of rainbow trout hepatocytes exposed to 200 μM BQ did not significantly decrease relative to controls through 24 h. Viability of cells exposed to 600 μM BQ decreased by 11% at 1.5 h, 35% at 6 h, and 65% at 24 h, as measured by exclusion of trypan blue dye (Fig. 2). Cell death determined by potassium leakage (data not shown) agreed with that measured by dye exclusion. Reduced GSH was greatly depleted (80%) in the cells exposed to 200 μM BQ by 1 h, with no further depletion noted at 7 h (p < 0.05), and some indication of recovering GSH intracellular concentrations noted at 24 h (Fig. 3A), although high variability was noted among replicate experiments. In contrast, no GSH could be detected within 15 min of cell exposure to 600 μM BQ (Fig. 2A). Likewise, GSSG in cells exposed to 600 μM BQ was undetectable 15 min following onset of exposure and did not appear for the duration of the experiment (Fig. 3B). However, cells exposed to 200 μM BQ showed a large increase in the relative amount of intracellular GSSG compared to controls. Following an initial increase of 200% at 0.25 to 1 h, intracellular GSSG eventually reached a plateau at ∼400% of control from 3 h to 7 h, but by 24 h there was no detectable intracellular GSSG (Fig. 3B). Note that measured concentrations of GSSG were highly variable. The concentration of reduced, unreacted PrSH in 200 μM BQ-exposed cells decreased by 20% at 1.5 h relative to control values of 237 ± 46 nmole GSH equivalents/106 cells (mean ± SE of five experiments; significantly different from control at p = 0.037), with 30 to 40% depletion noted from 6 through 24 h (p ≤ 0.01; Fig. 4). There was a more rapid (< 15 min) and dramatic reduction (55%) in PrSH following the addition of 600 μM BQ to isolated hepatocytes (significant at p = 0.003; Fig. 4). By 6 h, PrSH in these cells was extensively depleted (86%; significantly different from controls at p = 0.003). Thiol groups restored following addition of the reducing agent DTT represent that portion of the total PrSH loss caused by oxidation, presumably to protein-mixed disulfides, with the remaining PrSH depletion attributable to direct reaction, i.e., arylation of thiols. DTT treatment failed to restore any of the PrSH groups reacted during the 200 μM BQ exposures through 6 h (PrSH and DTT treatments not significantly different), indicating appreciable direct action of BQ. Yet, by 24 h, DTT completely restored PrSH in three of four 200 μM BQ experiments (data not shown), suggesting that oxidation was a large contributor to PrSH depletion at that time (p = 0.043; Fig. 4). In the 600-μM BQ treatment, DTT partially restored PrSH at all sampling times, evidenced by a significant difference in PrSH and DTT treatments at 0.25, 6, and 24 h (p = 0.01, 0.05, and 0.01, respectively, in paired t-tests). Appreciable PrSH loss remained (47% at 6 h and 69% at 24 h), indicating arylation played a major role in the disappearance of PrSH at the high dose (Fig. 4). Interestingly, after DTT treatment, the concentration of unrecoverable PrSH was similar between low and high BQ exposures through 6 h, indicting a similar degree of PrSH loss attributable to direct toxicant interaction. DISCUSSION The use of a sensitive, quantitative HPLC/EC method (Kolanczyk et al., 1999) allowed the measurement of extracellular and intracellular concentrations of both BQ and its reduced form HQ in the present study. A noted decrease of 45–71% BQ+HQ from nominal concentrations upon addition to suspension medium without hepatocytes was likely due to interactions of BQ (and/or HQ) with BSA, as has been shown previously with other organics (Belisario et al., 1994). Although total BQ+HQ remained relatively constant, an additional shift of the BQ to HQ equilibrium occurred upon addition of exposure medium to hepatocytes (Table 1). BQ has been shown to undergo one-electron reduction in the presence of NADH cytochrome b5 reductase or NADH: ubiquinone oxidoreductase with NADH as a cofactor (Powis and Appel, 1980), with two sequential one-electron reductions capable of producing HQ. Fish have been shown to readily oxidize both NADH and NADPH in interactions with redox cycling quinones such as menadione, in contrast to preferential utilization of only NADPH by rat hepatocytes (Di Giulio et al., 1989; Lemaire and Livingstone, 1997). It is also known that HQ can be readily oxidized to BQ, not only in mammals, but also in fish (Kolanczyk et al., 1999). Therefore, due to verification of HQ presence in the current exposures, as well as known interactions between BQ and HQ, the participation of HQ and/or BQ/HQ-GSH conjugates in the observed responses cannot be discounted, although similar exposures to nominal concentrations of HQ are required to determine its relative contribution to the observed effects. Interestingly, the rainbow trout 96-h LC50s for HQ and BQ are very similar [97 and 125 μg/l, respectively; (DeGraeve et al., 1980)]. Overall, it is difficult to compare the toxicant dynamics measured in the current study with previous work due to the paucity of measured toxicant concentrations in typical isolated rat hepatocyte exposures, with reported nominal concentrations serving as the default basis of comparison. The method to measure cellular PrSH in mammalian cells described by Cotgreave and Moldeus (1986) appeared to work well, with slight modifications, for the determination of cellular PrSH levels in isolated rainbow trout hepatocytes. The determination of intracellular PrSH and DTT-recoverable PrSH was based upon the binding of monobromobimane (mBBr) to protein sulfhydryl groups. This binding is very specific and reactive, resulting in highly fluorescent adducts (Fahey et al., 1981) that yield a detection limit suitable for the low concentration of PrSH anticipated in trout hepatocytes. Ten-fold lower GSH content in trout hepatocytes as compared to rat hepatocytes was previously observed (Table 2), with 3- to 7-fold less reduced GSH noted in trout compared with rat liver homogenates (Dalich and Larson, 1985; Wallace, 1989). The PrSH concentration measured in unexposed trout hepatocytes on nine occasions (representing values from 18 fish) was 240 nmole GSH equivalents/106 cells. This value is surprisingly similar to values reported for rat hepatocytes, although these varied from 115 to 240 nmole/106 cells (Table 2). What is striking is the large difference in GSH:PrSH ratio between the two species. In the present study, a GSH:PrSH ratio of 1:82 was observed for trout, but literature values indicate the same ratio to be between 1:2–1:6 for rats. Hepatocyte isolation has been shown to deplete GSH up to 30–50% in English sole hepatocytes (Jenner et al., 1990). If a similar depletion attributed to collagenase perfusion of the liver occurred in the present study, the GSH:PrSH ratio would still be substantially different than that determined for rat hepatocytes (which also were isolated with collagenase). Further investigation seems warranted to determine if the observed difference in GSH to PrSH balance between rats and trout indicates differential species susceptibility to reactive chemicals. Exposure of trout hepatocytes to 200 μM BQ did not result in the same degree of toxic insult as with 600 μM BQ. At the lower concentration, free protein sulfhydryl groups were depleted at a slower rate and to a lesser degree than at the high concentration, with a maximum low concentration PrSH depletion of 35–45%, with maintained cell viability and likely, therefore, some degree of protein function. Hepatocytes exposed to 600 μM BQ had nearly total PrSH loss with significant cell death, suggesting a loss of protein function. The loss of functional proteins leading to a loss of cellular function has previously been discussed for cells exposed to reactive quinones (Thor et al., 1988). It is possible that 600 μM BQ resulted in inactivation of Krebs's cycle dehydrogenases, with cells no longer capable of generating ATP (Moore et al., 1987, 1988). A rapid and total depletion of GSH, and the likely inactivation of glutathione-S-transferases (Dafre et al., 1996), occurred at the high BQ exposure, either through direct arylation, or more likely through both direct arylation and enhanced production of protein-mixed disulfides. This can be concluded from the observation that PrSH depletion due to direct arylation, i.e., the portion nonrecoverable upon DTT reduction, was similar in both the low and high exposures, yet lethality occurred only at the high concentration where more GSH depletion accompanied PrSH loss. Hepatocytes exposed to 200 μM BQ experienced a decline in GSH similar to the trend noted for PrSH. After an initial dramatic reduction of GSH (86%), although not as severe as seen in the hepatocytes exposed to 600 μM BQ, the concentration began to plateau, indicating that there was not enough BQ to arylate and inactivate all the cellular GSH. This left a small but apparently significant pool of GSH available for the detoxification of endogenous reactive oxygen generated by the cell and/or additional reactive oxygen possibly generated by BQ/HQ-glutathione conjugate-mediated redox cycling. Although the redox potential of BQ appears to limit its ability to directly cycle with O2, other BQ forms, i.e., BQ-SG or HQ-SG conjugates, may be able to cycle with O2 (Brunmark and Cadenas, 1989). Conjugation reactions with GSH are known to occur in trout hepatocytes (Parker et al., 1981). There is some evidence to suggest that BQ may induce some increase in reactive oxygen species (ROS). An apparent increase in ROS was noted in flounder microsomes exposed to BQ, although oxygen consumption was unaffected (Lemaire and Livingstone, 1997). In rat hepatocytes induced with phenobarbital or pretreated with a superoxide dismutase inhibitor, BQ was shown to induce superoxide formation, albeit at relatively low concentrations (Powis et al., 1981). The positive single electron reduction potential of BQ (+78 mV) (Wardman, 1989) favors a direct electron transfer from the semiquinone to target species, generally not resulting in interaction with molecular oxygen (–155 mV) and subsequent production of superoxide radical (Powis and Appel, 1980). Therefore, it is unlikely that BQ is directly producing superoxide. However, it is possible that the buildup of endogenously generated reactive oxygen within the cell may occur due to a breakdown of hepatocyte defense mechanisms with the drastic depletion of GSH. Alternatively, perhaps generation of glutathione thiyl radical species may be leading to the production of additional oxygen radicals and oxidative stress, dependent on the status of superoxide dismutase and catalase (Munday and Winterbourn, 1989). However, intracellular GSSG gradually increased over time out to 7 h in cells exposed to 200 μM BQ. The time course of this increase is inconsistent with what would be expected from a redox cycling compound such as menadione, where an immediate and very large spike in GSSG levels occurs followed by a equally large decrease as the GSSG is pumped out of the cells, shown to occur in rodents (Anari et al., 1995). The observed intracellular accumulation of GSSG may be consistent with the cell trying to compensate for the loss of active proteins while detoxifying the cellular-generated reactive oxygen with the GSH/GSSG redox pair (Di Monte et al., 1984; Lii et al., 1996). Thus, GSSG buildup may be due to the BQ-mediated arylation of protein pumps necessary for normal removal of GSSG. Arylation appears to be the primary mechanism by which BQ, at both 200 and 600 μM concentration, depleted hepatocellular PrSHs in rainbow trout. The BQ-induced cell death appeared to be concentration dependent; rainbow trout hepatocytes were capable of detoxifying 200 μM BQ, but were overwhelmed by 600 μM BQ. There was apparently a level of PrSH depletion, related to degree of GSH depletion, that the cells could withstand and still function. When cells were depleted beyond this level, cell death followed. A method is presented that allows the sensitive detection of PrSH loss in isolated trout hepatocytes exposed to an arylating quinone, BQ. This method can be further applied to detect the direct action of quinones on isolated trout hepatocytes, and will be useful to differentiate direct alkylation/arylation mechanisms from indirect mechanisms associated with formation of reactive oxygen species, leading to GSH depletion and associated sequelae. The ability to better distinguish reactive mechanisms of toxicity in this species is essential to developing predictive models of the susceptibility of aquatic organisms to reactive electrophile/proelectrophile modes of action (Bradbury, 1995; Hermens, 1990; Russom et al., 1997). This work furthers the development of mechanistically based structure-activity models used to assess the risk of exposure to chemicals for which little or no toxicity information exists. TABLE 1 Calculated and HPLC/EC Measured Concentrations of 1,4-Benzoquinone + 1,4-Hydroquinone (BQ+HQ) in Extracellular and Intracellular Fractions Samples during Exposure of Isolated Rainbow Trout Hepatocytes to Weighed Quantities of 1,4-Benzoquinone Dissolved in Cell Suspension Medium Containing BSA at 11°C   BQ + HQ  % BQ  BQ + HQ  % BQ   a Calculated concentration based on chemical weight and dilutions.  b Mean ± SE of four experiments.  c Concentration measured in cell medium without cells present.  d Concentration measured in cell medium 0.5 h after mixing with hepatocytes.  e 16% BQ noted at 30 min in one of four experiments.  f Concentration measured in cell medium 6 h after mixing with hepatocytes.  g Intracellular concentration measured 0.5 h after mixing with hepatocytes.  h Intracellular concentration measured 6 h after mixing with hepatocytes.  Target concentration  200 (μM)    600 (μM)    Nominala  198 ± 6b (μM)    594 ± 17 (μM)    Measuredc  57 ± 6 (μM)  14 ± 7  331 ± 29 (μM)  50 ± 4   Extracellular (0.5 h)d  44 ± 4 (μM)  0  247 ± 26 (μM)  0e  Extracellular (6 h)f  15 ± 7 (μM)  0  217 ± 25 (μM)  0   Intracellular (0.5 h)g  0.207 ± 0.042 (nmol/106 cells)  0  1.147 ± 0.102 (4) (nmol/106 cells)  0   Intracellular (6 h)h  0.106 ± 0.049 (nmol/106 cells)  0  1.139 ± 0.183 (4) (nmol/106 cells)  0    BQ + HQ  % BQ  BQ + HQ  % BQ   a Calculated concentration based on chemical weight and dilutions.  b Mean ± SE of four experiments.  c Concentration measured in cell medium without cells present.  d Concentration measured in cell medium 0.5 h after mixing with hepatocytes.  e 16% BQ noted at 30 min in one of four experiments.  f Concentration measured in cell medium 6 h after mixing with hepatocytes.  g Intracellular concentration measured 0.5 h after mixing with hepatocytes.  h Intracellular concentration measured 6 h after mixing with hepatocytes.  Target concentration  200 (μM)    600 (μM)    Nominala  198 ± 6b (μM)    594 ± 17 (μM)    Measuredc  57 ± 6 (μM)  14 ± 7  331 ± 29 (μM)  50 ± 4   Extracellular (0.5 h)d  44 ± 4 (μM)  0  247 ± 26 (μM)  0e  Extracellular (6 h)f  15 ± 7 (μM)  0  217 ± 25 (μM)  0   Intracellular (0.5 h)g  0.207 ± 0.042 (nmol/106 cells)  0  1.147 ± 0.102 (4) (nmol/106 cells)  0   Intracellular (6 h)h  0.106 ± 0.049 (nmol/106 cells)  0  1.139 ± 0.183 (4) (nmol/106 cells)  0  View Large TABLE 2 Glutathione and Protein Thiol Concentrations in Isolated Rat or Trout Hepatocyte Models Unexposed to Toxicants Organism  GSH nmol/106 cells  GSSG nmol/106 cells  PrSH nmol/106 cells  mg protein/106cells  PrSH nmol/mg protein  Ratio of GSH to PrSH  Referencea  Note. Mean ± standard deviation (n = number of experiments, references 6, 8; or number of observations, reference 7) are reported where possible; reference 6 reported as mean ± SEM.  a References: 1. d'Arcy Doherty et al., 1987; 2. Di Monte et al., 1984; 3. Albano et al., 1985; 4. Kyle et al., 1989; 5. Larsson et al., 1986; 6. Cotgreave and Moldeus, 1986; 7. Parker et al., 1981; 8. current study.  Rat  40  —  240  —  —  1:6  1   Rat  55  12  —  —  75–100  —  2   Rat  50  3–4  —  —  150  —  3   Rat  —  —  —  —  163–112  —  4   Rat  60  1.5–2  —  —  180  —  5   Rat  55.2 ± 0.5 (5)  1  115  2.6  44.6 ± 1.8 (3)  1:2  6   Trout  6.5 ± 2.5 (3)  —  —  —  —  —  7   Trout  2.9 ± 0.8 (12)  0.06 ± 0.03 (12)  240 ± 92 (9)  0.89 ± 0.03 (6)  251 ± 64 (5)  1:82  8  Organism  GSH nmol/106 cells  GSSG nmol/106 cells  PrSH nmol/106 cells  mg protein/106cells  PrSH nmol/mg protein  Ratio of GSH to PrSH  Referencea  Note. Mean ± standard deviation (n = number of experiments, references 6, 8; or number of observations, reference 7) are reported where possible; reference 6 reported as mean ± SEM.  a References: 1. d'Arcy Doherty et al., 1987; 2. Di Monte et al., 1984; 3. Albano et al., 1985; 4. Kyle et al., 1989; 5. Larsson et al., 1986; 6. Cotgreave and Moldeus, 1986; 7. Parker et al., 1981; 8. current study.  Rat  40  —  240  —  —  1:6  1   Rat  55  12  —  —  75–100  —  2   Rat  50  3–4  —  —  150  —  3   Rat  —  —  —  —  163–112  —  4   Rat  60  1.5–2  —  —  180  —  5   Rat  55.2 ± 0.5 (5)  1  115  2.6  44.6 ± 1.8 (3)  1:2  6   Trout  6.5 ± 2.5 (3)  —  —  —  —  —  7   Trout  2.9 ± 0.8 (12)  0.06 ± 0.03 (12)  240 ± 92 (9)  0.89 ± 0.03 (6)  251 ± 64 (5)  1:82  8  View Large FIG. 1. View largeDownload slide The structure of benzoquinone (BQ) and hydroquinone (HQ). FIG. 1. View largeDownload slide The structure of benzoquinone (BQ) and hydroquinone (HQ). FIG. 2. View largeDownload slide Viability of isolated rainbow trout hepatocyte exposed to 200 (square) or 600 (triangle) μM BQ. Data is expressed as percentage of cells no longer capable of excluding trypan blue dye relative to control cells (mean ± SE, n = 5). FIG. 2. View largeDownload slide Viability of isolated rainbow trout hepatocyte exposed to 200 (square) or 600 (triangle) μM BQ. Data is expressed as percentage of cells no longer capable of excluding trypan blue dye relative to control cells (mean ± SE, n = 5). FIG. 3. View largeDownload slide Change in intracellular concentrations of (A) GSH or (B) GSSG in isolated rainbow trout hepatocytes exposed to 200 (square) or 600 (triangle) μM BQ. Data shown are mean ± SE of six experiments, with the exception of the 24-h value, which is a mean of three experiments, expressed as percentage change relative to control values measured at the same time. FIG. 3. View largeDownload slide Change in intracellular concentrations of (A) GSH or (B) GSSG in isolated rainbow trout hepatocytes exposed to 200 (square) or 600 (triangle) μM BQ. Data shown are mean ± SE of six experiments, with the exception of the 24-h value, which is a mean of three experiments, expressed as percentage change relative to control values measured at the same time. FIG. 4. View largeDownload slide Decline in measurable total free cellular protein thiols in isolated rainbow trout hepatocyte exposed to 200 (square) or 600 (triangle) μM BQ for 24 h. Data shown are expressed relative to control cells, measured without (closed symbols, mean ± SE of five experiments) or with (open symbols, mean ± SE of four experiments) addition of the reducing agent dithiothreitol, representing depletion due to arylation plus oxidation, or arylation, respectively. Asterisk signifies significant difference determined at p< 0.05 when compared with control. Plus sign signifies significant difference from 200 μM BQ. Pound sign signifies significant difference from DTT treatment samples from same exposure concentration. FIG. 4. View largeDownload slide Decline in measurable total free cellular protein thiols in isolated rainbow trout hepatocyte exposed to 200 (square) or 600 (triangle) μM BQ for 24 h. Data shown are expressed relative to control cells, measured without (closed symbols, mean ± SE of five experiments) or with (open symbols, mean ± SE of four experiments) addition of the reducing agent dithiothreitol, representing depletion due to arylation plus oxidation, or arylation, respectively. Asterisk signifies significant difference determined at p< 0.05 when compared with control. Plus sign signifies significant difference from 200 μM BQ. 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TI - Depletion of Cellular Protein Thiols as an Indicator of Arylation in Isolated Trout Hepatocytes Exposed to 1,4-Benzoquinone
JF - Toxicological Sciences
DO - 10.1093/toxsci/55.2.327
DA - 2000-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/depletion-of-cellular-protein-thiols-as-an-indicator-of-arylation-in-WN8zbo3SR3
SP - 327
EP - 334
VL - 55
IS - 2
DP - DeepDyve
ER -