U.S. Environmental Protection Agency, National Health and Environmental Effects Laboratory, Mid-Continent Ecology Division, Duluth, Minnesota 55804
Received December 17, 1999; accepted February 22, 2000
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ABSTRACT |
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Key Words: glutathione; 1,4-hydroquinone; redox cycling; Oncorhynchus mykiss; monobromobimane.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 x 108 cells from two 2.55 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 x 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 x g, 2 min), resuspending in control (buffer IV) or toxicant solution (BQ in buffer IV) in round-bottom flasks (2 to 4 x 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 x 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 x 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 x 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 x 250 mm) column (Alltech, Deerfield, IL) with a guard column (4.6 mm x 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 x 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).
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RESULTS |
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DISCUSSION |
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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 1:21:6 for rats. Hepatocyte isolation has been shown to deplete GSH up to 3050% 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.
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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.
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ACKNOWLEDGMENTS |
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NOTES |
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