Chem. Res. Toxicol. 1996,8, 431-436
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Enzyme Induction by L-Buthionine (S,R)-Sulfoximinein Cultured Mouse Hepatoma Cells Howard G. Shertzer,* Vasilis Vasiliou, Rui-Ming Liu, M. Wilson Tabor, and Daniel W. Nebert Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056 Received October 21, 1994@ Induction of Phase I1 enzymes of the [Ah] gene battery by L-buthionine (S,R)-sulfoximine (BSO) and other agents was examined in mouse hepatoma Hepa-lclc7 cells. BSO, a nonelectrophilic inhibitor of y-glutamylcysteine synthetase (GCS), is routinely used to examine the toxicological implications of GSH depletion. Exposure to BSO for 24 h produced a 7585% depletion of GSH levels, proportional to the inhibition of GCS activity, as well as small increases in the UDP-glucuronosyltransferase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activities in Hepa-1 wild-type (wt)cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AHD4) enzyme activities, BSO produced larger increases (110% and 170%, respectively). The mechanisms of NMOl and AHD4 induction were examined further. In Hepa-1 wt cells, NMOl and AHD4 activities were increased by the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known inducing agents for these enzymes. However, NMOl and AHD4 were induced in Ah receptor nuclear translocationdefective mutant (c4) cells by BSO and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produced a concentration-dependent increase in intracellular cysteine levels, but not GSH levels, in the absence or presence of BSO. Furthermore, N-acetylcysteine had no effect on NMOl activity under any conditions examined, suggesting that GSH levelsper se, rather than change in overall thiol status, might be mediating increased NMOl activity. The increase in NMOl was accompanied by increased mRNA actinomycin D prevented the increases in both NMOl enzyme activity and mRNA levels. Conversely, N-acetylcysteine significantly increased AHD4 activity in the absence, but not the presence, of BSO. Although no increase in AHD4 mRNA was observed following BSO treatment, actinomycin D partially prevented the increase in enzyme activity. These results suggest that GSH depletion by BSO might be correlated with inducing NMOl and AHD4 activities. Furthermore, the data suggest that the electrophile response element (EpRE)-binding complex of the Nmol gene might differ from that of the Ahdl gene. Transcriptional activation appears to be responsible for the increased NMO1, and possibly in part for the increased AHD4 activity. A model is proposed whereby transcription of genes under the control of a n EpRE, such as that found in the regulatory region of all four [Ah] battery Phase I1 genes, might be controlled by the state of redox-active cysteinyl sulfur(s) contained in one or more EpRE-binding protein(s). We propose that the collection of genes that are under partial control of a n EpRE be termed the “electrophile response element’’ [EpREI gene battery.
Introduction Glutathione (GSH) is a cysteinyl tripeptide that is present at millimolar concentrations in most cell types and is essential for maintaining intracellular reducing conditions ( I ) . The protection that GSH affords cells and tissues against chemical and oxidative stress-mediated toxicity is well documented. The mechanisms for GSH protection involve the scavenging of reactive free radicals and electrophiles, reducing hydroperoxides, and maintaining the reduced states of protein thiols and lowmolecular-weight antioxidants, such as ascorbate and vitamin E (2-4). The consequences of intracellular GSH depletion are often used to examine the functions of GSH
* Corresponding author, at the Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, OH 45267-0056.Phone: (513)558-0522;Fax: (513)5584397;Internet:
[email protected]. Abstract published in Advance ACS Abstracts, March 15, 1995. @
0893-228xm5/2708-0431$09.00/0
in toxicity and disease (3). Many GSH-depleting agents are themselves antioxidants, electrophiles, free radicals, or enzyme inducing agents, and as such may obscure the specific role for GSH. However, the finding that the ratelimiting enzyme for GSH synthesis, y-glutamylcysteine synthetase (GCS),’ was inhibited irreversibly by sulfoximines such as L-buthionine (S,R)-sulfoximine(BSO)has made it possible to study the biochemical and toxicological implications of low GSH levels resulting from decreased rates of GSH synthesis (5-7). Abbreviations: AhRE, aromatic hydrocarbon response element; AHD4, cytosolic aldehyde dehydrogenase class 3;BSO, L-buthionine (S,R)-sulfoximine; DMSO, dimethyl sulfoxide; EpRE, electrophile response element; [EpREl, EpRE gene battery; GCS, y-glutamylcysteine synthetase; GST, glutathione S-transferase; NMO1, NAD(P)H: menadione oxidoreductase, (NAD(P)H:quinone acceptor oxidoreductase, quinone reductase, DT-diaphorase);tBHQ, tee-butylhydmquinone; UGT, UDP-glucuronosylTCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; transferase.
0 1995 American Chemical Society
432 Chem. Res. Toxicol.,Vol. 8, No. 3, 1995
Concomitant with GSH depletion, BSO is a weak inducing agent for UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (8). Specific forms of these enzymes, UGT form 1*06 (UGT1*06) and GST form A1 (GSTAl), as well as NAD(P)H:quinone acceptor oxidoreductase (NAD(P)H:menadione oxidoreductase (NMO1)) and cytosolic aldehyde dehydrogenase class 3 (AHD4) comprise the Phase I1 enzymes of the aromatic hydrocarbon-inducible [Ah]gene battery (9, 10). These genes can also be activated through an AP-1-like recognition site in the regulatory region termed the EpRE (also called antioxidant response element), that responds to chemical electrophiles o r reactive oxygen (10-15). We propose that these genes that are under partial control of an EpRE be termed the "electrophile response element" [EpREl gene battery. In this report we show that the [EpREl battery is coordinately induced following GSH depletion by BSO and that other thiols cannot replace GSH during GSH depletion in preventing these increases in enzyme activity. Furthermore, we show that this process is independent of the Ah receptor.
Materials and Methods Materials. All chemicals, reagents and enzymes used in this study were obtained from either the Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI), except as noted below. TCDD was purchased from Cambridge Isotope Laboratories (Woburn, MA). Caution: TCDD is a suspected human carcinogen and was handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (16). Cell Lines. The mouse hepatoma Hepa-lclc7 wild-type (wt) cells and the Ah receptor nuclear translocation-defective (c4) cells (17)were generous gifts of 0. Hankinson (UCLA, Los Angeles, CAI. The cells were cultured in modified Eagle's a-medium containing 5% fetal calf serum, 0.1% of gentamicin, and 26 mM NaHC03 a t 37 "C under 5% C02/95% air. The test compounds were dissolved in DMSO. Cells were incubated with TCDD (20 nM) or tBHQ (25 pM) for 24 h prior to harvest. For the experiments involving BSO, cells were treated with 20 pM BSO for 16 h followed by washing and the addition of fresh medium containing 20 pM BSO for another 8 h. The concentration of DMSO in the treated cells was less than 0.5%. No effect on cell viability was observed by treatment with either the vehicle or the test agents, as indicated by the lack of increase in fluorescence intensity in the presence of the membraneimpermeant DNA intercalating probe propidium iodide (18;data not shown). Preparation of SubcellularFractions. Cells were rinsed twice with ice-cold phosphate-buffered saline and scraped from flasks, and the cytosolic and microsomal fractions were isolated as modified from the procedure described previously (19). Briefly, the harvested cell suspension was centrifuged at 1500g for 5 min, and the cell pellet was resuspended in an ice-cold homogenization buffer consisting of 50 mM potassium phosphate, 1 mM EDTA, and 0.1 mM 2-mercaptoethanol (pH 7.5). The cell suspensions were sonicated on ice with a Kontes probetype sonicator (Kontes Micro-ultrasonic cell disrupter) until no whole cells remained, as judged by light microscopy. The cellfree suspension was centrifuged at 3000g for 10 min, and the supernatant fraction was centrifuged at 105000g for 1h. The soluble fraction was used for the AHD4, GST, and NMOl assays, while the microsomal pellet was resuspended in 0.3 mL of homogenization buffer and used for the CYPlAl and UGT assays. Assays. The activities of NMOl (EC 1.6.99.2) (20), GST as substrate (EC 1.2.3.4)(211, using l-chloro-2,4-dinitrobenzene CYPlAl as benzo[alpyrene hydroxylase (221, and AHD4 using NADPhenzaldehyde as substrate (23, 24) were assayed as described in the references cited. Microsomal UGT (EC 2.4.1.17) was assayed using p-nitrophenol as substrate (25), as modified
Shertzer et al. by Shertzer (26). Cytosolic reduced glutathione was determined spectrophotofluorometrically (271, and protein was measured by the bicinchoninic acid method (Pierce Chemical Co., Rockford IL), according to details supplied by the manufacturer. Specific activities are expressed in units/mg of protein. Cytosolic cysteine levels were assayed by modifications of the procedure described by Nardi et al. (28). Cells were washed with phosphate-buffered saline, harvested, and sonicated in 80 mM Tris-HC1 buffer containing 0.25 M sucrose, 5 mM MgC12, and 25 mM KCl (pH 7.4) with one 10 s burst at full power. After centrifugation, 1 mM dithiothreitol (final concentration) was added to an aliquot of freshly-prepared cell supernatant suspension or L-cysteine standard in 100 mM Tris-HC1(pH 7.6). A 40 pL aliquot of this mixture, or L-cysteine standard, was added to a microfuge tube containing 40 pL of derivatization mixture consisting of 50 mM N-ethylmorpholine hydrochloride (pH 8.4) and 10pL of 50 mM monobromobimane in CH3CN. After 5 min, 50 pL of 10%sulfosalicylic acid was added, the tubes were mixed and microfuged, and 80 pL of the supernatant solution was assayed for thiol-bimane fluorescence by HPLC. Isocratic HPLC was performed using a Waters M-45 solvent delivery system with a Model 501 pump and a Model U6K injector, fitted with a Waters Nova-Pak C-18 column. The mobile phase consisted of 10.5%CH30H in 0.25% HCOzH, pH 2.6, with a flow rate of 1.0 mUmin at 1500 psi. Fluorescence at E)470nnJEM45~~ was determined with a Hitachi F-2000 spectrofluorometer equipped with an 8 pL quartz flow cell. The column was washed with 200 pL of isopropyl alcohol before each set of determinations. Peak areas were integrated using SpectraCalc software (Galactic Industries, Salem, NH), and converted to nmol of cysteine equivalents from the integrated areas under the cysteine standard curve. For the determination of GCS activity, an aliquot of freshlyprepared cell extract was incubated at 37 "C in a reaction mixture containing 15 mM ATP, 20 mM MgC12, 1 mM EDTA, 50 mM KCl, 3 mM dithiothreitol, 3 mM L-cysteine, 15 mM L-glutamate, and 100 mM Tris-HC1 (final pH 7.8, final volume 0.3 mL). The reaction was stopped by adding 40 pL of the reaction mixture to the monobromobimane derivatization mixture, and the rate of formation of y-glutamylcysteine was determined by HPLC as described above. Standards for y-glutamylcysteine were synthesized from GSH (28) and derivatized with monobromobimane in parallel with the samples. The approximate peak positions on HPLC for the bimane adducts of cysteine and y-glutamylcysteine were 230 and 600 s, respectively. Northern Blot Analysis. RNA was isolated by the acid guanidinium thiocyanate method (29). Total RNA (10pg) was separated in formaldehyde-agarose gels and transferred to Nytran filters. Transfers were carried out for 2-4 h with the use of a semidry blotting apparatus (JKA BioTech, Copenhagen, Denmark) at
