Document DGjKB3q86J6DJGZkodY75MjgM

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 259, No. IL, November 15, pp. 29-37, 1987 Cytotoxicity of the Redox Cycling Compound Diquat in Isolated Hepatocytes: involvement of Hydrogen Peroxide and Transition Metals MARTHA S. SANDY,* PETER MOLDEUS,? DAVID ROSS,+ AND MARTYN T. SMITH*,' *Departmen.t of Biomedical and Environmental Health Sciences, School of Public Health, University Cala@nia; Berkeley, California 94720; TDepartment of Toxicology, Karol&&u In&it&et, Stockholm 104 01, Sweden; and $Molecular and Environmental Toxicology Program, School of Pharmacy, University of Colorado, Boulder, Colorado 80303 Received April 8, 1987, and in revised form July 15, 1987 of Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (0; ), hydrogen peroxide (H,O,), and hydroxyl radical (`OH), in the toxicity of diquat to BCNU-treatled isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both HzOz and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of 05 remains less clear, however, but studies with diethylenetriaminepentaa- cetic acid indicate that 0; is unlikely to significantly contribute to the reduction of Fe3' to Fe'+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H202/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection. 0 1987 Academic press, lnc. Numerouis foreign compounds are readily converted by one-electron reduction to free radical intermediates which then react with dioxygen. This reaction regenerates the parent compound and converts the dioxygen to superoxide anion radical (0;) ' (1). Thus, in the presence of a 1To whom correspondence should be addressed at : 322 Warren Hall, School of Public Health, University of California, Berkeley, CA 94720. `Abbreviations used: 0; , superoxide anion radi- cal; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; GSH, reduced glutathione; GSSG, glutathione disul- fide; `OH, hydroxyl radical; DEM, diethyl maleate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesul- supply of reducing equivalents, a small amount of such a chemical can generate large amounts of 0; by this redox cycling process (2). The toxicities of several redox cycling compounds, most notably the quinones menadione and adriamycin, have been studied extensively in isolated hepatocytes (3-6). Efforts to characterize the intracellular consequences of active oxygen generation in these studies have, however, been complicated by the fact that fonic acid; TBA, thiobarbituric dismutase; DMSO, dimethyl ylenetriaminepentaacetic aminetetraacetic acid. acid; SOD, superoxide sulfoxide; DTPA, diethacid; EDTA, ethylenedi- 29 0003-9861/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 30 SANDY ET AL. menadione and adriamycin participate in many other reactions, such as alkylation and conjugation. In the present study the action of another redox cycling compound, the herbicide l,l'-ethylene-2,2'-bipyridylium ion (diquat), has been investigated in isolated hepatocytes. Unlike menadione or adriamycin, diquat does not participate in alkylation or conjugation reactions, and is therefore a valuable investigative tool for the study of oxidative damage resulting solely as a consequence of active oxygen generation (7). We recently compared the relative abilities of diquat and other bipyridyl herbicides to generate active oxygen species in rat liver microsomes and found diquat to be the most potent (8). In addition, diquat was shown to be an effective inducer of oxidative stress in freshly isolated hepatocytes whose oxidative defenses had been compromised by pretreatment with low concentrations of 1,3-bis(2-chloroethyl)-lnitrosourea (BCNU), a relatively specific inhibitor of glutathione reductase (8). In BCNU-treated hepatocytes reduced glutathione (GSH) cannot be regenerated from the oxidized disulfide (GSSG), hence the ability of glutathione peroxidase to prevent the accumulation of hydrogen peroxide (HzOs) is impaired (8, 9). In BCNUtreated hepatocytes with inhibited glutathione reductase but normal glutathione levels, exposure to diquat results in rapid GSH oxidation, stimulation of lipid peroxidation, and loss of cell viability (8). This study extends this work and examines the importance of 05, HzOz, and the transition metals iron and copper in diquat cytotoxicity. MATERIALS AND METHODS Chemicals. Highly purified diquat (>99%) was a kind gift of Dr. L. L. Smith, I. C. I. plc (UK). BCNU was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, the National Cancer Institute (Bethesda, MD). Ebselen was a gift of A. Nattermann & Co. GmbH (Cologne, FRG). Des- ferrioxamine was obtained from Ciba-Geigy and manufactured deferoxiamine by Ben Venue Labs (Bedford, OH) as mesylate. Collagenase, superoxide dismutase, and catalase were from Boehringer (Mannheim, FRG). All other chemicals were of the highest grade available from local suppliers. Preparation of isolated rat hepatocytes. Male Spra- gue-Dawley rats weighing 200-240 g were allowed food and water ad lib. All rats received sodium phe- nobarbital (1 mM) in their drinking water for 5-10 days prior to use. Hepatocytes were isolated by collagenase perfusion as described by Moldeus et al. (lo), yielding 240-300 X lo6 cells per liver. Viability, as measured by the exclusion of 0.2% (w/v) trypan blue (10) was routinely 95-99% in freshly isolated preparations. Pretreatment with BCNU and diethyl maleate (DEM). Freshly isolated hepatocytes were pretreated with 50 PM BCNU for 20 min and then allowed to recover GSH levels in methionine (1 mM)-supple- mented medium, essentially as described previously (8, 11). Glutathione reductase inhibition, assayed ac- cording to Roos et aZ. (12) was 90-95% following BCNU pretreatment, and viability was decreased to approximately 75-80% postrecovery. Cellular GSH was depleted in some experiments by pretreating freshly isolated hepatocytes with 0.3 mM DEM for 30 min. DEM pretreatment lowered cellular GSH to ap- proximately 20% of control values. Hepatocyte incubations. All incubations were per- formed at lo6 cells/ml in rotating round-bottom flasks at 37C under an atmosphere of 95% Oz/5% CO, in equilibrated Krebs-Henseleit buffer, pH 7.4, containing 12.5 mM 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid (Hepes) (10, 13). Biochemical assays. Total levels of GSH and GSSG were determined in l-ml samples of cell incubate by HPLC following derivatization with iodoacetic acid and Sanger's reagent, as described in (14). Alterna- tively, GSH levels were determined as acid-soluble thiols by the method of Saville (15). Lipid peroxida- tion was assayed as thiobarbituric acid (TBA)-reac- tive products as previously described (8, 16). RESULTS Diquat undergoes rapid redox cycling in isolated hepatocytes, and creates a condi- tion of oxidative stress within the cell. Cytotoxicity is not readily manifested, however, unless a primary defense against oxidative stress, the glutathione peroxi- dase/reductase system, has been compro- mised. This is illustrated in Table I where the addition of 1.5 mM diquat to control (uncompromised) hepatocytes failed to significantly affect either cellular GSH levels or viability, as assessed at 15 and 60 min, respectively. Pretreatment of hepa- tocytes with DEM lowered cellular GSH to 20% of control levels, but did not signifi- cantly alter the time course of diquat cy- totoxicity (Table I). However, when hepa- CYTOTOXICITY OF DIQUAT IN ISOLATED HEPATOCYTES 31 TABLE I EFFECT OF DIQUAT ON GSH CONTENT AND VIABILITY OF CONTROL, DEM-PRETREATED, AND BCNU-PRETREATED HEPATOCYTES Treatment Control* Control + diquat" DEM" DEM + diquat" BCNUd BCNU + diquat" GSH" Toxicity (nmol/lOs cells) (% trypan blue uptake) No. expt. 0 min 15 min 0 min 60 min f4f5 50 46f 5 8 f Sf 16f 5 5 55 f 5 49 f 10 8k6 25 3110 3 10 f 2 11* 2 11 f 4 32f 1 3 10 f 2 14+ 3 11 f 4 27+ 4 15 42 k 2 40f 3 18 + 4 25f 5 15 40 + 2 7f 3 18 + 4 6'7 f 11 "Reduced glutathione was determined by HPLC after derivatization with iodoacetic acid and Sanger's reagent as described in (14). b Cells were isolated by collagenase perfusion and incubated in Krebs-Henseleit buffer (see Materials and Methods). `Upon isolati'on, cells were pretreated with 0.3 mM DEM for 30 min (see Materials and Methods). d Upon isolation, cells were pretreated with 50 pM BCNU for 20 min, followed by recovery of GSH levels (see Materials and Methods). ' 1.5 mM diquat. f Values represent Z f SD of 12 experiments. tocytes were pretreated with BCNU, an irreversible inhibitor of glutathione re- ductase, significant toxicity was manifest within 60 min (Table I). As previously re- ported (8), this loss of viability was de- pendent upon the redox cycling of diquat, and was preceded by a rapid depletion of cellular GSH (Fig. 1) and an equally rapid increase in GSSG. In order to examine the involvement of 0; and Hz02 in diquat cytotoxicity, super- oxide dismutase (SOD) and catalase were added to BCNU-compromised hepatocyte incubations. SOD, added at 733 U/ml, pro- vided no protection against diquat cyto- toxicity (Fig. 1). Addition of catalase at 650 U/ml also had no protective effect, al- though addition at 1300 U/ml did delay both GSH depletion and the expression of cytotoxicity (Fig. 1). The addition of boiled catalase had no protective effect (data not shown). Interestingly, the addition of SOD (733 U/ml) in combination with the lower concentratio'n of catalase (650 U/ml) af- forded greater protection against GSH loss than that provided by the higher cata- lase concentration alone. The expression of cytotoxicity was, however, only briefly delayed by the coaddition of SOD and cat- alase (Fig. 1). The seleno-organic compound 2-phenyl- 1,2-benzoisoselenazol-3(H)-one (ebselen; PZ51) has been demonstrated to possess both glutathione peroxidase-like activity and a separate antioxidant activity in TIME (mlnl 60 120 180 FIG. 1. Effect of SOD and catalase on diquat-in- duced depletion of GSH (A) and cytotoxicity (B). BCNU-pretreated hepatocytes, prepared as de- scribed under Materials and Methods, were incu- bated with no additions (O), or in the presence of 1.5 mM diquat, either alone (0), or with 733 U/ml SOD (0), 650 U/ml catalase (a), 1300 U/ml catalase (A), or 733 U/ml SOD and 650 U/ml catalase (X). Results shown are from one experiment typical of three. 32 SANDY ET AL. 100 0 ./.' 50 / TIME (mtnl 180 240 FIG. 2. Protective effect of ebselen and GSH on diquat-induced depletion of GSH (A) and cytotoxi- city (B). BCNU-pretreated hepatocytes, prepared as described under Materials and Methods, were incu- bated in the presence of 1.5 mM diquat, with either no further additions (0), 50 PM ebselen (Cl), 1 mM GSH with further 0.5 mM additions at 30, 60, and 90 min (A), or 50 MM ebselen and 1 mM GSH with further 0.5 mM additions at 30-min intervals (X). Results shown are from one experiment typical of three. studies with rat liver microsomes and hepatocytes (1'7, 18). As shown in Fig. 2, the addition of ebselen in combination with exogenously added GSH provided significant protection against both diquat-induced GSH loss and cytotoxicity in BCNU-compromised cells. When ebselen was added in the absence of exogenous GSH no protection was observed; similarly, GSH added alone also had no protective effect (Fig. 2). Formation of the short-lived, but highly reactive and potentially damaging `OH may result from either a direct interaction between 05 and HzOz or a transition metal-catalyzed reaction (19). The possible involvement of `OH in diquat cytotoxicity was investigated initially by adding two `OH scavengers, mannitol and di- methyl sulfoxide (DMSO), to diquat- treated hepatocytes. Mannitol addition at either 0.2 or 0.05 M did not provide signifi- cant protection against diquat cytotoxi- city (results not shown). Cells incubated in the presence of DMSO (1.`75%), on the other hand, were protected somewhat against diquat cytotoxicity during the ini- tial hour of incubation (Fig. 3B), but nei- ther GSH depletion, nor lipid peroxida- tion were affected by DMSO addition (Figs. 3A, 3C). The potential for an interaction between active oxygen species and transition metals in diquat cytotoxicity was then in- vestigated by addition of the iron chelator desferrioxamine. As shown in Fig. 4A, 100 PM desferrioxamine did not protect against GSH depletion and 1 IllM desfer- rioxamine only slightly delayed the deple- tion of GSH. Both concentrations of des- ferrioxamine did, however, inhibit lipid peroxidation, and both significantly de- layed the expression of diquat-induced cy- totoxicity (Figs. 4B, 4C). Desferrioxamine at 1 mM afforded no more protection against cytotoxicity than it did at 100 PM. Two other iron chelators, diethylene- triaminepentaacetic acid (DTPA) and eth- ylenediaminetetraacetic acid (EDTA), did not offer any significant protection against diquat cytotoxicity (Fig. 5). DTPA (1 mM) only slightly delayed diquat-in- duced GSH depletion, cytotoxicity, and lipid peroxidation. The addition of EDTA (1 mM) failed to protect against either GSH depletion or lipid peroxidation, and diquat-induced cytotoxicity was actually FIG. 3. Effect of DMSO on diquat-induced depletion of GSH (A), cytotoxicity (B), and accumula- tion of TBA-reactive products (C). BCNU-pretreated hepatocytes, prepared as described under mMMaterials and Methods, were incubated with 1.5 diquat in the absence (0) or presence (0) of DMSO (final concentration 1.75%). Results shown are from one experiment typical of three. CYTOTOXICITY OF DIQUAT IN ISOLATED HEPATOCYTES 33 FIG. 4. Protective effect of the iron chelator desferrioxamine against diquat-induced depletion of GSH (A), cytotoxicity (B), and accumulation of TBA-reactive products (C). BCNU-pretreated hepa- tocytes, prepared as described under Materials and Methods, were incubated in the presence of 1.5 mM diquat, with either no further additions (0), 100 WMdesferrioxamine (a), or 1 mM desferrioxa- mine (0). Results shown are from one experiment typical of three. slightly enhanced by its inclusion. Addition of an Fe'+/DTPA complex (150 PM FeS0&50 @M DTPA) also enhanced cytotoxicity, but interestingly, GSH depletion was delayed to almost the same extent as in the presence of 1 mM DTPA. The possible interaction of another transition metal, copper, with diquat-generated active oxygen species was then investigated. Copper (10 PM) was added as C&O4 to BCNU-compromised hepatocytes incubated in the presence of a lower and less toxic concentration (0.5 mM) of diquat. As shown in Fig. 6, the addition of 10 PM CL? dramatically enhanced the cytotoxicity of diquat, killing 80% of the hepatocytes within the first 30 min of incubation, whereas only 40% of the cells were dead after 30 min in the absence of Cu'+. Diquat-induced toxicity was enhanced by copper addition even in the presence of the previously protective iron chelator, des- ferrioxamine (Fig. 6). Although copper itself is cytotoxic to hepatocytes, the loss of cell viability following the addition of 10 PM C&O1 to control incubations was not expressed during the first 90 min of incubation (results not shown). As shown in Figs. 1 and 4, neither the addition of 100 PM desferrioxamine nor the addition of catalase at 650 U/ml had any significant effect on diquat-induced GSH depletion. However, when catalase and desferrioxamine were added together at these concentrations, a dramatic protection against the depletion of GSH was observed (Fig. 7). The extent to which GSH depletion was delayed was similar to that observed following the coaddition of SOD and catalase (Fig. 1). Although SOD/catalase did not provide prolonged protection against cytotoxicity, coaddition of desferrioxamine and catalase delayed diquat-induced cytotoxicity to a greater FIG. 5. Effect of DTPA and EDTA on diquat-induced depletion of GSH (A), cytotoxicity (B), and accumulation of TBA-reactive products (C). BCNU-pretreated hepatocytes, prepared as described under Materials and Methods, were incubated in the presence of 1.5 mM diquat, with either no further additions (0), 1 mM DTPA (O), 150 pM DTPA and 150 PM FeS04 (A), or 1 mM EDTA (X). Results shown are from one experiment typical of three. 34 SANDY ET AL. FIG. 6. Addition of the transition metal copper en- hances diquat-induced cytotoxicity. BCNU-pre- treated hepatocytes, prepared as described under Materials and Methods, were incubated in the pres- ence of 0.5 mM diquat, with either no further addi- tions (0), 10 @M CuSOd (Cl), or 10 NM CuS04 and 100 @M desferrioxamine (A). Results shown are from one experiment typical of three. extent than the single addition of either desferrioxamine or catalase (Fig. 7). DISCUSSION Diquat was employed here as a model redox cycling compound capable of generating large amounts of Oi and Hz02 within cells, and thus creating a condition of oxidative stress, without participating in alkylation or conjugation reactions. Isolated rat hepatocytes are well protected against oxidative stress, however, and diquat-induced cytotoxicity is not readily expressed unless cellular defenses against active oxygen are compromised. The pretreatment of hepatocytes with DEM diminished glutathione levels to approximately 20% of normal cellular GSH IeveIs, but these cells were far less susceptible to diquat-induced injury than BCNU-pretreated cells possessing normal levels of GSH but only trace amounts of glutathione reductase activity. In control and DEM-pretreated cells the ability of glutathione reductase to regenerate GSH from the oxidized form (GSSG) is apparently so great that merely catalytic amounts of glutathione are sufficient to detoxify Hz02 and other hydroperoxides via glutathione peroxidase. The demonstrated importance of this GSH-dependent detoxification system in protecting hepatocytes against diquat-induced cytotoxicity implicates HzOz as a major mediator of diquat toxicity within this in vitro model system. Recent studies by Eklow-Llstbom et al. (20) confirm the importance of GSH in protecting against diquat cytotoxicity in hepatocytes. The relative importance of the redox cycling products 0; and H202 in mediating diquat-induced cytotoxicity was studied by adding SOD and catalase to hepatocyte incubations. Although the efficacy of exogenously added SOD and catalase was limited by the inability of either enzyme to pass through the hepatocyte plasma membrane, extracellular enzyme activity would provide protection against active oxygen species present in the extracellular environment. Indeed, the ability of both the protonated form of O;, HOW, and HzOz to readily diffuse across membranes suggests that a component of the cytotoxic injury observed in our compromised hepatocyte system may be extracellular. The lack of protection afforded by exogenous SOD suggested that extracellular 0; does not contribute significantly to diquat cytotoxicity, although the possibility exists that added SOD was inactivated by H202, thus masking any protection. The small protection observed with the addition of catalase at the higher concentration (1300 U/ml) suggested that some HzOz is released from the cell and does contribute to cell death. A protective effect of extracellular catalase has previously been demonstrated in other studies performed with TIME ImInI FIG. 7. Enhanced protection against diquat-in- duced depletion of GSH (A) and cytotoxicity (B) by coaddition of desferrioxamine and cataiase. BCNU- pretreated hepatocytes, prepared as described under Materials and Methods, were incubated in the pres- ence of 1.5 mM diquat, with either no further addi- tions (0), 100 PM desferrioxamine (A), or 100 PM des- ferrioxamine and 650 U/ml catalase (X). Results shown are from one experiment typical of three. CYTOTOXICITY OF DIQUAT IN ISOLATED HEPATOCYTES 35 cultured cells exposed to an active oxygen challenge (21, 22). In addition to delaying the onset of diquat-induced cytotoxicity, high levels of catalase also delayed depletion of intracellular GSH. This sparing effect on GSH suggested that extracellular catalase activity favors H202 efflux from the cell, effectively lowering the intracellular H202 concentrations and thus the demand for GSH (Fig. 8). Although addition of catalase at a lower concentration (650 U/ml) had no effect upon diquat-induced GSH loss or cytotoxicity, coaddition of 650 U/ml catalase with SOD reduced the rate of GSH depletion and briefly delayed the expression of cytotoxicity. The mechanism by which this catalase/SOD combination so dramatiically delayed the oxidation of cellular glutathione is unknown but the following scenario seems most likely. The activity of extracellular SOD would favor 0; efflux as HOO', thereby reducing the amount of 0, present within the cell. Such a reduction in intracellular Op content would limit H202 formation within the cell, yet generation of HzOz outside the cell would be enhanced by the activity of extracellular SOD. Diffusion of this extracellular HsOz across the plasma membrane and into the cell would still result in a rapid 10~;s of cellular GSH, unless this HzOz influx was either prevented or diminished. Extracellular catalase activity could result in just such a diminution in H202 influx. Although the initial maintenance of G-SH by coaddition of catalase and SOD may explain the 30 min delay observed in the expression of diquat cytotoxicity, cellular GSH was nevertheless depleted during that 30 min period, and unabated cytotoxicity was expressed soon afterward (Fig. 1). Addition of ebselen, a synthetic compound possessing GSH peroxidase-like activity (17), protected against diquat cytotoxicity when present in conjunction with a supply of extracellular GSH. Coaddition of ebselen and GSH also delayed the diquat-induced depletion of cellular GSH, while no protection was observed with the addition of exogenous GSH in the absence of ebselen. These protective effects of ebselen suggest that HZOz is involved in diquat cytotoxicity, and have been further explored in Ref. (23). Hz02 can react with 0; in a HaberWeiss reaction (24) (Eq. [l]) to yield the highly reactive `OH, which may be the most damaging product of redox cycling (25, 26): 0; + HzOz --, O2 + `OH + OH-. [l] Alternatively, HzOz may participate in a transition metal-catalyzed Fenton reaction (Eq. [2]), also yielding `OH: Fe'+ + Hz02 + Fe3+ + `OH + OH-. [2] Although hydroxyl radical scavengers are not regarded as being either very efficient or specific, we tested the effects of mannitol and DMSO on diquat cytotoxicity. Only the addition of DMSO provided any protection against toxicity. The mini- FIG. 8. Scheme of postulated internal and external enzymes, thione peroxidase. interactions between diquat, diquat-derived active oxygen species, and transition metals. DQ, diquat; D&`, diquat radical; GPX, gluta- 36 SANDY ET AL ma1 protection afforded by DMSO does not preclude a role for `OH in the toxicity of diquat, however, since the scavenging ac- tion of DMSO may result in the formation of a methyl radical which could also con- tribute to cell damage, lipid peroxidation, and cytotoxicity (2'7). The site of formation of `OH within the cell may also have a sig- nificant effect upon the accessibility of `OH to scavengers, and the subsequent ef- ficacy of those scavengers (28). The dramatic protection observed fol- lowing desferrioxamine addition provided further evidence for the involvement of `OH or a closely related species in diquat toxicity. Desferrioxamine chelates intra- cellular iron in the Fe3+ state with an af- finity constant of 1031 (29), effectively blocking reduction (mediated by 05, the diquat radical, or other cellular reduc- tants) and thus preventing the participa- tion of iron in the Fenton reaction (Eq. [2]). EDTA, which chelates iron but does not prevent its reduction, can actually stimulate the Fenton reaction (30). The enhancement of cytotoxicity observed fol- lowing EDTA addition further supports a role for Fenton-type reactions in diquat toxicity. The marked effect of copper ion addition upon diquat-induced toxicity further es- tablished the importance of transition metals in this in vitro hepatocyte model. Cytotoxicity was greatly enhanced in the presence of added Cu'+. Not surprisingly, this effect was only slightly diminished by the addition of desferrioxamine, since desferrioxamine does not block the reduc- tion of copper from Cu2+ to Cu+. This sug- gests that desferrioxamine's principal protective action is indeed the chelation of iron, and not the inhibition of lipid peroxidation or the direct scavenging of `OH (31). In conclusion, these results indicate that HzOz and iron play important roles in the in vitro cytotoxicity of diquat, and suggest that `OH may be the ultimate toxin. The continued production of `OH via the Fen- ton reaction requires an adequate supply of both H202 and transition metals in the reduced state (e.g., Fe2+, Cu'). Continuous transition metal reduction is therefore es- sential. 0s can reduce Fe3+ to Fe2+ in chemical systems (32), but Fe3+ reduction via 0; does not appear to occur to any sig- nificant extent in diquat-treated hepato- cytes, since the addition of DTPA to che- late iron and slow the reduction of Fe3+ by OH (19) provided little protection against diquat cytotoxicity. Although DTPA-che- lated Fe3+ is relatively unreactive with 05, reaction with other reductants (e.g., ascorbic acid, reducing equivalents from NADPH-cytochrome P-450 reductase, or the diquat radical) to form the ferrous chelate can still occur. 0; may still play a role in the release of iron from cellular stores, however, and contribute to `OH generation indirectly (33, 34), although it has recently been shown that bipyridy- lium radicals can also do this (35). Further work is therefore needed to fully charac- terize the possible contributions of 0; to the cytotoxicity of the bipyridyl herbicide diquat, as well as for its congener para- quat. ACKNOWLEDGMENTS This work was supported by the National Founda- tion for Cancer Research, the Northern California Occupational Health Center, I.C.I. plc (UK), and the Swedish Medical Research Council. We thank Dr. D. Di Monte and Ms. P. Doane-Setzer for their assis- tance in some of the experiments. REFERENCES 1. Bus,J. S., AND GIBSON,J. 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