bioreactivity of glutathionyl hydroquinone with implications to benzene toxicity

Toxicology 150 (2000) 31–39

Bioreactivity of glutathionyl hydroquinone with implicationsto benzene toxicity

Sarfaraz Ahmad 1, Rashmi Agrawal, D.K. Agrawal, Gondi S. Rao *Industrial Toxicology Research Centre, PO Box 80, M.G. Marg, Lucknow 226 001, India

Received 29 February 2000; received in revised form 28 March 2000; accepted 16 May 2000

Abstract

Glutathionyl hydroquinone (GHQ), a highly reactive metabolite of benzene, has been implicated as a causativeintermediate of benzene toxicity. To substantiate, the bioreactivity of GHQ was investigated under in vitro and invivo conditions using end points, characteristic of benzene toxicity. Under in vitro conditions, the presence of GHQ:(a) linearly increased the release of aldehydic products from L-glutamate or deoxyuridine at GHQ concentrations of5–25 mM and from rat liver homogenates at GHQ concentrations of 50–250 mM; (b) cleaved plasmid pUC 18supercoiled DNA through a single strand nick to yield open circular relaxed DNA, and through a double strand cutto give out linear DNA at GHQ concentrations of 25–200 mM, with evidence of protection by catalase andsuperoxide dismutase; and (c) induced cross-linking and polymerization of lymphocyte nuclear DNA through in situgeneration of GHQ, which was protected by pretreatment of lymphocytes with N-ethylmaleimide. In vivo exposureof Swiss albino mice to GHQ (100 mg/kg, intraperitoneally once daily for 30 days) resulted in significant increase ofliver weight and inhibition of mitotic index in the bone marrow. The other test parameters, namely spleen weight,hematological indices, hepatic sulphahydryl content and nonenzymatic lipid peroxidation, and chromosomal aberra-tions in the bone marrow were, however, unaffected by GHQ treatment. The observations indicate pro-oxidant andcytotoxic potential of GHQ, mediated by the reactive oxygen species generated during the course of its auto-oxida-tion. Bioreactivity of GHQ with cellular macromolecules in vitro and inhibition of mitotic index of bone marrow onin vivo exposure have relevance to benzene toxicity, although in situ generation of GHQ at the site of action appearscritical in bringing about hematological and chromosomal effects that were probably spared due to rapid metabolicdisposition and, consequently, poor bioavailability of intraperitoneally administered GHQ. © 2000 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: Glutathionyl hydroquinone; DNA degradation; Plasmid pUC 18; Thiobarbituric acid reactive products; Lymphocyte;Mitotic inhibition

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* Corresponding author. Tel.: +91-522-220107; Fax: +91-522-228227.E-mail address: [email protected] (G.S. Rao).1 Present Address: MD 68, ECD, Biochemistry and Pathobiology Br., USEPA, NHEERL, Research Triangle Park, NC 27711,

USA.

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S. Ahmad et al. / Toxicology 150 (2000) 31–3932

1. Introduction

Chronic exposure of humans to benzene, animportant industrial chemical and environmentalpollutant, at work places and to animals underexperimental conditions results in progressive de-generation of bone marrow and blood dyscrasiasincluding lymphocytopenia, thrombocytopenia,pancytopenia, aplastic anemia and leukemia(Snyder et al., 1993). The toxicity of benzene isdependent upon its bioactivation, particularly inliver where cytochrome P450-2E1 is the principalenzyme involved in its oxidation to yield hydroxy-lated metabolites including phenol, hydroquinone(HQ), catechol, 1,2,4-benzenetriol and the ring-opened metabolites, namely trans,trans-mu-conaldehyde and trans,trans-muconic acid (Kalf,1987). Extensive research towards identification ofcausative metabolites has shown that while noneof the identified metabolites is individually com-petent, specific combinations, namely phenol andhydroquinone, can mimic certain characteristicsof benzene toxicity (Eastmond et al., 1987;Legathe et al., 1993; Chen and Eastmond, 1995).Regarding the mechanism of action, the polyphe-nolic metabolites of benzene accumulate againstthe concentration gradient in the bone marrow(Singh et al., 1994), which is rich in myeloperoxi-dase activity and transition metal ions, to facili-tate oxidation of HQ into quinoid derivatives andconsequent adduct formation with cellular macro-molecules (Levay et al., 1991, 1993) or the genera-tion of reactive oxygen species (ROS) through aredox cycle to cause hematotoxicity (Subrah-manyam et al., 1991a,b). The polyphenolicmetabolites and their quinoid derivatives are alsocapable of nucleophilic conjugation with glu-tathione (Lunte and Kissinger, 1983; Lau et al.,1988; Rao et al., 1988), and the presence ofthioether derivatives of benzoquinone (BQ) hasbeen demonstrated in the bone marrow (Brattonet al., 1997) and urine (Nerland and Pierce, 1990)of laboratory animals exposed to benzene and itsmetabolites. Species difference in benzene-inducedhematotoxicity (Zhu et al., 1995; Bratton et al.,1997), based on greater accumulation of glu-tathionyl-hydroquinone (GHQ) with higher chem-ical reactivity, namely auto-oxidation, arylation

(Brunmark and Cadenas, 1988; Lau et al., 1988;Hill et al., 1994) and higher yield of macromolec-ular adducts than its precursors (Thomas et al.,1991; Levay et al., 1991, 1993; Kleiner et al.,1998), implicated GHQ as a causative intermedi-ate of benzene toxicity. This assumption has beenvindicated through demonstration of the pro-oxi-dant potential of GHQ in terms of DNA degrada-tion by superoxide radicals generated in thecourse of its rapid auto-oxidation to glutathionylbenzosemiquinone (Rao, 1996), in vivo nephro-toxic (Lau et al., 1988; Hill et al., 1994) anderythrotoxic (Bratton et al., 1997) potential ofGHQ in rats, prevalence of low white blood cell(WBC) count in benzene-exposed human subjectspositive for glutathione S-transferase genotypes(GST TI and GST MI) that facilitate GHQ for-mation (Hsieh et al., 1999), and protection byNAD(P)H:quinone oxidoreductase I capable ofdetoxifying quinones (Zhu et al., 1995; Moran etal., 1999; Smith, 1999). This study was carried outto evaluate oxidative, cytotoxic and cytogeneticpotentials of GHQ, in vivo and in vitro, to sub-stantiate its causal relationship with benzenetoxicity.

2. Materials and methods

2.1. Materials

BQ and HQ were obtained from Aldrich (Mil-waukee, WI, USA). Glutathione reduced (GSH),L-glutamate monosodium, deoxyuridine, col-chicine, 5,5-dithio-bis(2-nitrobenzoic acid), 2-thio-barbituric acid, DNA, superoxide dismutase(bovine erythrocytes), catalase and Histopaque®-1077 (bovine liver) were obtained from Sigma (St.Louis, MO, USA). Plasmid pUC 18 supercoiledDNA (2655 base pairs (bp)) was obtained fromBangalore GENEI Pvt. Ltd. India. All otherchemicals were of analytical grade.

2.2. Methods

2.2.1. Preparation of GHQGHQ was prepared according to Rao (1996).

Briefly, equimolar concentration of BQ and GSH

S. Ahmad et al. / Toxicology 150 (2000) 31–39 33

were mixed in distilled water in 1:1 proportionby volume. The nucleophilic addition of GSHto BQ yields GHQ, and its authenticity wasconfirmed by the absorption maximum at 303nm. No absorption was observed at 248 nm,which is the absorption maximum for BQ.Free GSH was also not demonstrable by Ell-man’s reagent (Sedlak and Lindsay, 1968).

2.2.2. GHQ-induced release of thiobarbituric acidreacti6e products

Release of thiobarbituric acid reactive prod-ucts (TBAR) from L-glutamate or deoxyuridinewas followed according to the method of Gut-teridge (1981). Briefly, the reaction mixture (to-tal volume, 1.5 ml) contained 0.033 M sodiumphosphate buffer (pH 7.4) in 0.15 M NaCl, 4mM glutamate monosodium or 3.2 mM de-oxyuridine, and the reaction was started by theaddition of GHQ (5.0–25.0 mM). The contentswere incubated for 2 h at 37°C and the reac-tion was terminated by the addition of 1.0 mlof 10% (w/v) trichloroacetic acid. TBAR wasestimated as described earlier (Rao, 1996).Nonenzymatic lipid peroxidation in liver ho-mogenate was studied according to Singh et al.(1994).

2.2.3. GHQ-induced degradation of plasmid (pUC18) supercoiled DNA

The reaction mixture, in a total volume of50 ml, contained 20 mM phosphate-buffered sa-line (pH 7.4), 300 ng plasmid pUC 18 DNA,and 200 mM BQ or GSH or GHQ, with orwithout oxyradical scavengers. The contentswere mixed well and incubated for 3 h at37°C. After 3 h incubation, 5 ml solution con-taining 40 mM ethylenediamine tretraacetic acid(EDTA), 0.05% bromophenol blue tracking dyeand 50% (v/v) sucrose were added, and the so-lution was subjected to electrophoresis on 1%agarose gels for 3 h at 60 mA current. Thegels were stained with ethidium bromide (0.5mg/ml), visually evaluated by the bandwidthand their relative intensity of luminiscence, andwere photographed on an UV-transilluminator.

2.2.4. GHQ-induced damage to lymphocytenuclear DNALymphocytes from adult male albino Wistar ratswere isolated following exclusively the procedureof Boyum (1977). The lymphocytes were sus-pended in 0.1 M phosphate-buffered saline (pH7.4) and incubated with 0.5 and 1 mM concentra-tions of BQ or HQ for 3 h at room temperature.In order to deplete sulphahydryl groups,lymphocytes were incubated with 1 mM N-ethyl-maleimide, for 1 h, centrifuged and the pelletresuspended in the same buffer. Resuspendedlymphocytes, depleted of sulphahydryl groups,were incubated with BQ or HQ as described fornormal lymphocytes. Lymphocyte nuclear DNAdamage was evaluated by agarose gel elec-trophoresis (1%) using the procedure of Mahmu-toglu and Kappus, (1987).

2.2.5. GHQ-induced systemic and cytogeneticeffects

Healthy adult male albino Swiss mice (averageweight, 25 g), bred and raised by ITRC labora-tory animal facility, were used in this study. Theanimals were randomly grouped and housed inpolycarbonate boxes (six mice per box) with steelwire tops and rice husk bedding. They were main-tained in a controlled atmosphere of 12 h dark/light cycle, 2292°C temperature and 50–70%relative humidity with free access to pelleted feed(Ashirwad Laboratory Rodent Feed; Chandigarh)and fresh tap water.

Test animals (12) were intraperitoneally injectedwith freshly prepared aqueous solution of 100 mgGHQ/kg body weight (b.w.) daily for 30 days,while an equal number of mice were identicallytreated with normal saline to serve as referencecontrol. All animals were periodically observedfor general health and body weight changes. Atthe end of treatment period, animals wereweighed and six mice each from GHQ treatmentand reference control group were bled to death bycutting the jugular vein under ether anesthesia.Blood was collected in heparinized vials for hema-tological evaluation (Dacie and Lewis, 1968).Liver and spleen were surgically removed,cleansed of extraneous tissue and weighed. Por-tions of liver were homogenized in either 0.02 M


EDTA for estimation of total and protein-free SH(Sedlak and Lindsay, 1968) or in 0.15 M KCl fornonenzymatic lipid peroxidation (Singh et al.,1994).

The other six mice each from GHQ-treated andcontrol groups were intraperitoneally injectedwith an aqueous solution of 2.5 mg colchicine/kgb.w., 2 h prior to killing by cervical dislocation.Both the femurs were surgically removed fromeach mouse and their bone marrow was flushedout with Hank’s buffered salt solution (pH 7.2).The cells were dispersed by gentle aspiration, cen-trifuged at 200×g for 5 min and redispersed in a

hypotonic solution of 0. 075 M KCl. The prepara-tion was incubated at 37°C for 30 min to permitosmotic swelling of cells that were then harvestedby centrifugation, fixed in ice-cold Cornoy’s fluid,drop-spread on grease-free glass slides and stainedwith phosphate-buffered 5% Giemsa for micro-scopic (100× ) observation of metaphase chromo-somes (Adler, 1984). The mitotic index wascalculated as percentage of dividing cells fromapproximately 2000 cells per mouse. Chromosomeaberrations were scored in over 50 well-spreadmetaphase plates per mouse, and chromatid/isochromatid gaps were recorded but not includedin the percent frequency of aberrations.

3. Results

A concentration-dependent release of TBARfrom L-glutamate or deoxyuridine was observedin presence of GHQ (Table 1). Presence of BQ orGHQ also enhanced the rate of nonenzymaticlipid peroxidation in liver homogenates (Table 2).

Incubation of plasmid pUC 18 supercoiledDNA with GHQ produced extensive cleavage ofDNA, resulting in formation of an open circularrelaxed and linear form of DNA, which respec-tively moved slower than the supercoiled DNA on1% agarose gels (Fig. 1). Incubation mixturescomprised of pUC 18 DNA alone (lane a) or pUC18 DNA with 200 mM BQ (lane b) or pUC 18with 200 mM GSH (lane c) did not show any nicksin pUC 18 DNA. Incubation with GHQ con-verted almost all of the pUC 18 DNA to a majorfraction of open circular form through singlestrand nick, and to a minor fraction of linearform of DNA through a double strand scission(lane d). The degradation products of pUC 18DNA were identified by comparison with EcoRIdigested pUC 18 DNA, which characteristicallyproduced a double strand break giving rise to alinear form of DNA (lane e), and were used asreference standard. Incubation with GHQ (25–200 mM) linearly increased the degradation ofpUC 18 DNA (Fig. 2).

The effect of oxyradical scavengers on pUC 18DNA strand cleavage was investigated to charac-terize and quantitate the reactive oxygen species

Table 1Effect of varying concentration of GHQ on the release ofTBAR from L-glutamate or deoxyuridinea

TBAR formed (nmol per 2 h)Concentrtion of GHQ(mM)

L-Glutamate Deoxyuridine

5.0 0.2790.01 0.2390.060.5690.0510.0 0.4690.090.8190.1215.0 0.9090.07

20.0 1.1790.051.1590.081.2190.01 1.4090.0325.0

a Experimental conditions are as described in Section 2.Briefly, the assay system in a total volume of 1.5 ml contained0.033 M phosphate buffer (pH 7.4), 4.0 mM L-glutamatemonosodium or 3.2 mM deoxyuridine, and was initiated bythe addition of varying concentrations of GHQ. All values areaverages9S.E. of two sets of experiments conducted in dupli-cate.

Table 2Lipid peroxidation in rat liver homogenate in the presence ofGSH, BQ and GHQa

MDA (nmol/h per g liver)

None 13.991.713.791.0+GSH (250 mM)

+BQ (250 mM) 25.392.118.991.4+GHQ (50 mM)20.691.7+GHQ (100 mM)

+GHQ (150 mM) 22.991.824.791.6+GHQ (200 mM)

+GHQ (250 mM) 27.391.6

a All values are average of 9S.E. of two sets of experimentsconducted in duplicate.


Fig. 1. Agarose gel electrophoresis pattern of ethidium bro-mide stained pUC 18 DNA after treatment with BQ, GSH orGHQ (200 mM each). Lane (a), DNA alone; lane (b), DNA+BQ; lane (c), DNA+GSH; lane (d), DNA+GHQ; and lane(e), digestion of pUC 18 DNA with EcoRI. Reaction mixtureswere incubated at 37°C for 3 h. The positions of superocoiled(SC), linear (LIN) and open circular relaxed (OC) DNA areindicated. Experimental conditions as described in Section 2.

stantial protection was offered by catalase (lane d)and superoxide dismutase (lane c), while mannitol(lane e), thiourea (lane f) and sodium benzoate(lane g) rendered marginal protection againstDNA cleavage.

Fig. 4 depicts maximum damage to lymphocytenuclear DNA on treatment with 0.5 and 1.0 mMBQ (lanes b and c), and to a lesser extent onincubation with 0.5 and 1.0 mM HQ (lanes d ande) as compared with control (lane a). Lane (f) isthe DNA EcoRI digested molecular marker(21226–3530 bp) to serve as reference standard.DNA damage was not observed in N-ethyl-maleimide-treated lymphocytes exposed to boththe test concentrations of either BQ or HQ (Fig.5).

There were no significant gross abnormalities,body weight alterations or mortality in GHQ-treated mice as compared with reference controlsubjects. The relative weight of liver was signifi-cantly increased (26.8%) in GHQ-treated mice butspleen weight, hematological indices (hemoglobin,hematocrit, total and differential leucocyte count),total and nonprotein sulphahydryl contents inliver and total hepatic nonenzymatic lipid peroxi-dation were comparable with respective controls

Fig. 2. Cleavage of pUC 18 DNA by increasing concentrationsof GHQ. Lane (a), DNA only; lanes (b)–(f), DNA+GHQ(25, 50, 75, 100 and 200 mM). Reaction mixtures were incu-bated at 37°C for 3 h.

Fig. 3. Cleavage of pUC 18 DNA by GHQ in the presence ofoxyradical scavengers. Lane (a), DNA only; lane (b), DNA+GHQ; lanes (c)–(g), DNA+GHQ+SOD/catalase/mannitol/thiourea/sodium benzoate at 10-fold molar excessconcentration. Reaction mixtures were incubated at 37°C for 3h.

involved. The presence of superoxide dismutase,catalase, mannitol, thiourea and sodium benzoateexerted varied degrees of inhibition of GHQ-in-duced cleavage of pUC 18 DNA (Fig. 3). Sub-


Fig. 4. DNA damage by BQ and HQ in lymphocytes. Lane(a), Lymphocyte only; lane (b), lymphocyte+BQ (0.5 mM);lane (c), lymphocyte+BQ (1.0 mM); lane (d), lymphocyte+HQ (0.5 mM); lane (e), lymphocyte+HQ (1.0 mM); lane (f),DNA EcoRI digested marker.

(Table 3). The bone marrow mitotic index wassignificantly inhibited (19.2%) in the GHQ-treatedgroup of mice but their frequency of chromosomeaberrations was similar to reference control ani-mals (Table 3).

4. Discussion

The pro-oxidant potential of GHQ is demon-strated in the present study through linearly in-creased degradation of L-glutamate anddeoxyuridine, enhancement of nonenzymatic lipidperoxidation of rat liver homogenates, and cleav-age of plasmid pUC 18 supercoiled DNA onincubation with increasing concentrations ofGHQ in vitro. These reactions are known to becatalyzed by ROS, particularly the superoxideradical (Gutteridge, 1981; Li and Trush, 1993)generated in the course of GHQ auto-oxidation toglutathionyl benzosemiquinone (Rao et al., 1988).The involvement of superoxide radical is alsoevidenced in the present study by the protectionoffered by catalase and superoxide dismutaseagainst GHQ-induced cleavage of pUC18 DNA.These observations are consistent with our earlierreport of many-fold enhancements of iron-cata-lyzed bleomycin-dependent degradation of calf-thymus DNA by GHQ (Rao, 1996). Thedegradation of nuclear DNA in BQ- or HQ-treated lymphocytes is consistent with similar ef-fects of benzene and its metabolites in L5178YScells (Pellak-Walker and Blumer, 1986), HL60cells (Kolachana et al., 1993) and humanlymphocytes (Anderson et al., 1995). This indi-cates the role of cellular glutathione towards insitu generation of GHQ, which was effectivelyblocked by prior depletion of cellular thiols withN-ethylmaleimide, as also demonstrated in theglutathione-depleted rat liver cell line (RL-4)showing decreased cytotoxic response to chloro-dinitrobenzene (Bruggeman et al., 1988). The ef-fect was more pronounced with BQ, whichdirectly conjugates with glutathione to produceGHQ (Brunmark and Cadenas, 1988; Rao et al.,1988), than with HQ, which needs redox cyclingto yield BQ for subsequent generation of GHQ(Lunte and Kissinger, 1983; Lau et al., 1988;

Fig. 5. DNA damage by BQ and HQ in lymphocytes depletedof sulphahydryl group by N-ethylmaleimide. Lane (a),Lymphocytes only; lane (b), lymphocyte+BQ (0.5 mM); lane(c), lymphocyte+BQ (1.0 mM); lane (d), lymphocyte+HQ(0.5 mM); lane (e), lymphocyte+HQ (1.0 mM).


Thomas et al., 1991). These studies also demon-strate that in situ generated GHQ penetrates thenucleus to inflict DNA, as reported for in situgenerated glutathionyl benzosemiquinone, tocross the hepatocyte plasma membrane (Rao etal., 1988). The bioreactivity of GHQ is evidentlygreater than BQ or HQ, which independently

proved ineffective under the same experimentalcondition. Bioactivation of a variety of com-pounds after conjugation with glutathione is welldocumented (Anders and Dekant, 1998).

The single strand nicks (major damage) anddouble strand cut (minor damage) of pUC18DNA on direct addition, and degradation oflymphocyte nuclear DNA on in situ generation ofGHQ, could result from its high efficiency ofredox cycling, ROS generation or arylation/dele-terious modification of DNA bases (Lau et al.,1988; Kolachana et al., 1993; Hill et al., 1994;Bratton et al., 1997) with implications of benzenecarcinogenicity. Chromosomal anomalies includ-ing aneusomy and long arm deletion of chromo-somes 5 and 7 have been observed in peripheraland cultured human lymphocytes exposed to ben-zene and its metabolites (Anderson et al., 1995;Zhang et al., 1998a,b), with causal relationship tomyelodysplasia and acute myelogenous leukemia(Snyder et al., 1993).

In vivo bioreactivity of GHQ is evidenced inthe present study through increased liver weightand mitotic inhibition in the bone marrow ofexposed mice. The liver, being a primary site ofexposure and metabolic disposition of intraperi-toneally administered GHQ, appears to be adap-tively enlarged in response to its cytotoxicity andconsistent to its known nephrotoxic (Lau et al.,1988; Hill et al., 1994) and erythrotoxic (Brattonet al., 1997) potential. Mitotic inhibition, on theother hand, has connotation to both cytotoxicand cytogenetic effects of GHQ. Benzene and itsmetabolites have been reported to decrease totalbone marrow cellularity (Eastmond et al., 1987)and to interfere with the formation of mitoticspindle apparatus (Pfeiffer and Metzler, 1996),leading to abnormal chromosomal segregation(Chen and Eastmond, 1995) and aneusomy(Zhang et al., 1998a,b). The failure of intraperi-toneally administered GHQ to influence other testindices is attributed to its rapid metabolic disposi-tion and poor biodistribution to target tissues.This view is supported by an earlier report (Brat-ton et al., 1997) on poor distribution of intra-venously administered GHQ to bone marrow, itsfailure to cause erythrotoxicity and the necessityof generation of GHQ at the site of action foreffective biological response.

Table 3Systemic and cytogenetic effects of intraperitoneally injectedGHQ in micea

GHQ-treatedTest parameters Referencecontrol group group

Relati6e organ weights (g/100 g b.w.)Liver 4.7390.36 6.0090.15*Spleen 0.6290.08 0.8090.10

HematologyHemoglobin (g/dl) 13.9090.65 14.4090.32

41.8091.07Hematocrit (%) 40.5091.1091179209Total WBC count 80009582

(per mM3)Differential leucocyte count (%)Neutrophil 24.091.9 27.093.4

71.093.371.591.9Lymphocyte1.89 0.42.39 0.6Eosinophil

Monocyte 1.390.4 0.390.2Basophil NilNil

Hepatic sulphahydryl content (mmol/100 g tissue)Total 3.2590.13 3.2790.05

0.7390.07Nonprotein 0.8590.05

25.094.6 33.894.5Nonenzymatic lipidperoxidation(nmol/h per g li6er)

Cytogenetic indices in bone marrow2.409 0.09Mitotic index (%) 1.9490.06*

2/336Metaphases 3/674(aberrant/totalscored per group)

Chromosome aberrationsNilBreaks/fragmentation 2NilExchanges Nil

Chromatid aberrationsGaps 42

NilBreaks/fragmentation NilNilExchanges Nil

Frequency of 0.4590.130.6090.16aberration (%)

a All values are group means9SE from six mice. * PB0.05as compared with reference controls.


It is concluded from the present study thatGHQ is a potent pro-oxidant capable of inducingmacromolecular degradation, cytotoxicity and mi-totic effects, in vitro and in vivo. In as much assimilar events lead to characteristic manifestationof myelotoxicity and carcinogenicity on exposureto benzene, GHQ qualifies to be designated as acausative intermediate of benzene toxicity. Itshigh chemical reactivity and rapid metabolic dis-position, and consequently poor biodistribution,nevertheless necessitate in situ generation at thetarget sites to elicit full potential of benzene-spe-cific hematotoxic and genotoxic effects.

Acknowledgements

S.A. is grateful to the Council of Scientific andIndustrial Research for the award of a SeniorResearch Fellowship, and R.A. to the IndianCouncil of Medical Research for the award of aSenior Research Fellowship. Thanks are due toRam Surat for technical assistance, Lakshmi Kantfor computer assistance, and Ram Lal formicrophotography.

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bioreactivity of glutathionyl hydroquinone with implications to benzene toxicity

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