isolation and characterization of two benzene-derived ... · formed as a reactive metabolite of...

Vol. 1, 307-313, May/june 1992 Cancer Epidemiology, Biomarkers & Prevention 307

Isolation and Characterization of Two Benzene-derivedHemoglobin Adducts in Vivo in Rats1

Assieh A. Melikian,2 Agasanur K. Prahalad, andStuart Coleman

American Health Foundation, Naylor Dana Institute for Disease

Prevention, Valhalla, New York 10595

Abstract

The present study was aimed at the characterization ofthe major adducts formed by reaction of themetabolites of [‘4C]benzene with rat hemoglobin invivo. Groups of 12-week-old male Fisher rats receivedi.p. injections of a single dose of 10 mmol/kg bodyweight or three equal daily subdoses of 3.3 mmol/kgbody weight of [‘4Cjbenzene. High-performance liquidchromatographic analysis of strong acid hydrolysates ofthe [‘4C]benzene-modified globin indicated that thetwo major adducts in rats cochromatographed withsynthetic S-(2,5-dihydroxyphenyl)cysteine and S-phenylcysteine. These adducts were converted toO,O’,S-tris-acetyl-3-thiol-hydroquinone and S-phenylthioacetate, which were then characterized bygas chromatography/mass spectrometry. The majorradioactive adduct peaks accounted for 60-75% ofthe total radioactivity associated with rat globin.Characterization of the S-(2,5-dihydroxyphenyl)cysteineadduct provides evidence that p-benzoquinone isformed as a reactive metabolite of benzene. Formationof the S-phenylcysteine adduct indicates that benzeneoxide and/or a hydroxycyclohexadienyl free radical isformed as an active intermediate upon i.p. injection ofbenzene into rats.

Introduction

That benzene is toxic and leukemogenic has been estab-lished through epidemiologic evidence (1, 2). Chronicexposure of humans to benzene vapors suppresses bonemarrow functions and causes anemia, chromosomal ab-errations, and leukemia (3-5). Chronic oral toxicity stud-ies with B6C3F1 mice and Fisher 344/N rats indicate thatbenzene is a multipotential carcinogen. It induces neo-plastic lesions at eleven sites in rodents. Carcinoma ofthe Zymbal gland is the predominant tumor type inducedin rats (6-9).

Among the organic chemicals known to be carcino-genic to humans, benzene is produced in the greatest

volume (10). Humans are exposed to this compound inboth occupational and environmental settings. Benzeneis a constituent of petroleum and gasoline and is presentin other consumer products. Furthermore, it is also aproduct of combustion. Studies carried out by the Na-tional Aeronautics and Space Administration indicate thatabout 400 of 5000 consumer products tested emittedbenzene ranging from 0.01 to 140 zg/g (11). Tobaccosmoke and other emissions from the combustion of or-ganic matter contain benzene. Accordingly, the majorsources of benzene exposure to the general populationare: cigarette smoke; environmental tobacco smoke; au-tomobiles; and fuel and gasoline pumps (12, 13). On thebasis of an Environmental Protection Agency study whichassessed the total exposure of 400 people in eight cities,the major source of exposure to benzene for more than50 million smokers in the United States is the mainstreamsmoke of cigarettes (smoke generated during puff draw-ing) (14).

Recent epidemiological studies, both case-controland large prospective follow-up, have shown a positiveassociation between cigarette smoking and leukemia(15-19). The relative risks observed in the reported stud-ies were greater for myeloid leukemia than for otherforms of leukemia in men. Since chronic exposure tobenzene causes acute myeloid leukemia, it is likely thatthe benzene present in tobacco smoke can make asignificant contribution to the increased risk of cigarettesmokers for leukemia.

Although the ultimate toxic and carcinogenic metab-olite of benzene and its mechanism(s) of action in carci-nogenesis are not known, it is well established that met-abolic activation of benzene is a prerequisite for itstoxicity. Several of its metabolites, such as benzene ox-ide, BQ,3 and muconaldehyde, are active electrophilesthat can react with cellular macromolecules and formadducts (see Fig. 1; Refs. 5, 20-23). Sun et a!. (24) havedemonstrated that benzene-denived Hb adducts accu-mulate linearly in rats and mice given up to 3 daily dosesof 0.5 mmol benzene/kg body weight. This observationsuggests that benzene-denived adducts with Hb can beused as biomankers of benzene exposure. The biologicalmonitoring of benzene exposure is of great importancefor the prevention of occupational diseases caused bybenzene and for clarifying the relationship between ben-

Received 10/21/91.1 Supported by Grants CA44377 and CA29580 from the National Cancer

Institute.2 To whom requests for reprints should be addressed, at American HealthFoundation, Naylor Dana Institute for Disease Prevention, One Dana

Road, Valhalla, NY 10595.

3 The abbreviations used are: BQ, benzoquinone; HQ, hydroquinone;HQ-cysteine, S-(2,5-dihydroxyphenyl)-i-cysteine; O,O’,S-tris-acetyl-3-S-HQ, O,O’,S-tris-acetyl-3-thiohydroquinone; S-phenylmercapturic acid, 5-phenyl-N-acetylcysteine; TFA, trifluoroacetic acid; H PLC, high-perform-

ance liquid chromatography; GC, gas chromatography; MS, massspectrometry; Hb, hemoglobin; NMR, nuclear magnetic resonancespectrometry.

on June 26, 2021. © 1992 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

Qe

OH

� benzene oxide

,, 9.’

,‘7 ..

F

00H00

hydrooycyciohsoad#{232}enyi biphenyi

umeMeji OH

�OHN

4.4.--

0: �‘

HCO

/;�

HCO

aidehyde

OH OH

��OH

�

Ez::��LOHp-biezoqu,0one o-hydroiy-

benooqeinons

Fig. 1. Metabolic pathways of benzene for the formation of active

intermediates (#{176})and active electrophiles.

308 Identification of Benzene-Globin Adducts in Rats

zene exposure from cigarette smoke and leukemia casesin smokers. The present work was undertaken to isolateand characterize the major benzene-denived Hb adductsin rats in vivo. The incentive for determining the structureof benzene-Hb adducts is the eventual development ofa biochemical dosimeter for the human uptake ofbenzene.

Materials and Methods

Chemicals

[14C]Benzene (1 12 mCi/mmol) was purchased fromChemsyn Science Laboratories (Lenexa, KS). Reverse-phase HPLC determined its purity to be >98%. Unla-beled benzene was obtained from Bundick and Jackson(Muskegon, Ml) and was used to dilute [14C]benzene tothe desired specific activity. Benzoquinone (BQ), meth-anesulfonic acid, thiophenol, S-phenylthioacetate, andacetamidoacrylic acid were purchased from AldrichChemical Co. (Milwaukee, WI), and L-cysteine was ob-tamed from Sigma Chemical Co. (St. Louis, MO).

Animals

Male F344/N rats were obtained from Charles RiverBreeding Laboratories (North Wilmington, MA). Theywere 12 weeks old at the onset of experiments.

Animal Treatment

Two groups of 10 rats each were given i.p. injections ofa single dose of 10 mmol (1 mCi) [‘4C]benzene/kg bodyweight in 0.5 ml corn oil, or they were given three dailysubdoses of 3.3 mmol (0.33 mCi) [4C]benzene/kg bodyweight. The rats were sacrificed 24 h after the last dosing;upon cardiac puncture, blood samples were collectedinto EDTA-containing vacutainers. Thus, both in the sin-gle-dose administration or with three daily treatments ofequal subdoses each rat received a total of 10 mmol (1mCi) [14C]benzene.

Isolation of Globin from Blood

Globin was isolated from blood samples by methodsdescribed previously (25). The blood samples were cen-tnifuged at 2500 rpm at 4#{176}Cfor 10 mm; the plasma wasremoved. RBC were washed three times with 1 volume

of physiological saline. Then RBC were lysed by additionof 1 volume deionized H2O and 1 volume of 0.57 Mphosphate, pH 6.5. The hemolysate was centrifuged at25,000 x g at 4#{176}Cfor 25 mm. The supernatant wasdialyzed against H2O at 4#{176}C.Globin was precipitated bythe dropwise addition of the Hb solution to 20 volumes

0 of ice-cold acetone containing l% HCI, with vigorous

I I stirring. The globin precipitate was filtered and washedwith ice-cold acetone. An aliquot of each globin sample

I I was subjected to liquid scintillation counting to quanti-

0 tate the radioactivity associated with protein. Another4.4-dipk.noqu.non. aliquot was analyzed by HPLC (system 1). One-mI frac-

tions were collected, and the radioactivity of each frac-tion was measured by liquid scintillation counting.

Hydrolysis of Globin Samples to Amino Acids

Samples of 15 mg globin each were hydrolyzed with 3ml 6 N HCI at 110#{176}Cfor 24 h in vacuo. The resultingsolution was evaporated to dryness; the residue wasdissolved in 100 �zl of phosphate buffer, pH 7, andanalyzed by HPLC (system 2). One hundred fractions of0.5 ml each and 40 fractions of 1 ml each were collectedand subjected to liquid scintillation counting. Aliquots ofa mixture of S-phenylcysteine and HQ-cysteine stand-ards were added to all samples as tracers.

Synthesis of HQ-Cysteine

HQ-cysteine was synthesized by a procedure describedpreviously (26). Ten mmol (1.21 g) L-cysteine in 60 ml

H2O were reacted with 11 mmol (1.18 g) BQ in 30 mlmethanol at room temperature for 1 mm. Unreacted BQwas removed by extraction with ethyl acetate; theaqueous layer was treated with activated charcoal (0.2 g)and then filtered and evaporated to dryness. Ten mlmethanol and 100 ml acetone were added to the dryresidue, and the mixture was stored overnight at -20#{176}C.The precipitate which contained mostly cystine was re-moved by filtration; the filtrate was evaporated to drynessand the residue was dissolved in methanol and purifiedby HPLC system 2, from which it eluted with a retentiontime of 17.7 mm. The NMR spectrum was identical withthat published in the literature for HQ-cysteine (26).

Synthesis of 5-PhenylcysteineThis compound was synthesized by acid hydrolysis of S-phenylmencaptunic acid (Fig. 2) (27). For the synthesis ofS-phenylmercaptunic acid, thiophenol (1.8 g; 16.4 mmol)was suspended in 20 ml of freshly distilled dioxane alongwith acetamidoacrylic acid (1.94 g; 15 mmol) and 0.4 mlof pipenidine, flushed with N2, and refluxed for 3 h. Thesolvent was removed, and the residue was partitionedbetween ether and NaHCO, solution. The aqueous layerwas neutralized, extracted with ether, and acidified topH 1 -2, following which a precipitate of crude S-phen-

ylmercaptunic acid was formed. The precipitate was crys-talized from aqueous methanol (Fig. 2) and was charac-tenized by its NMR and mass spectra [360 MHz NMR (d,,-dimethyl sulfoxide): t5 1.8 (3H, S, CH,), 3.15 and 3.35(2H, dd, cystf3and cystfl’, J,1,1. 13.6 Hz, J�,,,,,.,, = 4.95,



rI��SH

�. 1�:;:L�� C00HCH2-CH

“NHAc

S-phenylmercapturicacid

0O � HS CH2- -CH‘NH2

0

Cancer Epidemiology, Biomarkers & Prevention 309

COOH+ H2C=C(

NHAc

thiophenol acetamidoacrylicacid

_________________ � H�

iI”�’i methanesulfonic acid (� COOH� � acetic anhydride

S-phenylthioacetate NH2S-phenylcysteine

Fig. 2. Synthesis of S-phenylmercaptunic acid, S-phenylcysteine, and S

phenylthioacetate.

8.95 Hz), 4.35 (1H, m, cysta), 7.25-7.35 (5H, m, aro-matic), 8.3 (1H, d, NH, J,,,NH = 7.92 Hz), 12.9 (1H, broad,COOH)]. The major components of the fragmentationpattern of MS were as follows: m/z 239 (Md’); 180(C6H5SCH2CHCOOH); 1 23 (C6H5SCH2). S-Phenyl-cysteine was obtained from acid hydrolysis (6N HCI; 110#{176}C;12 h in vacuum) of S-phenylmercaptunicacid and characterized by its NMR and mass spectra [360MHz NMR (d�,-dimethyl sulfoxide): e5 3.25, 3.45 (2H, dd,cystfl and cystf3’), 4.05 (1H, t, cysta, L, � and J�, �‘ = 5.6and 5.96), 7.3-7.5 (5H, m, aromatic), 8.6 (3H, broad,NH3�). Mass spectral data established the molecular ionas 197 (M’�) and the major fragment as 123 (C6H5SCH2).

Derivatization of HQ-Cysteine and 5-PhenylcysteineAdducts

For further characterization of HQ-cysteine and S-phe-nylcysteine, both synthetic standards and biological sam-pIes were denivatized prior to GC/MS analysis by acety-lation with acetic anhydnide and methanesulfonic acid(Figs. 2 and 3; Refs. 26, 28). In this procedure, 0.4 ml ofacetic anhydnide and 0.02 ml of methanesulfonic acidwere added to the dry synthetic sample of HQ-cysteine,

or biological samples obtained from 15 mg globin. Themixture was heated for 40 mm at 100#{176}Cand then cooled,and 1 ml of water was added. The resulting mixture wasextracted 3 times with 1 ml of benzene and purified byHPLC prior to GC analysis and characterization by MS.

GC/MS Analyses

GC/MS analyses of synthetic standard samples of O,O’,S-tris-acetyl-3-S-HQ on S-phenylthioacetate, as well as ace-tylated globin adducts collected from HPLC (Fig. 4, Peaksb and c), were performed on a Hewlett-Packard model5890 GC, operated in the splitless mode, with an injec-tion port temperature of 250#{176}C,and an oven tempera-tune program from 70#{176}Cto 275#{176}Cwas run for 30 mm. ADB-5 J & W Scientific (Folsom, CA) fused silica capillarycolumn (60 x 0.25 mm) was used. The column outlet wasinserted directly into the ion source of a Hewlett-Packardmodel 5988A mass spectrometer. MS conditions were asfollows: ion source temperature, 200#{176}C;emission cur-rent, 300 ptA; electron energy, 70 eV.

OH

1�:L ,COOHS-CH2-CH

OH ‘NH2

HQ-Cysteine

methanesulfonic acidacetic anhydride

O-C-CH3

�

O-C-CH3

0O,0S-tris-acetyl-3-S-HQ

Fig. 3. Synthesis of HQ-cysteine and 0,0’ ,S-tnis-acetyl-3.S-hydroquinone.

HPLC

Three solvent systems were used routinely throughoutthese studies.

System 1. Solvent A, 0.1% TFA in water; solvent B, 0.1%TFA in acetonitnile. A program was run with a lineargradient from 70:30 to 20:80 (solvent A:solvent B) for 100mm. The flow rate was 1 mI/mm. A 10-�zm Krackelen(Albany, NY) reverse-phase C18 Vydac column (25 cm x4.6 mm) was used in this system.

System 2. Solvent A, 5 volumes acetonitnile:0.05 volumeTFA:94.45 volumes H2O; solvent B, 94.45 volumes ace-tonitnile:0.05 volume TFA:5 volumes H2O. A programwas run with a linear gradient from 100:0 to 70:30 (sol-vent A:solvent B) for 30 mm followed by a linear gradient70:30 to 0:100 (solvent A:solvent B) for 10 mm. The flowrate was 1 mI/mm. A 5-�cm Beckman-Altex (San Ramon,CA) Ultrasphere ODS column (25 cm x 4.6 mm) wasused.

System 3. A program was run on a Beckman UltraspheneODS column with a linear gradient from 100:0 to 75:25H2O:methanol for 15 mm, followed by isocratic elutionwik 75:25 H2O:methanol for 5 mm and by a lineargradient from 75:25 to 100:0 H2O: methanol. The flowratewas 1.5 mI/mm.

Results

Dosage of [‘4C]Benzene and Levels of Binding of ItsMetabolites to Globin from Rats. Table 1 shows the levelsof binding of metabolites of [14C]benzene to the globinof rats measured 24 h after i.p. injections of a single doseof 10 mmol [4C]benzene/kg body weight in 0.5 ml cornoil, as well as those measured 24 h after administrationofthe last ofthree daily doses of 3.3 mmol [14C]benzene/kg body weight. In dose-response studies, the levels of[14C]benzene-denived globin adducts have shown an in-crease with the dose; the highest dose reported was 10



Table 1 Binding of {4C]benzene metabolites to the globin in F334/N-

Rats’

=0

U-

5

600 -

450 -

300 -

150

Il-i--- .j_ ___1i I L...r.....� i

20 40 60 80 100 120 140-4 0.5 mm Fractions ��-1 mm Fractions-*-

Fraction Number

Fig. 4. Radiochromatogram obtained upon HPLC analysis of O,S-acety-lated derivatives ofthe major [4C]benzene-denived globin adducts (peaks

6 and 7 of Figure 6) from rats 24 h after i.p. injection of 10 mmol/kg bodyweight [4C]benzene.

3 10 Identification of Benzene-Globin Adducts in Rats

0

C-CH3

9�’S#.C�CH3

“C-CH3

0”

b

Id

mmol/kg body weight (24). Therefore, this dose waschosen for the present study so as to afford the highestpossible level of globin adducts for their characterization.When [14C]benzene was administered in three subdoses,the level of binding of the metabolites of [14C]benzenewas 2.1-fold greater than upon that single administrationof the same total dose.

Fig. 5 illustrates the HPLC elution profile of globmnsamples from rats 24 h after [14C]benzene injection. This

chromatographic system separates a and f3 chains ofglobin and isolates heme and any free (unbound) metab-olites from globin. Fig. SB indicates that the major radio-active peak coeluted with the major protein UV peak,most likely corresponding to fi chains as described in theliterature (29) (Fig. SA). This observation suggests thatmost of the radioactivity associated with rat globmn was

covalently bound.

Characterization of the Major [14C]Benzene-derivedGlobin Adducts in Rats. Fig. 6 depicts the HPLC elutionprofile of a strong acid hydnolysate of globin from ratstreated with a single dose of [14C]benzene. Fig. 6 alsodemonstrates that there are two major radioactive peaksin this hydnolysate. Peak 6 of Fig. 6 cochnomatographedwith synthetic HQ-cystemne, and peak 7 coeluted withauthentic S-phenylcysteine. A standard HQ-cysteinesample was prepared by the reaction of BQ with cysteineas shown in Fig. 3 (26). The S-phenylcysteine sample wassynthesized using the approach described by Hanzlik et

a!. (27) for preparation of S-(p-bromophenyl)mercaptunic

acid. In this procedure, adding thiophenol to acetami-doacrylic acid in the presence of a mild base catalystresulted in S-phenylmercaptunic acid, which then washydrolyzed to S-phenylcysteine, as shown in Fig. 2. Both

synthetic S-phenylmercaptunic acid and S-phenylcys-teine samples were then characterized by NMR and MSas described in “Materials and Methods.”

Dose of [4C]benzene

pmol adduct/mg

globin”

10 mmol/kg body weight (single injec- 155 ± 15tion)

3 x 3.3 mmol/kg body weight (3 daily 324 ± 32injections)

a The vehicle used in this protocol was 0.5 ml corn oil/rat. Blood samples

were taken 24 h after last dosing.

b Mean ± SD, for n = 10.

Peaks 6 and 7 of Fig. 6, corresponding to HQ-

cysteine and S-phenylcystemne, respectively, compriseabout 37% and 23%, respectively, of the total radioactiv-ity associated with globmn. Furthermore, the HPLC elutionprofiles of acid hydrolysates of the globmn sample ob-

tamed from rats treated with three subdoses of {14CJbenzene were similar to those in Fig. 6. The percentagesof radioactivity associated with Peaks 6 and 7 were 46%and 29%, respectively (data not shown).

In order to identify the HQ-cysteine and S-phenyl-cysteine adducts, the derivatization procedure describedby Bakke (28) and Pascoe et a!. (26) was used. In thisprocedure, using acetic anhydride and methanesulfonicacid, the S-CH2 bond of the cysteinyl residue is cleaved,

and dehydroalanmne is released from the cystemnylmoiety. As shown in Fig. 3, HQ-cysteine is denivatized to

0,0’ ,S-tnis-acetyl-3-S-HQ, and S-phenylcysteine is ace-tylated to S-phenylthioacetate, as illustrated in Fig. 2.When a standard sample of HQ-cysteine was denivatized

by the above procedure, it yielded a single peak at 34.36mm on GC analysis, which gave the mass spectrumshown in Fig. 7A. Characteristic features of this spectrumare the M”' ion at m/z 268 and major fragment ions at m/z 226[268-(CH2=C=O)]�; m/z 1 84{226-(CH2=C=O)}”; and m/z 142[184-(CH2-C=O)]”, which resultfrom successive losses of the ketene from the parent ion.

Similarly, derivatization of authentic samples of S-phenylcysteine yielded a peak at 14 mm on GC analysis,which cochnomatographed with the purchased S-phen-

ylthioacetate sample, and gave the mass spectrum shownin Fig. 8A. Characteristic features of this spectrum are the

M’� ion at m/z 1 52 and a major fragment ion at m/z

110[152-(CH2=C=O)]�, which is due to the loss ofketene from the parent ion. For further identification ofadducts corresponding to HQ-cysteine and S-phenylcys-

teine adducts in HPLC analysis, products of Peaks 6 and7 of Fig. 6 were collected from HPLC and acetylated with

acetic anhydnide and methanesulfonic acid and then

analyzed by HPLC (Fig. 4). The major radioactive Peaksb and c of Fig. 4 coeluted with synthetic standard deny-atives of HQ-cysteine and S-phenylcysteine, respec-

tively. For further characterization, products of Peaks b

and c of Fig. 4 were collected from HPLC and analyzedby GC/MS. The mass spectra reproduced in Figs. 7B and8B were recorded at the correct retention time for theexpected O,O’,S-tris-acetyl-S-HQ and S-phenylthioace-tate, respectively. Thus, on the basis of HPLC, CC andmass spectral characteristics, the two major fl4C]ben-

zene-denived globmn adducts were identified as HQ-cys-teine and S-phenylcystemne.



EC

a,C,,C00�C,,a,

0C.,a,

a,

C.,,C0

C.,C�

U-

a-

=0

U-

S

0

600 -

450 -

300 -

150

12 3 4gLI_ I

9

Cancer Epidemiology, Biomarkers & Prevention 3 1 1

8 16 24 32 40

Fta�ion Number(1 mmFta�oi�)

Fig. 5. Chromatograms obtained upon reverse-phase HPLC analysis ofrat globin 24 h after i.p. injection of 10 mmol/kg body weight [‘4C]

benzene. A, UV detector response; B, radiochromatogram.

Discussion

It has been suggested that the formation of metabolitesof benzene which can react with critical macromoleculesmay be responsible for the toxicity and leukemogenicactivity of benzene. The initial step in benzene metabo-

lism is believed to be oxidation to benzene oxide by amixed-function oxygenase complex (30, 31) and/or for-mation of a hydroxycyclohexadienyl free radical by in-sertion of a hydnoxyl radical (Fig. 1; Ref. 32). Benzeneoxide may react with cellular macromolecules, glutathi-one, or other cellular nucleophiles. The glutathione con-jugate is converted to S-phenylmercaptunic acid andexcreted in the urine (33). Alternatively, benzene oxideor hydnoxycyclohexadienyl free radical may be con-vented to phenol and to a number of hydroxylated spe-cies, such as catechol and HQ, which can be oxidizedto BQ. On the basis of comparative toxicity of metabo-lites of benzene in bone marrow cells, clastogenicity inhuman lymphocytes, and capacity to form covalent ad-ducts with macromolecules, BQ was hypothesized to bean attractive candidate as a reactive intermediate in ben-zene toxicity (34). However, there are no reports ofdetection of free BQ in in vivo systems following benzeneadministration. Identification of an adduct of HQ tocysteinyl residues of globin in the present study indicatesthat benzene indeed forms reactive BQ or its semiqui-none precursor in vivo. Nerland and Pierce (35) havedetected S-HQ-N-acetyl-cystemne in the urine of ratstreated with benzene or phenol, which also indicatesthat BQ or a corresponding semiquinone are formed invivo in rats (35).

4;%S�CH2’CH

OHNH2

HO-cysteine

�“�S�CH2�CH

I6/

J_._. - .U I I I I I I

20 40 60 80 100 120 140-� 0.5 mm Fractions ��-1 mm Fractions �

Fraction Number

Fig. 6. Radiochromatogram obtained upon HPLC analysis of strong acidhydrolysates of rat globin 24 h after i.p. injection of 10 mmol/kg bodyweight [‘4C]benzene.

Detection of S-phenylcystemne in the present studycan be interpreted as reflecting the intermediacy of ben-zene oxide or a cyclohexadienol free radical in the bio-transformation of [14C]benzene. S-Phenylcysteine hasbeen detected in the urine of the workers who wereexposed to high levels of benzene in their workplace andin rats during the administration of benzene (33, 36). Thisresult also points to the fact that benzene oxide and/or acyclohexadienol free radical are active intermediates ofbenzene.

On the basis of the results of this study, it may beconcluded, therefore, that the covalent binding of [‘4C]benzene metabolites exhibits a remarkably high degreeof selectivity for -SH groups. This suggests that proteinswhich are rich in free thiol functional groups are theprimary target sites for benzene metabolites generatedintracellularly, a phenomenon which may play a role inthe pathology of benzene-induced toxicity.

That repeated smaller doses of i.p. administeredbenzene led to a 2.1-fold greater level of globin adductformation in rats than the same dose given by a singleinjection is related to the fact that benzene metabolismbecomes saturated at about 0.5 mmol/kg body weight(37), which is below the levels of dosage in our expeni-ment; that the Hb binding sites for benzene metabolitesare still available at the dose used in the current studyand did not become saturated; and that the major globinadduct appears to be stable and can accumulate. Sun eta!. (24) also observed a cumulative effect in hemoglobinadduct formation upon repeated benzene administrationP.O. of 0.5 mmol/kg body weight to rats, which surpassedthe level of adduct formation predicted from a singledose of 1.5 mmol/kg body weight.

These findings suggest that the benzene metaboliteadducts formed with globin can be used as a measure ofcumulative low-level exposure to benzene if adduct for-mation in humans follows pathways similar to those seenin rats. Determination of the alkylation products of blood



l’12

A 0II

0-C-CH3

MW=268 c�i S-�-CH3O-�-CH3

0

184

85 113_i� , 167 �-i--i--. i

�‘2=a,

C

0,

0)

40 80 120 160 200 240 280

2000

>0

C,)Ca)

C

a)

a)

‘Jo1600

1400

1200

1000

800

600

400

200

40 811 120 160 200 240 280 40

hemoglobin and serum albumin are currently consideredto be one of the most promising techniques for monitor-ing the uptake of environmental carcinogens in humans(38-40).

In conclusion, we have established that HQ-cysteineand S-phenylcysteine are the two major globmn adductsformed upon i.p. injection of benzene into rats.

3 12 Identification of Benzene-Globin Adducts in Rats

360000

320000

280000

240000

200000

160000

120000

80000

40000

3000

2600

43

B i�2200

1800

1400

1000 184

600 2�

200n

72 ��

114126ill .

162 ��.

,268

I

m/e

Fig. 7. Mass spectra of O,S-acetylated hydnoquinone cysteine. A, syn-thetic standard of HQ-S,O,O’-tnis-acetate; B, spectrum recorded fromGC peak at 34.36 mm. from denivatized rat globin hydrolysates obtained

from rats 24 h after i.p. injection of 10 mmol/kg body weight [‘4C]benzene.

References1. Rinsky, R. A., Smith, A. B., Hornung, R., Filloon, T. G., Young, R. I.,

Okun, A. H., and Landnigan, P. 1. Benzene and leukemia. An epidemio-logic risk assessment. N. EngI. J. Med., 316: 1044-1050, 1987.

2. Aksoy, M. Malignancies due to occupational exposure to benzene.

Am. J. Ind. Med., 7: 395-402, 1985.

3. Goldstein, B. D. Clinical hematotoxicity of benzene. In: M. A. MehI-man led.), Advances in Modern Environmental Toxicology. Vol. 4. Can-

cinogenicity and Toxicity of Benzene, pp. 51 -61 . Princeton, NJ: PrincetonScientific PublishingCo., Inc., 1983.

4. Dean, B. I. Recent findings on the genetic toxicology of benzene,

toluene, xylenes and phenols. Mutat. Res., 154: 153-181, 1985.

5. KaIf, G. F., Post, C. B., And Snyder, R. Solvent toxicology: recent

advances in the toxicology of benzene, the glycol ethers, and carbon

tetrachlonide. Ann. Rev. Pharmacol., 27: 399-427, 1987.

6. Huff, I. F. Toxicology and Cancinogenesis Studies of Benzene in F344Rats and B6C3F, Mice (Gavage Studies), Technical Report Series no. 289,

CAS no. 71-43-2. Research Triangle Park, NC: National Toxicology Pro-gram, 1986.

14000

12000

10000

8000

6000

4000

110

AMW-152

-1�:�L 0II

S-C-CH3

5�V . � � 10:,-‘.-I � 137

1� ‘ .

152

1i54.

60 80 100 120 140 160 180 2�

110

B

69

61

ij� I I.138 152

I� 160 10 100 120 140 160 180 200

m/e

Fig. 8. Mass spectra of S-acetylated S-phenylcysteine. A, synthetic stand-

and of S-phenylthioacetate; B, spectrum recorded from CC peak at 14mm from denivatized globin hydrolysates obtained from rats 24 h after

i_p. injection of 10 mmol/kg body weight [‘4C]benzene.

7. Maltoni, C., Conti, B., and Cotti, C. Benzene: a multipotential carcin-ogen. Results of long-term bioassays I)enformed at the Bologna InstituteofOncology. Am. I. Ind. Med., 4: 589-630, 1983.

8. Huff, I. E., Haseman, I. K., De Manini, D. M., Eustis, S., Manonpot, R.R., Peters, A. C., Pershing, R. L., Chnisp, C. C., and )acobs, A. C. Multiple-site cancinogenicity of benzene in Fisher 344 rats and B6C3F mice.

Environ. Health Penspect., 82: 125-163, 1989.

9. Cronkite, E. P., Bullis, I. E., lnoue, T., and Drew, R. I. Benzeneinhalation produces leukemia in mice. Toxicol. AppI. Phanmacol., 75:358-361, 1984.

10. Top 50 Chemicals Production Reaches Record High, Chemical andEngineering News, pp. 1 1-1 5, April 10, 1989.1 1 . Ozkaynak, H., Ryan, P. B., Wallace, L. A., Nelson, W. C., and Behan,

I. V. Sources and emission rates of organic chemical vapors in homes andbuildings. In: Indoor Air ‘87: Proceedings of the 4th International Confer-ence on Indoor Air Quality and Climate, August 17-21. Berlin: Institutefor Water, Soil, and Air Hygiene, 1987.

12. Wallace, L. A. The exposure of the general population to benzene.In: M. A. Mehlman led.), Advances in Modern Environmental Toxicology.Benzene: Occupational and Environmental Hazards-Scientific Update,pp. 113-130. Princeton, N): Princeton Scientific Publishers, Inc., 1989.

13. Hoffman, D., Brunnemann, K. D., and Hoffmann, I. Significance of

benzene in tobacco cancinogenesis. In: M. A. Mehlman ed.), Advancesin Modern Environmental Toxicology. Benzene: Occupational and Envi-nonmental Hazard-Scientific Update, pp. 99-112. Princeton, N): Prince-ton Scientific Publishers, Inc., 1989.

14. Wallace, L. A., Pellizzani, E. D., Hartwell, T. D., Spanacino, C., Whit-

more, R., Sheldon, L., Zelon, H., and Pennitt, R. The TEAM study: personalexposure to toxic substances in air, drinking water, and breath of 400

residents of New Jersey, North Carolina, and North Dakota. Environ.Res., 43: 290-307, 1987.



Cancer Epidemiology, Biomarkers & Prevention 313

15. Kinlen, L. J., and Rogot, E. Leukemia and smoking habits amongUnited States veterans. Br. Med. J., 297: 657-659, 1988.

16. Severson, R. K., Davis, S., Heuser, L., et al. Cigarette smoking andacute nonlymphocytic leukemia. Am. I. Epidemiol., 132: 418-422, 1990.

17. McLaughlin, J. K., Hrubec, Z., Linet, M. S., et al. Cigarette smokingand leukemia (Letter). J. NatI. Cancer Inst., 81: 1262-1263, 1989.

18. Garfinkel, L., and Boffetta, P. Association between smoking andleukemia in two American Cancer Society prospective studies. Cancer(Phila.), 65: 2356-2360, 1990.

19. Brownson, R. C., Chang, J. C., and Davis, J. R. Cigarette smoking andniskofadult leukemia. Am.). Epidemiol., 134: 938-941, 1991.

20. KaIf, G. F. Recent advances in metabolism and toxicity of benzene.Cnit. Rev. Toxicol., 18: 141-159, 1987.

21. Latniano, L., Witz, G., Goldstein, B., and Jeffrey, A. M. Chromato-

graphic and spectrophotometnic characterization of adducts formed dur-ing the reaction of tnans,tnans-muconaldehyde with ‘4C-deoxyguanosine

5-phosphate. Environ. Health Perspect., 82: 249-251, 1989.

22. Reddy, M. V., Bleicher, W. T., Blackburn, G. R., and Mackerer, C. R.DNA adduction by phenol, hydroquinone or benzoquinone in vitro butnot in vivo; nuclease P1 -enhanced 32P-postlabeling of adducts as labelednucleoside biphosphates, dinucleotides and nucleoside monophos-phates. Carcinogenesis (Lond.), 1 1: 1349-1357, 1990.

23. Levay, G., Pongracz, K., and BodelI, W. J. Detection of DNA adductin HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-postlabeling. Carcinogenesis (Lond.), 12: 1 182-1 186, 1991.

24. Sun, I. D., Medinsky, M. A., Birnbaum, L. S., Lucier, G., and Hender-

son, R. F. Benzene hemoglobin adducts in mice and rats: characterizationand formation and physiological modeling. Fundam. AppI. Toxicol., 15:468-479, 1990.

25. Ascoli, F., Fanelli, M. R., and Antonini, E. Preparation and propertiesof apohemoglobin and reconstituted hemoglobin. Methods Enzymol.,76: 72-87, 1981.

26. Pascoe, G. A., Calleman, C. J., and Baille, T. A. Identification of 5-(2,5-dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-

cysteine as urinary metabolites of acetaminophen in the mouse. Evidence

for p-benzoquinone as reactive intermediate in acetaminophen metabo-lism. Chem.-BioI. Interact., 68: 85-98, 1988.

27. Hanzlik, R. P., Weller, P. E., Desai, J., Zheng, J., Hall, L. R., andSlaughter, D. E. Synthesis of mercapturic acid derivatives of putative toxicmetabolites of bromobenzene. J. Org. Chem., 55: 2736-2742, 1990.

28. Bakke, J. E. Conversion of prosthetic moieties of glutathione pathway

conjugates to the corresponding S-acetates. Biomed. Mass Spectrom., 9:74-77, 1982.

29. Schroeder, W. A., Shelton, J. B., Shelton, J. R., Huynh, V., andTeplow, D. B. High performance liquid chromatography separation ofthe globular chains of non-human hemoglobin. Hemoglobin, 9:461-482,1985.

30. Jenina, D., Daly, J., Witkop, B., zaltzman-Nirenberg, P., and Uden-friend, S. Role of the arene oxide-oxepin system in the metabolism ofaromatic substances. I. In vitro conversion of benzene oxide to a pre-mercaptunic acid and dihydrodiol. Arch. Biochem. Biophys., 128: 176-183, 1968.

31. Gonasun, L. M., Witmer, C. M., Kocsis, J. J., and Snyder, R. Benzenemetabolism in mouse liver microsomes. Toxicol. AppI. Pharmacol., 26:

398-406, 1973.

32. Hanzlik, R. P., Hogberg, K., and Judson, C. Microsomal hydroxylation

of specifically deuterated monosubstituted benzene. Evidence for directaromatic hydroxylation. Biochemistry, 23: 3048-3055, 1984.

33. Stommel, P., Muller, G., Stucker, W., Verkoyen, C., Schobel, S., and

Norpoth, K. Determination of S-phenylmercapturic acid in the urine-

an improvement in the biological monitoring of benzene exposure.

Carcinogenesis (Lond.), 10: 279-282, 1989.

34. Jowa, L., Winkle, S., KaIf, G., Witz, G., and Snyder, R. Deoxyguano-

sine adducts formed from benzoquinone and hydroquinone. In: J. J.Kocsis, D. J. Jollow, C. M. Witmer, J. 0. Nelson, and R. Snyder (eds.),Biological Reactive Intermediates Ill: Mechanisms of Action in Animal

Models and Human Disease, pp. 825. New York: Plenum Press, 1986.

35. Nerland, D. E., and Pierce, W. M., Jr. Identification of N-acetyl-S-

(2,5-dihydroxyphenyl)-i-cysteine as urinary metabolite of benzene,

phenol, and hydroquinone. Drug Metab. Dispos., 18: 958-961, 1990.

36. Jongeneelen, F. J., Dirven, H. A. A. M., Leijdekkers, C-M., andHenderson, P. T. S-Phenyl-N-acetylcysteine in urine of rats and workersafter exposure to benzene. J. Anal. Toxicol., 1 1: 100-104, 1987.

37. Sabourin, P. J., Chen, B. T., Luicer, G., Birnbaum, L. S., Fisher, E.,and Henderson, R. F. Effect of dose on the absorption and excretion of

[‘4C]benzene administered orally or by inhalation in rats and mice.Toxicol. AppI. Pharmacol., 87: 325-336, 1987.

38. Skipper, P. L., and Tannenbaum, S. R. Protein adducts in the molec-

ular dosimetry ofchemical carcinogens. Carcinogenesis (Lond.), 1 1: 507-518, 1990.

39. Ehrenberg, L., Hiesche, K. D., Osterman-Bolkar, S., and Wennberg,

I. Evaluation of the genetic risks of alkylating agents: tissue doses in themouse from air contaminated with ethylene oxide. Mutat. Res., 24: 83-103, 1974.

40. Neumann, H. G. Analysis of hemoglobin as a dose monitor for

aklylating and arylating agents. Arch. Toxicol., 55: 1 -6, 1984.



1992;1:307-313. Cancer Epidemiol Biomarkers Prev A A Melikian, A K Prahalad and S Coleman hemoglobin adducts in vivo in rats.Isolation and characterization of two benzene-derived

Updated version

http://cebp.aacrjournals.org/content/1/4/307

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cebp.aacrjournals.org/content/1/4/307To request permission to re-use all or part of this article, use this link


http://cebp.aacrjournals.org/content/1/4/307http://cebp.aacrjournals.org/cgi/alertsmailto:[email protected]://cebp.aacrjournals.org/content/1/4/307http://cebp.aacrjournals.org/

isolation and characterization of two benzene-derived ... · formed as a reactive metabolite of...

Documents