-
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.
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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,
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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
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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.
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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
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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.
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AMW-152
-1�:�L 0II
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152
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