[CANCER RESEARCH 45, 2471-2477,
June 1985]
Sister Chromatid Exchange Induction in Human Lymphocytes Exposed to
Benzene and Its Metabolites in Vitro1
Gregory L. Erexson,2 James L. Wilmer, and Andrew D. Kligerman3
Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709
ABSTRACT
mas are the predominant neoplasias found in humans exposed
to benzene, other types of cancers such as Zymbal's gland and
Previous in vivo studies have shown that low-dose benzene
exposure (10 to 28 ppm for 4 to 6 h) in mice can induce sister
chromatid exchange (SCE) in peripheral blood B-lymphocytes
and bone marrow as well as micronuclei in bone marrow poly
chromatic erythrocytes. Because benzene is metabolized to a
variety of intermediate compounds and two of these, catechol
and hydroquinone, have been reported to be potent SCE-induc-
hepatocellular carcinomas are found more often in rodents ex
posed to benzene (7-10). Great variability exists in the sensitivity
ers, it is possible that other known and proposed metabolites
could have chromosome-damaging effects in lymphocytes. In
duced SCE frequencies, mitotic indices, and cell cycle kinetics
were quantitated in human peripheral blood T-lymphocytes ex
posed to benzene, phenol, catechol, 1,2,4-benzenetriol, hydro
quinone, 1,4-benzoquinone, or frans,irans-muconic acid. Three
proposed metabolites of phenol, 4,4'-biphenol, 4,4'-diphenoquinone, and 2,2'-biphenol, which can be generated by a phenolhorseradish peroxidase-hydrogen peroxide system were also
examined. Benzene, phenol, catechol, 1,2,4-benzenetriol, hydro
quinone, and 1,4-benzoquinone induced significant concentra
tion-related increases in the SCE frequency, decreases in mitotic
indices, and inhibition of cell cycle kinetics. Based on the slope
of the linear regression curves for SCE induction, the relative
potencies were as follows: catechol > 1,4-benzoquinone >
hydroquinone > 1,2,4-benzenetriol > phenol > benzene. On an
induced SCE per pu basis, catechol was approximately 221
times more active than benzene at the highest concentrations
studied, frans.frans-Muconic acid had no significant effect on the
cytogenetic parameters analyzed. 2,2'-Biphenol induced a sig
nificant increase in SCE only at the highest concentration ana
lyzed, and 4,4'-biphenol caused a significant increase in SCE
frequency that was not clearly concentration related. However,
both 2,2'- and 4,4'-biphenol caused significant cell cycle delay
and mitotic inhibition. 4,4'-Diphenoquinone caused only a signif
icant decrease in mitotic activity. These data indicate that in
addition to phenol, di- and trihydroxybenzene metabolites play
important roles in SCE induction. Furthermore, the results sug
gest either that benzene alone can induce SCE or, a more likely
possibility, that mononuclear leucocytes have a limited capability
to activate benzene.
INTRODUCTION
Benzene is generally accepted as being a carcinogen in hu
mans (1-5) and rodents (6-10). Although leukemias and lympho1This research was funded by the Chemical Industry Institute of Toxicology, a
privately funded institute.
2 Present address: Environmental Health Research and Testing, Inc., P. 0. Box
12199, Research Triangle Park, NC 27709. To whom requests for reprints should
be addressed.
3 Present address: Environmental Health Research and Testing, Inc., P. O. Box
12199, Research Triangle Park, NC 27709.
Received 1/21 /85; revised 3/11/85; accepted 3/12/85.
of individuals to benzene exposure. Factors such as age, geno
type, ¡mmunocompetence, and life-style complicate assessment
of the probable causes of benzene toxicity (11, 12). Also, the
length of exposure to and dose of benzene makes the interpre
tation of epidemiológica! studies difficult (13, 14). Thus, the
effects of low-level exposure to benzene remain an important
although controversial issue (15-17).
Benzene is thought to be metabolized through a benzene
oxide intermediate by cytochrome P-450 in the liver (18), and
most of the benzene oxide spontaneously rearranges to form
phenol (19) (Chart 1). Most of the phenol is conjugated and
excreted, but further ring oxidation can form hydroquinone (20),
which spontaneously oxidizes to 1,4-benzoquinone. Also, ben
zene oxide is conjugated with glutathione by epoxide transferase
to yield the inactivation product phenylmercapturic acid (21). In
addition, lymphocytes could conceivably convert benzene oxide
to benzene glycol by the action of epoxide hydrolase, which is
found in human lymphocytes (22). Dehydrogenation of the glycol
can then yield predominately catechol and, upon further oxidation
of the glycol, frans ,frans-muconic acid (21). A small amount of
catechol is ring oxidized to 1,2,4-benzenetriol, but most of the
catechol is conjugated and excreted (23).
Because benzene is metabolized primarily in the liver and the
sites of toxicity are principally in the hematopoietic tissues,
transport of benzene or its active metabolites by blood is re
quired. Further metabolism of benzene or its known metabolites
can occur in the bone marrow (24, 25). Studies of rodents
injected with either [3H]- or [14C]benzene have shown that a
metabolite(s) binds covalently to rat liver DNA (26) and mitochondrial DNA from mouse liver and bone marrow (27,28). [14C]Benzene is metabolized and bound irreversibly to macromolecules when incubated with rat liver microsomes in the presence
of a NADPH-generating system (19). Tunek ef a/. (19) also found
that the metabolites bind predominately to microsomal protein
and to a lesser extent, RNA. They suggested that a metabolite
of phenol rather than benzene oxide was responsible for the
binding. Sawahata and Neal (29) proposed that phenol is metab
olized to 2,2'-biphenol and 4,4'-biphenol by myeloperoxidase in
the bone marrow (Chart 1). They demonstrated that horseradish
peroxidase or bone marrow homogenate (free of mature eryth
rocytes) in the presence of hydrogen peroxide could metabolize
phenol to 2,2'-biphenol, 4,4'-biphenol, and 4,4'-diphenoquinone.
Previous studies have shown that benzene induces SCE4 in
mouse bone marrow (30, 31 ). Other investigators have failed to
detect a statistically significant increase in SCE frequency in
4 The abbreviations used are: SCE, sister chromatid exchange; MNL, mononu
clear leukocyte; AHH, aryl hydrocarbon hydroxylase; DMSO, dimethyl sulfoxide.
CANCER RESEARCH VOL. 45 JUNE 1985
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BENZENE-INDUCED
SCE IN HUMAN LYMPHOCYTES
benzene
trans,trans-rnuconic
acid
DOH
^
benzene glycol
p^
pvOH
cis.cis-muconic
«cid
•CO,
epoxide
^¡^^OH
hydrol.se
1,4-benzosemiquinone
b.hydroow.M
?
catechol
hydroquinone
OOH
1,2,4-benzenetriol
2-hydroxy-1,4-benzosemiquinone
spontaneous
4
2-hydroxy-1,4-benzoquinone
4.4'-biphenol
4,4'-dlphenoquinone
OH
Chart 1. Schematic diagram of the known and proposed metabolism of benzene. The figure is a modified and composite representation of the metabolic pathways
for benzene as reported by Rusch et al. (21 ), Irons er al. (52), and Sawahata et al. (64).
peripheral blood lymphocytes of humans after occupational ex
posure to benzene (32-34). However, mice exposed to benzene
concentrations as low as 10 ppm for 6 h exhibited an increased
SCE frequency in peripheral blood B-lymphocytes and increased
increased concentrations of AHH in MNLs are found after mito
genic stimulation and concurrent exposure to aromatic hydro
carbons (39-41 ), chemical treatment at 24 h would coincide with
an optimal time for potential metabolism.
numbers of micronucleated polychromatic erythrocytes in their
bone marrow (35). These results suggest that benzene or its
metabolites in low concentrations in vivo are responsible for the
chromosome-damaging effects.
Morimoto and Wolff (36) found that catechol, hydroquinone,
and to a lesser extent phenol induced SCE in phytohemagglutinin-stimulated lymphocytes. Benzene did not induce any in
crease in SCE but showed some cytotoxicity at 5 HIM and
inhibited growth at 250 mw. Morimoto ef al. (37) showed that rat
liver S-9 augmented SCE induction in human lymphocytes ex
posed to phenol, catechol, and hydroquinone during a 2-h pulse
treatment at 40 to 42 h of 3-day cultures. In addition, Morimoto
(38) reported that benzene induced SCE in human lymphocytes
only after incubation of the cells in the presence of rat liver S-9.
Therefore, to understand further the chromosome-damaging
effects of benzene, SCE induction in human T-lymphocytes was
investigated after exposure in vitro to benzene, 6 known metab
olites of benzene (phenol, catechol, 1,2,4-benzenetriol, hydro
quinone, 1,4-benzoquinone, and frans,frans-muconic acid) and 3
proposed metabolites of phenol (2,2'-biphenol, 4,4'-biphenol,
and 4,4'-diphenoquinone).
The purpose of this study was to
determine
stimulation
toxicity of
treatment
MATERIALS
RESEARCH
METHODS
Blood Processing and Lymphocyte Culture Technique. Heparinized
whole blood (35 to 60 ml) samples were drawn by venipuncture from the
same healthy adult male for all experiments to alleviate any possible
donor-to-donor variability. No attempt was made in these experiments
to survey the variability known to exist in human AHH inducibility; rather,
the relative potencies of benzene and its known and proposed metabo
lites were studied. Whole blood was processed on Ficoll-Paque (Phar
macia Fine Chemicals, Piscataway, NJ) density gradients, and MNLs
were cultured as described previously (42). Two to 4 MNL cultures/
treatment were established by inoculating 108 MNLs into 1.9 ml of
complete medium composed of RPM11640,10%
heat-inactivated
fetal
bovine serum, 100 units of penicillin, and 100 ng of streptomycin sulfate/
ml, and an additional 292 MQ.of u-glutamine/ml. T-lymphocytes were
stimulated to divide with 8 ng concanavalin A/ml. The cultures were
incubated at 37°C in a humidified CO2 atmosphere for 24 h. 5-Bromo2'-deoxyuridine (5 J/M) was added at 24 h. The test compounds were
added separately to cultures over the following concentration ranges (in
tiu): benzene (5 to 7000); phenol (5 to 3000); catechol (5 to 500); 1,2,4benzenetriol (5 to 500); hydroquinone (5 to 500); 1,4-benzoquinone (5 to
500); fra/7s,f/-ans-muconic acid (5 to 500); 4,4'-biphenol (0.1 to 500);
4,4'-diphenoquinone
(0.1 to 100); or 2,2'-biphenol (5 to 500). The
if human lymphocytes exposed 24 h after mitogenic
(G,-S phase) would be more sensitive to the genobenzene and its metabolites rather than if chemical
was begun at the start of culture (G0-G,). Because
CANCER
AND
cultures were harvested at 72 h following a 4-h treatment with demecolcine(1.35fiM).
Chemicals.
Phenol (99+%),
catechol (99+%),
1,2,4-benzenetriol
VOL. 45 JUNE 1985
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BENZENE-INDUCED
(99%), hydroquinone
(99+%),
and 1,4-benzoquinone
SCE IN HUMAN LYMPHOCYTES
(98%) were pur-
to cultures immediately prior to incubation. All test chemicals were added
to the cultures in 2CM aliquots.
Slide Preparation, Cytogenetic, and Statistical Analyses. The slides
were prepared as described previously, coded, and stained using a
modified fluorescence-plus-Giemsa
technique (43, 44). Fifty second-
Chased from Aldrich Chemical Company (Milwaukee, Wl) and dissolved
in RPMI 1640 medium. Benzene was obtained from Fisher Scientific
Company (Raleigh, NC) and also dissolved in RPMI 1640. trans,transMuconic acid (Aldrich), 4,4'-biphenol (Sigma Chemical Company, St.
Louis, MO), and 2,2'-biphenol (Sigma) were dissolved initially in DMSO
(Fisher) and diluted with RPM11640
0.33% DMSO. 4,4'-Diphenoquinone
division metaphases, 200 consecutive metaphases, and 2000 nuclei
were analyzed from each treatment for SCE frequency, cell cycle kinetics,
and mitotic index, respectively, unless noted otherwise. All cytogenetic
data were tested for normality and then subjected to a one-way analysis
of variance with the level of significance chosen as 0.05 (45). A onetailed Dunnett's multiple range test was used to compare the SCE
to achieve a final concentration of
(Pfaltz and Bauer, Inc., Stamford,
CT) was also dissolved initially in DMSO and diluted with RPM11640 to
obtain a final concentration of 0.8% DMSO. The test chemical stock
solutions were prepared and sterilized (except benzene and phenol)
using 0.22-A¡mMillex-GS filter units (Millipore Corporation, Bedford, MA).
frequency of each concentration of chemical tested to the concurrent
control or pooled control if warranted (45, 46). Unear equations were
Because of their volatility, benzene and phenol were prepared and added
Table 1
Effect of benzene and its known in vivo water-soluble metabolites on the SCE frequency, mitotic activity, and cell cycle kinetics of human
peripheral blood T-lymphocytes exposed in vitro
The removal of blood, treatment of MNLs, culture of lymphocytes, harvest, and slide preparation were as described in "Materials and Methods."
Fifty second-division metaphases, 2000 nuclei, and 200 consecutive metaphases were analyzed for SCE, mitotic index, and cell cycle kinetics,
respectively, unless noted otherwise.
(%)ChemicalRPM1
Cell cycle kinetics
tion
(¿¿M)5505001000500070005505007001000300055070100300550701003005005507010030055070100300SCEs/metaphase8.68
(%)5.99
index
division17.0 division41.0
division30.0
division12.0
controlBenzene*Phenol*Catechol*1
1640
,2,4-Benzenetrtor'Hydroquinone*1
,4-Benzoquinone*Concentra
c9.45±0.306'
0.42°5.65
±
±2.6°19.5 ±5.744.5
±1.05"9.88
0.2810.94
±
0.41s11.
±
0.354.60
±
0.574.53
±
0.46e4.05
±
±0.718.0
±2.821.0
±2.2"27.0
±0.0310.98
58
±0.3713.22
0.9310.52
±
0.212.95
±
±1.062.85
0.644.70
±
5.735.5
±
3.535.0
±
0.021.0
±
0.1711.
±
±0.5113.47
08
0.95*13.10
±
0.283.95
±
0.212.73
±
0.95"2.45
±
±1.416.5
2.128.0
±
8.2e40.5
±
0.5916.56
±
0.3419.50
±
'10.42 ±0.71
0.072.05
±
0.210.20
±
0.004.1
±
2.160.0
±
2.896.0
±
0.022.5
±
0.2013.61
±
0.93e15.12
±
±0.072.18
5
±0.17*1.75
6.447.0
±
6.2e69.0
±
±0.1123.00
0.00"Cytotoxic9.50
±
±0.210.85
0.075.50
±
5.691±
^±0.0"21.0
0.20"1±
0.32e14.04
3.92 ±
0.145.23
±
0.49"4.30
±
±2.816.5
3.8e19.0
±
0.7915.52
±
0.143.1
±
0.6222.27
±
±0.350.1
5
±1.27"Cytotoxic10.32±0.074.00
5
±0.014.0
±1.443.0
0.0*42.0
±
±0.231
0.53e12.96
2.84 ±
0.282.30
±
0.37e1.50
±
4.241±
6.2e42.5
.0±
±0.4515.92
7Cytotoxic12.36
±0.1
40.70±0.1
0.003.50
±
2.158.5
±
2.122.5
±
3.427.5
±
5.38.5
±
3.541.0
±
±1.440.0
4.845.5
±
6.441±
±2.146.5
.5
2.147.5
±
3.530.5
±
3.531.0
±
±4.122.0
7.117.0
±
±4.216.0
±2.824.0
0.710.5
±
±5.08.0
2.85.5
±
4.96.0
±
2.82.5
±
0.77.5
±
3.543.5
±
0.744.5
±
2.652.0
±
0.037.0
±
4.24.0
±
0.046.0
±
±1.429.5
2.121.0
±
±7.17.5
2.13.0
±
±1.426.5
3.510.5
±
0.76.5
±
3.15.0
±
±1.441.0
±2.929.0
7.09.0
±
0.046.0
±
6.410.0
±
±4.72.0
±1.427.0
±1.42.0
±1.86.0
4.241±
±6.635.5
.5
7.847.0
±
4.946.0
±
0.035.5
±
2.830.0
±
4.930.5
±
±1.430.0
.411.0
±1
±0.017.5
4.212.0
±
±5.415.0
2.89.0
±
4.25.0
±
2.141±
±4.946.5
.5
2.136.5
±
2.147.0
±
±4.913.0
±3.68.0
4.25.0
±
0.025.5
±
±1.44.5
3.13.0
±
0.05.0
±
0.4017.52
±
±1.49"16.74
0.573.20
±
6.419.0
±
2.851.0
±
0.723.0
±
±1.47.0
0.50*2.00
±
±2.4*18.0
±5.143.0
4.527.0
±
2.412.0
±
±0.8219.08
0.281.95
±
±2.135.5
2.839.0
±
±1.420.5
±6.45.0
±0.51CytotoxicMitotic
±0.21First
±3.5Second ±1.4Third
±2.1Fourth
±2.8
* The test chemical is significantly different from the concurrent control at P < 0.05, using a one-way analysis of variance for the SCE, mitotic
index, and cell cycle kinetics data, respectively.
6 Mean ±SD among cultures.
0 A total of 225 second-division metaphases, 9000 nuclei, and 900 consecutive metaphases were analyzed for SCE, mitotic index, and cell
cycle kinetics, respectively.
" Using a one-tailed Dunnett's multiple range test for the SCE data, 5 MM benzene and 1,2,4,-benzenetriol
are not significantly different from
the concurrent control at P < 0.05.
*A total of 100 second-division metaphases, 4000 nuctei, and 400 consecutive metaphases were analyzed for SCE, mitotic index, and cell
cycle kinetics, respectively.
A total of 3 second-division metaphases.
' A total of 9 second-division metaphases and 50 consecutive metaphases.
A total of 25 second-division metaphases and 50 consecutive metaphases.
CANCER RESEARCH
VOL. 45 JUNE 1985
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BENZENE-INDUCED
SCE IN HUMAN LYMPHOCYTES
derived for benzene and each known metabolite examined that showed
a significant concentration-related increase in SCE frequency (45).
RESULTS
Cytogenetic Analysis of Benzene and Its Known Metabo
lites. Benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, and 1,4-benzoquinone induced significant concentrationdependent increases in the SCE frequency in human T-lymphocytes (Table 1). Based on the slopes of the linear regression
curves for SCE induction, the relative potencies were as follows:
catechol > 1,4-benzoquinone > hydroquinone > 1,2,4-benzene
triol > phenol > benzene (Chart 2). At the lowest concentration
examined (5 MM), 1,4-benzoquinone was the most potent SCE
inducer of all the compounds examined (Table 1). frans,frans Muconic acid did not increase the SCE frequency significantly
(Table 2). Benzene, phenol, catechol, 1,2,4-benzenetriol, hydro
quinone, and 1,4-benzoquinone caused significant depression of
mitotic activity and inhibition of cell cycle progression (Table 1).
frans,frans-Muconic acid did not significantly affect the mitotic
activity or cell cycle progression (Table 2).
Cytogenetic Analysis of the Proposed Metabolities of Ben
zene. 2,2'-Biphenol and 4,4'-biphenol induced a slight but sta
tistically significant increase in SCE frequency (Table 2). How
ever, the magnitude of the response was minimal compared to
the response seen with benzene, phenol, catechol, 1,2,4-ben
zenetriol, hydroquinone, and 1,4-benzoquinone. 1,4-Diphenoquinone did not cause a statistically significant increase in SCE
(Table 2). 2,2'-Biphenol, 4,4'-biphenol, and 4,4'-diphenoquinone
caused a significant reduction in mitotic activity, and both of the
biphenols significantly inhibited cell cycle progression (Table 2).
DISCUSSION
These results demonstrate that benzene, phenol, catechol,
1,2,4-benzenetriol, hydroquinone, and 1,4-benzoquinone induce
zz
SCEs in human T-lymphocytes
from MNL cultures exposed in
vitro without any additional activating system. It is interesting to
note the marked differences in SCE potencies of the compounds
studied (Chart 2). On an induced SCE per >M basis, catechol
was approximately 221 times more active than benzene at the
highest concentrations studied. Results from the present study
on SCE induction, inhibition of mitotic activity, and cell cycle
progression with catechol and hydroquinone correlate well with
the data of Morimoto and Wolff (36). Both studies report the
following: (a) catechol is a more potent SCE inducer than is
hydroquinone; (b) catechol and hydroquinone are about equal in
decreasing mitotic activity; and (c) catechol is more effective
than hydroquinone in inhibiting cell cycle progression. The pres
ent study shows that 1,4-benzoquinone (the oxidized metabolite
of hydroquinone) is more potent than catechol at inducing SCE
at 5 and 50 MM.In general, catechol depressed the mitotic activity
and slowed the cell cycle progression more effectively than did
1,4-benzoquinone. 1,2,4-Benzenetriol is the least cytotoxic of
the 4 most reactive metabolites (catechol, 1,2,4-benzenetriol,
hydroquinone, and 1,4-benzoquinone). Thus, these 4 metabolites
might be responsible for most of the in vivo genotoxicity asso
ciated with exposure to benzene.
Few reports exist on the mutagenic effects of the major
metabolites of benzene (phenol, catechol, and hydroquinone),
and no data are available regarding the mutagenicity of its
remaining known and proposed metabolites. Exposure to phenol,
catechol, and hydroquinone in the Ames' Salmonella test with or
without S-9 activation did not increase the number of revenants
(47, 48). However, Gocke ef al. (49) found that phenol with S-9
and hydroquinone without S-9 were positive in the Ames test
when using ZLM medium instead of Vogel-Bonner medium. Also,
hydroquinone has been shown to induce micronuclei in mouse
bone marrow (49,50) whereas catechol does not (50). Bulsiewicz
(51) found a significant increase in chromatid aberrations in
mouse spermatogonia and primary spermatocytes following p.o.
exposure to phenol. Thus, mammalian systems detect the mu
tagenic effects of the metabolites of benzene, whereas the
results from prokaryotic systems are equivocal.
Quinone metabolites formed by the oxidation of catechol and
hydroquinone are toxic to lymphoid cells and bone marrow (52-
20
55). Quiñonesand semiquinones have been implicated as the
ultimate reactive metabolites of benzene in the liver (20), in
human lymphocytes (37, 38), and in rabbit bone marrow nuclei
(56). Irons ef al. (52) implicated 1,4-benzoquinone as being the
16
ultimate toxic metabolite of benzene but did not rule out the
potential importance of 1,4-benzosemiquinone. They also dem
onstrated that of the metabolites studied (hydroquinone, 1,4benzoquinone, and catechol), 1,4-benzoquinone was the most
potent inhibitor of blast transformation in phytohemagglutininstimulated rat spleen lymphocytes. Their data on the mitogenic
responsiveness after treatment with hydroquinone and 1,4-ben
in
8«
10
200
500
1000 '
3000 5000 7000
CONCENTRATION
(uM)
Chart 2. Linear regression equations of the SCE induction curves for benzene
and each known metabolite that showed a significant concentration-related in
crease in SCE. The best-fit linear equation and squared correlation coefficient for
each compound are as follows: (•),
catechol (CT), y = 0.124x + 8.6, r* = 0.91;
(A), 1,4-benzoquinone(BO),y = 0.092x + 10.7, r2 = 0.85; (•).
hydroquinone(HO),
y = 0.063X + 9.3, r2 = 0.95; (O), 1,2,4-benzenetriol(BT), y = 0.043x + 10.27, r2
= 0.93; (A), phenol (PH), y = 0.0032x + 10.9, r2 = 0.84; (D), benzene (BZ), y =
0.0004X + 9.9, r" = 0.62.
CANCER
RESEARCH
zoquinone correlate well with our data on mitotic inhibition
caused by these metabolites. Irons et al. (52) showed that 0.4
MM 1,4-benzoquinone and 2 /IM hydroquinone inhibited mitogenesis in rat splenocytes, whereas up to 10 /¿M
catechol had no
effect. In contrast with the study of Irons ef al. (52), the present
study shows that 5 UM catechol in addition to hydroquinone and
1,4-benzoquinone inhibited mitotic activity. Hydroquinone and
1,4-benzoquinone have also been shown to be highly toxic to
the colony formation
of mouse bone marrow stromal cells,
VOL. 45 JUNE 1985
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BENZENE-INDUCED
SCE IN HUMAN LYMPHOCYTES
Table 2
Effect of known and proposed DMSO-solublebenzene metabolites on the SCE frequency, mitotic activity, ano cell cycle kinetics of humanperipheral blood Tlymphocytes exposed in vitro
The removal of blood, treatment of MNLs, culture of lymphocytes, harvest, and slide preparation were as described in "Materials and Methods." Fifty second-division
metaphases, 2000 nuclei, and 200 consecutive metaphases were analyzed for SCE, mitotic index, and cell cycle kinetics, respectively, unless noted otherwise.
(%)Chemical0.33%
controlfrans.rrans-Muconic
DMSO
Cell cycle kinetics
(%)4.30
index
division17.0
(MM)5SO500550701003005000.10.55.07.010.050.0SCEs/metaphase9.27
S365.15
±1.
±4.9"20.0
*9.82±0.48a'
add2,2'-Biphenolc4,4'-BiphenolcConcentration
0.069.52
±
0.2310.06
±
0.629.76
±
0.355.45
±
0.215.40
±
0.283.25
±
0.628.90
±
0.2510.1
±
±0.4510.06
2
±0.4810.48
±0.06"No
0.494.10
±
±0.212.50
0.854.00
±
0.281.95
±
±0.211.00
±0.143.55
division9.46
second
±1.203.45
0.659.22
±
0.212.45
±
0.5911.62±0.72*<>9.78
±
3e1.40
±0.1
division33.0
division8.0
5.744.0
±
7.129.5
±
4.36.5
±
±0.019.5
±0.722.5
0.722.0
±
±1.747.0
0.244.0
±
2.847.5
±
0.725.0
±
0.025.0
±
5.624.5
±
0.78.5
±
4.98.5
±
3.56.0
±
±2.829.0
±8.532.0
±15.535.5
±3.541.0
±10.6100.0
±0.028.0
0.748.0
±
2.841.0
±
±1.442.5
3.547.0
±
4.936.0
±
3.519.0
±
±4.221
±9.220.5
.5
0.710.5
±
4.935.5
±
2.14.0
±
.45.5±1
4.91.5
±
±0.71.5
±0.70.5
±7.122.5
±0.738.5
6.4"44.5
±
0.0810.46
±
0.59"CytotoxicMitotic
±
±0.002.00
±0.00First
4.231±
±12.042.0
±2.147.0
.5
7.113.0
±
2.940.0
±
±6.715.0
±12.057.5
8.540.5
±
2.81.5
±
±3.5Seconddivision42.0
±3.5Third
±0.7Fourth
0.74.0
±
5.71.5
±
±1.00.5
0.70.5
±
±0.7
0.80% DMSO control
10.52 ±1.47
2.75 ±0.64
19.0 ±1.4
40.5 ±0.7
37.5 ±2.1
3.0 ±0.0
4,4' -Diphenoquinone'0.10.51.05.050.070.09.66
0.258.98
±
±1.108.82
0.8810.1
±
±0.2311.
6
±0.51Cytotoxic3.05
56
0.784.45
±
0.073.70
±
±1.133.35
0.070.65
±
±0.0731.0
±4.217.5
±2.124.5
±9.216.0
±1.434.0
±8.530.0
7.130.5
±
7.826.0
±
0.032.0
±
±1.443.5
±6.438.0
12.748.0
±
±1.448.0
±11.345.0
±1.422.5
±2.11.0
±1.44.0
±1.41.5
±2.17.0
±1.4
* Mean ±SD among cultures.
A total of 175 second-divisionmetaphases,7000 nuclei, and 700 consecutive metaphaseswere analyzedfor SCE, mitotic index, and cell cycle kinetics, respectively.
c The test chemical is significantly different from the concurrent control at P < 0.05, using a one-way analysis of variance for the SCE, mitotic index, and cell cycle
kinetics
data,one-tailed
respectively.
Usinga
Dunnett s multiple range test for the SCE data, 300 pM 2,2'-biphenol and 5 and 10 MM4,4'-biphenol are significantlydifferent from the concurrent
control at P < 0.05.
8 A total of 100 second-divisionmetaphases, 4000 nuclei, and 400 consecutive metaphaseswere analyzedfor SCE, mitotic index, and cell cycle kinetics, respectively.
' The test chemical is significantly different from the concurrent control at P < 0.05, using a one-way analysisof variance for the mitotic index data only.
whereas catechol is considerably less toxic (57). In general, the
present results on the Cytotoxic effects of the dihydroxy and
quinone metabolites of benzene are in agreement with other
recent studies, but catechol might have a more prominent role
in toxicity to human lymphocytes compared to rodent lympho
cytes.
Gerner-Smidt and Friedrich (58) showed that benzene did not
induce SCE in human T-lymphocytes stimulated with phytohe-
between the Morimoto and Wolff (36) and present studies, (a)
They added benzene and phenol at culture initiation when the
lymphocytes were in G0-G,, whereas we added these com
magglutinin. Their finding was most likely attributable to the
experimental protocol used. In their study, benzene (0.195 to
19.5 HIM) was mixed with serum and injected into corked incu
bation flasks in which the whole blood lymphocytes were cultured
under reduced oxygen tension for 72 h. A significant decrease
in mitotic activity and inhibition in cell cycle progression was not
seen after exposure of up to 19.5 DIM. These results contrast
with the findings of Morimoto and Wolff (36) and the present
study where aerobic-culture methodologies were used, and sig
nificant cytotoxicity was seen with benzene concentrations of 1
mW and 50 UM, respectively.
Morimoto and Wolff (36) did not observe an increase in the
SCE frequency in phytohemagglutinin-stimulated human lympho
cytes from whole blood cultures treated with up to 5 m.Mbenzene
and saw only a small increase after treatment with 1 mu phenol.
There are at least 4 possible reasons for the discrepancies
benzene (59), it is probable in the present study that benzene
and phenol could be stimulating their own metabolism. Rüdiger
er al. (60) demonstrated that benzo(a)pyrene induced its own
metabolism in human lymphocytes as shown by an increased
SCE frequency, (b) Cytogenetic lesions induced immediately
prior to S phase might have little or no time to be repaired, and
consequently, SCE induction should be greater (61). (c) Because
of their high volatility, benzene and phenol might have evaporated
before DNA synthesis in the Morimoto and Wolff (36) study, (d)
Differences could exist in the SCE response between FicollPaque-separated MNLs and whole-blood cultures.
pounds 24 h after mitogenic stimulation when lymphocytes
would be blast transformed and in the G,-S phase. Because
cytochrome P-450 (AHH) activity increases during blast transfor
mation of human MNLs in the presence of aromatic hydrocar
bons (39-41), and aryl-4-hydroxylase is an enzyme that oxidizes
Because the liver contains much higher concentrations of AHH
than do hematopoietic tissues (62), use of a postmitochondrial
supernatant should be an efficient method to activate benzene
to genotoxic intermediates. In this regard, Morimoto (38) found
that 5 mu benzene in the presence of 10% rat liver S-9 induced
CANCER RESEARCH VOL. 45 JUNE 1985
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BENZENE-INDUCED
SCE IN HUMAN LYMPHOCYTES
some studies on workers exposed to atmospheric benzene. The possible
influence of age. Eur. J. Cancer, 6: 49-55,1970.
15. Goldstein, B. D., and Snyder, C. A. Benzene leukemogenesis. Environ. Sci.
Res., 25: 277-289,1982.
16. Goldstein, B. D. Benzene is still with us. Am. J. Ind. Med., 4: 585-587,1983.
17. Hasetey,D. (ed.). InsideE. P. A., Vol. 5, No. 45, p. 13. Washington, DC: Inside
Washington Publishers, 1984.
18. Gonasun, L. M., Witmer, C., Kocsis.J. J., and Snyder, R. Benzenemetabolism
in mouse liver microsomes. Toxicol. Appi. Pharmacol.,26: 398-406,1973.
19. Tunek, A., Platt, K. L., Bentley, P., and Oesch, F. Microsomal metabolism of
benzene to species irreversibly binding to microsomal protein and effects of
modificationof this metabolism. Mol. Pharmacol.,14: 920-929,1978.
20. Tunek, A., Platt, K. L., Przybyski, M., and Oesch, F. Multi-step metabolic
activation of benzene. Effect of Superoxidedismutase on covalent binding to
microsomalmacromoiecules,and identificationof glutathioneconjugates using
high pressure liquid chromatography and field desorpttonspectometry. Chem.Biol. Interact., 33:1-17,1980.
21. Rusch, G. M., Leong, B. K. J., and Laskin, S. Benzenemetabolism.J. Toxicol.
Environ. Health, 2 (Suppl.y 23-36,1977.
22. Glatt, H. R., Wölfe),T., and Oesch, F. Determination of epoxide hydrolase
activity in whole cells (human lymphocytes) and activation by benzoflavones.
Biochem. Biophys. Res. Commun., 110: 525-529,1983.
23. Teisinger, J., Bergerova-Fiserova, V., and Kudma, J. The metabolism of
benzene in man. Frac. Lek., 4:175-188,1952.
24. Irons, R. D., Dent,J. G., Baker, T. S., and Rickert, D. E. Benzeneis metabolized
and covalently bound in bone marrow in situ. Chem.-Biol. Interact., 30: 241245,1980.
25. Snyder, R., Sammelt, D., Witmer, C., and Kocsis, J. J. An overview of the
problem of benzene toxicity and some recent data on the relationship of
benzene metabolism to benzene toxicity. Environ. Sci. Res., 25: 225-240
1982.
26. Lutz, W. K., and Schlatter, C. H. Mechanism of the carcinogenic action of
benzene: irreversiblebinding to rat liver DNA. Chem.-Biol. Interact., 18: 241245, 1977.
27. Gill, D. P., and Ahmed, A. E. Covalent binding of ["C]benzene to cellular
organelles and bone marrow nucleic acids. Biochem. Pharmacol.,30: 11271131,1981.
28. Kalf, G. F., Rushmore, T., and Snyder, R. Benzene inhibits RNA synthesis in
mitochondria from liver and bone marrow. Chem.-Biol. Interact., 42:353-370,
1982.
29. Sawahata, T., and Neal, R. A. Horseradish peroxidase-mediatedoxidation of
phenol. Biochem. Biophys. Res. Commun., 109: 988-994,1982.
30. Tice, R. R., Costa, D. L., and Drew, R. T. Cytogenetic effects of inhaled
benzene in murine bone marrow: induction of sister chromatic)exchanges,
chromosomal aberrations, and cellular proliferation inhibition in DBA/2 mice.
Proc. Nati. Acad. Sci. USA, 77: 2148-2152,1980.
31. Ttee,R. R., Vogt, T. F., and Costa, D. L. Cytogenetic effects of inhaledbenzene
in murine bone marrow. Environ. Sci. Res., 25: 257-275,1982.
32. Clare, M. G., Yardley-Jones,A., MacLean,A. C., and Dean, B. J. Chromosome
analysisfrom peripheralblood lymphocytesof workers after an acute exposure
to benzene.Br. J. Ind. Med., 41: 249-253,1984.
33. Sarto, F., Geminato, l.. Pinton, A. M., Brovedani, P. G., Merier, E., Peruzzi,
M., Bianchi, V., and Levis, A. G. A cytogenetic study on workers exposed to
tow concentrations of benzene.Carcinogenesis(Lond.), 5: 827-832,1984.
34. Watanabe, T., Endo, A., Kato, Y., Shima, S., Watanabe, T., and Ikeda, M.
Cytogenetics and cytokinetics of cultured lymphocytes from benzene-exposed
workers. Int. Arch. Occup. Environ. Health, 46: 31-41,1980.
35. Erexson, G. L, Wilmer, J. L., and Kligerman,A. D. Inductionof sister chromatid
exchanges and micronuclei in mate DBA/2 mice after inhalation of benzene.
Environ. Mutagen., 6:408,1984.
36. Morimoto, K., and Wolff, S. Increase of sister chromatid exchanges and cell
cycle perturbations of cell division kinetics in human lymphocytes by benzene
metabolites. Cancer Res., 40:1189-1193,1980.
37. Morimoto, K., Wolff, S., and Koizumi, A. Induction of sister-chromatid ex
changes in human lymphocytes by microsomal activation of benzene metab
olites. Mutât.Res., Õ79:355-360,1983.
38. Morimoto, K. Induction of sister chromatid exchanges and cell cycle division
delays in human lymphocytes by microsomal activation of benzene. Cancer
Res., 43; 1330-1334,1983.
39. Bast, R. C., Jr., Okuda, T., Ptotkin, E., Tarane, R., Rapp, H. J., and Gelboin,
H. V. Developmentof an assay for aryl hydrocarbon [benzo(a)pyrene]hydroxylase in human peripheral blood monocytes. Cancer Res., 36; 1967-1974,
1976.
40. Busbee, D. L., Shaw, C. R., and Cantrell, E. T. Aryl hydrocarbon hydroxylase
induction in human leukocytes. Science (Wash. DC), 778: 315-316,1972.
41. Whittock, J. P., Jr., Cooper, H. L., and Gelboin, H. V. Aryl hydrocarbon
(benzopyrene)hydroxylase is stimulated in human lymphocytes by mitogens
and benz(a)anthracene.Science(Wash. DC), 777: 618-619,1972.
42. Wilmer, J. L., Erexson, G. L., and Kligerman,A. D. Implicationsof an elevated
sister-chromatid exchange frequency in rat lymphocytes cultured in the ab
sence of erythrocytes. Mutât.Res., 709: 231-248,1983.
43. Kligerman, A. D., Wilmer, J. L., and Erexson, G. L. Characterization of a rat
6.2 SCEs/metaphase in human lymphocytes. It is interesting to
note that in the present study, 7 mw benzene induced 4.5 SCEs/
metaphase without addition of any exogenous activating system.
Also, normal individuals can have either low (53%), intermediate
(37%), or high (10%) AHH inducibility after exposure to polycyclic
aromatic hydrocarbons (63). Thus, it is possible that benzene
treatment of T-lymphocytes from additional individuals could
yield results different from those reported in the Morimoto (38)
and the present studies.
Results obtained from the present study suggest that the
metabolism of phenol to an SCE-inducing intermediate is not
mediated by myeloperoxidase. 2,2'-Biphenol, 4,4'-biphenol, and
4,4'-diphenoquinone were marginal SCE inducers compared to
benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone,
and 1,4-benzoquinone. Phenol (1 mw) induced about 8 SCEs/
cell in the present study, whereas 2,2'-biphenol, 4,4'-biphenol,
or 4,4'-diphenoquinone induced about one SCE/cell at the high
est concentrations that could be analyzed. Although the phenol
metabolites proposed by Sawahata and Neal (29) are inducing
few SCEs/cell, the biphenolic metabolites could be contributing
to bone marrow cytotoxicity in vivo. The present results suggest
that phenol is either being metabolized further by AHH or alone
can induce SCE.
In conclusion, the present study demonstrates that in addition
to phenol, di- and trihydroxybenzene metabolites play important
roles in SCE induction. Furthermore, the results suggest that
either benzene alone can induce SCE or, a more likely possibility,
that MNLs have a limited capability to activate benzene.
ACKNOWLEDGMENTS
We thank Douglas A. Neptun for drawing the blood samples, James S. Bus,
Byron E. Butterworth, and Richard D. Irons for reviewing the manuscript, and Linda
Smith and Joanne Quate for typing the manuscript.
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Sister Chromatid Exchange Induction in Human Lymphocytes
Exposed to Benzene and Its Metabolites in Vitro
Gregory L. Erexson, James L. Wilmer and Andrew D. Kligerman
Cancer Res 1985;45:2471-2477.
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