[CANCER
RESEARCH
40,1189-1193,April1980)
0008-5472/80/0040-0000$02.00
Increase of Sister Chromatid Exchanges and Perturbations of Cell Division
Kinetics in Human Lymphocytes by Benzene Metabolites1
Kanehisa Morimoto2 and Sheldon Wolff
Laboratory of Radiobiology and Department of Anatomy, University of California at San Francisco, San Francisco, California 94143
regarded as a sensitive indicator of mutagenic carcinogens (5,
ABSTRACT
Benzene, which has been associated with human cancers,
is metabolized
to produce
several
major
metabolites
that
could
be responsible for the biological effects. Tests have now been
carried out on human lymphocytes in culture to determine if
benzene or its metabolites, phenol, catechol, and hydroquin
14, 26, 33, 40). By the use of these techniques in human
lymphocytes treated in vitro, we are now able to show that,
although benzene itself does not increase SCE's or delay cell
turnover, its metabolites, catechol, hydroquinone, and phenol,
do.
one, inducecytogeneticchangesand affectthe cell cycle. The
results indicate that benzene itself does not induce sister
chromatid exchanges or affect cell cycle kinetics over a wide
rangeof doses. Phenolhas an effect only at very high doses.
On the other hand, catechol is a potent compound that induces
sister chromatidexchangesand delayscell divisionvery read
MATERIALS AND METHODS
Whole blood (0.2 ml) from a healthy adult man was added to
S ml of Roswell Park Memorial Institute Tissue Culture Medium
1640 containing 15% fetal calf serumand 1% PHAM (Grand
Island Biological Co., Grand Island, N. Y.). The medium also
contained 20 @M
BrdUrd and 20 mM 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid buffer (pH 7.3). The cultures
were incubated at 37°for various periods of time in complete
darkness. Two hr before fixation, Colcemid (2 x 10@ M final
INTRODUCTION
concentration) was added. The cells were then collected by
Benzene, which has a direct association with human cancer, centnifugation, exposed to 0.075 M KCI hypotonic solution for
e.g., leukemia (12, 32), is converted metabolically into phenolic 4 mm to spread the chromosomes and hemolyze the RBC's,
compounds in the body. The principal metabolites are phenol, and fixed 3 times in methanol:acetic acid (3:1 ). Drops of a
catechol,
and hydnoquinone
(7,30,32).A recentexperiment concentrated suspension of cells were placed on microslides,
(21)has suggestedtheoccurrenceofan unspecified
invivo which were allowed to air dry. The slides were then stained by
covalent interaction of benzene metabolites with matliven DNA, a modification of the fluorescent plus Giemsa (39) technique to
and, because benzene itself did not induce SCE's3 in human obtain harlequin chromosomes. The slides were stained for 15
Hoechst 33258 per ml in Sorensen's
lymphocytes in vitro, it has been postulated (1 1) that the mm in a solution of 5 @.tg
buffer,
pH
6.8.
The
slides
were then washed, dried, mounted
metabolites might be the mutagenic substances. Because
many mutagens are carcinogens (1), the identification of those with the buffer under a coverslip, and exposed to light from a
metabolites of benzene that are mutagenic might give possible 100-watt high-pressuremercuryburner(625 J/sq m/sec) for
4 mm. The slides were then stained for 10 mm in a 3% Giemsa
insights into the mechanisms involved in the induction of ben
zene-associated leukemia. Some experiments have already solution made in the same Sorensen's buffer. Cells dividing for
been carried out on the ability of benzene and its metabolites the first (Xl), second (X2), and third or more (X3+) time in
to induce chromosome aberrations (7, 32, 41 ). Because the culture can be determined in such preparations (35, 38). Xl
metabolites of benzene are cytotoxic, affect the morphology of cells contain chromosomes with both sister chromatids stained
uniformly dark. X2 cells contain only harlequin chromosomes
cell nuclei, and at high concentrations decrease cell prolifera
tion (10, 30, 32), these aberration studies often did not show with one chromatid darkly stained and its sister chromatid
lightly stained, whereas X3+ cells contain some harlequin
a clear dose response, non did they allow a comparative anal
chromosomes
and other chromosomes with both sister chro
ysis of the types of aberrationsinducedby the variousmetab
olites. It has therefore been difficult to distinguish between the matids stained uniformly lightly. SCE's were analyzed in 75
second-division metaphases for each point. Three hundred
cytotoxicity, clastogenicity, and mutagenicity of these chemi
cells were scored to determine the percentage of Xl , X2, and
cals(7,41).
Methods for producing harlequin chromosomes (18, 27) now X3+ cells. Three thousand cells were scored to determine the
mitotic index.
enable us to investigate accurately disturbances of cell turn
In a second experiment designed to test the effects of
over rates (6, 35, 37) as well as SCE induction, which is
benzene and its metabolites, cells from 72-hr cultures only
I Work
performed
under
the auspices
of the United
States
Department
of
were studied. In this experiment, 50 cells were scored for
Energy.
SCE's, 300 cells for the determination of the proportion of Xl,
2 Permanent
address:
Department
of Public
Health,
Faculty
of Medicine,
University of Tokyo, Tokyo 113, Japan. Fellow of the Japan Society for the X2, and X3+ cells, and 2000 cells at each point for the mitotic
PromotIon of Science.
index. Each compound to be tested (Aldrich, Milwaukee, Wis.)
ily. Hydroguinone is also potent, but less so than catechol.
Thus, the formation of catechol and hydroquinone is the most
likely cause of benzene toxicity.
3 The
abbreviations
used
are:
SCE,
sister
chromatid
exchange;
hemagglutlnln; BrdUrd, bromodeoxyurldine.
ReceivedNovember,15, 1979; acceptedJanuary14, 1980.
PHA,
phyto
wasfirst dissolvedin completeculturemediumto givea 25 mM
concentration. Because benzene is highly volatile, it was dis
APRIL 1980
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research.
1189
K. Morimotoand S. Wolff
solved by mixing in complete medium in a capped test tube,
which was then immersed in an ultrasound cleaner. Just before
incubation, aliquots of these freshly made solutions were added
to the cultures to give the appropriate final concentration.
For SCE's, the standard error of the mean was determined
by the use of Poisson variances. The standard error of the
mean for the percentages of cells in Xl , X2, and X3+ divisions
was determinedby the useof binomialvariances.
RESULTS
When cells from untreated cultures were examined at inter
vals from 48 to 72 hr after initiation of incubation, it was found
that the relative numbers of Xl , X2, and X3+ cells shifted with
increasing culture times (Chart I b; Table 1). At 48 hr after
stimulation with PHA, 65.7% of the dividing lymphocytes were
dividing for the first time, 32.7% were dividing for the second
time, and only I .6% were dividing for the third time. At subse
quent fixation times, it was noted that the frequency of Xl
metaphases decreased continuously and that of the X3+ me
taphases increased. The relative frequency of X2 metaphases
peaked at 56 hr, when 59% of all metaphases observed were
cells that were dividing for the second time. By 72 hr, most of
the cells were dividing for a third time. At later sampling times,
an occasional fourth-division cell was found. These control
data are not unlike those previously reported for untreated
cells (6, 35, 38).
The data indicate that when G0 lymphocytes are stimulated
to divide by exposure to PHA, the resultant cultures contain
1S.C
w
U
10.0
a.
I
I
I
J
J
I
I
I
I
@S2
56
60
64
LU
0..
LU
‘I,
5.0
U
U)
@
80
60
40
20
@
68
72
CULTURETIME(hr;
cells that have divided a different number of times. The yield of
base-line SCE's found in second-division cells, however, is
independent of the culture time. Thus, cells that required 48 hr
from the beginning of the culture period to divide twice had the
same number of SCE's as cells that took up to 72 hr to reach
the second mitosis (Chart 1a; Table 1). Because the base-line
level of SCE's is dependent upon the amount of BrdUrd in the
medium (17, 39), the data indicate that the cell culture time
does not affect the sensitivity to BrdUnd as measured by the
induction of SCE's.
When the cells were exposed to benzene and its major
metabolites, it was found that catechol was the most cytotoxic
(Table 2) in that it, more than the others, delayed cell turnover
times (Chart 2) and induced SCE's (Chart 3). A clearly dose
dependent delay in cell turnover times was manifested as a
change in the distribution of Xl , X2, and X3+ mitoses in
cultures fixed at 72 hr after initiation. For instance, an exposure
to 1.6 X 10_6 M catechol did not change the distribution very
much from what it was in cultures not exposed to the chemical,
but an exposure to 4 x 1O@ M catechol gave 40% Xl , 49%
X2, and 11% X3+ metaphases (Chart 2), which corresponds
to the distribution seen in untreated cells at 54 hr (Chart 1). It
therefore appears that 4 x 10@ M catechol leads to an I 8-hr
delay in cell turnover times.
Benzene does not affect the relative proportions of cells
unless it is administered at very high concentrations. Even
then, treatment with 5 x 1O@ M benzene gives a distribution
at 72 hr that is approximately that seen in untreated cells at 64
hr, indicating only an 8-hr delay.
Phenol and hydroquinone are both more active than ben
zene, but less active than catechol.
The activity is also evident in the dose curves of SCE's
observed in second-division cells from 72-hr cultures (Chart 3).
Benzene did not increase SCE's in any of the concentrations
used. Phenol gave a slight increase only at the highest con
centration, whereas hydnoquinone and catechol gave marked
increases, with catechol being the more potent compound. The
data in Charts 2 and 3 and Table 2, when compared with the
data in Chart 1 and Table 1, lead to estimates of concentrations
of benzene and its metabolites required to induce either a
doubling in the number of SCE's, a 12-hr delay in cell cycle
times, on a 50% decrease in mitotic index (Table 3). The ratios
of these concentrations can be used as estimates of the relative
potency of the compounds.
DISCUSSION
Chart 1. The frequency of 5CE's (a) and the percentage of Xl , X2, and X3+
cells (b) after various times in culture. Bars, SE.
The results indicate that the metabolites of benzene, rather
1
lymphocytesCulture
ges of Xl, X2, and X3+ cells, and mitotic indices in unt
SCE's/ce!l,
percentaTable
fixed at different sampling timesreated
human
of cellsMitotic
time
No. of
x@X3+485CE's%
(hr)
index
(%)SCE's/cell
xi
52
501
463
±0308
6.17 ±0.29
65.7 ±2.7
46.7 ±2.9
32.7 ±2.7
50.7 ±2.9
56
60
64
68
3.3‘-a
72
584
548
472
483
5006.68
7.79 ±0.32
30.3 ±2.7
58.7 ±2.8
7.31 ±0.31
17.7 ±2.2
44.0 ±2.9
6.29 ±0.29
6.44 ±0.29
6.67 ±0.30
6.7 ±1.4
5.3 ±1.3
4.3 ±1.2
32.3 ±2.7
22.7 ±2.4
19.0 ±2.31.6
2.7
11.0
38.3
61.0
±0.7
±0.9
±1.8
±2.8
±2.7
72.0 ±2.6
76.7 ±2.50.8
1.1
1.4
1.9
2.6
3.1
Mean ±S.E.
1190
CANCER
RESEARCH
VOL. 41
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Effect of Benzene Metabo!ites on SCE and Cell Cycle
Table 2
SCE's/cell, percentages of 72-hrincubation
Xl, X2, and X3+ cells, and mitotic indices in humanlymphocytesafter
metabolitesConcentrawith benzene or one of its
% of cells
hon of
indextreated
chemicalsMitotic
X2X3+(%)Control
(M)
No. of SCE's
SCE's/cell
2.53.4Benzene4.0
356
7.12 ±0.38a
57 ±1.3
2.43.12.0
x i0@
2.73.31.0
x 10@
343
6.86 ±0.38
327
2.81.45.0
x i0@
336
2.80.62.5
X i03
307
growthPhenol8.0
x 10_2
No
6.54 ±0.36
6.72 ±0.37
6.14 ±0.35
2.73.44.0
X 10@6
314
2.73.82.0
X iO@
344
2.83.91.0
X i0@
381
1.90.95.0
X i0@
490
growthCatechol1.6
X i0'3
No
6.28
6.88
7.62
9.80
2.73.58.0
x 106
362
2.93.74.0
x l0@
321
X i0@
774
0.71.OxlO-3
2.0
x i0@
938b
NogrowthHydroquinone1.6
2.53.88.0
x 106
2.72.94.0
x 10_c
2.91.72.0
X i0'@
2.70.91.0
X iO@
growtha
x i0@
Mean
348
365
559
1071
No
Xl
18.3 ±2.576.0
±
2.7 ±0.9
19.3 ±2.278.0
8.0 ±1.6
15.3 ±2.1
8.0 ±1.6
23.7 ±2.568.3
20.3 ±2.364.3
33.3 ±2.758.7
±
±
±0.35
±0.37
±0.39
±0.44
8.7
6.0
9.7
29.0
±1.6
±1.4
±1.7
±2.6
22.3
25.7
27.3
57.3
±2.469.0
±2.568.3
±2.663.0
±2.913.7
±
7.24
6.42
15.48
40.78
±0.38
±0.36
±0.56
±1.33
7.3
11.7
38.3
74.8
±1.5
±1.9
±2.8
±2.5
23.3
32.3
50.0
24.8
±2.869.3
±2.756.0
±2.9
±2.511.7 0.4
±
±
±1.9
±0.41.4
6.96
7.30
11.18
21.42
±0.37
±0.38
±0.47
±0.65
4.7
11.0
21.0
24.3
±1.2
±1.8
±2.3
±2.5
20.0
20.3
29.3
42.4
±2.375.3
±2.368.7
±2.649.7
±2.933.3
±
±
±
±
±
±
±
±
±
± SE.
Only 23 cells were scored for SCE's because this concentration was highly toxic.
LU
0#)
aLU
Chart 2. The percentage of Xl , X2, and X3+ cells
in cultures exposed to benzene or one of its metabo
lites. Bars, SE.
u--,,' @
-/@ -
X2
X3+
r@i
@Xi
_______
I II
@.6 8.01 4.0 I 2.0
106
10-5
10@
l0-@
CONCENTRATION(M)
than benzene itself, are the cytotoxic substances. Of those
tested, catechol was the most potent in inducing both cell cycle
delays and SCE's. Because the base-line SCE level did not
vary between slow-growing and fast-growing control cells, it is
apparent that the increased yield of SCE's obtained with cat
echol is a direct effect of the chemical and not a reflection of
its merely changing cell turnover rates so that cells with differ
ing base-line yields would be sampled.
We have also noted that catechol inhibits the repair of
radiation-induced chromosome breaks much more efficiently
than does benzene.4
4 K.
Morimoto
and
S.
Wolff,
unpublished
data.
The results of previous ‘@
â‘.tional
€˜
and cytogenetic studies
with benzene and its me;dbolites have been quite variable,
depending in large part on the systems studied. For instance,
benzene itself is negative in the Ames Salmonella test, even
when applied with an S-9 metabolic activating system (7). In
addition, benzene only rarely induces real chromatid breaks
(23) in cultured human lymphocytes, although it does induce
numerous gaps in the chromosomes (16, 23). In animal exper
iments in vivo, where benzene is metabolized into phenolic
compounds, benzene treatment can lead to both chromatid
and chromosome deletions in bone marrow cells (9, 15, 28),
and phenol, which is mutagenic in Escherichia coli (8) and
Drosophila (13), has been found to induce chromatid breaks in
APRIL1980
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1980 American Association for Cancer Research.
K. Morimoto and S. Wolff
50
.
I
“@
1
I
I
I
I
quinone, which is a relatively active compound, could contnib
ute to the mutagenic and cytotoxic effects of benzene in the
body.
F
CATECHOL
40
LU
U
30
aLU
REFERENCES
I
/YDROQUINONE
20
U
U)
-
,@
PHENOL
10
@
-
*.-w@--@
1 —.‘#-l
I
0
â€1̃.6
10-6
-
@l
8.0'
10-5
I
4.0
I
I
‘2.0
I
I
5.0
10-s
CONCENTRATION(M)
Chart 3. The frequency
of SCE's in cells exposed to benzene or one of its
metabolites. The standard error of the mean is not shown because it is less than
6% of the mean at each point.
Table 3
Estimated
concentratio of benzene and its metabolites require to
induceEffectConcentration
cns
ytogenetic and cytotoxic effectsd
(M)Hydro
PhenolBenzeneSCE
Catechol
quinone
doubling
12-hr delay of cell cy
cle
50% reduction in mi
totic index3
x i05
2 x iO@
9 x 1O@
1 x iO@
5 x i0@
3 x iO@
4 x iO@
7 x i0@8
x iO@
spermatogonia and primary spermatocytes of mature mice (4).
Phenol, which is a known tumor-promoting agent (3), also
inhibits both DNA repair and replicative synthesis (29) and
inhibits the repair of radiation-induced chromosome breaks at
less than one-tenth the concentration required for benzene
(22—24).Although the metabolites of benzene can induce
chromatid aberrations in plants (19, 20, 31), phenol is not very
active in this respect (19, 20). Another metabolite, hydroquinone, does not induce forward mutations in Micrococcus py
ogenes. However, since hydnoquinone has been found to inhibit
the growth of DNA polymerase-deficient bacteria (2, 7), it is
possible that in bacteria hydnoquinone does indeed attack DNA
but that the damage is repairable.
A numberof studieshavebeencarriedoutonthe metabolism
and cytotoxicity of benzene (7, 32, 34, 36). For example,
metabolic studies showed that in 15 human subjects, of the
benzene originally retained, 62% was later eliminated in the
urine as phenol, 6.3% was eliminated as catechol, and 2.4%
was eliminated as hydroquinone (34, 36). Basically similar
results were reported in animal experiments (32). Although
there is a considerable amount of data on the mutagenic and
cytotoxic effects of phenol, which is the first major metabolite
of benzene, our data indicate that the mutagenic and cytotoxic
effects of benzene in vivo are more likely to be caused by
catechol, which is by far the most active in the induction of
SCE's and the inhibition of cell turnover rates in vitro. Nomi
yama (25), who studied hematopoietic effects of benzene me
tabolites in rats, has also suggested that when these sub
stances were administered at dose levels similar to the meta
bolic yields, the formation of catechol was the most likely cause
of chronic benzene toxicity. Our data also indicate that hydno
1192
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1193
Increase of Sister Chromatid Exchanges and Perturbations of
Cell Division Kinetics in Human Lymphocytes by Benzene
Metabolites
Kanehisa Morimoto and Sheldon Wolff
Cancer Res 1980;40:1189-1193.
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