Alcohol & Alcoholism Vol. 32, No. 2, pp. 145-152, 1997
ETHANOL INDUCES TRANSIENT ARREST OF CELL DIVISION
(G2 + M BLOCK) FOLLOWED BY GoA^ BLOCK: DOSE EFFECTS OF
SHORT- AND LONGER-TERM ETHANOL EXPOSURE ON CELL CYCLE
AND CELL FUNCTIONS
KEIKO MIKAMI*, TAKESHI HASEBA and YOUKICHI OHNO
Department of Legal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan
(Received 21 March 1996; m revised form 18 September 1996; accepted 27 September 1996)
Abstract — To study the cytophysiological effects of ethanol systematically, L929 cells, a fibroblastic
cell line derived from mouse connective tissue, were exposed to various concentrations of ethanol (12.5,
50, 100 and 200 mM) for short (3 and 6 h) and longer (24 or 26 h) durations. Ethartol-induced cellular
responses were analysed by a combination of the following assays: number of cells, amounts of DNA
and protein, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cell cycle.
Ethanol dose-dependently suppressed these cellular functions, except that 12.5 mM exposures for both 6
and 26 h increased the amount of protein in spite of almost no change in other cellular functions,
compared to the control. The most marked dose-dependency was observed in a reduction of formazan
product in an M i l assay after both 6 and 26 h exposures to ethanol, being independent of the number of
cells and probably reflecting dose-dependent depression of mitochondrial respiration. A G2 + M block in
the cell cycle, an inhibition of cell division, was induced after short-term exposures (3 and 6 h) to 100
and 200 mM ethanol, but the block was released before 24 h had passed. Alternatively, prolonged
exposures (24 h) to 50-200 mM ethanol induced a (VG! block, resulting in a decrease in the amount of
DNA below the control value. Moreover, the percentage of the S phase was decreased gradually and
dose-dependently throughout the 24 h exposure. Thus, high concentrations of ethanol (50, 100 and
200 mM) perturbed the cell cycle progression by causing both a transient G2 + M block (an inhibition of
mitosis) and a continuous Go/G| block, though the latter was masked by the G2 + M block during shortterm exposure. The cells seem finally to acquire some tolerance to ethanol so as to pass through mitosis,
but much less tolerance to pass through the checkpoint from the G, to the S phase, which results in a
decline in proliferation.
INTRODUCTION
Ethanol is a principal constituent of alcoholic
drinks and its acute and chronic effects have been
extensively investigated in various fields using
humans, animals and cultured cells. Cell cultures,
such as hepatocytes and astrocytes, have been
used to examine the effects of ethanol, and it has
been elucidated that exposures to ethanol
(20-217 mM) for several days suppressed cell
proliferation (Higgins, 1987; Cook et al., 1990a;
Adickes et al., 1990, 1993; Davies and Cox, 1991;
Devi et al., 1993) and the amount or synthesis of
DNA (Guerri et al., 1990; Snyder et al., 1992;
Adickes et al., 1993; Devi et al., 1993; Wimalasena, 1994). A variety of cell functions, such as
•Author to whom correspondence should be addressed.
cell integrity (Acosta et al., 1986; Devi et al.,
1993), protein synthesis (Acosta et al., 1986;
Kennedy and Mukerji, 1986a,i>; Guerri et al.,
1990; Adickes et al., 1993; Lokhorst and Druse,
\993a,b) and cell cycle (Higgins, 1987; Guerri et
al., 1990; Cook et al., 1990o,b; Cook and Keiner,
1991) were also suppressed by ethanol. The
reduction in cell proliferation rate by ethanol has
so far been attributed to an inhibition of DNA
synthesis and an accumulation of cells in the
Go^G] phase of the cell cycle. The duration of
ethanol exposure in these studies was, however,
mostly of the order of days. We previously
investigated how ethanol affected the cell functions of cultured neonatal myocardial cells, which
are non-proliferative, in a 24 h period and partially
succeeded in elucidating its chronotropic
(Mashimo, 1985) and morphological (Mikami et
al., 1990, 1993) effects, especially mitochondrial
145
© 1997 Medical Council on Alcoholism
146
K. MIKAMI el al.
alterations.
It is also important to clarify how a small
quantity of ethanol affects cells. Few investigators,
however, have attempted a comprehensive examination of various levels of ethanol ranging from
the physiologically detectable to the toxic.
L929 is a murine proliferative cell line like a
fibroblast, and the cell metabolizes ethanol much
less than the hepatocyte, because the cell does not
have a Class I alcohol dehydrogenase (ADH) (our
unpublished data). Class I ADH is a key enzyme
involved in ethanol metabolism due to its low Km
for ethanol and exists abundantly in the liver. We
selected the L929 cell as representative of general
somatic cells to examine the direct effects of
ethanol excluding those seconding to metabolism.
We tried to study comprehensively dose- and
time-dependent effects of ethanol on the cell
functions of L929 cells, with particular emphasis
on action appearing in the short time phase of the
investigation.
MATERIALS AND METHODS
an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay] were performed to check the linearity with the number of
cells.
Ethanol exposure
Cells cultured for about 17 h in MicroWell
Plates or Petri dishes were exposed to ethanol
(Wako, Osaka, Japan) by the addition of an
ethanol medium containing three times the desired
final concentration of ethanol (12.5, 50 and
200 raM for number of cells, amounts of DNA
and protein, and MTT assays, and 12.5, 50, 100
and 200 mM for cell cycle). Namely the volume of
the added medium was half that of the culturing
medium (50 (il for a MicroWell Plate, 1.5 ml for a
Petri dish). To the control culture was added a
medium containing water instead of ethanol. The
plates and dishes were covered with sheets of
sterilized sticky plate seal (Sumitomo Bakelite
Co., Tokyo, Japan) to prevent evaporation of the
ethanol. Then, the cells were exposed to ethanol
for 6 or 26 h. For the analysis of the cell cycle,
exposure periods of 3, 6 and 24 h were used.
Cell culture
NCTC Clone 929 (L929), a cell line derived
from mouse connective tissue, was obtained from
the Japanese Cancer Research Resources Bank
(JCRB, Tokyo, Japan). The L929 cells were
maintained in minimum essential medium with
Earle's salts (MEM, GIBCO, Grand Island, USA)
supplemented with 10% heat-inactivated horse
serum (GIBCO) in a humidified atmosphere of
5% CO 2 in air at 37°C. The cells were inoculated in
MicroWell Plates (96 wells, Nunclon, Nunc,
Roskilde, Denmark) at a standard concentration
of 6 x 103 cells/100 ul/well in order to examine the
effects of ethanol on cell proliferation and biochemical indices. The cells used for analysis of the
cell cycle were inoculated in Petri dishes (diameter 60 mm, Nunclon, Nunc) in 3 ml of the cell
suspension at a concentration of 1.2 x 105 cells/ml.
Determination of ethanol concentration
The ethanol concentration of the medium was
measured according to a previously reported
method using a Perkin-Elmer Head Space
Analyzer 8500 (Haseba et al., 1993). The sample
medium (100 (il) was transferred and sealed off in
a vial containing 0.5 ml of 0.1% «-propanol
(Wako) as an internal standard.
Number of cells
Cells in MicroWell Plates were photographed
through an optical microscope at the same
magnification and the number of cells appearing
in the photograph (one photograph per culture)
was counted. The total number counted was
> 1000 per photograph in the ethanol experiments
and >500 in linearity measurements (more than
five cultures for each point).
Linearity of measurements
A series of dilute (0.5 and 0.8 x standard
concentration) or concentrated (1.2 and 1.5 x standard concentration) samples of cells were inoculated into MicroWell Plates. The number of cells
was counted at 23 and 43 h, and biochemical
examinations [amounts of DNA and protein, and
Amount of DNA
The amount of DNA per well was determined
according to the method reported by Adams
(1990) with a slight modification. Briefly, cells
were gently washed once with Hanks' solution
buffered with 20 mM HEPES and incubated in
50 nl of 0.2% SDS in ETN solution (10 mM
ALCOHOL AND CELL CYCLE AND FUNCTION
EDTA, 10 mM Tris-HCl, 100 mM NaCl, pH 7.0)
at 37°C for 20 min to dissolve them. Then, the
solution was mixed with 2.45 ml Hl'N containing
lOOng/ml Hoechst 33258 (bis-benzimide trihydrochloride, Sigma, St Louis, MO, USA) plus
5 ng/ml RNase (Wako), and incubated at 37°C for
20 min in the dark. The fluorescence was measured
at 470 run with a fluorescence spectrophotometer
(FP-770F, Japan Spectroscopic Co., Tokyo, Japan)
using an excitation wavelength of 355 nm.
Amount of protein
The cells were gently washed once with Hanks'
solution and dried. The amount of protein per well
was determined by adding Coomassie protein
assay reagent (100 ul; Pierce, Rockford, USA)
(Bradford, 1976) to each well of a MicroWell
Plate. The plate was incubated at room temperature and shaken occasionally for > 15 min. Finally,
the optical density was measured with a microplate reader (Model 3550, Bio-Rad, Hercules,
USA) at a wavelength of 595 nm.
MTT assay
The assay using MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide, Sigma)
was performed according to the method of
Mosmann (1983) with a slight modification. The
MTT solution [5 mg/ml in calcium- and magnesium-free Dulbecco's phosphate-buffered saline
(PBS)] was added to a well 3h before the
cessation of the experiment. The volume was a
tenth of that of the culture medium. After a 3 h
incubation the supernatant was removed, and
100 nl of dimethylsulphoxide (DMSO, Sigma)
was then added immediately to each well, and the
plate was shaken to dissolve the formazan crystals.
The optical density of each well was measured
with a microplate reader (Bioreader FS-340,
Sigma Seiki Inc., Tokyo, Japan) using a test
wavelength of 540 nm and a reference wavelength
of 690 nm.
Analysis of cell cycle
The cells were gently washed once with cold
PBS and treated with 0.8 ml of Hanks' solution
containing 0.05% trypsin and 0.53 mM EDTA
(GIBCO) at 37°C for 5 min. The detached cells
were transferred to an ice-cooled glass centrifuge
tube containing 0.5 ml of horse serum, and
centrifuged at 4°C and 150 g for 5 min. The
147
cells were washed with cold PBS by centrifugation, and finally resuspended in 70% cold ethanol
and fixed overnight at 4°C.
The fixed cells were washed by centrifugation
(4°C, 300 g, 5 min) with cold PBS. The final cell
pellet was incubated with 0.5 ml of 2% RNase
(Wako)/PBS at 37°C for 10 min. After pipetting
with a Pasteur pipette, the suspension was filtered
through a nylon mesh sheet (300 mesh/inch) to
eliminate any cell aggregates. To the filtrate,
0.05 ml of 100 ug/ml propidium iodide (PI,
Sigma)/PBS was added. The Pi-stained suspension
was shielded from the light and stored on ice till
the time of measurement.
Flow cytometric measurements were performed
for ~ 10000-20000 cells/sample with a FACScan™ flow cytophotometer (Becton Dickinson,
San Jose, USA) equipped with a 488-nm argon
laser. To estimate the percentage of the cell
population in each phase of the cell cycle (Go/Gi,
S and G2 + M), an analysis of the histogram of the
relative DNA content was carried out with
CellFIT™ software (Becton Dickinson) employing
the SOBR (Sum of Broadened Rectangles) model.
Statistics
In each culture series, the values obtained from
the examinations (number of cells, amounts of
DNA and protein, and MTT assay) after ethanol
exposure (6 and 26 h) were converted to percentages of the control values, which were obtained
from ethanol-untreated cells after 6 h of exposure.
Most of the experiments were carried out in three
separate series (one series to determine the number
of cells) of more than triplicate cultures per series.
For analysis of the cell cycle, four separate series
of single culture per series were carried out.
Statistical analyses were performed using Student's Mest. Results were considered statistically
significant when P<0.05.
RESULTS
Ethanol exposure
It was confirmed by the growth curve of L929
cells (data not shown) that the cells were exposed
to ethanol during their logarithmic growth phase
from 17 to 43 h after inoculation. The growth
curve was obtained from cultures incubated for up
to 65 h after inoculation of the cells at the standard
K. M1KAMI et al.
148
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26 h
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6h
26 h
6h
26 h
Hours of Exposure to Ethanol
Fig. 1. Cytophysiological responses of L929 cells exposed continuously to ethanol.
Exposure to concentrations of ethanol of 0 ( • ) , 12.5 (D), 50 (J3) and 200 (•) mM was for 6 and 26 h. (a) The number of
cells; (b) the amount of DNA; (c) the amount of protein; (d) the amount of formazan produced in the M i l assay. The
values are percentages of the mean of the control (0 mM) of each assay at 6 h after the start of exposure [means ± SD of 5
(a), >9 (b), >10 (c and d) values]. •P<0.01, |/ > <0.001 compared with the corresponding time control group.
concentration, the same concentration as in the
ethanol-exposure experiments. The doubling time
of L929 cells in the logarithmic phase was
estimated to be ~ 15 h.
When cells were incubated for 26 h in a
medium containing ethanol, almost no decline in
the ethanol concentration of the medium was
detected by gas chromatography, if the plates or
dishes were covered with sticky plate seals.
Allowing for measurement error, the decrease of
200 mM ethanol in the medium was thus <3% of
the original level.
Number of cells
A continuous 6 h exposure to ethanol at
concentrations of 12.5 and 50 mM did not induce
changes in the number of cells per well compared
to the control (0 mM ethanol), though there was a
slight decrease (6%) at a concentration of 200 mM
which was not significant (Fig. la). Significant
reduction below the control value occurred only
after a 26 h exposure at a concentration of
200 mM. The number of cells was 27% below
the control level.
Amount of DNA
The amount of DNA in ethanol-untreated cells
exhibited a one-to-one correspondence with the
number of cells (data not shown), the slope of the
curve being ~ 1.
After a continuous 6 h exposure to ethanol, the
amount of DNA did not change compared to the
control level regardless of the ethanol concentration used (Fig. lb). However, a 26 h exposure to
50 or 200 mM ethanol reduced the amount of
DNA by 11% and 34%, respectively, compared to
the control (Fig. lb).
Amount of protein
The amount of protein in ethanol-untreated cells
ALCOHOL AND CELL CYCLE AND FUNCTION
was approximately proportional to the number of
cells in the range of numbers used in these
experiments, though the correlation was slightly
parabolic (data not shown).
The amount of protein in cells exposed to
12.5 mM ethanol was 13% above the control at
6 h, and 9% above at 26 h (Fig. lc). However, the
amount of protein after 26 h of exposure to
200 mM ethanol was 11% below the control.
MTT assay
The amount of formazan produced in ethanoluntreated cells in the MTT assay was approximately proportional to the number of cells in the
range of numbers used in these experiments,
though showing slightly parabolic correlation
(data not shown).
Continuous exposure to ethanol decreased the
amount of formazan produced in the MTT assay
below the control dose-dependently after both 6
and 26 h exposures: exposures to 12.5, 50 and
200 mM ethanol decreased it at 6 h by 9%, 23%
and 59%, respectively, and at 26 h by 3%, 21%
and 67%, respectively (Fig. Id). Even after a
short-term (6 h) exposure, the amount of formazan
decreased markedly and dose-dependently, while
the numbers of cells were almost the same in all
examined concentrations of ethanol at that time
(Fig. la).
3
6
Hours of Exposure to Ethanol
24
Cell cycle
At 0 h the percentages of the Go/G^ S and Fig. 2. Changes in the percentage of phase populations in
G2 + M phases were 36.9%, 49.0% and 14.1%, the cell cycle of L929 cells exposed continuously to
ethanol.
respectively. Short-term (3 h) exposure to 100 and Exposure to concentrations of ethanol of 0 ( • ) , 12.5 (E3)>
200 mM ethanol increased the percentage of the 50 (J3), 100 (•) and 200 (•) mM was for 3, 6 and 24 h. (a)
G2 + M phase (a G2 + M-phase block) (Fig. 2c) and Go/G,, (b) S and (c) G2 + M. Values are means ± SD of four
decreased that of the S phase (Fig. 2b) in the cell separate experiments. */'<0.05) **P<Q.0\, t^<0.001,
the corresponding time control
cycle compared to the control (the S phase after a t f < 0.0001 compared with
group.
100 mM exposure, P = 0.056 vs control), but
scarcely changed that of the Go/G] phase (Fig.
2a). A 6h exposure to 50-200mM ethanol also but produced a dose-dependent block in the G(/Gj
induced a G2 + M block accompanied by a dose- phase, that is, an increase in the GQIG\ phase
dependent increase in the G2 + M phase (for accompanied by decreases in the S and G2 + M
50 mM exposure, P = 0.051 vs control) (Fig. 2c) phases compared to the control (Fig. 2a, b, c,
and a dose-dependent decrease in the S phase respectively).
compared to the control (Fig. 2b). At this time, the
percentage of the GQ/GI phase showed a tendency
DISCUSSION
to be increased, but statistical significance was
shown only after 100 mM exposure (Fig. 2a).
The present study using L929 cells has demonProlonged (24 h) exposure to 50-200 mM ethanol strated the suppressive and dose-dependent action
released the cell accumulation in the G2 + M phase, of ethanol (12.5-200 mM) on DNA content,
150
K. MIKAMI et al.
formazan production (Fig. 1) and cell cycle (Fig.
2). However, cell proliferation was suppressed
only after a 26 h exposure to a high dose (Fig. la);
moreover, the protein content was increased at a
low dose (Fig. lc). No decline in proliferation in
12.5 and 50 mM ethanol at 26 h (Fig. la) was
observed and this is probably attributable to the
brevity of the exposure; the number of cells might
decrease after another day, at least for 50 mM
exposure. This presumption is derived from the
decreases in the DNA content at 26 h (Fig. lb) and
in the percentage of the S phase in the cell cycle at
6 h and thereafter (Fig. 2b). Accordingly, the
amount of DNA and the percentage of the S phase
are useful and predictive for examination of the
effect of a chemical substance on cell proliferation
in a shorter period, compared with the number of
cells.
The low dose of ethanol (12.5 mM) increased
the amount of protein after both 6 and 26 h
exposures by ~ 10% above controls (Fig. lc), in
spite of no changes in the number of cells (Fig.
la), the amount of DNA (Fig. lb) and the cell
cycle (Fig. 2). Concerning the effects of ethanol
on protein synthesis, consistent results have not
been obtained so far. Many investigators have
reported a decrease in [3H]leucine incorporation
(Guerri et al., 1990; Adickes et al., 1993) or in
protein content (Acosta et al., 1986; Kennedy and
Mukerji, 1986a; Guerri et al., 1990; Lokhorst and
Druse, 1993a,/?) by various cultured murine cells
in response to 33—120 mM ethanol. However,
some have reported an increase in protein content
(Acosta et al., 1986; Kennedy and Mukerji,
1986a) or in the incorporation of amino acids
(Heitman et al., 1987; Snyder et al., 1992) upon
exposure to low (11-37 mM) as well as high (85
and 200 mM) concentrations of ethanol. Kennedy
and Mukerji (1986a) demonstrated that 11 mM
ethanol increased protein, RNA and DNA content
per culture dish of mouse astrocytes after 11 days,
though the protein/DNA ratio was decreased. The
protein content was influenced by the timing of
ethanol exposure relative to critical events of
astrogliogenesis (Kennedy and Mukerji, 1986fc).
These findings together with ours suggest that
ethanol may stimulate protein synthesis, or
decrease protein degradation or secretion in the
cultured cells under certain conditions, though its
mechanism remains to be elucidated.
In the M i l assay, yellow water-soluble MTT is
transformed into dark blue water-insoluble formazan by succinate dehydrogenase (Slater et al.,
1963), a key enzyme involved in mitochondrial
respiration. And so, the present study might
indicate that ethanol suppressed mitochondrial
respiratory function even at low concentrations
(Fig. Id). Many investigators have reported
similar suppressive effects of ethanol on the
mitochondrial respiration of hepatocytes (Cederbaum and Rubin, 1975; Thayer and Rubin, 1979;
Spach and Cunningham, 1987; Montgomery et al.,
1987; Devi et al., 1994). The latter authors
discovered a 23% reduction in succinate dehydrogenase activity of cultured fetal rat hepatocytes
exposed to 54 mM ethanol for 24 h. The reduction
was comparable to that in formazan produced in
our MTT assay (50 mM, 26 h; Fig. Id) and also to
that in an MTT assay of fetal rat hepatocytes,
which cells showed no decline in proliferation at
that time (43 mM, 24 h; Devi et al., 1993).
We found that 100 and 200 mM ethanol
inhibited cell division after 3 and 6h exposures,
as shown by a G2+M block (Fig. 2) with almost
no effect on the number of cells (Fig. la). This
finding is attributable to our focus on short-term
exposure. The cell accumulation in the G2+M
phase had been released at 24 h (Fig. 2c). There
was a possibility that the G 2 +M block still
occurred at 24 h, but did not manifest itself as an
accumulation in the G2 + M phase due to the
decrease in cycling cells, as shown by the
decreases in the S-phase cells after 24 h exposure
to high concentrations of ethanol (Fig. 2b). It is,
however, likely that the G2 + M block was released
before 24 h had passed, because the ratios of the
cell population in the G2 + M phase to that in the S
phase were almost the same at 24 h regardless of
the ethanol dose.
One possible mechanism of the G 2 +M block is
that ethanol inhibits the construction of the cell
division machinery by increasing the fluidity of
cell membrane (Taraschi and Rubin, 1985) and/or
by inhibiting the assembly of cytoskeleton
(Matsuda et al, 1979; Worman, 1990), thereby
preventing cells from preparing for cell division.
The G2 + M block had been released at 24 h,
probably because the cells had by then acquired
some degree of tolerance to ethanol in the
membranes and/or cytoskeleton (Taraschi and
Rubin, 1985; Mikami et al., 1990).
On the other hand, it is said that cells exposed to
ALCOHOL AND CELL CYCLE AND FUNCTION
some chemical substances arrest in the G2 phase to
repair damaged DNA, thus delaying progress into
the M phase. An additional possible mechanism of
the G2 + M block seems an involvement of
perturbed G2 cyclin kinetics (our unpublished
data). The G2 cyclin is a key protein involved in
the cell cycle progression through the G2 and M
phases.
Apoptosis was induced in ethanol-exposed
chick embryos, an in-vivo model of fetal alcohol
syndrome (Cartwright and Smith, 1995); in the
present study, we did not observe any sub-peaks
before the G^Gi peak in the DNA histogram (data
not shown). The sub-peak usually indicates
apoptosed cells. There is a possibility that cells
vulnerable to ethanol, such as dividing cells, fail to
repair damaged DNA during the G2 + M arrest and
fall into apoptotic death.
After 24 h exposures to 50, 100 and 200 mM
ethanol, a Go/Gj block was observed (Fig. 2).
Several investigators elucidated a similar Go/G,
block using various cultured cells exposed to
60-200 mM ethanol for 6 h to 4 days (Higgins,
1987; Cook et al., l990a,b; Guerri et al., 1990;
Cook and Keiner, 1991). A possible mechanism of
the Go/G] block due to ethanol may be associated
with the perturbation of cell cycle kinetics
including Gi cyclins which control passage
through the restriction point.
As far as the percentage of the GQIG\ -phase
population is concerned, the G^G] block did not
seem to occur before 24 h (Fig. 2a), though the
increase in the percentage of the G^G]-phase cells
was only observed after a 6 h exposure to 100 mM
ethanol. However, the percentage of the S-phase
cells had already started to decrease at 3 h in
200 mM ethanol and at 6 h in 50 and 100 mM
ethanol (Fig. 2b). That is, a Go/Gi block might
have already started at 3 h (200 mM) or at 6 h (50
and 100 mM), but was masked by the G2 + M
block, which occurred at the same time. Therefore,
the apparent percentage of the Go/G ]-phase cells
seemed not to change at 3 and 6 h, except for the
100 mM exposure. After a 24 h exposure, the
G2 + M block had been released and the Go/Gi
block was unmasked to manifest itself as a dosedependent increase in the percentage of the Go/G r
phase cells (Fig. 2a).
In conclusion, high concentrations of ethanol
(50, 100 and 200 mM) perturbed the cell cycle
progression by causing both a transient G2 + M
151
block (an inhibition of mitosis) and a continuous
Go/G! block (an inhibition of DNA synthesis),
though the latter was masked by the G2 + M block
during a short-term exposure. The cells seem
finally to acquire some tolerance to ethanol so as
to pass through mitosis, but much less tolerance to
pass through the checkpoint from the Gj to the S
phase, which results in a decline in cell proliferation.
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