Effects of Acetone and Phenol on Established Cell Lines Cultivated in Vitro
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Effects of Acetone and Phenol on Established
Cell Lines Cultivated in Vitro*
DONALDM. PACEANDALICEELLIOTT!
(Institute for Cellular Research, Department of Physiology, University of Nebraska, Lincoln, Nebraska)
SUMMARY
Two substances, acetone and phenol, common constituents of cigarette smoke,
have been studied with reference to their short-term effects on mouse fibroblast and
human skin cells cultivated in vitro.
Concentrations of acetone, 10.0 mg/ml and above, are toxic to both types of cells.
The skin cells are more sensitive to acetone effects than the fibroblast cells. Although
5.0 mg acetone/ml is not toxic to fibroblasts within a 10-day period, it is noticeably so
to the skin cells.
Phenol in concentrations of 0.2 mg/ml and above is very toxic and kills the cells
within an hour or 2. In concentrations of 0.1 and 0.16 mg/ml some of the Strain L
cells are still living after 10 days, but there is a definite inhibitory effect indicated;
only 15 per cent survived at this time.
The skin cells appear to be less sensitive to the effects of phenol than fibroblast cells,
as indicated by those surviving in 0.16 mg/ml at the end of the experiment—15 per
cent of the Strain L cells, 57 per cent of the skin cells.
This investigation is a continuation of studies inhibitory effect within 1 minute on ciliary activity.
made on the effects of certain tobacco constituents This was followed within 16 minutes by a slight
on tissue cells grown in vitro (13) and concerns accelerating effect. Knowledge concerning the
short-term experiments made with acetone and germicidal or antiseptic effects of phenol upon
phenol. various types of living cells is common. Recent
Phenolic constituents of cigarette smoke have studies suggest that they may be mitotic poi
been reported by several investigators, and anal sons (1).
ysis reveals varying quantities of these substances Since the high toxicity of phenol has been
in different types of tobacco (6, 15). recognized for years, and since investigations sug
Some workers (2, 3, 4, 16) have reported pro gest its possible role as a carcinogen or co-carcino
duction of papillomas and carcinomas on the skin gen, it seemed desirable to determine its effects
of mice treated with phenol and related substances, upon cells in vitro.
but others (10, 17) did not observe this. Buyske et al. (5) reported the presence of ace
Roe et al. (16) noted that, although the phenolic tone in cigarette smoke, the quantity varying
fraction of cigarette smoke alone applied to skin with different types of tobacco.
of mice did not produce tumors, if the same were Little appears in the literature concerning the
applied along with 9,10-dimethyl-l,2-benzanthra- effects of acetone upon cells grown in vitro. Smith
cene, tumors appeared. Falk, Tremer, and Kotin et al. (18), in determining cytotoxicity of anti
(9) have shown that phenol (1-2.1 mg.) has a slight biotics, chemicals, and solvents in respect to tissue
* These investigations were supported by grants from the cells, found the dosage of acetone necessary to
Tobacco Industry Research Committee and the Cooper Foun produce 50 per cent inhibition (ID6o) to be 5,000
dation through the University of Nebraska Foundation (Lin mg/ml. Acetone has been employed as a solvent
coln, Nebraska). for carcinogenic hydrocarbons used in studying
t Present address: Department of Biology, Hope College, appearance of malignancy in tissue cells in vitro.
Holland, Michigan.
However, carcinogenic capacity has not been re
Received for publication August 18, 1961. ported for acetone (10, 17).
107
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Since acetone occurs in a relatively high con maximum population and extent of proliferation
centration and is used extensively as a solvent for Strain L cells exposed to 2.0 mg and 5.0 mg
in investigations upon carcinogenicity of certain acetone/ml were slightly, though not significantly,
substances, an investigation of acetone was also below those of the controls (Table 1, Exp. A).
undertaken to determine its toxicity and effects In the cells exposed to 10.0 mg/ml, there was a
upon the morphology and proliferation of cells definite inhibition—the maximum population was
in vitro. not quite one-half that of the controls. After 8
days' exposure, 99 per cent of the cells in the
MATERIALS AND METHODS control and in the 2.0-mg. cultures, 97 per cent
Strain L cells (NCTC clone 929) and human of those in the 5.0-mg., and 81 per cent of those
skin cells (NCTC clone 2414) were used in these in the 10.0-mg. cultures, were still alive.
experiments. The nutrient medium employed for Recognizable morphological changes were evi
culturing was NCTC 109, supplemented with 10 dent in the cells in 10.0 mg. acetone. These changes
per cent horse serum (7, 8, 11). Prodecures for were not well defined until 7 or 8 days, at which
maintaining and replicating both stock and ex time the cells appeared larger and more granular
perimental cultures were the same as those de than normal. At the termination of the experiment
scribed by Pace and Elliott (13). (14 days) another check on viability was made.
The replicate method of culture as modified At this time the controls, the cells in 2.0 mg. and
by Pace and Aftonomos (12) was employed, and in 5.0 mg. acetone were approximately 100 per
approximately every 48 hours control and experi cent viable; the 10.0-mg. cells, 92 per cent. In
mental cultures in Carrel flasks (1) were randomly other words, there were more viable cells in 10.0
selected and sacrificed for counting, by means mg. acetone after 14 days than at 8 days.
of the Coulter Electronic Counter, Model A. Dur In the second experiment (Experiment B) there
ing the first 48-hour period the cells were allowed were approximately twice the number of cells
to "adjust" to the medium, following which in at the time they were subjected to the acetone
terval they were subjected to the conditions of than in the first experiment. There was very little,
the experiment. From time to time viability of the if any, effect on the cells in 2.0 mg. and 5.0 mg.
cells was ascertained by means of erythrosin B, acetone. However, the cells in 10.0 mg. never
as recommended by Phillips and Terryberry (14). reached as great a maximum population as those
For this purpose, three different samples were in lower concentrations. The cells in the control
obtained from each flask for observation. In each and 5.0-mg. cultures reached a maximum popula
sample, 400 cells were examined in order to record tion about triple that of the cells in 10.0 mg.
the number of living cells as compared with the The maximum reached by the cells in 2.0 mg.
number of dead cells. acetone was not actually known for this experi
The desired concentrations of acetone or phenol ment, since the cells began to slough off the glass
for the experimental solutions were prepared (a) by before the 10th day. They may have actually
adding the necessary quantity of acetone directly reached a greater maximum than in the other
to the culture medium, prepared in the same tests. From direct observations it appeared so.
manner as acetaldehyde solutions (13); or (6) in In a third experiment with Strain L cells (Ex
the case of phenol, by first diluting it in balanced periment C) the results of the two previous ex
salt solution before adding it to the culture medi periments were substantiated. Acetone had very
um. The latter step was taken to prevent the little, if any, effect on these cells in concentrations
phenol from coagulating the proteins present in of 5 mg/ml or less, but when the cells were exposed
the medium. to 10.0 mg/ml growth was retarded.
Human skin cells.—Inthe first experiment, the
RESULTS results were somewhat erratic, owing to the fact
ACETONE that rather extensive sloughing took place in all
In preliminary experiments, in which prolifera cultures within 5 or 6 days after the experiment
tion of the cells was observed in several rather was started (Table 1, Experiment D). However,
low concentrations of acetone—0.02, 0.2, 0.5, and even in this short time, proliferation of the cells
1.0 mg/ml—no significant results, favorable or exposed to 5.0 mg acetone/ml was inhibited, and
otherwise, were observed. Consequently, higher those exposed to 10.0 mg/ml were markedly in
concentrations, consisting of 2.0, 5.0, and 10.0 hibited. The cells in 2.0 mg. acetone proliferated
mg acetone/ml, were used. Three different experi at about the same rate as did those of the controls.
ments were conducted for each cell line. In the second experiment (E) of this series,
Strain L cells.—In the first experiment, the maximum populations in control, 2.0-mg., and
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and Phenol Effects on Cells 109
5.0-mg. cultures were not attained until 13 days Even after 2 days' exposure, many of the cells
after initial exposure. The average total number in the 10.0-mg. cultures were rounded and slough
of cells in the controls and 2.0-mg. cultures was ing. The sheet of cells that formed on the floor
approximately equal; proliferation in those exposed of the flask was never as densely packed as in the
to 5.0 mg. acetone was inhibited. After 3 days, the controls, and much cellular debris was present.
cells exposed to 10.0 mg. acetone revealed an "un The attached cells, at first, closely resembled
healthy" appearance; many cells were rounded, those of the controls; later, however, they ap
granular, and considerable cellular debris was pres peared more granular and larger. After 11 days
ent in the cultures. This rather poor condition the difference in numbers was very pronounced,
of the cells continued throughout the experiment and only 31 per cent of the cells in the 10.0-mg.
at this concentration. On about the 10th day of cultures were viable as compared with over 90
exposure the cells in 5.0 mg. acetone appeared per cent of those in the controls.
similar to those in the 10.0 mg. Those in 2.0
mg. acetone resembled the cells in the controls. PHENOL
In Experiment F (Table 1), only the 10.0 mg As in the previous experiments, Strain L and
acetone/ml was used to compare with the control. human skin cells were used for these tests. To
TABLE 1
GROWTHOFSTRAINL (NCTC 929) ANDHUMANSKINCELLS(NCTC 2414)
IN MEDIUMCONTAININGDIFFERENTCONCENTRATIONS
OFACETONE
Initial cell number per ml. in A—363,000;B—368,000;C—437,000;D—450,000;E- 479,000; F—348,000.Each figure repre-
sents an average count for three cultures selected at random.
CENTINCREASE
TRATION
OFACETONE(MO
EXPERIMENTCONCEN NO. CELLS IN MILLIONS PER ML.PER ATMAXIMUM
/ML)Av.
Strain L (NCTC 9a9)
day.423aaa.880"au.893ttuu7tli
day3.510a. day1.2703.5941.14412th
day4.1663.5803.7892.11014th
day3.6893.8593.9681
day5 day5
Ac0251002510025101st
749I8th
9513.0381.68910th
7985.3095.6023.91421st
6925.3755.4003.72588581283839932325633310354950
Human skin cells (NCTC 2414)
day.795"KU.491"uu.6593d
day1 day2.8872.206Util day3.0781.91913th day3.1922.9692.082.51081894775505053244367235
4851.5051
JJgF02510025100101st
1. 72.8529th
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obtain concentrations of phenol that could be used an hour or 2, and all cells, Strain L and skin,
experimentally, so that its effects on cells culti exposed to concentrations of 0.5 mg. and above
vated in vitro could be tested, preliminary experi were dead within 24 hours. Those in 0.2 mg
ments were carried out with concentrations of phenol/ml were dead within 48-72 hours.
phenol varying from 10.0 to 0.2 mg/ml, all of Strain L cells.—Since the cells proved to be
which were toxic. This effect was apparent within affected in concentrations of phenol as low as
TABLE 2
GROWTH
OFSTRAIN
L (NCTC929)ANDHUMANSKINCELLS
(NCTC2414)
INMEDIUMCONTAINING
DIFFERENTCONCENTRATIONS
OFPHENOL
Initial cell number per ml. in A—278,000;B—315,000; C—328,000; D—315,000; E—357,000; F—374,000; G—314,000;
H—848,000.Each figure represents an average count for three cultures selected at random.
CENTINCREASE
EXPERIMENTCONCEN-TBATIONOFPHENOL(HO/ML)Av. NO. TELLS IS MILLIONS PEU ML.PER ATMAXIMUM
Strain L cells (NCTC 9ÃŽ9)
day2.5221
day.390a.uu.624UIItÃ-a.700Uuu.570ua6th day3.2803.3943.1302.8512.5693
day3.686.885.788lOth day3.4814.1143.4072.5964.2173.4132.768793955774566426444402
BC\sD00.0250.050.1000.0250.050.100.1600.050.100.1600.100.161st
1982
6302.4871.9489th
210.700Tth
Human skin cells (NCTC 24H)
day1.1381.2471.166.9096th day2.1652.3822.3231.7961.9239th
day1.5861.7261.7331.6697th
day.236UUa.740atiu**.522ilUU.658aa3d day2.8821.97910th
day2.7502 day3.2902.5082.2931.0651.02342819322221414316053038033
6511.247llth
FH00.0250.050.1000.0250.050.100.1600.050.100.1600.100.161st
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and Phenol Effects on Cells 111
0.2 mg/ml, tests were carried out in which they reveal that detectable toxic effects of phenol ap
were subjected to lower concentrations (Table peared in much lower concentrations than they
2). As may be noted, 0.16 mg phenol/ml has a did with acetone.
definite inhibitory effect on proliferation in Strain All the cells of both Strain L and skin exposed
L cells, although they were still living after 10 to 0.5 mg. acetone and above were dead within
days. The same is true for cells in 0.1 mg/ml, 24 hours. Both were markedly affected within
although the effect is not nearly so noticeable. 24 hours after exposure to 0.2 mg/ml, and within
In fact, in the latter condition no morphological 72 hours all were dead. It appears as though this
alterations could be noted by ordinary microscopic concentration is near the lower limits that will
examination. However, quite detectable differ produce a rather prompt action—i.e., within 24-48
ences were evident in the cells in 0.16 mg.— hours.
they appeared large and vacuolated, soon became In concentrations of 0.1 mg and 0.16 mg phenol/
rounded, and later disintegrated. After 10 days' ml, both types of cells survived for about 10 days,
exposure only 15 per cent of the cells in both although proliferation was definitely inhibited.
0.1 mg. and 0.16 mg. phenol were viable—many When viability of Strain L cells was checked after
of them had disintegrated, as indicated by the 7 days, it was found that about 95 per cent of the
large amount of cellular debris in these cultures. cells in the 0.1-mg. and 0.16-mg. concentrations
Although a concentration of 0.05 mg/ml apparent were alive. The number of cells, however, were
ly had no effect on the cells, 0.025 mg. proved somewhat less than those of the controls and those
to be favorable—proliferation at this concentra in lower concentrations. After being subjected
tion was actually greater than for the cells in the to phenol for a period of 10 days, 99 per cent
control cultures. of the controls, but only about 15 per cent of those
Human skin cells.—Although an injurious effect in the 0.1- and 0.16-mg. groups, were viable.
was observed on the skin cells in 0.2 mg phenol/ml, On the other hand, in human skin cells, 90 per
they proved to be less sensitive to phenol at lower cent of the controls, 66 per cent of the cells in
concentrations than did the Strain L cells. The 0.05 mg., and 57 per cent of those in 0.1 mg.
results presented in Table 2 indicate that concen and 0.16 mg. phenol were alive.
trations of 0.025 or 0.05 mg/ml have little, if Both cell types seemed to be relatively unaf
any, effect (Experiments E and F). fected by a concentration of 0.025 mg phenol/ml.
The cells exposed to 0.1 mg. or 0.16 mg. phenol However, from observations made in these investi
never attained a maximum population as great gations, it appears that concentrations between
as that in the controls. It is interesting to note, 0.05 mg. and 0.1 mg. might be capable of produc
however, that those in 0.16 mg. reached a maxi ing adverse effects, if the period of exposure is
mum within 6 days—more quickly than the others. sufficiently lengthy.
At the termination of the experiment, however Primarily because of its protein-coagulating
(10th day), the cells in the control cultures were capacity, phenol has long been used as a germicidal
much more numerous than in either 0.1 or 0.16 agent. Perhaps the deleterious effects upon the
mg phenol/ml. cells might be explained in part by alteration of
From time to time, a check was made on via the protein in the medium or within the cells,
bility, with erythrosin B. After 7 days (Experiment or, since, phenol also acts as a mitotic poison,
F), 89 per cent of the cells in the controls, 66 it could retard cell division and proliferation by
per cent of those in 0.05 mg phenol/ml, but only this means.
57 per cent of those in both 0.1 mg. and 0.16 Acetone proved to be less toxic than phenol.
mg. were viable. A concentration of 2.0 mg/ml, or even 5.0 mg/ml,
As in the case of the Strain L cells, several days' appeared to have negligible, if any, effect upon
exposure were required to produce morphological either Strain L cells or human skin cells. There
changes in the cells subjected to 0.16 mg phenol/ was, however, a noticeable difference in the effects
ml. The cells in 0.16 mg. were affected more than of acetone on fibroblasts as compared with skin
any of the others—they were rounding up and cells. The skin cells were more sensitive to the
detaching within a few days after exposure. Those acetone. In all the experiments completed, the
that remained attached were very granular in human skin cells in 5.0 mg acetone/ml, or above,
appearance. were definitely inhibited, but those in 2.0 mg.
were apparently not affected.
DISCUSSION Strain L cells, however, seemed to be relatively
In these short-term experiments, in which phe unaffected by 5.0 mg/ml. Ten mg acetone/ml
nol and acetone were investigated, the results was definitely harmful to both types of cells.
Downloaded from cancerres.aacrjournals.org on March 13, 2021. © 1962 American Association for Cancer Research.112 Cancer Research Vol. 22, January 1962
It is interesting to note that, although 10.0 6. COMMINS,B. T., and LINDSEY,A. J. Some Phenolic
mg acetone/ml was harmful and that maximum Constituents of Cigarette Smoke. Brit. J. Cancer, 10:
504-6, 1956.
population was only about one-half that of the
7. EVANS,V. J.; BRYANT,J. C.; FIORAMONTI, M. C.; MC
controls, 81 per cent of the cells were viable after QUILKEN,W. T.; SANFORD,K. K.; and EARLE, W. R.
8 days (as compared with 99 per cent for the con Studies of Nutrient Media for Tissue Cells in Vitro.
trols). Of even greater interest is the fact that after I. A Protein-free Chemically Defined Medium for Cultiva
14 days' exposure, the cells of the control cultures tion of Strain L Cells. Cancer Research, 16:77-86, 1956.
were still 99 per cent viable and those in 10.0 8. EVANS,V. J.; BRYANT,J. C.; McQuiLKEN, W. T.; FIORA
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Downloaded from cancerres.aacrjournals.org on March 13, 2021. © 1962 American Association for Cancer Research.Effects of Acetone and Phenol on Established Cell Lines
Cultivated in Vitro
Donald M. Pace and Alice Elliott
Cancer Res 1962;22:107-112.
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