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The cytotoxicity of some organic solvents
ENVIRONMENTAL
RESEARCH
The
7, 183-192
( 1974)
Cytotoxicity B.
of Some
HOLMBERG’
AND
Organic T.
Solvents
MALMFOR~
Section of Occupational Toxicology, Department of Occupational National Board of Occupational Safety and Health, Stockholm, Received
August
Health, Sweden
10, 1973
The cytotoxicity of 33 organic solvents was determined using Ehrlich-Landschutz diploid (ELD) ascites tumour cells during short-time in vitro incubations. Among the organic solvents studied, tetralin, crotonic aldehyde, formaldehyde, and acrolein were highly toxic to ELD cells. Benzyl chloride, carbon tetrachloride, methyl ethyl ketone, and 1,1,1,2-tetrachloroethane were found to be moderately toxic. Tumour cells treated with crotonic aldehyde were reinoculated into mice. The growth capacity of the inoculated population was shown to be dependent on the frequency of surviving cells after treatment with the organic solvents indicating that true cell mortality was studied. INTRODUCTION
Organic solvents are widely used in industrial work. The cytotoxicity of most of the solvents is regarded to be low according to the absence of effects on the liver of experimental animals. Only for a few solvents, namely, carbon disulfide, carbon tetrachloride, and chloroform hepatotoxic effects have been established. In vitro experiments for evaluating the cytotoxicity of chemicals occurring in the environment have been performed for air pollutants (Rounds and Bils, 1965), pesticides (Litterst and Lichtenstein, 1971; Kolberg et aZ., 1971), solvents (Smith et al., 1959; Franceschini, 1964; Pace and Elliott, 1962) as well as for anesthetic agents (Corssen and Sweet, 1966; Corssen and Sweet, 1967; Rosenberg and Wahlstriim, 1971). In these studies the effects of added agents on cell growth and/or cell behaviour were observed. The present report describes the acute cytotoxicity of organic solvents as studied in vitro with Ehrlich-Landschiitz diploid (ELD) ascites tumour cells. A dye exclusion test was used for estimating the frequency of cells in a stage of irreversible cell injury leading to cell death. Such acute cytotoxicity studies can throw some light on the mechanism underlying the biologica effects of organic solvents. MATERIALS
AND
METHODS
Ehrlich-Landschiitz (ELD) diploid ascites tumour cells were propagated in outbred albino NMRI mice. Tumour cell populations were withdrawn not earlier than 1 week after the inoculation. Cells from several mice were pooled and centrifuged at 1566g for 10 min. The supernatant ascites fluid was discarded. ’ National Board of Occupational Safety Section of Occupational Toxicology, P. 0. ‘Present address: Astra Pharmaceutical Sweden.
and Health, Department of Occupational Health, Box S-10026 Stockholm 34, Sweden. AB, Toxicology Laboratories, S-151 85 .%dertllje, 183
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
acetate
50 100
50 100
chloride
Benzyl
iso-Butyl
3.0 5.0
100 200
alcohol
Benzyl
50 100
0 1.0
50 100
Benzene
Bromochloromethane
1.5 2.0
50 100
Amylacetate
2.0 1.0
7.0 6.0
5.0 4.5
2.0 1.0
50 100
alcohol
Ally1
0 1.0 1.5 1.0 1.5 2.5
0.0
1 5 10 25 50 100
ppm
PERCENTAGE
Acrolein
Solvent
THE
7.0 7.5
4.5 8.5
0.5 2.0
3.5 4.5
2.5 2.0
1.0 0 1.5 2.0 2.5 4.5
1.0
OF DEAD -
5.5 10.0
1.5 2.5
4.5 6.5
4.5 9.0
1.5
ELD
CELLS
0.5 2.0
4.0 6.0
5.0 8.5
4.5 4.5
4.0 5.5
2.0 3.5
1.5 1.5 3.0 6.0 7.0 10.5
2.0
0 1.5
7.5 10.0
4.0 4.5
6.0 5.5
1.5 2.5
1.5 2.0 6.5 17.0 8.5 17.0
2.5
Incubation
TABLE 1 DURING INCUBATION
2.5 4.0
1.5 3.0
1.0 2.0
9.5 12.5
5.0 4.5
5.5 5.0
3.5 4.5
73.5 48.0 50.5
11.0 18.5
4.0
SOLVENTS
4.5 5.0
8.0 12.0
4.5 5.5
7.5 5.5
53.5 60.5
7.0 14.0 20.5
3.5
OF ORGANIC
3.5 5.0
7.5 12.0
6.5 4.5
5.0 5.0
2.5 3.0
3.0 5.5 11.0 29.5 31.0 29.5
3.0
time
IN PRBSENCI~
2.0 3.5
9.5 13.5
72.5 81.0
8.0 20.5 44.0
4.5
3.0 3.0
4.0 6.0
11.0 14.5
5.0 5.5
4.5 4.0
2.5 3.5
z c z 3 z
5
E E g
66.0 85.0 75.0 83.5 3.5 4.0
g
47.0
7.5
5.0
50 100
50 100
50 100 200
50 100
50 100
Cumene
1,2-Dichloroethane
Ethanol
Ethylbenzene
1 5 10 25 50 100
aldehyde
tetrachloride
Carbon
50 100
Crotonic
disulfide
Carbon
50 100
50 100
ether
n-Butyl
50 100
Chloroform
acetate
n-Butyl
2.0 3.0
0 2.0
2.0 3.5 2.0
3.5 2.5
1.0 2.0 1.0 1.0 3.0 3.0
1.0 2.0
6.5 7.5
4.0 4.0
1.0 0.5
1.5 2.0
1.5 3.0
3.0 2.0
3.0 2.5 2.0
3.5 3.5
0.5 1.0 1.0 3.5 2.0 3.5
1.5 4.0
10.5 13.0
3.5 4.5
2.5 4.0
3.0 3.0
3.0 4.0
3.0 5.0
11.5 15.0
8.0 6.5
3.0 3.0
3.5 3.0
3.5 4.5 2.5
4.0 6.0
1.5 1.0 1.5 3.0 4.5 24.0
4.0 5.0
12.5 16.0
6.0 8.0
3.0 3.0
4.0 4.0
7.0 11.0
6.5 8.5
1.5 2.0 1.5 4.5 5.0 26.0
2.5 7.0
13.5 14.5
6.0 6.0
2.5 1.5
1.5 3.0
3.5 3.5
4.0 4.5
7.5 6.5 6.0
4.5 5.0
1.5 2.5 4.0 6.0 5.0 39.5
3.0 5.0
13.0 15.5
6.0 9.0
2.0
5.0 4.5
4.0 3.5
6.5
6.5 12.5
2.0 2.0 3.5 4.5 18.5
3.0 6.0
9.5 18.0
4.3 9.0
5.5 7.0
3.5
7.5 7.0 7.5
4.5 7.0
2.0 2.0 4.0 7.0 68.0 95.0
3.0 4.0
9.0 11.5
4.5 8.5
6.0 6.0
3.0 3.5
9.0 84.0 95.5
6.0 4.5
15.0 13.0
5.0 9.0
5.0 7.0
9.0 7.5 6.5
5.0 18.0
1.5 2.0 3.5 7.5 100.0 100.0
4.5 3.0
12.5 18.5
6.5 10.0
1.0 2.5
3.0 2.5
1.0 2.0
2.0 4.5
50 100
50 100
50 100
50 100
50 100
50 100
Methylethylketone
Nitrobenzene
n-Octane
n-Propylacetate
Styrene
1,1,1,2-Tetrachloroethane
6.0 5.5
5.0 4.0
0.5 1.5
2.0 3.0
2.0 4.0
50 100
Furfural
0.0
5.0 6.0
--
50 100
ppm
Formaldehyde
Solvent
4.5 8.0 9.0 9.5
4.5 5.5
8.0 10.0
4.5 5.5
5.0 5.0 0 2.0
4.0 4.5
4.0 4.0
0.5 0.5
5.0 8.5
1.5
5.5 5.0
1.5 3.5
8.5 30.5
1.0
12.5
6.0 7.5
0.5 1.5
5.5 5.5
4.0 4.5
7.5 10.0
2.5 3.5
21.0 25.5
2.0
TABLE
9.5 12.0
6.0 8.0
0.5 1.5
2.5 2.5
12.5” 13.50
23.0 27.5
2.5
Incubation
1 (Continued)
16.0
6.5 9.0
0.5 1.0
6.0 5.0
6.0 7.0
10.5” 10.56
1.5 5.5
28.5 31.5
3.0
time
8.0 14.5
6.5 9.5
1.0 1.5
4.0 4.5
8.@ 17.5a
3.5 6.0
25.5 26.5
3.5
9.5 14.0
7.5 10.5
1.0 1.5
5.5 6.5
1.5 2.0
11.00 12.5a
3.0 7.0
22.5 26.0
4.0
9.5 15.0
6.5 10.0
6.5 6.5
3.5 7.5
24.5 30.0
4.5
11.5 19.5”
7.5 10.0
0.5 2.0
6.0 7.0
4.5 4.5
10.5& 14.oa
23.0 35.5
5.0
50 100
25 50 75 100
50 100
50 100
50 100
50 100
Tetrachloroethylene
Tetralin
Toluene
1,1,2-Trichloroethane
Trichloroethylene
o-Xylene
2.3
2.0 2.5
3.0 2.5
0 0
6.0 5.0
3.0 3.0 3.0 4.0
3.5 2.5
0 2.0
2.6
2.5 3.0
2.5 3.5
0.5 1.0
4 (5 4.0
2.5 5.5 4.5 11.0
4.0 4.5
1.5 2.0
a Sample contained cell fragments. * Mean values of between 23 and 53 estimations.
Control*
50 100
1,1,2,2-Tetrachloroethane
3.2
4.5 4.0
3.0 3.5
0 0
4.5 6.0
7.0 9.5 11.5 44.5
1.0 8.5
1.5 5.5
3.1
3.0 3.5
3.5 5.0
0 0.5
9.0 8.5
6.5 15.0 17.0 41.5
3.0 6.0
3.0
3.5
4.0 5.5
4.0 5.5
1.0 0.5
4.5 8.0
42.5
16.0
5.0 10.5
2.0 2.0
3.6
3.5 5.0
5.5 6.0
0.5 1.0
6.0 8.0
8.0 16.0 17.5 40.5
7.5 9.5
15.0 8.5
4.5 5.5
3.6
1.0 0.5
0 0.5
3.5
2.5 4.5
7.5 8.5
15.0 18.0 24.5 67.0
5.5 7.5
1.5 2.0
6.5 8.0
14.5 16.0 21.5 56.5
6.5 10.5
2.0
4.2
7.0 6.5
0 0.5
7.5 9.0
15.5 18.5 28.0 64.5
3.5 4.5
4.2
2.0 4.5
12.0 8.5
95.5
18.5
5.5 9.0
2.5 2.5
188
HOLMBERG
AND
MALMFORS
The cells were washed once by resuspending in solvent-free incubation medium at a cell concentration of about 1. 106 cells/ml. After washing, the cells were resuspended at the same final cell concentration in Eagle’s suspension medium containing 10% heat-inactivated calf serum, antibiotics, and glutamine (Eagle, 1959). Cell concentrations were determined in a Biirker hemocytometer. The organic solvents were added to the incubation medium prior to the addition of cells. The cell suspensions were divided into subpopulations incubated in separate tubes, one for each sampling time in both control and experimental series. Cells were incubated up to 5 hr at 37 2 1°C under constant stirring in sealed 3-ml glass tubes filled to the top. Cell suspensions were not aerated during the incubation. Samples for estimation of the frequency of injured cells was usually taken every half hour. One drop of the cell suspensions was smeared on microscope slides for estimation of the frequency of irreversibly injured cells. A few drops of 2% Lissamine green B (Gurr Ltd., Colour Index No. 73) dissolved in physiological saline were added. 200 cells were counted in each sample as soon as possible after the addition of dyestuff and the frequency of cells diffusely stained by Lissamine green was taken as an index on cell injury. Cell fragments were not included in the cell count. Cell populations treated with 50 ppm crotonic aldehyde for 5 hr were reinoculated intraperitoneally into five mice, 0.3 ml, i.e., a total number of 3. lo5 cells, was injected into each animal. The total body weight of injected mice was recorded during 4 weeks after inoculation. The total body weight of mice was used as a rough measure on tumour growth capacity. Control cells were incubated for 5 hr in absence of solvent. RESULTS
The frequency of irreversibly injured cells after incubation with different organic solvents can be seen in Table 1. Most of the organic solvents tested did not within 5-hr incubation time induce an increase in the frequency of irreversibly injured cells which was differing from the values for nontreated cells. Benzyl chloride, carbon tetrachloride, cumene, methyl ethyl ketone, styrene and 1,1,1,2-tetrachloroethane in 100 ppm concentrations did, however, induce a cellular injury which showed up as a frequency of diffusely stained cells ranging between 10 and 20% after 5 hr. Formaldehyde, which is used as a fixation agent in histological work, was deleterious to ELD cells at 100 ppm concentration after 1-hr incubation, and at 50 ppm after 2-hr incubation. The frequency of irreversibly injured cells at the end of 5hr incubation did not further increase remarkably compared to the frequency at l- and 2-hr incubation, Acrolein, crotonic aldehyde, and tetralin, on the other hand, induced at 100 ppm concentration a cellular injury which progresses until the entire ELD population is irreversibly injured within 5-hr of incubation. Acrolein did even induce a significant increase in the frequency of injured cells after 3.5hr incubation at a concentration of 5 ppm. In order to demonstrate that the increased permeability of ELD cells toward
THE
CYTOTOXICITY
OF
SOME
ORGANIC
SOLVENTS
189
43 l41 39 x 0 37 2 2 35.F p 33-0” j
31-
5 2
29-
--crotonic -control
old&y&
FIG. 1. Change in total body weight with time of NMRI mice intraperitoneally inoculated with 3.106 cells at Day zero. Cells in experimental series were incubated for 5 hr at 37°C in presence of 50 ppm crotonic aldehyde prior to injection. Control cells were incubated without solvent. Each point represents mean values from five mice. Asterisks indicate days of death of animals. The last asterisk represents two dead animals.
Lissamine green induced by organic solvents was in a stage of irreversible cellular injury preceding cell death, ELD populations treated with crotonic aldehyde were reinoculated into mice. The total body weight of mice, intraperitoneally inoculated, was recorded as a rough measure of tumour growth. AS can be seen in Fig. 1, the body weight of mice inoculated with nontreated ELD cells increased up to a maximum 20 days after inoculation. After that time the carcass weight decreased owing to a general cachexia preceding the extinction of the animal population. Treatment of ELD cell populations with 50 ppm crotonic aldehyde for 5 hr in vitro seemedto result in complete inhibition of tumour growth after reinoculation. DISCUSSION
The diffuse staining of the cytoplasm by so-called vital dyes is widely used as a criterion of cellular injury (Hoskins, Meynell, and Sanders, 1956; Hanks and Wallace, 1958; Holmberg, 1961; Geczy and Baumgarten, 1970; Kaltenbach, Kaltenbach, and Lyons, 1958; Pappenheimer, 1917; Phillips and Terryberry, 1957). The penetration of vital dyes into the cell has also been taken by some authors as an absolute criterion of cell death. The penetration of the negatively charged nontoxic triphenylmethane dye Lissamine green is an event parallelled by irreversible morphological and biochemical changes in the cell (Holmberg, 1961). Thus, diffusely stained cells show a refraction change in the microscope characteristic of cellular injury. This change in refraction is associated with visible extrusion of cytoplasmic material ( Holmberg, 1960). In connection with, or just
190
HOLMBERG
ArjD
MALMFORS
preceding the penetration of dye stuffs, cell membranes do show the so-called blebbing phenomenon (Bessis, 1964; Biggers, 1964; Hartveit, 1962; Holmberg, 1960; Kay, 1965; King et al., 1959) indicating cellular injury. Cells permeable to dyestuffs, used for vital staining, do also show a decrease in oxygen consumption (Kaltenbach, Kaltenbach, and Lyons, 1958; Phillips and Terryberry, 1957) as well as a decrease in the per-cell activities of cytoplasmic enzymes indicating a loss of vital cytoplasmic components (Holmberg, 1961). Penetration of dyestuffs into cells cannot be taken indiscriminately as an indication of cell death, in that sense that the stained cell generally should be considered metabolically inert or even incapable of cell division (Harris, 1966; Kay et al., 1965). It could be possible, for instance, that a presumed cytotoxic chemical only enhances the transport of the dyestuff into the cell without causing irreversible cellular injury. Time-lapse cinemicrographic studies earlier performed do, however, show that the undulating movement of the cell membrane of in uitro-cultivated strain L cells is irreversibly inhibited in the Lissamine greenstained cells. Stained L cells have also lost their active directed amoeboid movement on the glass surface ( Holmberg, 1960). Moreover, stained L cells have not been observed to divide in vitro, nor has stained ELD cells been demonstrated to multiplicate in tivo (Holmberg, 1961). This is in accordance with what has been found with the vital dyes trypan blue, eosin, and triphenyltetrazolium chloride used for estimating the frequency of irreversibly injured cells (Hoskins, Meynell, and Sanders, 1956). Earlier observations indicate that an increase in the permeability of cells to Lissamine green is an indication of cell death or at least of an irreversible cell injury immediately preceding complete cell death. The inoculation experiments with solvent-treated ELD-populations presently reported do corroborate this statement. The diffuse staining of ELD cells with Lissamine green after treatment with organic solvents is thus not an artefact, but a true measure of the acute cytotoxicity of the tested agents. The present studies show that the aldehydes acrolein, crotonic aldehyde, and formaldehyde, as well as tetralin, are highly biologically reactive toward ELD cells. These organic solvents seem to be more cytotoxic during short-time in vitro experiments than, for instance, the established hepatotoxic agents carbon disulfide, carbon tetrachloride, and chloroform. This might indicate that the cytotoxic effect presently observed is due to the toxic properties of the solvents per se and not to toxic effects of metabolites. Now, the present results should not be taken as an indication of a high toxicity in vivo of acrolein, crotonic aldehyde, formaldehyde, or tetralin. Although there appear some instances of good correlation between the effects of various chemicals on cells in vitro with effects seen clinically in humans (Cline, 1967; Everett et al., 1951) or in animals (Smith, Grady, and Northam, 1963) it appears as if the toxicity data obtained in vitro does not, as a general rule, correlate well with the effect of these agents in whole animal studies (Smith, Grady, and Northam, 1963). Acute cytotoxicity experiments obtained in vitro are, however, most useful as rapid screening methods for potentially deleterious agents. Acute cytotoxicity data obtained in vitro are also important as a first step toward an understanding
THE
CYTOTOXICITY
OF
SOME
ORGANIC
191
SOLVENTS
of the mechanisms of action of deleterious substances on the cellular and subcellular level. The highly cytotoxic solvents acrolein, crotonic aldehyde, formaldehyde, and tetralin should be further studied with respect to their probable cell growthinhibiting effects and their interaction with important cellular functions, such as the adhesion properties to the glass substrate in in vitro cultures and other membrane characteristics. The cell growth-inhibiting effect of crotonic aldehyde, as observed with the solvent-treated tumour populations, suggests an antitumour under investigation. capacity of the highly cytotoxic solvents. This is presently ACKNOWLEDGMENT This Sweden.
investigation
was
supported
by a grant
from
“Riksbankens
Jubileumsfond,”
Stockholm,
REFERENCES ( 1964). Studies on cell agony and death: An attempt at classification.In BESSIS, M. Injury” (A. V. S. de Reuck and J, Knight, Eds.), pp. 287316. Ciba Foundation “Cellular Symp., Churchill, London. Brm~~s, J. D. ( 1964). The death of cells in normal multicellular organisms. In “CeHular Injury” (A. V. S. de Reuck and J. Knight, Eds.), pp. 329-349. Ciba Foundation Symp., Churchill, London. CLINE, M. J. ( 1967). Prediction of in vivo cytotoxicity of chemotherapeutic agents by their effect on malignant leukocytes in vitro. Blood 34 176-188. CORSSEN, C., AND SWEET, R. B. ( 1967). Effects of halogenated anesthetic agents on selectively starved cultured human liver cells. Anesth. A&g. (Cleveland) 46, 575-588. CORSSRN, G., SWEET, R. B., AND CHENOWETH, M. B. ( 1966). Effects of chloroform, halothane and methoxyfiurane on human liver cells in vitro. Anesthesiology 27, 155-162. EAGLE, H. ( 1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432+437. EVERETT, E. T., POMERAT, C. M., Hu, F. N., AND LIVINCOOD, C. S. ( 1951). Tissue culture studies on human skin. I. A method of evaluating the toxicity of certain drugs employed Iocally on the skin. Texas Rep. Biol. Med. 9, 281-291. FUNCESCHINI, M. ( 1964). L’azione de1 Diossano e della Talidomide sull’accrescimento in coltura organotipica della tibia di embrione di pollo. Spetimentale 114, l-17. GE~ZY, A. F., AND BAIJMGARTEN, A. (1970). Th e validity of the eosin technique as an index of viability. Exp. Cell Res. 62, 356-358. HANKS, J. H., AND WALLACE, J. H. ( 1958). Determination of cell viability. Proc. Sot. Exp. Biol. Med. 98, 188-192. HARRIS, M. (1966). Criteria of viability in heat-treated cells. Exp. Cell Res. 44, 658-661. HARTVEIT, F. ( 1962 ). Cellular injury in untreated Ehrlich’s ascites carcinoma. Btit. J. Cancer 1’6, 556-561. HOLMBERG, B. ( 1960). T’ lme -1 apse cinµgraphy on the permeability of strain L-cells to Lissamine green. Res. Film 3, 366-368. HOLMBERG, B. ( 1961). On the permeability to Lissamine green and other dyes in the course of cell injury and cell death. Erp. CeZZ Res. 22, 40~114. HOSIUNS, J. M., MEYNELL, G. G., AND SANDERS, F. K. ( 1956). A comparison of methods for estimating the viable count of a suspension of tumour cells. Ezp. Cell Res. 11, 297-305. KALTENBAcH, J. P., KALTENBACH, M. H., AND LYONS, W. B. ( 1958). Nigrosin as a dye for differentiating live and dead ascites cells. Erp. Cell Res. 15, 112-117. KAY, E. R. M. (1965). The effects of Tween 80 on the in vitro metabolism of cells of
the Ehrlich-Lettrk KING,
D. W.,
ascites carcinoma.
PAULSON,
S. R., F’UCKETT,
Cancer Res. 25, 764-769. N. L.,
AND KREBS,
A. T.
(1959).
Cell
death
IV.
192
HOLMBERG
AND
MALMFORS
The effect of injury on the entrance of vital dye in Ehrlich tumor cells. Amer. J. PuthoZ. 35, 1067-1079. KOLBERC, J., HELGELAND, K., JONSEN, J., AND TJELTVEIT, 0. (1971). The herbicide 2,4dichlorophenoxyacetic acid. I.: Effects on L cells. Actu Ph~rmuc~l. ToxicoZ. 29, 81-86. LI-I-IXRST, C. L., AND LICHTENSTEIN, E. P. (1971). Effects and interactions of environmental chemicals on human cells in tissue culture. Arch. Enuiron. Health. 22, 454-459. PACE, D. M., AND ELLIOTT, A. (1962). Effects of acetone and phenol on established cell lines cultivated in vitro. Cancer Res. 22, 107-112. PAPPENHEIMER, A. M. (1917). Experimental studies upon lymphocytes. I. The reactions of lymphocytes under various experimental conditions. J. Erp. Med. 25, 633-650. PHILLIPS, H. J., AND TERRYBERRY, J. E. Counting actively metabolizing tissue cul( 1957). tured cells. Exp. Cell Res. 13, 341347. ROSENBERG, P. H., AND WAHLSTROM, T. ( 1971). Hepatotoxicity of halothane metabolites in vivo and inhibition of fibroblast growth in vitro. Acta Phumnacol. Toxicol. 29, 9-19. RODIDS, D. E., AND BILS, R. F. ( 1965). Effects of air pollutants on cells in culture. Arch. Environ. Health 10, 251-259. SMITH, C. G., LUMMIS, W. L., AND GRADY, J. E. ( 1959). An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals, and solvents. Cancer Res. 19, 847-852. SMITH, C. G., GRADY, J. E., AND NORTHAM, J. I. ( 1963 ). Relationship between cytotoxicity in vitro and whole animal toxicity. Cancer Chemother. Rep. 30, 9-12.
RESEARCH
The
7, 183-192
( 1974)
Cytotoxicity B.
of Some
HOLMBERG’
AND
Organic T.
Solvents
MALMFOR~
Section of Occupational Toxicology, Department of Occupational National Board of Occupational Safety and Health, Stockholm, Received
August
Health, Sweden
10, 1973
The cytotoxicity of 33 organic solvents was determined using Ehrlich-Landschutz diploid (ELD) ascites tumour cells during short-time in vitro incubations. Among the organic solvents studied, tetralin, crotonic aldehyde, formaldehyde, and acrolein were highly toxic to ELD cells. Benzyl chloride, carbon tetrachloride, methyl ethyl ketone, and 1,1,1,2-tetrachloroethane were found to be moderately toxic. Tumour cells treated with crotonic aldehyde were reinoculated into mice. The growth capacity of the inoculated population was shown to be dependent on the frequency of surviving cells after treatment with the organic solvents indicating that true cell mortality was studied. INTRODUCTION
Organic solvents are widely used in industrial work. The cytotoxicity of most of the solvents is regarded to be low according to the absence of effects on the liver of experimental animals. Only for a few solvents, namely, carbon disulfide, carbon tetrachloride, and chloroform hepatotoxic effects have been established. In vitro experiments for evaluating the cytotoxicity of chemicals occurring in the environment have been performed for air pollutants (Rounds and Bils, 1965), pesticides (Litterst and Lichtenstein, 1971; Kolberg et aZ., 1971), solvents (Smith et al., 1959; Franceschini, 1964; Pace and Elliott, 1962) as well as for anesthetic agents (Corssen and Sweet, 1966; Corssen and Sweet, 1967; Rosenberg and Wahlstriim, 1971). In these studies the effects of added agents on cell growth and/or cell behaviour were observed. The present report describes the acute cytotoxicity of organic solvents as studied in vitro with Ehrlich-Landschiitz diploid (ELD) ascites tumour cells. A dye exclusion test was used for estimating the frequency of cells in a stage of irreversible cell injury leading to cell death. Such acute cytotoxicity studies can throw some light on the mechanism underlying the biologica effects of organic solvents. MATERIALS
AND
METHODS
Ehrlich-Landschiitz (ELD) diploid ascites tumour cells were propagated in outbred albino NMRI mice. Tumour cell populations were withdrawn not earlier than 1 week after the inoculation. Cells from several mice were pooled and centrifuged at 1566g for 10 min. The supernatant ascites fluid was discarded. ’ National Board of Occupational Safety Section of Occupational Toxicology, P. 0. ‘Present address: Astra Pharmaceutical Sweden.
and Health, Department of Occupational Health, Box S-10026 Stockholm 34, Sweden. AB, Toxicology Laboratories, S-151 85 .%dertllje, 183
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
acetate
50 100
50 100
chloride
Benzyl
iso-Butyl
3.0 5.0
100 200
alcohol
Benzyl
50 100
0 1.0
50 100
Benzene
Bromochloromethane
1.5 2.0
50 100
Amylacetate
2.0 1.0
7.0 6.0
5.0 4.5
2.0 1.0
50 100
alcohol
Ally1
0 1.0 1.5 1.0 1.5 2.5
0.0
1 5 10 25 50 100
ppm
PERCENTAGE
Acrolein
Solvent
THE
7.0 7.5
4.5 8.5
0.5 2.0
3.5 4.5
2.5 2.0
1.0 0 1.5 2.0 2.5 4.5
1.0
OF DEAD -
5.5 10.0
1.5 2.5
4.5 6.5
4.5 9.0
1.5
ELD
CELLS
0.5 2.0
4.0 6.0
5.0 8.5
4.5 4.5
4.0 5.5
2.0 3.5
1.5 1.5 3.0 6.0 7.0 10.5
2.0
0 1.5
7.5 10.0
4.0 4.5
6.0 5.5
1.5 2.5
1.5 2.0 6.5 17.0 8.5 17.0
2.5
Incubation
TABLE 1 DURING INCUBATION
2.5 4.0
1.5 3.0
1.0 2.0
9.5 12.5
5.0 4.5
5.5 5.0
3.5 4.5
73.5 48.0 50.5
11.0 18.5
4.0
SOLVENTS
4.5 5.0
8.0 12.0
4.5 5.5
7.5 5.5
53.5 60.5
7.0 14.0 20.5
3.5
OF ORGANIC
3.5 5.0
7.5 12.0
6.5 4.5
5.0 5.0
2.5 3.0
3.0 5.5 11.0 29.5 31.0 29.5
3.0
time
IN PRBSENCI~
2.0 3.5
9.5 13.5
72.5 81.0
8.0 20.5 44.0
4.5
3.0 3.0
4.0 6.0
11.0 14.5
5.0 5.5
4.5 4.0
2.5 3.5
z c z 3 z
5
E E g
66.0 85.0 75.0 83.5 3.5 4.0
g
47.0
7.5
5.0
50 100
50 100
50 100 200
50 100
50 100
Cumene
1,2-Dichloroethane
Ethanol
Ethylbenzene
1 5 10 25 50 100
aldehyde
tetrachloride
Carbon
50 100
Crotonic
disulfide
Carbon
50 100
50 100
ether
n-Butyl
50 100
Chloroform
acetate
n-Butyl
2.0 3.0
0 2.0
2.0 3.5 2.0
3.5 2.5
1.0 2.0 1.0 1.0 3.0 3.0
1.0 2.0
6.5 7.5
4.0 4.0
1.0 0.5
1.5 2.0
1.5 3.0
3.0 2.0
3.0 2.5 2.0
3.5 3.5
0.5 1.0 1.0 3.5 2.0 3.5
1.5 4.0
10.5 13.0
3.5 4.5
2.5 4.0
3.0 3.0
3.0 4.0
3.0 5.0
11.5 15.0
8.0 6.5
3.0 3.0
3.5 3.0
3.5 4.5 2.5
4.0 6.0
1.5 1.0 1.5 3.0 4.5 24.0
4.0 5.0
12.5 16.0
6.0 8.0
3.0 3.0
4.0 4.0
7.0 11.0
6.5 8.5
1.5 2.0 1.5 4.5 5.0 26.0
2.5 7.0
13.5 14.5
6.0 6.0
2.5 1.5
1.5 3.0
3.5 3.5
4.0 4.5
7.5 6.5 6.0
4.5 5.0
1.5 2.5 4.0 6.0 5.0 39.5
3.0 5.0
13.0 15.5
6.0 9.0
2.0
5.0 4.5
4.0 3.5
6.5
6.5 12.5
2.0 2.0 3.5 4.5 18.5
3.0 6.0
9.5 18.0
4.3 9.0
5.5 7.0
3.5
7.5 7.0 7.5
4.5 7.0
2.0 2.0 4.0 7.0 68.0 95.0
3.0 4.0
9.0 11.5
4.5 8.5
6.0 6.0
3.0 3.5
9.0 84.0 95.5
6.0 4.5
15.0 13.0
5.0 9.0
5.0 7.0
9.0 7.5 6.5
5.0 18.0
1.5 2.0 3.5 7.5 100.0 100.0
4.5 3.0
12.5 18.5
6.5 10.0
1.0 2.5
3.0 2.5
1.0 2.0
2.0 4.5
50 100
50 100
50 100
50 100
50 100
50 100
Methylethylketone
Nitrobenzene
n-Octane
n-Propylacetate
Styrene
1,1,1,2-Tetrachloroethane
6.0 5.5
5.0 4.0
0.5 1.5
2.0 3.0
2.0 4.0
50 100
Furfural
0.0
5.0 6.0
--
50 100
ppm
Formaldehyde
Solvent
4.5 8.0 9.0 9.5
4.5 5.5
8.0 10.0
4.5 5.5
5.0 5.0 0 2.0
4.0 4.5
4.0 4.0
0.5 0.5
5.0 8.5
1.5
5.5 5.0
1.5 3.5
8.5 30.5
1.0
12.5
6.0 7.5
0.5 1.5
5.5 5.5
4.0 4.5
7.5 10.0
2.5 3.5
21.0 25.5
2.0
TABLE
9.5 12.0
6.0 8.0
0.5 1.5
2.5 2.5
12.5” 13.50
23.0 27.5
2.5
Incubation
1 (Continued)
16.0
6.5 9.0
0.5 1.0
6.0 5.0
6.0 7.0
10.5” 10.56
1.5 5.5
28.5 31.5
3.0
time
8.0 14.5
6.5 9.5
1.0 1.5
4.0 4.5
8.@ 17.5a
3.5 6.0
25.5 26.5
3.5
9.5 14.0
7.5 10.5
1.0 1.5
5.5 6.5
1.5 2.0
11.00 12.5a
3.0 7.0
22.5 26.0
4.0
9.5 15.0
6.5 10.0
6.5 6.5
3.5 7.5
24.5 30.0
4.5
11.5 19.5”
7.5 10.0
0.5 2.0
6.0 7.0
4.5 4.5
10.5& 14.oa
23.0 35.5
5.0
50 100
25 50 75 100
50 100
50 100
50 100
50 100
Tetrachloroethylene
Tetralin
Toluene
1,1,2-Trichloroethane
Trichloroethylene
o-Xylene
2.3
2.0 2.5
3.0 2.5
0 0
6.0 5.0
3.0 3.0 3.0 4.0
3.5 2.5
0 2.0
2.6
2.5 3.0
2.5 3.5
0.5 1.0
4 (5 4.0
2.5 5.5 4.5 11.0
4.0 4.5
1.5 2.0
a Sample contained cell fragments. * Mean values of between 23 and 53 estimations.
Control*
50 100
1,1,2,2-Tetrachloroethane
3.2
4.5 4.0
3.0 3.5
0 0
4.5 6.0
7.0 9.5 11.5 44.5
1.0 8.5
1.5 5.5
3.1
3.0 3.5
3.5 5.0
0 0.5
9.0 8.5
6.5 15.0 17.0 41.5
3.0 6.0
3.0
3.5
4.0 5.5
4.0 5.5
1.0 0.5
4.5 8.0
42.5
16.0
5.0 10.5
2.0 2.0
3.6
3.5 5.0
5.5 6.0
0.5 1.0
6.0 8.0
8.0 16.0 17.5 40.5
7.5 9.5
15.0 8.5
4.5 5.5
3.6
1.0 0.5
0 0.5
3.5
2.5 4.5
7.5 8.5
15.0 18.0 24.5 67.0
5.5 7.5
1.5 2.0
6.5 8.0
14.5 16.0 21.5 56.5
6.5 10.5
2.0
4.2
7.0 6.5
0 0.5
7.5 9.0
15.5 18.5 28.0 64.5
3.5 4.5
4.2
2.0 4.5
12.0 8.5
95.5
18.5
5.5 9.0
2.5 2.5
188
HOLMBERG
AND
MALMFORS
The cells were washed once by resuspending in solvent-free incubation medium at a cell concentration of about 1. 106 cells/ml. After washing, the cells were resuspended at the same final cell concentration in Eagle’s suspension medium containing 10% heat-inactivated calf serum, antibiotics, and glutamine (Eagle, 1959). Cell concentrations were determined in a Biirker hemocytometer. The organic solvents were added to the incubation medium prior to the addition of cells. The cell suspensions were divided into subpopulations incubated in separate tubes, one for each sampling time in both control and experimental series. Cells were incubated up to 5 hr at 37 2 1°C under constant stirring in sealed 3-ml glass tubes filled to the top. Cell suspensions were not aerated during the incubation. Samples for estimation of the frequency of injured cells was usually taken every half hour. One drop of the cell suspensions was smeared on microscope slides for estimation of the frequency of irreversibly injured cells. A few drops of 2% Lissamine green B (Gurr Ltd., Colour Index No. 73) dissolved in physiological saline were added. 200 cells were counted in each sample as soon as possible after the addition of dyestuff and the frequency of cells diffusely stained by Lissamine green was taken as an index on cell injury. Cell fragments were not included in the cell count. Cell populations treated with 50 ppm crotonic aldehyde for 5 hr were reinoculated intraperitoneally into five mice, 0.3 ml, i.e., a total number of 3. lo5 cells, was injected into each animal. The total body weight of injected mice was recorded during 4 weeks after inoculation. The total body weight of mice was used as a rough measure on tumour growth capacity. Control cells were incubated for 5 hr in absence of solvent. RESULTS
The frequency of irreversibly injured cells after incubation with different organic solvents can be seen in Table 1. Most of the organic solvents tested did not within 5-hr incubation time induce an increase in the frequency of irreversibly injured cells which was differing from the values for nontreated cells. Benzyl chloride, carbon tetrachloride, cumene, methyl ethyl ketone, styrene and 1,1,1,2-tetrachloroethane in 100 ppm concentrations did, however, induce a cellular injury which showed up as a frequency of diffusely stained cells ranging between 10 and 20% after 5 hr. Formaldehyde, which is used as a fixation agent in histological work, was deleterious to ELD cells at 100 ppm concentration after 1-hr incubation, and at 50 ppm after 2-hr incubation. The frequency of irreversibly injured cells at the end of 5hr incubation did not further increase remarkably compared to the frequency at l- and 2-hr incubation, Acrolein, crotonic aldehyde, and tetralin, on the other hand, induced at 100 ppm concentration a cellular injury which progresses until the entire ELD population is irreversibly injured within 5-hr of incubation. Acrolein did even induce a significant increase in the frequency of injured cells after 3.5hr incubation at a concentration of 5 ppm. In order to demonstrate that the increased permeability of ELD cells toward
THE
CYTOTOXICITY
OF
SOME
ORGANIC
SOLVENTS
189
43 l41 39 x 0 37 2 2 35.F p 33-0” j
31-
5 2
29-
--crotonic -control
old&y&
FIG. 1. Change in total body weight with time of NMRI mice intraperitoneally inoculated with 3.106 cells at Day zero. Cells in experimental series were incubated for 5 hr at 37°C in presence of 50 ppm crotonic aldehyde prior to injection. Control cells were incubated without solvent. Each point represents mean values from five mice. Asterisks indicate days of death of animals. The last asterisk represents two dead animals.
Lissamine green induced by organic solvents was in a stage of irreversible cellular injury preceding cell death, ELD populations treated with crotonic aldehyde were reinoculated into mice. The total body weight of mice, intraperitoneally inoculated, was recorded as a rough measure of tumour growth. AS can be seen in Fig. 1, the body weight of mice inoculated with nontreated ELD cells increased up to a maximum 20 days after inoculation. After that time the carcass weight decreased owing to a general cachexia preceding the extinction of the animal population. Treatment of ELD cell populations with 50 ppm crotonic aldehyde for 5 hr in vitro seemedto result in complete inhibition of tumour growth after reinoculation. DISCUSSION
The diffuse staining of the cytoplasm by so-called vital dyes is widely used as a criterion of cellular injury (Hoskins, Meynell, and Sanders, 1956; Hanks and Wallace, 1958; Holmberg, 1961; Geczy and Baumgarten, 1970; Kaltenbach, Kaltenbach, and Lyons, 1958; Pappenheimer, 1917; Phillips and Terryberry, 1957). The penetration of vital dyes into the cell has also been taken by some authors as an absolute criterion of cell death. The penetration of the negatively charged nontoxic triphenylmethane dye Lissamine green is an event parallelled by irreversible morphological and biochemical changes in the cell (Holmberg, 1961). Thus, diffusely stained cells show a refraction change in the microscope characteristic of cellular injury. This change in refraction is associated with visible extrusion of cytoplasmic material ( Holmberg, 1960). In connection with, or just
190
HOLMBERG
ArjD
MALMFORS
preceding the penetration of dye stuffs, cell membranes do show the so-called blebbing phenomenon (Bessis, 1964; Biggers, 1964; Hartveit, 1962; Holmberg, 1960; Kay, 1965; King et al., 1959) indicating cellular injury. Cells permeable to dyestuffs, used for vital staining, do also show a decrease in oxygen consumption (Kaltenbach, Kaltenbach, and Lyons, 1958; Phillips and Terryberry, 1957) as well as a decrease in the per-cell activities of cytoplasmic enzymes indicating a loss of vital cytoplasmic components (Holmberg, 1961). Penetration of dyestuffs into cells cannot be taken indiscriminately as an indication of cell death, in that sense that the stained cell generally should be considered metabolically inert or even incapable of cell division (Harris, 1966; Kay et al., 1965). It could be possible, for instance, that a presumed cytotoxic chemical only enhances the transport of the dyestuff into the cell without causing irreversible cellular injury. Time-lapse cinemicrographic studies earlier performed do, however, show that the undulating movement of the cell membrane of in uitro-cultivated strain L cells is irreversibly inhibited in the Lissamine greenstained cells. Stained L cells have also lost their active directed amoeboid movement on the glass surface ( Holmberg, 1960). Moreover, stained L cells have not been observed to divide in vitro, nor has stained ELD cells been demonstrated to multiplicate in tivo (Holmberg, 1961). This is in accordance with what has been found with the vital dyes trypan blue, eosin, and triphenyltetrazolium chloride used for estimating the frequency of irreversibly injured cells (Hoskins, Meynell, and Sanders, 1956). Earlier observations indicate that an increase in the permeability of cells to Lissamine green is an indication of cell death or at least of an irreversible cell injury immediately preceding complete cell death. The inoculation experiments with solvent-treated ELD-populations presently reported do corroborate this statement. The diffuse staining of ELD cells with Lissamine green after treatment with organic solvents is thus not an artefact, but a true measure of the acute cytotoxicity of the tested agents. The present studies show that the aldehydes acrolein, crotonic aldehyde, and formaldehyde, as well as tetralin, are highly biologically reactive toward ELD cells. These organic solvents seem to be more cytotoxic during short-time in vitro experiments than, for instance, the established hepatotoxic agents carbon disulfide, carbon tetrachloride, and chloroform. This might indicate that the cytotoxic effect presently observed is due to the toxic properties of the solvents per se and not to toxic effects of metabolites. Now, the present results should not be taken as an indication of a high toxicity in vivo of acrolein, crotonic aldehyde, formaldehyde, or tetralin. Although there appear some instances of good correlation between the effects of various chemicals on cells in vitro with effects seen clinically in humans (Cline, 1967; Everett et al., 1951) or in animals (Smith, Grady, and Northam, 1963) it appears as if the toxicity data obtained in vitro does not, as a general rule, correlate well with the effect of these agents in whole animal studies (Smith, Grady, and Northam, 1963). Acute cytotoxicity experiments obtained in vitro are, however, most useful as rapid screening methods for potentially deleterious agents. Acute cytotoxicity data obtained in vitro are also important as a first step toward an understanding
THE
CYTOTOXICITY
OF
SOME
ORGANIC
191
SOLVENTS
of the mechanisms of action of deleterious substances on the cellular and subcellular level. The highly cytotoxic solvents acrolein, crotonic aldehyde, formaldehyde, and tetralin should be further studied with respect to their probable cell growthinhibiting effects and their interaction with important cellular functions, such as the adhesion properties to the glass substrate in in vitro cultures and other membrane characteristics. The cell growth-inhibiting effect of crotonic aldehyde, as observed with the solvent-treated tumour populations, suggests an antitumour under investigation. capacity of the highly cytotoxic solvents. This is presently ACKNOWLEDGMENT This Sweden.
investigation
was
supported
by a grant
from
“Riksbankens
Jubileumsfond,”
Stockholm,
REFERENCES ( 1964). Studies on cell agony and death: An attempt at classification.In BESSIS, M. Injury” (A. V. S. de Reuck and J, Knight, Eds.), pp. 287316. Ciba Foundation “Cellular Symp., Churchill, London. Brm~~s, J. D. ( 1964). The death of cells in normal multicellular organisms. In “CeHular Injury” (A. V. S. de Reuck and J. Knight, Eds.), pp. 329-349. Ciba Foundation Symp., Churchill, London. CLINE, M. J. ( 1967). Prediction of in vivo cytotoxicity of chemotherapeutic agents by their effect on malignant leukocytes in vitro. Blood 34 176-188. CORSSEN, C., AND SWEET, R. B. ( 1967). Effects of halogenated anesthetic agents on selectively starved cultured human liver cells. Anesth. A&g. (Cleveland) 46, 575-588. CORSSRN, G., SWEET, R. B., AND CHENOWETH, M. B. ( 1966). Effects of chloroform, halothane and methoxyfiurane on human liver cells in vitro. Anesthesiology 27, 155-162. EAGLE, H. ( 1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432+437. EVERETT, E. T., POMERAT, C. M., Hu, F. N., AND LIVINCOOD, C. S. ( 1951). Tissue culture studies on human skin. I. A method of evaluating the toxicity of certain drugs employed Iocally on the skin. Texas Rep. Biol. Med. 9, 281-291. FUNCESCHINI, M. ( 1964). L’azione de1 Diossano e della Talidomide sull’accrescimento in coltura organotipica della tibia di embrione di pollo. Spetimentale 114, l-17. GE~ZY, A. F., AND BAIJMGARTEN, A. (1970). Th e validity of the eosin technique as an index of viability. Exp. Cell Res. 62, 356-358. HANKS, J. H., AND WALLACE, J. H. ( 1958). Determination of cell viability. Proc. Sot. Exp. Biol. Med. 98, 188-192. HARRIS, M. (1966). Criteria of viability in heat-treated cells. Exp. Cell Res. 44, 658-661. HARTVEIT, F. ( 1962 ). Cellular injury in untreated Ehrlich’s ascites carcinoma. Btit. J. Cancer 1’6, 556-561. HOLMBERG, B. ( 1960). T’ lme -1 apse cinµgraphy on the permeability of strain L-cells to Lissamine green. Res. Film 3, 366-368. HOLMBERG, B. ( 1961). On the permeability to Lissamine green and other dyes in the course of cell injury and cell death. Erp. CeZZ Res. 22, 40~114. HOSIUNS, J. M., MEYNELL, G. G., AND SANDERS, F. K. ( 1956). A comparison of methods for estimating the viable count of a suspension of tumour cells. Ezp. Cell Res. 11, 297-305. KALTENBAcH, J. P., KALTENBACH, M. H., AND LYONS, W. B. ( 1958). Nigrosin as a dye for differentiating live and dead ascites cells. Erp. Cell Res. 15, 112-117. KAY, E. R. M. (1965). The effects of Tween 80 on the in vitro metabolism of cells of
the Ehrlich-Lettrk KING,
D. W.,
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PAULSON,
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AND KREBS,
A. T.
(1959).
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AND
MALMFORS
The effect of injury on the entrance of vital dye in Ehrlich tumor cells. Amer. J. PuthoZ. 35, 1067-1079. KOLBERC, J., HELGELAND, K., JONSEN, J., AND TJELTVEIT, 0. (1971). The herbicide 2,4dichlorophenoxyacetic acid. I.: Effects on L cells. Actu Ph~rmuc~l. ToxicoZ. 29, 81-86. LI-I-IXRST, C. L., AND LICHTENSTEIN, E. P. (1971). Effects and interactions of environmental chemicals on human cells in tissue culture. Arch. Enuiron. Health. 22, 454-459. PACE, D. M., AND ELLIOTT, A. (1962). Effects of acetone and phenol on established cell lines cultivated in vitro. Cancer Res. 22, 107-112. PAPPENHEIMER, A. M. (1917). Experimental studies upon lymphocytes. I. The reactions of lymphocytes under various experimental conditions. J. Erp. Med. 25, 633-650. PHILLIPS, H. J., AND TERRYBERRY, J. E. Counting actively metabolizing tissue cul( 1957). tured cells. Exp. Cell Res. 13, 341347. ROSENBERG, P. H., AND WAHLSTROM, T. ( 1971). Hepatotoxicity of halothane metabolites in vivo and inhibition of fibroblast growth in vitro. Acta Phumnacol. Toxicol. 29, 9-19. RODIDS, D. E., AND BILS, R. F. ( 1965). Effects of air pollutants on cells in culture. Arch. Environ. Health 10, 251-259. SMITH, C. G., LUMMIS, W. L., AND GRADY, J. E. ( 1959). An improved tissue culture assay. II. Cytotoxicity studies with antibiotics, chemicals, and solvents. Cancer Res. 19, 847-852. SMITH, C. G., GRADY, J. E., AND NORTHAM, J. I. ( 1963 ). Relationship between cytotoxicity in vitro and whole animal toxicity. Cancer Chemother. Rep. 30, 9-12.