- Email: [email protected]
Studies with carcinogens and tumor-promoting agents in cell culture
Q
Experimental
572
1068
by
Academic
Press
Inc.
Cell Research 49, 572-583 (19C8)
STUDIESWITHCARCINOGENSANDTUMOR-PROMOTING AGENTSINCELLCULTURE A. SIVAK Laboratory Medicine,
and B. L. VAN
of Organic Chemistry New York University
DUUREN
and Carcinogenesis, Institute of Environmental Medical Center, New York, N.Y. 10016, USA
Received April 24, 1967
As
early as 1939, Creech [4] reported an increase of cell proliferation and chromosome abnormalities in mouse embryo fibroblasts treated with dibenz(a,h)anthracene-choleic acid as compared to untreated controls and to cells treated with phenanthrene or acenaphthene-choleic acid. Subsequently, Earle [9, lo] found that 3-methylcholanthrene could give rise to stable morphological alterations in a line of mouse embryo fibroblasts; however, the spontaneous changes that occurred precluded the direct implication of the carcinogen as the transforming agent. More recently, several groups of investigators have showed that carcinogenic aromatic hydrocarbons could induce heritable alterations in rodent cells in culture and that these transformed cell lines were tumorigenic in appropriate hosts [2, 3, I-21. These findings suggest that tissue culture systems may be of value in the study of cellular response to chemical agents, particularly the phenomenon of initiation and promotion in tumor induction and the screening of potential carcinogenic and promoting agents, e.g. from tobacco leaf and cigarette smoke condensate. The tumor-promoting activity of croton resin and of phorbol esters derived from croton resin [23, 241 and of tobacco leaf and smoke components [25] on mouse skin has been the subject of several reports from this laboratory; therefore, it was of interest to study the effects of these materials in cell culture. This communication describes the toxic and, in some cases, transforming effects of phorbol esters, fractions derived from cigarette smoke condensate and tobacco leaf extracts, and various carcinogens in several populations of mouse and hamster cells. MATERIALS
AND
METHODS
The 3T3 line of mouse fibroblasts and its polyoma- and SV40-transformed sublines were obtained through the courtesy of Dr G. J. Todaro and Dr H. Green, Department of Pathology, New York University School of Medicine. The behavior of this cell line as grown in our laboratory closely paralleled that described by Todaro and Green [19, 20, 211. Experimental
Cell Research 49
Chemicd
cctrcinogen
erects
in rodent
cell culture
558
In addition to these cell lines, primary and secondary mouse embryo (Swiss, HA/ICR) and Syrian hamster embryo cell cultures were prepared by the techniques described by Youngner [27]. The medium employed was Dulbecco’s modification of Eagle’s formulation containing 10 per cent calf serum [8]. Cultures were fed twice weekly with 4 ml of medium, and they were maintained by serial passage of monolayer cultures at lo5 cells per plate. Polystyrene culture dishes (Falcon 3002, 60 x 15 Plates for cloning or for the determination of mm) were employed throughout. lransformed areas were washed with phosphate-buffered saline (PBS), stained either with Giemsa stain for 45 minutes after methyl alcohol fixation, or with Harris’s hcmatoxylin after fixation in 10 per cent formalin-PBS. Karyological analyses were carried out essentially according to the method of Moorhead rt a/. 1161. Experiments to determine the tumorigenicity of the various populations of cells under study were carried out as follows: (a) Subcutaneous injection in adult hamsters; (b) subcutaneous injection in l-day-old hamsters; (c) implantation in cheek pouches of cortisonized hamsters [ll]; (d) subcutaneous injection in cortisonized mice [‘il. Test maferials.--Aromatic hydrocarbons, with the exception of chrysene, were obtained from commercial sources and were purified by column chromatography and/or recrystallization. Purity was confirmed by thin-layer chromatography, melting points and ultra-violet absorption spectra. A pure sample of chrysene (free of linear benzcarbazole) was generously supplied by Dr iK. 1’. Buu-Hoi (Centre National de la Recherche Scientifique, Paris, France). The preparation of a purified phorbol ester fraction (CRA) from Croton tiglium L. seeds has been described in our earlier work [231. Keconstituted tobacco leaf extract and whole cigarette tar were obtained by procedures which we have reported elsewhere [25]. The concentrations used in the various experiments are reported with the results of individual experiments. In early studies, the substances to be tested were dissolved in 95 per cent ethyl alcohol to yield a final concentration of 0.1 per cent ethyl alcohol in the culture medium. Subsequently, dimethylsulfoxide (experimental drug grade, Crown Zellerbach Corp.) was used as the solvent at the same concentration, although 1 per cent dimethylsulfoxide did not inhibit the cloning efficiency of 3T3 cells. Control media for appropriate experiments contained comparable solvent concentrations. The test materials were usually added 24 h after plating in transformation or long-term growth studies and at the time of plating for cloning studies.
RESULTS To.xicity
of hydrocnrbons
The toxic effect of 7,12-dimethylbenz(a)anthracene (DMKA) on the cellular proliferation of untransformed and polyoma virus-transformed cells of the 3TR line is shown in Fig. 1. At a level of 0.001 pg/ml DPtlB,4 no substantial inhibition was observed n-ith either line of cells. At the higher (loses of 0.01 and 0.1 pg/ml the behavior of the polyomatransformeti cells is of interest in view of the various reports that ascribe to transformed cells an increase in resistance to the toxic eflects of carcinogenic 37
~ 681805
Experimental
Cell Research 49
574
A. Sivak and B. I,. van Duuren
aromatic hydrocarbons [l, 6, 261. After exposure for 4 days, the virustransformed cells appeared to be more resistant to DMBA than cells of the 3T3 line. On the other hand, between 4 and 8 days when the cells were growing at a logarithmic rate, the results indicate that the polyoma-transformed line was more sensitive to DMBA toxicity than 3T3 cells. Similar results
Fig. I.-Effect of 7,1X-dimethylbenz(a)allthracene on growth of untransformed and polyomatransformed 3T3 mouse fibroblasts. q , 3T3 polyoma transferred; W, 3T3, untransformed. Abscissa: DMBA concentration in medium, pg/ml; ordinate: growth, % of control.
were observed after 11 days when both cultures began to decrease in growth rate. 3T3 Cultures were exposed for several weeks during their growth phase to an apparently non-toxic dose of DMBA and were subsequently cloned. The results of this experiment are shown in Table 1. Thus, exposure of 3T3 cultures to this carcinogen had no apparent effect on the immediate rate of growth of mass cultures; however, the ability of single cells from these exposed populations to grow out as colonies was reduced when the cells were transplanted at low density. Exposure of 3T3 cells to benzo(a)pyrene (BP) at 0.1 pg/ml resulted in a similar inhibitory effect on subsequently cloned populations. The toxicity of 3T3 cells to BP appeared to be the result of a change that persisted over at least three serial transplants. After exposure of a growing population to 0.1 ,ug/ml BP for 1, 4 or 7 days followed by a recovery period in normal medium, the cloning efficiencies were increasingly depressed as a function of exposure time as shown in Table 2. Furthermore, a similar pattern was still evident after two subsequent transplants representing more than twenty generations. Colonies of 3T3 cells exhibiting unusual morphology were seen on occasion; however, clones with heritable characteristics of transformed cells, that could be attributed to carcinogen exposure, were not found. Experimenlal
Cell Research 49
Chemical Toxicity
carcinogen
effects in rodent
575
cell culfrrre
of promoters
Studies on the effect of CRA on cellular proliferation of 3T3 cells and their polyoma-transformed variants revealed that 1 pg/ml caused no inhihition of growth. However, exposure of 3T3 cells to CRA at this concentration resulted in a substantial reduction of cloning efficiency, when the exposed cells were subsequently plated in control medium. This effect is similar to that observed with the hydrocarbons. ?LRLE
1. E~eet
of e~pos~~re to carcinogens and promoters efficiency of 3T3 mouse fibroblasts.
on sabse~aen~
eroding
Per cent cloning efficiency. 28 days exposure, Expt 1
20 days exposure, Expt 2
14 days exposure, Expt 3
Control
35.5 (35-36)
11.7 (9.6-13.5)
18 (17-18.5)
carcinogens DMBA (0.001 pg/ml) BP (0.1 pg/ml)
15 (12-18) 7.4 (6.5-8.3)
7.3 (6.5-8)
Promoters CRA (1 +ag/ml) Cigarette smoke condensate
18.5 (17-20)
(1 m/ml) Tobacco leaf extract
6.5 (3.9-7.8)
(1 pg/ml)
11.3 (8.7-13.5)
Under similar conditions, exposure to reconstituted tobacco leaf extract at 1 pg/mI in the medium caused no reduction in subsequent cloning efficiency. Whole cigarette smoke condensate, at the same dose, reduced the cloning efficiency by 50 per cent (Table 1). TABLE 2. To~icif~
fo 3T3
mouse ~~rob~as~s.
Recovery time (days)
Clones/100 cells immediately after treatment
Clones/1000 cells 20 generations after treatment
0
-
1 4 7
19 16 13
22.0 21.6 18.6 11.3
hfonolayer 80 51 23
Treatment time (days)
of beI~:o(a)p~rene
0.1 pg benzo(a)pyrene
per ml medium.
Experimental Cell Research 49
576
A. Siuak and B. L. van Durrren
Fig. %.-Morphology of hamster embryo cultures after 55 days in cuiture Phase contrast, x 40. L4, Gntreated; R, benzo(a)pyrcne-treated.
(10 generations).
As with the carcinogens, morphological alterations \vere usually not ohserved in the 3T3 cultures exposed to tLlIllor-proln~~ting agents. On occasion, however, exposure to the phorhol ester fraction CRA gave rise to dense, multilayered clones in cultures of 3T3. This phenomenon has been deseribed elsewhere [ 17 1. Txansformcrtion
studies
Hamster.-Seven different attempts were made to induce a neoplastic transformation with chemical carcinogens in primary or secondary hamster embryo cultures. Of these, only one series of experiments gave rise to lines of cells that induced tumors when injected subcutaneously into new-horn hamsters as \vell as into cheek pouches of cortisonized young adult hamsters. In the series that gave rise to tumorigenic cells a secondary culture of hamster embryo cells near monolayer was chemically treated for eight days. In addition to the carcinogens ISI’ and DMBA (0.1 ~~g~rnl), pyrene (1 .O pg/ml), which is non-carcinogenic, was also tested. After 6 generations and 29 days Experimental
Cell Resecrrch 49
Chemical
cnrcinogen
erects
in rodent
ii77
cell culture
in culture, inoculation of treated and untreated cells into newborn hamsters yielded no tumor-bearing animals after 9 months. After 7 to 9 generations in culture, cells exposed to BP and DSIBB began to exhibit a loss of contact inhibition and showed a tendency to form dense, multilayered clones (Fig. 2). After 22 generations (98 days) in culture, the karyotypes of the cells were T:\IsI,I:
3. Krrryologicnl
rrnalysis
of hcrmster embryo trentmen t.
Chromosome Treatment
-----
Benzo(a)pyrene, uncloned
--’
Bcnzo(a)pyrene, c!oned DMBA,
uncloned
number
x 35 37 41 42 43 44 45 46 47 4X 58
Control
22 14
-
-
131 -
-
-
131--
1113 1 -
15
fibroblnsts
-
1
--
1
12--1
3 -
2 -
1
Tetraploid
after ccrrcinogen
No. of cells countetl
Pen cent tetraploid
1
23
4.3
15
55
27.3
.7
-I6
10.9
3
22
13.6
analyzed; the results are sho\vn in Table 3. The uncloned population of BP-exposed cells exhibited an increase in the number of tetraploid karyotypes as compared to controls. The cloned BP-treated cells and the uncloned population of DhlBA-treated cells gave the same results. Furthermore, the transformed populations showed a considerable variation in chromosome number (3&M), around the normal diploid number of -M. At the same time that the karyological studies were performed, populations of uncloned control-, BP-, and DXIBA-treated cells were injected subcutaneously into young adult hamsters. No tumors were evident after eight months. \\‘ithin a few additional generations, the control and pyrene-exposed cells degenerated. Thus, the life-time of the hamster embryo cells that were not transformed into lines in this particular experiment was about 23 generations and 105 days in culture, which is an unusually long lifetime for a strain of hamster cells. The BP-transformed line (THE-BP) was carried for further experiments. After 34 generations, THE-BP cells were injected again into newborn hamExperimental
Cell Research 49
A. Sivak and B. L. van Duuren sters, and within 20 weeks palpable tumors that grew rapidly appeared in the injected animals. Subsequently, a cloned and uncloned population of THE-BP cells were examined for tumorigenicity in the hamster cheek pouch and both populations gave rise to tumors in high yield (Table 4). The tumors that gre\v after injection into newborn hamsters were histologically Tanr.~
4. Tumoriyenicity
of henro(cr)pyrene-treated
hamster embryo
fibroblasts. Generations at time of test 7 7 22 34 62
Methoda Newborn, S.C. Adult, S.C. Adult, S.C. Newborn, S.C. Adult, Cheek pouch
Tumor
yield
0 0 0 5/7 S/5 cloned 3/J uncloned
Time to first tumor
20 weeks 22 days 52 days”
Termination (months) 8 8 8 8 2 2
a S.C. = subcutaneous. 0.5-l .O x lo6 cells inoculated. b None seen at 32 days.
identified as fibrosarcomas. Tumors growing in hamster cheek pouches also were fibrosarcomas (Fig. 3). In the other six attempts to induce transformation, control and treated cultures degenerated within 6 to 10 generations with no emergence of lines. In general, the main efrect of the carcinogens on the hamster embryo cell cultures \vas a toxicity that was manifested by granulation of cytoplasm and detachment of cells from the culture dish. In these experiments, the materials that were used in addition to DRIBA and BP \\-ere chrysene (1 ,ug/ml), CHA (1 pg/ml), tobacco leaf extract (1 pg/ml) and cigarette smoke condensate (1 ,ug/ml). The times of exposure ranged from 4 to 8 days and both primary and secondary hamster embryo cultures were used. Transformation
studies
Moose.-Among three separate attempts to induce transformation in mouse embryo fibroblasts with all of the materials listed agove, two resulted in degeneration of cells by 12 generations in culture. In a third experiment, evidence of loss of contact inhibition was observed in control as well as BP-treated cultures after 92 days and about 15 generations in culture. In one experiment, using mouse fibroblasts derived from one-day-old mouse femoral Experimental
Cell Research 49
Fig. J.-Fibrosareomas fibroblasts. Hematoxylin
induced in hamsters by benzo(a)pyrene-transformed hamster embryo and eosin, x 150. A, Subcutaneous sarcoma; B, cheek-pouch sarcoma. Erperimenlal
Cell Research 49
580
A. Sun.4 rend B. L. van Durtren
muscle tissue, alteration to a line was noted at about 16 generations (86 days in culture) in control, pyrene- and HP-treated cells. There appeared to be no change in morphoIogy of the control cells, and the only evidence of formation of a line was the ability of the control cells to grow at low density (lo2 cells per plate). The pyrene- and BP-treated cells assumed the morphology shown in Fig. 4, and these morphological characteristics appeared to breed true. At 54 generations in culture, the cells from the three lines were injected into adult cortisonized mice, and after 12 months, there were no palpable tumors. DISCUSSION
Toxicity Differences between untransformed and transformed cell populations to the toxic effects of aromatic hydrocarbon carcinogens have been observed in a variety of experimental situations [l, 2, 6, 15, 261. Notably, Diamond [6] and Berwald and Sachs [2] compared normal populations of rodent cells and their viral- and chemically-induced transformants, respectively. In both cases, the transformed populations were markedly more resistant to the toxic effects of BP than comparable untransformed cells. On the other hand, Experimental
Celf Kesenreh 49
Chemical cnrrinoyen
effects in rodent cell cttltrtre
581
the results obtained in the present study with DMBA and two lines of 3T3 mouse fibroblasts suggest that generalizations concerning the differences in carcinogen toxicity to normal and neoplastic cells [l, 6, 261 be viewed with some caution. The results obtained in the present study suggest that the inhibition of growth of mass cultures as an index of toxicity is a relatively insensitive parameter when compared to cloning efficiency. The results obtained with 3T3 cells exposed to DXIRA are particularly instructive in this respect. At a ~(~ncelltratioll of 0.001 pg/ml there was no depression of growth of the exposed population; however, if these cells were s~ihseqLIeI~tly cloned, a substantial reduction in cloning efficiency was observed. Rerwald and Sachs [‘L] showed that 131’.transformed hamster embryo cells were 1000 times more resistant to the toxic effect of RP on cloning efficiency \vhcn compared to untransformed cells. These effects were evident 150 days after the carcinogen was removed. \\‘hen a recovery period of 13 to 19 days was introduced between exposure to BP and cloning in our experiments with 3T3 mouse fibroblasts, the toxic effect was still observed and persisted through at least twenty subsequent cell generations. These results suggested that a permanent change in the sensitivity of the cell pop~llation to BP had occurred, altho~lgll properties characteristic of neoplastic fells in culture lx-ere not observed. Similar elfects were observed on the cloning efficiency of 3’1’3 cells previously exposed to CRA and cigarette smoke condensate, and the two materials \vere approximately equally toxic. On the other hand, whole tobacco leaf extract was not toxic at the dose employed (1 lug/ml). It is worthy of notice that the promoting activity of CRA is 1000 times as great, on a weight basis, as cigarette smoke condensate when tested by mouse skin painting [a,?]. These results suggest that tumor promotion in viuo probably does not involve a selection brought about by some general toxicity of the promoting agent, since tobacco leaf extract was not toxic to 3T3 cells at the dose employed, and its potency as a promoter is similar to that of the smoke condensate 1251. Trrrnsfornmtion Cntil quite recently, there has been no convincing evidence that aromatic hydrocarbon carcinogens could induce a neoplastic change in a population of cells in culture. Several investigators [2, 3, 14, 151 have reported on their successful efforts to isolate neoplastic cells from populations of cells in culture following exposure to RP and other carcinogens. Our experiments indicate that the occurrence of transformation in a hamster cell culture following carcinogen exposure is a rare event. The work of Huberman and Sachs E~perimen~u~ Ceil Research 49
552
A. Sivnk
and R. L. van Lhrren
[ISI also suggests this, and moreover, their results clearly show that not all cells of a population are susceptible to the induction of transformation by BP. With reference to the analytical methods used in assessing transformation in vitro, Gottlieb-Stematsky and co-workers [12, 131 have shown that cloning exerts a selective efrect which permits the outgrowth of neoplastic cells from hamster embryo populations in preference to normal cell types which predominate in mass cultures. Furthermore, morphological properties usually attribL~te~~ to neoplastic cells in culture, especially loss of contact inhibiti~~n, do not appear to be completely reliable markers [*5, 181. Another factor in the analysis of tumorigenicity of cultured cell lines is the duration of cultivation after the transforming event until in ~&IO assay. with Todaro et al. [22] found that hamster embryo fibroblasts transformed poiyoma virus in vitro did not reach their “full neoplastic potential” in viva, i.e., ability to produce tumors, until the cells had grown for more than 40 in our own work, hamster embryo cells generations in culture. Similarly, that showed altered morphology typical of transformed populations 7 generations after BP treatment did not give rise to tumors in uino. Ho\\-ever, after the cells had grown in culture for 31 generations, they readily produced tumors when injected into new-born hamsters. On the basis of the available evidence, it is not possible to state with ccrtainty whether selective or direct mutagenic events or both obtain \vhen a population of cells is exposed to a chemical carcinogen. However, although the mechanisms are not clear, it has been established that chemical carcinogens, in addition to their l~re~l~)~linating toxic effects, can give rise to transformed variant cells in vitro that ultimately have the potential to grow as tumors in uivo. The refinement of in vitro techniques for the induction of transformation by chemical carcinogens should provide a valuable additional tool to study mechanisms of carcinogenesis at the cellular level. SUMMARY
Transformation into permanent cell lines of hamster embryo fibroblasts was induced by benzo(a)pyrene and 7,12-dimethylbenz(a)antbracene. Cells of the transformed lines had altered karyotypes, and the benzo(a)pyreneinduced line was tul~or~ge~lie in hamsters. Several lines of mouse ~broblasts of altered morphology were obtained following pyrene and benzo(a)pyrene exposure, but these cells were not tumorigenic when injected in cortisonized mice. 7,12-Dimethylbenz(a)anthracene inhibited the cellular proliferation of an Experimental
Cell Research 49
Chemictrl
carcinogen
effects in rodent
cell culture
583
untransformed line (3T3) of mouse fibrohlasts to a lesser estent than in a polyoma Tirus-transformed variant of the same line. At doses that were not toxic to growth of mass cultures, exposure to 7,l X-dimethylhenz(a)anthracene caused a reduction in the subsequent cloning efficiency of the 3T3 line. Similar inhibitory effects \vere obtained with tobacco smoke condensate, a phorbol ester fraction from Croton tiglium L. and benzo(a)pyrene. The tosic cfrect of bcnzo(a)pyrene \yas demonstrable 20 generatior,s after esposure. So transformation was observed in the 3T3 cell line with any chemical treatment. The toxic and transforming effects of carcinogens in cell culture systems is discussed. This work was supported by Contract PH43-64-938, grant CA-06989 from the National Cancer Institute, National Institutes of Health and grant No. ES-00014 from the Bureau of State Services, U.S. Public Health Service. The authors acknowledge the aid and helpful criticism of Drs Marvin Kuschner, and Leo Orris and the excellent technical assistance of Miss Frances Ray. REFERENCES 1. ALFKEU, L. .J., GLOBERSOS, A., BERW.%LD, Y. and Pnmx, R. T., Ibit. .I. Cancer 18, I59 (1964). 2. BEHWALD, Y. and SACHS, L., J. Satl Cancer Inst. 35, 641 (1965). 3. BORENFREUND, E., KRIM, M., SAXDERS, F. I<., STERNBERG, S. S. and BWOICH, A., I'roc. Sufl Acad. Sci. 56, 672 (1966). 3. CREECH, E. M. H., Am. J. Cancer 35, 191 (1939). 5. DEFENDI, V., LEIMA~, J. and KRAEMER, P., Virology 19, 592 (1963). 6. DIA~IOND, L., .I. Cellular Camp. I-‘hysio/. 66, 183 (1965). 7. DURAS-RE~NALS, M. L. and STASLEI', B., Science 134, 1984 (1962). 8. DULBECCO, R. and Fmxmas, G., Virofogy 8, 396 (1959). 9. EAXLE, 11'. R., J. Nat! Cancer Inst. 4, 165 (1943). IO. Emm, W. R., SCIULLI~~, E. L. and SKELTOS, E., J. Gaff Cancer Inst. 10, 1067 (1950). 11. F'OLEY, G. E. and HANDLER, A. H., p'roc. Sot. Ezpff Biof. Med. 94, 661 (1957). 12. C~~~LIE~-STE~~.~TSK’, T. and SHILO, R., Virology 22, 314 (1964). 13. Go~~I.I~u-STE~~.~TSKY, T., Yasrr, A. and GOZITH, A., .I. 1Yuff Cancer Insf. 36, 477 (1966). 1-l. HEII~ELRERGER, C. and II'PE, P. T., Science 155, 214 (1966). 15. HUBERMAN, E. and SACIIS, L., Proc. Saff Acad. Sci. 56, 1123 (1966). 16. RIOORWXD, P. S.,~OWELL, P. C.,I\IELLRIAS,\\:. J., BATTIPS, D. M. and HVSQERFORD, D. A., Expff Cell Kes. 20, 613 (1960). 17. SIVAK, h. and VAN L)UURES, B. I,., Scirnce 157, 1444 (196i). 18. STOKER, >I., Virology 24, 165 (1964). 19. TODARO, G. J. and GREEN, H., .J. Ceff Rid. 17, 299 (1963). 10. ~~ - Virology 24, 393 (1964). 21. ~~ Science 147, 513 (1965). 22. TODARO, G. J., NILIIAUSES, K. and GREEN, H., Cancer Res. 23, 825 (1963). 23. \'.m DUURIZN, B. I,., LI\N~SETII, L., SIVAK, 4. and ORRIS, L., Cancer Res. 26, 1729 (1966). 24. \:AS D~UHF.N, B. L. and ORRIS, L., Cancer Res. 25, 1871 (1965). 25. V.4s Duun~s-, B. L., SIVAI(, A., SEG.~L, A., ORRIS, L. and LANGSETII, L., J. Gaff Cancer Inst. 37, 519 (1966). 26. VASILIF.~, J. M. and GUELSTEIS, V. I., J. Saff Cancer Inst. 31, 1123. (1963) 27. YOUSGSER, J. S., Proc. Sot. Expff Biof. Med. 85, 202 (1954).
Experimental
Cell Research 49
Experimental
572
1068
by
Academic
Press
Inc.
Cell Research 49, 572-583 (19C8)
STUDIESWITHCARCINOGENSANDTUMOR-PROMOTING AGENTSINCELLCULTURE A. SIVAK Laboratory Medicine,
and B. L. VAN
of Organic Chemistry New York University
DUUREN
and Carcinogenesis, Institute of Environmental Medical Center, New York, N.Y. 10016, USA
Received April 24, 1967
As
early as 1939, Creech [4] reported an increase of cell proliferation and chromosome abnormalities in mouse embryo fibroblasts treated with dibenz(a,h)anthracene-choleic acid as compared to untreated controls and to cells treated with phenanthrene or acenaphthene-choleic acid. Subsequently, Earle [9, lo] found that 3-methylcholanthrene could give rise to stable morphological alterations in a line of mouse embryo fibroblasts; however, the spontaneous changes that occurred precluded the direct implication of the carcinogen as the transforming agent. More recently, several groups of investigators have showed that carcinogenic aromatic hydrocarbons could induce heritable alterations in rodent cells in culture and that these transformed cell lines were tumorigenic in appropriate hosts [2, 3, I-21. These findings suggest that tissue culture systems may be of value in the study of cellular response to chemical agents, particularly the phenomenon of initiation and promotion in tumor induction and the screening of potential carcinogenic and promoting agents, e.g. from tobacco leaf and cigarette smoke condensate. The tumor-promoting activity of croton resin and of phorbol esters derived from croton resin [23, 241 and of tobacco leaf and smoke components [25] on mouse skin has been the subject of several reports from this laboratory; therefore, it was of interest to study the effects of these materials in cell culture. This communication describes the toxic and, in some cases, transforming effects of phorbol esters, fractions derived from cigarette smoke condensate and tobacco leaf extracts, and various carcinogens in several populations of mouse and hamster cells. MATERIALS
AND
METHODS
The 3T3 line of mouse fibroblasts and its polyoma- and SV40-transformed sublines were obtained through the courtesy of Dr G. J. Todaro and Dr H. Green, Department of Pathology, New York University School of Medicine. The behavior of this cell line as grown in our laboratory closely paralleled that described by Todaro and Green [19, 20, 211. Experimental
Cell Research 49
Chemicd
cctrcinogen
erects
in rodent
cell culture
558
In addition to these cell lines, primary and secondary mouse embryo (Swiss, HA/ICR) and Syrian hamster embryo cell cultures were prepared by the techniques described by Youngner [27]. The medium employed was Dulbecco’s modification of Eagle’s formulation containing 10 per cent calf serum [8]. Cultures were fed twice weekly with 4 ml of medium, and they were maintained by serial passage of monolayer cultures at lo5 cells per plate. Polystyrene culture dishes (Falcon 3002, 60 x 15 Plates for cloning or for the determination of mm) were employed throughout. lransformed areas were washed with phosphate-buffered saline (PBS), stained either with Giemsa stain for 45 minutes after methyl alcohol fixation, or with Harris’s hcmatoxylin after fixation in 10 per cent formalin-PBS. Karyological analyses were carried out essentially according to the method of Moorhead rt a/. 1161. Experiments to determine the tumorigenicity of the various populations of cells under study were carried out as follows: (a) Subcutaneous injection in adult hamsters; (b) subcutaneous injection in l-day-old hamsters; (c) implantation in cheek pouches of cortisonized hamsters [ll]; (d) subcutaneous injection in cortisonized mice [‘il. Test maferials.--Aromatic hydrocarbons, with the exception of chrysene, were obtained from commercial sources and were purified by column chromatography and/or recrystallization. Purity was confirmed by thin-layer chromatography, melting points and ultra-violet absorption spectra. A pure sample of chrysene (free of linear benzcarbazole) was generously supplied by Dr iK. 1’. Buu-Hoi (Centre National de la Recherche Scientifique, Paris, France). The preparation of a purified phorbol ester fraction (CRA) from Croton tiglium L. seeds has been described in our earlier work [231. Keconstituted tobacco leaf extract and whole cigarette tar were obtained by procedures which we have reported elsewhere [25]. The concentrations used in the various experiments are reported with the results of individual experiments. In early studies, the substances to be tested were dissolved in 95 per cent ethyl alcohol to yield a final concentration of 0.1 per cent ethyl alcohol in the culture medium. Subsequently, dimethylsulfoxide (experimental drug grade, Crown Zellerbach Corp.) was used as the solvent at the same concentration, although 1 per cent dimethylsulfoxide did not inhibit the cloning efficiency of 3T3 cells. Control media for appropriate experiments contained comparable solvent concentrations. The test materials were usually added 24 h after plating in transformation or long-term growth studies and at the time of plating for cloning studies.
RESULTS To.xicity
of hydrocnrbons
The toxic effect of 7,12-dimethylbenz(a)anthracene (DMKA) on the cellular proliferation of untransformed and polyoma virus-transformed cells of the 3TR line is shown in Fig. 1. At a level of 0.001 pg/ml DPtlB,4 no substantial inhibition was observed n-ith either line of cells. At the higher (loses of 0.01 and 0.1 pg/ml the behavior of the polyomatransformeti cells is of interest in view of the various reports that ascribe to transformed cells an increase in resistance to the toxic eflects of carcinogenic 37
~ 681805
Experimental
Cell Research 49
574
A. Sivak and B. I,. van Duuren
aromatic hydrocarbons [l, 6, 261. After exposure for 4 days, the virustransformed cells appeared to be more resistant to DMBA than cells of the 3T3 line. On the other hand, between 4 and 8 days when the cells were growing at a logarithmic rate, the results indicate that the polyoma-transformed line was more sensitive to DMBA toxicity than 3T3 cells. Similar results
Fig. I.-Effect of 7,1X-dimethylbenz(a)allthracene on growth of untransformed and polyomatransformed 3T3 mouse fibroblasts. q , 3T3 polyoma transferred; W, 3T3, untransformed. Abscissa: DMBA concentration in medium, pg/ml; ordinate: growth, % of control.
were observed after 11 days when both cultures began to decrease in growth rate. 3T3 Cultures were exposed for several weeks during their growth phase to an apparently non-toxic dose of DMBA and were subsequently cloned. The results of this experiment are shown in Table 1. Thus, exposure of 3T3 cultures to this carcinogen had no apparent effect on the immediate rate of growth of mass cultures; however, the ability of single cells from these exposed populations to grow out as colonies was reduced when the cells were transplanted at low density. Exposure of 3T3 cells to benzo(a)pyrene (BP) at 0.1 pg/ml resulted in a similar inhibitory effect on subsequently cloned populations. The toxicity of 3T3 cells to BP appeared to be the result of a change that persisted over at least three serial transplants. After exposure of a growing population to 0.1 ,ug/ml BP for 1, 4 or 7 days followed by a recovery period in normal medium, the cloning efficiencies were increasingly depressed as a function of exposure time as shown in Table 2. Furthermore, a similar pattern was still evident after two subsequent transplants representing more than twenty generations. Colonies of 3T3 cells exhibiting unusual morphology were seen on occasion; however, clones with heritable characteristics of transformed cells, that could be attributed to carcinogen exposure, were not found. Experimenlal
Cell Research 49
Chemical Toxicity
carcinogen
effects in rodent
575
cell culfrrre
of promoters
Studies on the effect of CRA on cellular proliferation of 3T3 cells and their polyoma-transformed variants revealed that 1 pg/ml caused no inhihition of growth. However, exposure of 3T3 cells to CRA at this concentration resulted in a substantial reduction of cloning efficiency, when the exposed cells were subsequently plated in control medium. This effect is similar to that observed with the hydrocarbons. ?LRLE
1. E~eet
of e~pos~~re to carcinogens and promoters efficiency of 3T3 mouse fibroblasts.
on sabse~aen~
eroding
Per cent cloning efficiency. 28 days exposure, Expt 1
20 days exposure, Expt 2
14 days exposure, Expt 3
Control
35.5 (35-36)
11.7 (9.6-13.5)
18 (17-18.5)
carcinogens DMBA (0.001 pg/ml) BP (0.1 pg/ml)
15 (12-18) 7.4 (6.5-8.3)
7.3 (6.5-8)
Promoters CRA (1 +ag/ml) Cigarette smoke condensate
18.5 (17-20)
(1 m/ml) Tobacco leaf extract
6.5 (3.9-7.8)
(1 pg/ml)
11.3 (8.7-13.5)
Under similar conditions, exposure to reconstituted tobacco leaf extract at 1 pg/mI in the medium caused no reduction in subsequent cloning efficiency. Whole cigarette smoke condensate, at the same dose, reduced the cloning efficiency by 50 per cent (Table 1). TABLE 2. To~icif~
fo 3T3
mouse ~~rob~as~s.
Recovery time (days)
Clones/100 cells immediately after treatment
Clones/1000 cells 20 generations after treatment
0
-
1 4 7
19 16 13
22.0 21.6 18.6 11.3
hfonolayer 80 51 23
Treatment time (days)
of beI~:o(a)p~rene
0.1 pg benzo(a)pyrene
per ml medium.
Experimental Cell Research 49
576
A. Siuak and B. L. van Durrren
Fig. %.-Morphology of hamster embryo cultures after 55 days in cuiture Phase contrast, x 40. L4, Gntreated; R, benzo(a)pyrcne-treated.
(10 generations).
As with the carcinogens, morphological alterations \vere usually not ohserved in the 3T3 cultures exposed to tLlIllor-proln~~ting agents. On occasion, however, exposure to the phorhol ester fraction CRA gave rise to dense, multilayered clones in cultures of 3T3. This phenomenon has been deseribed elsewhere [ 17 1. Txansformcrtion
studies
Hamster.-Seven different attempts were made to induce a neoplastic transformation with chemical carcinogens in primary or secondary hamster embryo cultures. Of these, only one series of experiments gave rise to lines of cells that induced tumors when injected subcutaneously into new-horn hamsters as \vell as into cheek pouches of cortisonized young adult hamsters. In the series that gave rise to tumorigenic cells a secondary culture of hamster embryo cells near monolayer was chemically treated for eight days. In addition to the carcinogens ISI’ and DMBA (0.1 ~~g~rnl), pyrene (1 .O pg/ml), which is non-carcinogenic, was also tested. After 6 generations and 29 days Experimental
Cell Resecrrch 49
Chemical
cnrcinogen
erects
in rodent
ii77
cell culture
in culture, inoculation of treated and untreated cells into newborn hamsters yielded no tumor-bearing animals after 9 months. After 7 to 9 generations in culture, cells exposed to BP and DSIBB began to exhibit a loss of contact inhibition and showed a tendency to form dense, multilayered clones (Fig. 2). After 22 generations (98 days) in culture, the karyotypes of the cells were T:\IsI,I:
3. Krrryologicnl
rrnalysis
of hcrmster embryo trentmen t.
Chromosome Treatment
-----
Benzo(a)pyrene, uncloned
--’
Bcnzo(a)pyrene, c!oned DMBA,
uncloned
number
x 35 37 41 42 43 44 45 46 47 4X 58
Control
22 14
-
-
131 -
-
-
131--
1113 1 -
15
fibroblnsts
-
1
--
1
12--1
3 -
2 -
1
Tetraploid
after ccrrcinogen
No. of cells countetl
Pen cent tetraploid
1
23
4.3
15
55
27.3
.7
-I6
10.9
3
22
13.6
analyzed; the results are sho\vn in Table 3. The uncloned population of BP-exposed cells exhibited an increase in the number of tetraploid karyotypes as compared to controls. The cloned BP-treated cells and the uncloned population of DhlBA-treated cells gave the same results. Furthermore, the transformed populations showed a considerable variation in chromosome number (3&M), around the normal diploid number of -M. At the same time that the karyological studies were performed, populations of uncloned control-, BP-, and DXIBA-treated cells were injected subcutaneously into young adult hamsters. No tumors were evident after eight months. \\‘ithin a few additional generations, the control and pyrene-exposed cells degenerated. Thus, the life-time of the hamster embryo cells that were not transformed into lines in this particular experiment was about 23 generations and 105 days in culture, which is an unusually long lifetime for a strain of hamster cells. The BP-transformed line (THE-BP) was carried for further experiments. After 34 generations, THE-BP cells were injected again into newborn hamExperimental
Cell Research 49
A. Sivak and B. L. van Duuren sters, and within 20 weeks palpable tumors that grew rapidly appeared in the injected animals. Subsequently, a cloned and uncloned population of THE-BP cells were examined for tumorigenicity in the hamster cheek pouch and both populations gave rise to tumors in high yield (Table 4). The tumors that gre\v after injection into newborn hamsters were histologically Tanr.~
4. Tumoriyenicity
of henro(cr)pyrene-treated
hamster embryo
fibroblasts. Generations at time of test 7 7 22 34 62
Methoda Newborn, S.C. Adult, S.C. Adult, S.C. Newborn, S.C. Adult, Cheek pouch
Tumor
yield
0 0 0 5/7 S/5 cloned 3/J uncloned
Time to first tumor
20 weeks 22 days 52 days”
Termination (months) 8 8 8 8 2 2
a S.C. = subcutaneous. 0.5-l .O x lo6 cells inoculated. b None seen at 32 days.
identified as fibrosarcomas. Tumors growing in hamster cheek pouches also were fibrosarcomas (Fig. 3). In the other six attempts to induce transformation, control and treated cultures degenerated within 6 to 10 generations with no emergence of lines. In general, the main efrect of the carcinogens on the hamster embryo cell cultures \vas a toxicity that was manifested by granulation of cytoplasm and detachment of cells from the culture dish. In these experiments, the materials that were used in addition to DRIBA and BP \\-ere chrysene (1 ,ug/ml), CHA (1 pg/ml), tobacco leaf extract (1 pg/ml) and cigarette smoke condensate (1 ,ug/ml). The times of exposure ranged from 4 to 8 days and both primary and secondary hamster embryo cultures were used. Transformation
studies
Moose.-Among three separate attempts to induce transformation in mouse embryo fibroblasts with all of the materials listed agove, two resulted in degeneration of cells by 12 generations in culture. In a third experiment, evidence of loss of contact inhibition was observed in control as well as BP-treated cultures after 92 days and about 15 generations in culture. In one experiment, using mouse fibroblasts derived from one-day-old mouse femoral Experimental
Cell Research 49
Fig. J.-Fibrosareomas fibroblasts. Hematoxylin
induced in hamsters by benzo(a)pyrene-transformed hamster embryo and eosin, x 150. A, Subcutaneous sarcoma; B, cheek-pouch sarcoma. Erperimenlal
Cell Research 49
580
A. Sun.4 rend B. L. van Durtren
muscle tissue, alteration to a line was noted at about 16 generations (86 days in culture) in control, pyrene- and HP-treated cells. There appeared to be no change in morphoIogy of the control cells, and the only evidence of formation of a line was the ability of the control cells to grow at low density (lo2 cells per plate). The pyrene- and BP-treated cells assumed the morphology shown in Fig. 4, and these morphological characteristics appeared to breed true. At 54 generations in culture, the cells from the three lines were injected into adult cortisonized mice, and after 12 months, there were no palpable tumors. DISCUSSION
Toxicity Differences between untransformed and transformed cell populations to the toxic effects of aromatic hydrocarbon carcinogens have been observed in a variety of experimental situations [l, 2, 6, 15, 261. Notably, Diamond [6] and Berwald and Sachs [2] compared normal populations of rodent cells and their viral- and chemically-induced transformants, respectively. In both cases, the transformed populations were markedly more resistant to the toxic effects of BP than comparable untransformed cells. On the other hand, Experimental
Celf Kesenreh 49
Chemical cnrrinoyen
effects in rodent cell cttltrtre
581
the results obtained in the present study with DMBA and two lines of 3T3 mouse fibroblasts suggest that generalizations concerning the differences in carcinogen toxicity to normal and neoplastic cells [l, 6, 261 be viewed with some caution. The results obtained in the present study suggest that the inhibition of growth of mass cultures as an index of toxicity is a relatively insensitive parameter when compared to cloning efficiency. The results obtained with 3T3 cells exposed to DXIRA are particularly instructive in this respect. At a ~(~ncelltratioll of 0.001 pg/ml there was no depression of growth of the exposed population; however, if these cells were s~ihseqLIeI~tly cloned, a substantial reduction in cloning efficiency was observed. Rerwald and Sachs [‘L] showed that 131’.transformed hamster embryo cells were 1000 times more resistant to the toxic effect of RP on cloning efficiency \vhcn compared to untransformed cells. These effects were evident 150 days after the carcinogen was removed. \\‘hen a recovery period of 13 to 19 days was introduced between exposure to BP and cloning in our experiments with 3T3 mouse fibroblasts, the toxic effect was still observed and persisted through at least twenty subsequent cell generations. These results suggested that a permanent change in the sensitivity of the cell pop~llation to BP had occurred, altho~lgll properties characteristic of neoplastic fells in culture lx-ere not observed. Similar elfects were observed on the cloning efficiency of 3’1’3 cells previously exposed to CRA and cigarette smoke condensate, and the two materials \vere approximately equally toxic. On the other hand, whole tobacco leaf extract was not toxic at the dose employed (1 lug/ml). It is worthy of notice that the promoting activity of CRA is 1000 times as great, on a weight basis, as cigarette smoke condensate when tested by mouse skin painting [a,?]. These results suggest that tumor promotion in viuo probably does not involve a selection brought about by some general toxicity of the promoting agent, since tobacco leaf extract was not toxic to 3T3 cells at the dose employed, and its potency as a promoter is similar to that of the smoke condensate 1251. Trrrnsfornmtion Cntil quite recently, there has been no convincing evidence that aromatic hydrocarbon carcinogens could induce a neoplastic change in a population of cells in culture. Several investigators [2, 3, 14, 151 have reported on their successful efforts to isolate neoplastic cells from populations of cells in culture following exposure to RP and other carcinogens. Our experiments indicate that the occurrence of transformation in a hamster cell culture following carcinogen exposure is a rare event. The work of Huberman and Sachs E~perimen~u~ Ceil Research 49
552
A. Sivnk
and R. L. van Lhrren
[ISI also suggests this, and moreover, their results clearly show that not all cells of a population are susceptible to the induction of transformation by BP. With reference to the analytical methods used in assessing transformation in vitro, Gottlieb-Stematsky and co-workers [12, 131 have shown that cloning exerts a selective efrect which permits the outgrowth of neoplastic cells from hamster embryo populations in preference to normal cell types which predominate in mass cultures. Furthermore, morphological properties usually attribL~te~~ to neoplastic cells in culture, especially loss of contact inhibiti~~n, do not appear to be completely reliable markers [*5, 181. Another factor in the analysis of tumorigenicity of cultured cell lines is the duration of cultivation after the transforming event until in ~&IO assay. with Todaro et al. [22] found that hamster embryo fibroblasts transformed poiyoma virus in vitro did not reach their “full neoplastic potential” in viva, i.e., ability to produce tumors, until the cells had grown for more than 40 in our own work, hamster embryo cells generations in culture. Similarly, that showed altered morphology typical of transformed populations 7 generations after BP treatment did not give rise to tumors in uino. Ho\\-ever, after the cells had grown in culture for 31 generations, they readily produced tumors when injected into new-born hamsters. On the basis of the available evidence, it is not possible to state with ccrtainty whether selective or direct mutagenic events or both obtain \vhen a population of cells is exposed to a chemical carcinogen. However, although the mechanisms are not clear, it has been established that chemical carcinogens, in addition to their l~re~l~)~linating toxic effects, can give rise to transformed variant cells in vitro that ultimately have the potential to grow as tumors in uivo. The refinement of in vitro techniques for the induction of transformation by chemical carcinogens should provide a valuable additional tool to study mechanisms of carcinogenesis at the cellular level. SUMMARY
Transformation into permanent cell lines of hamster embryo fibroblasts was induced by benzo(a)pyrene and 7,12-dimethylbenz(a)antbracene. Cells of the transformed lines had altered karyotypes, and the benzo(a)pyreneinduced line was tul~or~ge~lie in hamsters. Several lines of mouse ~broblasts of altered morphology were obtained following pyrene and benzo(a)pyrene exposure, but these cells were not tumorigenic when injected in cortisonized mice. 7,12-Dimethylbenz(a)anthracene inhibited the cellular proliferation of an Experimental
Cell Research 49
Chemictrl
carcinogen
effects in rodent
cell culture
583
untransformed line (3T3) of mouse fibrohlasts to a lesser estent than in a polyoma Tirus-transformed variant of the same line. At doses that were not toxic to growth of mass cultures, exposure to 7,l X-dimethylhenz(a)anthracene caused a reduction in the subsequent cloning efficiency of the 3T3 line. Similar inhibitory effects \vere obtained with tobacco smoke condensate, a phorbol ester fraction from Croton tiglium L. and benzo(a)pyrene. The tosic cfrect of bcnzo(a)pyrene \yas demonstrable 20 generatior,s after esposure. So transformation was observed in the 3T3 cell line with any chemical treatment. The toxic and transforming effects of carcinogens in cell culture systems is discussed. This work was supported by Contract PH43-64-938, grant CA-06989 from the National Cancer Institute, National Institutes of Health and grant No. ES-00014 from the Bureau of State Services, U.S. Public Health Service. The authors acknowledge the aid and helpful criticism of Drs Marvin Kuschner, and Leo Orris and the excellent technical assistance of Miss Frances Ray. REFERENCES 1. ALFKEU, L. .J., GLOBERSOS, A., BERW.%LD, Y. and Pnmx, R. T., Ibit. .I. Cancer 18, I59 (1964). 2. BEHWALD, Y. and SACHS, L., J. Satl Cancer Inst. 35, 641 (1965). 3. BORENFREUND, E., KRIM, M., SAXDERS, F. I<., STERNBERG, S. S. and BWOICH, A., I'roc. Sufl Acad. Sci. 56, 672 (1966). 3. CREECH, E. M. H., Am. J. Cancer 35, 191 (1939). 5. DEFENDI, V., LEIMA~, J. and KRAEMER, P., Virology 19, 592 (1963). 6. DIA~IOND, L., .I. Cellular Camp. I-‘hysio/. 66, 183 (1965). 7. DURAS-RE~NALS, M. L. and STASLEI', B., Science 134, 1984 (1962). 8. DULBECCO, R. and Fmxmas, G., Virofogy 8, 396 (1959). 9. EAXLE, 11'. R., J. Nat! Cancer Inst. 4, 165 (1943). IO. Emm, W. R., SCIULLI~~, E. L. and SKELTOS, E., J. Gaff Cancer Inst. 10, 1067 (1950). 11. F'OLEY, G. E. and HANDLER, A. H., p'roc. Sot. Ezpff Biof. Med. 94, 661 (1957). 12. C~~~LIE~-STE~~.~TSK’, T. and SHILO, R., Virology 22, 314 (1964). 13. Go~~I.I~u-STE~~.~TSKY, T., Yasrr, A. and GOZITH, A., .I. 1Yuff Cancer Insf. 36, 477 (1966). 1-l. HEII~ELRERGER, C. and II'PE, P. T., Science 155, 214 (1966). 15. HUBERMAN, E. and SACIIS, L., Proc. Saff Acad. Sci. 56, 1123 (1966). 16. RIOORWXD, P. S.,~OWELL, P. C.,I\IELLRIAS,\\:. J., BATTIPS, D. M. and HVSQERFORD, D. A., Expff Cell Kes. 20, 613 (1960). 17. SIVAK, h. and VAN L)UURES, B. I,., Scirnce 157, 1444 (196i). 18. STOKER, >I., Virology 24, 165 (1964). 19. TODARO, G. J. and GREEN, H., .J. Ceff Rid. 17, 299 (1963). 10. ~~ - Virology 24, 393 (1964). 21. ~~ Science 147, 513 (1965). 22. TODARO, G. J., NILIIAUSES, K. and GREEN, H., Cancer Res. 23, 825 (1963). 23. \'.m DUURIZN, B. I,., LI\N~SETII, L., SIVAK, 4. and ORRIS, L., Cancer Res. 26, 1729 (1966). 24. \:AS D~UHF.N, B. L. and ORRIS, L., Cancer Res. 25, 1871 (1965). 25. V.4s Duun~s-, B. L., SIVAI(, A., SEG.~L, A., ORRIS, L. and LANGSETII, L., J. Gaff Cancer Inst. 37, 519 (1966). 26. VASILIF.~, J. M. and GUELSTEIS, V. I., J. Saff Cancer Inst. 31, 1123. (1963) 27. YOUSGSER, J. S., Proc. Sot. Expff Biof. Med. 85, 202 (1954).
Experimental
Cell Research 49