In Vitro Cell. Dev. Biol. 28A:267-272, April 1992 © 1992 Tissue Culture Association 0883-8364/92 $01.50+0,00

NEOPLASTIC EXPRESSION IN MURINE CELLS INDUCED BY HALOGENATED HYDROCARBONS

KATHRYN SCHULTZ, LAGNAJITA GHOSH, AND SIPRA BANERJEE 1

Department o.f Molecular Biology, Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195

(Received 29 August 1991; accepted 1 November 1991)

SUMMARY

The neoplastic expression in mouse embryo fibroblasts exposed to 1,2-dibromoethane and its chloroanalogue, 1,2- dichloroethane in vitro, was examined. Both substances are widely used as fumigants for carpet and upholstery, as gasoline additives, and as organic solvents. Both are known to be highly toxic, mutagenic, and carcinogenic agents. C3H10T1/2 cells treated with these haloalkanes exhibited altered morphology and were selected further by cloning in soft agar. Soft agar clones were found to induce a 100% multitumor occurrence in the nude mouse model. These results suggest that this pair of mutagens have altered the normal phenotype of mouse embryo cells, and these cells have become neoplastic. These neoplastic cell lines will be useful as an in vitro model to study the role of genetic changes in the transformation processes induced by halogenated hydrocarbons.

Key words: in vitro transformation; fibroblasts; focus assay; anchorage-independent phenotype; halogenated hydro- carbons.

INTRODUCTION

The haloalkanes 1,2-dibromoethane (DBE) and 1,2-diehloroeth- ane (DCE) are two of the largest volume chemicals manufactured and used all over the worm (Davidson et al., 1982; Fishbein, 1979). Both are extensively used as pesticides, as lead scavengers in gasoline, and as fumigants for soil, fruit, grain, and vegetables (Davidson et al., 1982; Fishbein, 1979). DCE is used in the produc- tion of vinyl chloride, a potent human liver carcinogen, and of trich- loroethylene, an analogue of vinyl chloride. DBE and DCE are also used as intermediates in the synthesis of dyes and pharmaceuticals as well as solvents for resins, gums, waxes, and chemicals. DBE and DCE have been found in ground water around the nation and as contaminants in flour and biscuits. These halocompounds are known to be highly toxic to various organs in animals and humans (Davidson et al., 1982; Ott et al., 1980; Storer et al., 1984; Wong et al., 1982). Human deaths due to acute exposure to DBE have been reported (Letz et al., 1981). Both substances are mutagenic (Crespi et al., 1985; Davidson et al., 1982; Fishbein, 1979) and induce a high incidence of tumors and carcinomas in rodents (NCI, 1978; NTP, 1982; VanDuuren et al., 1979; Wong et al., 1982). On the basis of extensive production, uses, environmental disper- sion, toxicity in animals and humans, and sufficient evidence of carcinogenicity in animals, DBE and DCE have been recognized as human health hazards. Consequently, OSHA has recently lowered the allowable limits of DBE and DCE to which workers may be exposed.

DBE and DCE have been reported to bind covalentty to proteins and DNA of target organs (Banerjee and Van Duuren, 1979; Ban-

1 To whom correspondence should be addressed.

erjee et al., 1979; Banerjee, et al., 1980; Banerjee and Van- Duuren, 1983; Guengerich et al., 1980; Shih and Hill, 1981; Sundheimer et al., 1982). Both microsomal activation and glutathi- onc-S-transferase-catalyzed GSH conjugation pathways are thought to play a vital role in generating toxic, teratogenic, mutagenic, and carcinogenic metabolites (Banerjee, 1988; Cmarik et al., 1990). A major DNA adduct, identified as S-[2-NV-guanyl)ethyl] glutathione, was detected in both in vitro and in vivo systems (Inskeep et al., 1986; Ozawa and Guengericb, 1983). A report from our laboratory previously showed that the transcription and replicative activities of liver nuclei using nuclear DNA as template are suppressed signifi- cantly during DNA damage induced by DCE (Banerjee, 1988). DCE has been shown to enhance the transformation of Syrian ham- ster embryo cells induced by SA7 adenovirus (Hatch et al., 1983).

The present study was undertaken to determine whether expo- sures of C3H 10T1/2 C 18 cells, widely used as an in vitro system to study in vivo carcinogenesis (Chan and Little, 1979; Heidelburger et al., 1983), to DBE and DCE can alter normal phenotype to neoplastic phenotype. The neoplastic phenotype of cells treated with DBE or DCE was determined by several criteria: a) altered morphology, b) ability to divide and form colonies in semisolid me- dium, and c) tumorigenicity in the nude mouse model.

MATERIALS AND METHODS

Cell line and cell culture, The C3H10T1/2 Clone 8, mouse embryo fibroblast cell line, was originally provided to us by Dr. I. B. Weinstein, Columbia University, New York. This cell line was developed and estab- hshed by the late Charles Heidelberger to study the mechanisms of carcino- genesis in an in vitro model (Heidelberger et al., 1983; Meyer, 1983). Cells, Passages 7 to 9, were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated (at 56 ° C for 30 min) fetal bovine serum (GIBCO, Grand Island, NY), L-glutamine, penicillin

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268 SCHULTZ ET AL.

TABLE 1

CYTOTOXICITYAND MORPHOLOGIC TRANSFORMATION OF C3H10T1/2 CL8 CELLS BY DBEAND DCE

Transforming Ax~tivity b Cytotoxic Type II Foci/ Type lII Foci/ Types 11 and Ill Foci/ Foci/Dish

Concentration, Activity, ° Significam~, ~ /.~/ml RCE, % No. Dishes No. Dishes Total No. Dishes P

Acetone control d 100 7/40 0 /40 7/40 0.2 DBE

25 79 20/40 8 /40 28/40 0.7 NS e 100 68 38/40 20/40 58/40 1.5 <0.01 200 53 42/40 45/40 87/40 2.2 <0.001 400 29 ND f ND ND ND

DCE 25 85 ND ND ND ND

200 66 27/24 3 /24 30 /24 1.3 <0.05 400 52 26/24 4 /24 30 /24 1.3 <0.02 600 46 37 /24 25/24 62/24 2.6 <0.01

BaP 2 54 31 /40 51/40 82/40 2.1 <0.001

° Expressed as relative cloning efficiency, number of colonies in treated dishes/number of colonies in control dishes × 100; cloning efficiency of untreated cells was 21 to 31%.

b Total number of foci obtained from two transformation assays using 20 and 12 replicative dishes for the DBE and DCE groups, respectively, per assay.

c Statistical significance compared with acetone control on the basis of foci/dish using a two-sample t test. d Medium contained 0.5% acetone. "NS: not significant, P > 0.05. S ND: not determined.

(100 U/ml), and streptomycin sulfate (100 #g/ml) at 37 ° C in humidified, 5% CO2-air atmosphere as we have used earlier (Banerjee and Segal, 1986). Cells were routinely examined for mycoplasma by Hoechst fluores- cent staining (Freshney, 1984), and cells with no detectable mycoplasma were used.

Determination of cytotoxicity of DBE and DCE. For the cytotoxieity assay, DBE and DCE (purchased from Aldrich Chemical Co., 99%+ pure) were dissolved in acetone. Serial dilutions were then made with DMEM. All the solutions were freshly made and filter-sterilized using 0.2-/,tin filters attached to sterile syringes just before addition to the cells. Two hundred C3H10T1/2 cells were seeded in each 60-mm tissue culture petri dish (Falcon) 24 h before exposure to chemicals as described (Banerjee and Segal, 1986). DBE or DCE was added to the ceils incubated for 48 h at 37 ° C in a humidified COz incubator. After incubation, cells were washed with phosphate buffered saline (PBS) to remove chemicals, fresh medium was added, and the cells were allowed to grow for 7-10 days with one medium change. Bcnzo(a)pyrine (BaP) was used as a positive control, and medium containing the appropriate concentration of acetone served as a negative control. Cells were fixed in 70% methanol and stained with 10% Giemsa. Colonies containing more than 20 cells were counted. The cytotoxic activity of DBE, DCE, or BaP was expressed as relative cloning efficiency (%) of cells treated with chemical in comparison to control.

Determination of morphologic transformation. For the transformation assay, 2 × 103 cells were seeded in a 60-mm dish and allowed to attach for 24 h. DBE, DCE or BaP at different concentrations (selected from the cytotoxicity assay) were added in a 5-ml vol to the cells, and the cells were exposed to these chemicals for 48 h. As a negative control, cells were incubated in 5 ml medium only. Cells were also incubated in the presence of medium containing acetone, which served as a solvent control. After 48 h exposure, the medium containing chemicals was removed, cells were washed twice with PBS, and the fresh medium containing 10% serum was added. Medium was changed twice weekly until cells reached confluency, then weekly using 5% serum. Cells were allowed to express their trans- formed phenotypes for 6 wk. Cells were then fixed, stained with Giemsa, and scored microscopically for type II and type III following the recom- mended criteria (Cancer Res., 1985). Cytotoxicity assays were carried out in parallel to transformation assays.

Determination of anchorage-independent growth of DBE- and DCE- treated cells. To determine whether DBC- and DCE-treated cells that have lost contact inhibition have become anchorage independent, type III foci were picked from dishes using a stainless steel cloning cylinder (Banerjee et

al., 1986) and grown initially in 35-mm petri dishes (Falcon) using DMEM containing 10% serum and antibiotics. Cells were further passaged 3 to 5 times and mass cultured before they were used for anchorage-independent growth in soft agar assays. The protocol for the assay using 0.33% agar on a 5 nd base layer of 0.5% agar was as previously described (Banerjee and Segal, 1986; Freshney, 1984). As a control, C3H10T1/2 cells, the parent cell line, were used side by side to determine anchorage dependence. Six randomly selected cell tines whose transformations had been induced by DBE or DCE were used in the agar assay. The anchorage-independence was determined by the growth of cells under the conditions of this assay and quantitated by the number of colonies of at least 50 cells.

Agar-positive clones were isolated individually from dishes using a Pas- teur pipette and were made unicellular suspension in DMEM medium by repeated pipetting to separate the clone from agar. Anchorage-independent cells were then grown in 150-cm 2 tissue culture flasks to confluence to test their tumorigenic activities.

Determination of tumorigenic properties of DBE- and DCE-transformed cells. Agar clones, 1 to 5 × 106 cells suspended in PBS buffer, were inoculated subcutaneously at the back of five male athymic Nu/Nu mice (Harlan Sprague-Dawley). A group of five mice was injected with normal C3H10T1/2 cells serving as negative control, with another group of mice left untreated to determine any spontaneous tumor occurrence. Animals were examined weekly. Tumors were excised, weighed, fixed in 10% for- maline, paraffin blocked, stained with hematoxylene and eusin, and histo- pathologically diagnosed.

RESULTS AND DISCUSSION

Results presented in this study provide evidence for a dose-de- pendent cytotoxic effect of DBE and DCE in mouse embryo cells. Observations are summarized in Table ] . With the increase in the

dose of DBE, 25 to 4 0 0 #g /ml , the lethality was increased from 20 to 7 0 % , whereas a ] 5 to 5 4 % increase in lethal effect o f DCE was

observed when the dose was increased from 25 to 6 0 0 / ~ g / m l in C 3 H 1 0 T 1 / 2 cells. On the basis of its cytotoxic effect, DBE at a concentration range of 25 to 2 0 0 t t g /ml was selected to test the transforming activity, as 4 0 0 t tg /ml dose seemed to be highly toxic for the cells. On the basis of the cytotoxic effect of DCE, doses between 200 and 6 0 0 # g / m l were chosen for the transformation

IN VITRO TRANSFORMATION: DBE AND DCE 269

A

i ' : ( ....

B

Or,, D

FIG. 1. Focus-formation assay of C3H10T1/2 cells transformed by DBE, DCE, and BczP. Cells stained with Giemsa after six wk exposure to chemicals and macroscopically represented as dish A, 10T1/2 cells treated with acetone only; dish B, 10T1/2 cells treated with DBE; dish C, 10T1/2 cells treated with DCE; and dish D, lOT1/2 cells treated with BaP.

assay. Thus, both haloalkanes were found to inhibit the cellular growth significantly. Results also showed that DBE is relatively more toxic than DCE, probably due to its active bromine group. DBE and DCE are known to be highly toxic to various tissues such as those in the liver and kidney and in the respiratory, cardiac, and reproductive organs in different species (Ott et al., 1980; Wong et al., 1982) as well as in a hepatocyte tissue culture system (Chang et al., 1985).

Shown in Fig. 1 are typical dishes with cells stained with Giemsa representing acetone control (A), DBE-treated (B), DCE-treated (C), and BaP-treated (D) cells, the latter serving as a positive con- trol. The morphology of the foci of C3H10T1/2 cells treated with DBE and DCE exhibited similar characteristics to foci resulting from cells treated with BaP, a known transforming agent, e.g.,

unstained colonies easily visible by naked eye, stained intensely with Giemsa, loss of contact inhibition property, muhilayered, re- fractory, disoriented, distinct focus boundaries, criss-crossed, some- times chording, and invasive to neighboring flat, monolayer normal cells as shown in Figs. 2 and 3.

To determine the transforming activity of the halohydrocarbons, cells were exposed to DBE and DCE at concentrations of 25 to 200 #g/ml and 200 to 600 gg/ml, respectively. Results (shown in Ta- ble 1) demonstrate a direct dose-dependent increase of transform- ing activity of DBE in C3H10T1/2 cells. The transforming activity expressed as foci per dish was increased consistently with the in- crease of DBE concentration from 25 to 200 #g/ml. Similarly, the activity in DCE-treated cells increased with the dose as the concen- tration was increased from 200 to 600 ~tg/ml. At a concentration of

270 SCHULTZ ET AL.

FIg. 2. Morphology of IOTI/2 cells transformed by DBE using X40 magnification.

200 #g/ml, DBE induced approximately two foci per dish, whereas a concentration of 600 #g/ml was required for DCE having similar effects on cells. This result indicates that DBE probably has more transforming activity than DCE.

To select transformed colonies further, DBE- and DCE-indueed foci were tested for their abilities to grow independently of anchor- age. Results presented in Table 2 demonstrate that both DBE- and DCE-transformed cells formed discrete colonies, indicating that cells were competent for anchorage-independent growth, whereas the untreated parent 1 0 T ] / 2 cells did not grow in soft agar. The expressions of the anchorage-independent phenotype in both DBE-

FIG. 3. Morphology of a representative focus of 10T1/2 cells trans- formed by DBE using ×100 magnification.

and DCE-transformed cells seem to be similar. Inasmuch as six randomly selected transformed cell lines were positive in agar as- says, we did not test the other 14 lines further. We have used these six lines only for our study. Most of the eukaryotic cells are able to divide in vitro only upon attachment to an artificial surface, a prop- erty known as anchorage-dependent growth. When cells are trans- formed either by virus or chemicals, they lose this normal pheno- type and can grow either by anchorage or by suspension in semi- solid medium such as agar. Anchorage-independent growth is considered to be a sensitive and specific marker for transformed

TABLE 2

EXPRESSION OF ANCHORAGE-INDEPENDENT AND TUMORIGENIC PHENOTYPE IN DBE AND DCE

TRANSFORMED CELLS

Grow~ i. Soft Agar

Colonies/ Colony Forming Cells No. of Dishes ~ Efficiency b Tumorigenicity ~

N o t r e a t m e n t - - - - 0/5 Untreated C3H10T1/2 0/18 - - 0/5 DBE 3.1 × 103/18 0.43 + 0.07 10/10 DCE 2.2 × 103/18 0.3 + 0.2 10/10

Total number of clones are from six independent cell lines with three replicate dishes/subculture.

b Expressed as number of agar clones per number of cells used × 100. Expressed as number of mice with tumors per total number of mice

injected with control and transformed cells.

IN VITRO TRANSFORMATION: DBE AND DCE 271

fibroblasts and epithelial cells (Barrett et al., 1979; Greiner et at., 1981; MacPherson and Montagneir, 1964; San et al., 1979).

Table 2 also shows the tumorigenic activities of agar clones of DBE- and DCE-transformed cells. Considering the cost of animals and their maintenance, we have randomly selected two agar cloned cell lines from each DBE- and DCE-treated group for their tumori- genic properties. A 100% occurrence of tumor in nude mice was observed in all four agar cell lines; 50% of these mice developed several tumors (three to six tumors per mouse). The tumors weighed 2 to 4.5 g and were diagnosed as fibrosarcomas. The groups of mice that were inoculated with untreated 10T1/2 cells or that had no treatment seemed to be healthy; no tumor was observed even after 1 yr, according to later autopsy. A strong relationship between ability to grow in soft agar and tumorigenicity in animals has been suggested (Barrett et at., 1979; Meyer, 1983; Shin et at., 1975). In our study, we have established a relationship between the anchor- age-independent growth of morphologically transformed cells and tumorigenicity in nude mice. Thus, these results confirm that DBE- and DCE-treated cells become transformed and acquire anchorage- independence and a neoplastic phenotype.

The transforming activity of DBE has not been reported. DCE is known to enhance viral transformation of SHE cells (Hatch et al., 1983) although it was found to be inactive in a Balb/c3T3 cell transformation assay (Tu et at., 1985). Our results demonstrate that DCE is capable of transforming 10T1/2 cells which are anchorage- independent and tumorigenic. An exactly similar cytotoxic effect was obtained using 25 ttg/ml DCE in both 10T1/2 and Balb/c3T3 cell assays, although the experimental methodologies were differ- ent. The inactiveness of DCE as reported by Tu et al. is probably due to not exposing cells to higher dosages of DCE (higher than 50 #g/ml). To test the transforming activity of DCE, as we have ob- served earlier (Banerjee and VanDuuren, 1979) that DCE cova- lenfly interacts with protein and DNA at a significant lower level than DBE, 10T1/2 cells were exposed in our study to much higher levels of DCE, 200 to 600 gg/ml. At these concentrations, DCE was responsive to transform 10T1/2 cells.

It is well recognized that xenobiotics, either directly or upon metabolic activation, bind covalently to cellular DNA and form DNA adducts. These adducts can be removed by endogeneous repair mechanisms. Instead of error-free repair, if the adducts go through error-prone repair (for example, mutation, rearrangement, dele- tion) the normal phenotype of the cells will be altered. Thus, forma- tion and removal of DNA adducts of DBE and DCE will be one of the critical steps in the transformation processes initiated by them. One of the major challenges of the study of toxicity, mutagenicity, teratogenicity, or carcinogenesis is to identify target gene(s) which, when altered, may change normal cells to a transformed state. It will be interesting and important to study the molecular events involved in growth and proliferation of DBE- and DCE-mediated trans- formed cells. The relevance of growth factors and their receptors, oncogenes, and tumor suppressor genes in the transformation pro- cesses and tumor development are well documented (Balmain and Brown 1988; Bradshaw, 1986; Klein, 1988; Sager, 1989; Wein- berg, 1989; Weinstein, 1988). Expressions of transforming genes and anti-transforming genes in cells and tumors transformed by carcinogenic halogenated hydrocarbons remain to be studied.

ACKNOWLEDGEMENTS

The authors thank Ms. Christine Kassuba for editing and Ms. Laura Tripe# for preparation of the manuscript.

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