Initiation of solC3H10T12 cell transformation by formaldehyde

325

Cancer Letters, 13 (1981) 325-331 o Elsevier/North-Holland Scientific Publishers Ltd.

INITIATION OF C3H/lOT% FORMALDEHYDE

CELL TRANSFORMATION

BY

DANIEL L. RAGAN and CRAIG J. BOREIKO* Chemical Industry Institute of Toxicology, North Carolina 27709 (U.S.A.)

P.O. Box 12137,

Research

Triangle Park,

(Received 1 April 1981) (Revised version received 18 May 1981) (Accepted 20 May 1981)

SUMMARY

The effects of formaldehyde were evaluated in the C3H/lOT% Cl 8 cell transformation system. Treatment of the cells with 0.1-2.5 pg/ml of formaldehyde alone did not result in significant rates of transformation. If formaldehyde treatment was followed by continuous treatment with 0.1 pg/ ml of the tumor promoter 120-tetradecanoyl phorbol-13-acetate (TPA), transformed foci were produced. Methanol and formic acid lacked significant transforming activity under either treatment regimen. The results suggest that formaldehyde is an initiating agent for C3H/lOT% Cl 8 transformation. The fact that some compounds may act solely as initiators should be considered when this transformation system is used to study chemicals which may interact with cells by mechanisms similar to that of formaldehyde.

INTRODUCTION

Formaldehyde is an important commodity chemical with multiple industrial applications [ 5,141. Industrial exposures to formaldehyde are widespread, as are exposures of the general public to formaldehyde from such sources as vehicle exhaust, smog, smoking and certain types of home insulation [ 41. These exposures are of concern in view of formaldehyde’s high chemical reactivity [14] and the fact that formaldehyde has been demonstrated to exert genotoxic effects, albeit weak and somewhat variable, on mammalian cells, yeast, fungi, Drosophila and bacteria [ 1,4]. More importantly, chronic exposure of rats to high concentrations of formaldehyde vapor has recently been reported to induce squamous cell carcino-

*To whom all correspondence

should be sent.

326

mas of the nasal cavity [ 131. There is thus a need to understand the mechanism of formaldehyde carcinogenesis and to evaluate the potential carcinogenic risk it poses for humans. C3H/lOT1/2 Cl 8 mouse embryo fibroblasts [ 111 have been widely used for the detection and study of chemical carcinogens and radiation [ 71. In some instances, the transformation of these cells can be demonstrated to mimic the multi-step nature of in vivo carcinogenesis [12] in that the process of transformation can be separated into the 2 discrete steps of initiation and promotion [ 71. For example, treatment of the cells with subthreshold doses of certain carcinogens [ 7,8] or radiation [6,9] produces low or negligible rates of transformation unless treated cells are subsequently exposed to tumor promoters. The C3H/lOTS transformation bioassay is not, however, generally conducted in the presence of tumor promoters. This leads to the theoretical possibility that compounds which possess only initiating ability would remain undetected by the C3H/lOT1/2 transformation system. In recognition of this theoretical limitation, we have investigated both the ability of formaldehyde to transform C3H/lOT1/2 cells and its ability to act as an initiating agent for the process of transformation. MATERIALS

AND METHODS

C3H/lOT’/z Cl 8 cells [ll], between passages 6 and 12, were used as described by Reznikoff et al. [ 111. The cells were cultured in Basal Medium Eagles with Earle’s Salts (Grand Island Biological Co., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Reheis, Kankakee, IL). All studies and stocks were maintained in the absence of antibiotics and stock cultures were certified to be PPLO free by Flow Laboratories (McLean, VA). Transformation studies were conducted following a modification of the basic protocols of Reznikoff et al. [lo]. Unless otherwise indicated, 2000 cells from logarithmically growing stock cultures were seeded into 60-mm plastic petri dishes (Corning Glassworks, Corning, NY). Cytotoxicity was assessed in parallel dishes simultaneously seeded with 200 cells. The dishes were incubated for 24 h at 37°C in a well-humidified incubator under an atmosphere of 5% CO*. Test compounds were then added to the dishes by micropipet. Formaldehyde (37% w/w; Fisher) and formic acid (EastmanKodak, Rochester, NY) were diluted as necessary with normal Dulbecco’s phosphate-buffered saline (PBS; GIBCO) and added in 25-~1 aliquots. Methanol (HPLC grade; Fisher) was added undiluted in volumes sufficient to achieve the desired final concentration. Each experiment contained negative controls treated only with 25 ~1 of PBS and positive controls treated with 1.0 pg/ml or 0.1 pg/ml of 3-methylcholanthrene (MCA; Eastman-Kodak). MCA controls were treated by first dissolving the MCA in HPLC grade acetone (Fisher), adding this solution to a large volume of medium (0.2%

327

final concentration of acetone), and replacing the plating medium in the di.shes with the MCA containing medium. After an incubation of 24 h, the treatment medium was replaced with fresh medium. Cytotoxicity dishes (46 per treatment group) were fixed and stained with Giemsa 7-9 days after treatment and colonies counted to assess cytotoxicity. Transformation dishes (24..- -48 per treatment group) were fed twice weekly with fresh complete medium until confluent, and once a week thereafter. Before the renewal of medium on day 5 post-treatment, each treatment group was split in 2 as described by Mondal et al. [ 81. One set of dishes received complete medium for the remainder of the experiment. The other set was continuously provided with medium containing 0.1 pg/ml TPA which had first been dissolved in HPLC grade acetone. The final concentration of acetone in the medium was 0.2%. Furthermore, in accordance with the bioassay modification introduced by Bertram [ 21, the concentration of fetal bovine serum in the medium was reduced to 5% in all experimental groups with each renewal of medium performed from day 15 posttreatment to the termination of the experiment. After 6 weeks of incubation, the transformation dishes were fixed and stained with Giemsa. The dishes were then scored for the presence of Type II and Type III foci using the morphological criteria of Reznikoff et al. [lo]. Where appropriate, the statistical significance of results was determined using a Chi-square analysis of the 2 X 2 contingency table, with Yates adjustment. RESULTS

The cytotoxicity of methanol, formaldehyde and formic acid were established in preliminary experiments (data not shown). These data were used to select concentrations for use in transformation studies. Formaldehyde produced significant cytotoxicity for C3H/lOT% cells over a narrow concentration range (0.1-2.5 pg/ml), with an LD,, concentration of approximately 1 pg/ml (Table 1). A single treatment of cells with formaldehyde was generally not sufficient to produce the transformed state (Table 1). The single focus observed following treatment with 2.5 pg/ml of formaldehyde was not statistically significant at the P I 0.05 level. The results obtained when formaldehyde treatment was followed by continuous treatment with the tumor promoter TPA, were dramatically different. Formaldehyde concentrations of 0.1-1.0 pg/ml produced transformed foci in TPAtreated dishes, with focus production increasing in a dose-dependent fashion. Focus production then declined at 2.5 pg/ml of formaldehyde. Only 1 or 2 foci were generally observed per dish, with type II foci outnumbering type III foci by a 4 : 1 ratio. The frequencies of focus production per cell surviving formaldehyde treatment at 0.1, 0.5, 1.0 and 2.5 pg/ml were 9.2 X lo-‘, 4.8 X 10e4, 1.6 X 10m3 and 3.7 X 10m5, respectively. The ability of methanol and formic acid to transform C3H/lOT% cells, or to initiate C3H/lOT% transformation, was also studied (Table 2). Neither

323

TABLE

1

PRODCCTION

OF TRANSFORMED

Compound’ concentration

Formaldeh) de 0.1 pig/ml 0.5 Hg:ml 1.0 pg/ml 2.5 ug/mlb Controls 1 pg/ml MCA 0.1 ug/ml MCA

FOCI

Treatment

Protocol

TPX

4 TPA

Surviving fraction relative to solvent controlC

0131 o/15 Oi-15 1133

2149 14/6’ie 1 9/65e l/40

0.95 0.79 0.56 0.12

5 * + +

_d

9/23e

0.89 0.90

* 0.05 + 0.03

0162

1.0

33110’ 0116 o/52

PBS

BY FORMALDEHYDEa

a Data expressed as (No. of dishes with type II or Type III foci)/total Results are the compilation of 5 experiments. Not all formaldehyde were included in each experiment. b A 1 O-fold excess of cells was plated to compensate for toxicity. ‘= S.E.M. dNot done. ‘Statistically significant at the P < 0.05 level. TABLE

0.04 0.05 0.14 0.02

no. of dishes. concentrations

2

PRODUCTION

OF TRANSFORMED

Compound/ concentration

BY METHANOL

Treatment

Protocol

-TPA

t TPA

Formic 100 500 1000

acid pg/ml pg/ml pglml

Oil9 O/24

Methanol 5 10 20 30 100

mglml mglml mgiml mglml mg/mlb

Controls PBS 1 pg/ml MCA 0.1 bg/ml MCA -.

FOCI

AND

FORMIC

ACIDa

Surviving fraction relative to solvent control’

O/8

o/55 o/55 o/15

0.93 0.70 0.02

+ 0.03 * 0.04 t 0.003

O/4 O/27 0132 O/18 013

0113 3154 O/42 O/24 o/21

1.06 0.95 0.92 0.67 0.04

i + + f *

0126 21122 612.5

Oi63 _d

1.00 0.93 0.98

+ 0.03 + 0.04

16137

a Data expressed as in Table 1. Results are the compilation of 3 experiments compound. Not all doses were included in each experiment. bA 1 O-fold excess of cells was plated to compensate for toxicity. ‘* S.E.M. dNot done.

0.03 0.04 0.05 0.06 0.02

for each

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compound demonstrated significant transforming or initiating activity. A small number of foci were observed in dishes treated with 10 mg/ml of methanol and TPA. However, the number of foci observed was not statistically significant at the P I 0.05level, nor were dose-dependent increases in focus production observed. Thus, while the appearance of a few foci in methanol treated dishes is surprising in view of the history of low spontaneous transformation frequency for the cells [ 71, it would not appear to constitute a positive result. DISCUSSION

These studies indicate that formaldehyde is an initiating agent for C3H/lOTV2 transformation, producing significant numbers of transformed foci only if formaldehyde treatment is followed by continuous treatment with the tumor promoter TPA. Thus, the initiating activity of formaldehyde is similar to, although weaker than, that of ultraviolet light which is also reported by some investigators [ 91, but not others [ 31, to require the addition of tumor promoters to transform C3H/lOTS cells. As has been observed in the TPA-enhanced transformation of X-ray initiated cells [ 61, type II foci were the predominant focus type produced by formaldehyde. The possibility that treatment with concentrations of formaldehyde greater than 2.5 pg/ml might produce transformed foci without the addition of TPA cannot be ruled out, however. Formaldehyde is extremely cytotoxic and preliminary cytotoxicity studies indicated that less than 0.01% of the cells survive 24-h treatment with 4.0 pg/ml of formaldehyde (data not shown). Quantitative studies would be extremely difficult to perform at such cytotoxic concentrations. Both methanol and formic acid lacked significant transforming or initiating activity. These latter observations are important for several reasons. The formaldehyde experiments det.ailed here utilized commercial formaldehyde solutions which contain approximately 10% methanol. A lack of initiating activity by methanol at concentrations 105-fold higher than that present during formaldehyde treatment, suggests that this contaminant is not responsible for the observed initiation of transformation. Furthermore, treatment of C3H/lOTYz cells with methanol concentrations similar to that present during formaldehyde treatment (0.25 pg/ml) also lack initiating activity (data not shown). These studies do not, however, preclude the possibility of a synergistic interaction between methanol and formaldehyde. The lack of initiation of transformation following treatment with formic acid indicates that initiation by formaldehyde is not due to the oxidation of formaldehyde monomer to formate, the predominant route of formaldehyde metabolism in vivo [14]. Thus, the initiating activity of formaldehyde solutions appears to be mediated by formaldehyde. The molecular mechanism for the initiation of transformation by formaldehyde remains obscure, but initiation could be the result of formaldehyde’s ability to react with amino groups in nucleic acids and/or proteins [ 11.

330

Our results have several practical implications. First, they suggest that formaldehyde-induced carcinogenesis in rodents may entail a multi-step process. Specifically, the induction of carcinomas by formaldehyde may require the presence of promotional influences. The identity of hypothetical promoting agents in formaldehyde carcinogenesis remains to be established, but several candidates have been suggested, including formaldehyde itself [ 131. Evidence of multi-step processes in formaldehyde carcinogenesis is of importance to the risk assessment process, because it influences the choice of statistical models used to estimate carcinogenic risk. Of course, initiation and promotion phenomena exhibit strong tissue and species specificity [ 121 and caution must be observed in drawing parallels between the transformation of C3H/lOT% cells and the induction of squamous cell carcinomas in rodents. Finally, these observations demonstrate that the C3H/lOT% transformation assay may be useful as a model system for the study of formaldehyde carcinogenesis. The system should also possess utility for the study of other compounds that might interact with cells by mechanisms similar to that of formaldehyde (e.g., acetaldehyde). Our results further suggest that the study of such compounds be conducted using transformation protocols which have been expanded to include the exposure of chemically-treated cells to tumor promoters. REFERENCES Auerbach, C., Moutschen-Dahmen, M. and Moutschen, J. (1977) Genetic and cytogenetical effects of formaldehyde and related compounds. Mutat. Res., 38, 317--362. Bertram, J.S. (1977) Effects of serum concentrations on the expression of carcinogeninduced transformation in the C3H/lOT% Cl 8 cell line. Cancer Res., 37, 514-523. Chan, G.L. and Little, J.B. (1976) Induction of oncogenic transformation in vitro by ultraviolet light. Nature, 264, 442-444. Formaldehyde - An assessment of its health effects (1980) A report prepared for the Consumer Product Safety Commission by the Board on Toxicology, National Academy of Sciences, Washington, DC. Gerberich, H.R., Stautzenberger, A.L. and Hopkins, W.C. (1980) Formaldehyde. In: Kirk-Othmer Encyclopedia of Chemical Technology, 3rd edn., Vol. 11, 231-250. John Wiley and Sons, New York. Kennedy, A.R., Mondal, S., Heidelberger, C. and Little, J.B. (1978) Enhancement of X-ray transformation by 12-O-tetradecanoyl-phorbol-13-acetate in a cloned line of C3H mouse embryo cells. Cancer Res., 38, 439-443. Mondal, S. (1980) The C3H/lOT% Cl 8 mouse embryo cell line: its use for the study of carcinogenesis and tumor promotion in cell culture. In: Advances in Environmental Toxicology, Vol. 1. Mammalian Cell Transformation by Chemical Carcinogens, pp. 181-211. Editors: N. Mishra, V. Dunkel, M. Mehlman. Senate Press, Princeton Junction, New Jersey. Mondal, S., Brankow, D.W. and Heidelberger, C. (1976) Two-stage chemical oncogenesis in cultures of C3H/lOT% cells. Cancer Res., 36, 2254-2260. Mondal, S. and Heidelberger, C. (1976) Transformation of C3H/l OT% Cl 8 mouse embryo fibroblasts by ultraviolet radiation and a phorbol ester. Nature, 260, 710-711.

331 10 Reznikoff, C.A., Bertram, J.S., Brankow, D.W. and Heidelberger, C. (1973) Quautitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res., 33,3239-3249. 11 Reznikoff, C.A., Brankow, D.W. and Heidelberger, C. (1973) Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res., 33, 3231-3238. 12 Scribner, J.D. and Siiss, R. (1978) Tumor initiation and promotion. Int. Rev. Exp. Pathol., 18, 137-198. 13 Swenberg, J.A., Kerns, W.D., Mitchell, R.I., Gralla, E.J. and Pavkov, K.L. (1980) Induction of squamous cell carcinomas of the rat nasal cavity by inhalation exposure to formaldehyde vapor. Cancer Res., 40, 3398-3402. 14 Walker, J.F. (1975) Formaldehyde, 3rd edn. Robert E. Krieger Publishing Co., Huntington, NY.