Journal of Toxicology and Environmental Health

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Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates M. Toraason , J. S. Bohrman , E. Elmore , G. Wyatt , D. McGregor , S. E. Willington & W. Zajac To cite this article: M. Toraason , J. S. Bohrman , E. Elmore , G. Wyatt , D. McGregor , S. E. Willington & W. Zajac (1991) Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates, Journal of Toxicology and Environmental Health, 34:1, 95-102, DOI: 10.1080/15287399109531550 To link to this article: http://dx.doi.org/10.1080/15287399109531550

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INHIBITION OF INTERCELLULAR COMMUNICATION IN CHINESE HAMSTER V79 CELLS BY FRACTIONATED ASPHALT FUME CONDENSATES

Journal of Toxicology and Environmental Health 1991.34:95-102.

M. Toraason, J. S. Bohrman Centers for Disease Control, National Institute for Occupational Safety and Health, Division of Biomedical and Behavioral Science, Cincinnati, Ohio E. Elmore, G. Wyatt NSI Technology Services Corp., Research Triangle Park, North Carolina D. McGregor, S. E. Willington, W. Zajac Inveresk Research International, Ltd., Musselburgh, Scotland

Asphalt fume condensate is a skin carcinogen in mice, yet this complex mixture contains relatively low levels of known carcinogenic initiators. Consequently, its biological activity has been attributed to the presence of cocarcinogenic or tumor-promoting agents. One of several proposed mechanisms of tumor promotion is inhibition of intercellular communication. In an attempt to determine if asphalt fume has tumorpromoting potential inhibition of intercellular communication was measured in V79 cells exposed to fractionated asphalt fume condensate. Fume from air-blown Arabian crude asphalt was trapped and separated into five fractions by preparative-scale highpressure liquid chromatography. The parent fume condensate and the five fractions inhibited intercellular communication in a concentration-dependent fashion, with a minimum effective concentration of 2.5 µg/ml for the most potent fraction. Cytotoxicity assays were performed at the same time and concentrations as the metabolic cooperation assays. Cytotoxic responses paralleled the inhibition of intercellular communication.

INTRODUCTION A variety of products derived from coal, shale, and crude petroleum have been found to be tumorigenic (Biles et al., 1988; Bingham and Falk, 1969; Clark et al., 1988). The tumorigenicity of petroleum products derived at high temperatures is attributed to increased production of poRequests for reprints should be sent to M. Toraason, Centers for Disease Control, National Institute for Occupational Safety and Health, Division of Biomedical and Behavioral Science, 4676 Columbia Parkway, Cincinnati, OH 45226.

95 Journal of Toxicology and Environmental Health, 34:95-102, 1991 Copyright © 1991 by Hemisphere Publishing Corporation

Journal of Toxicology and Environmental Health 1991.34:95-102.

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lyaromatic hydrocarbons (IARC, 1984; Wallcave et al., 1971). Asphalt has generally been considered nontumorigenic due to the low temperatures involved in its production (IARC, 1984; Wallcave et al., 1971). Concern over the potential carcinogenic hazard in the roofing industry arising from the high temperatures attained during application of asphalt led the National Institute for Occupational Safety and Health (NIOSH) to test coal tar and asphalt fume condensates in a mouse skin painting bioassay (Niemeier et al., 1988). Although asphalt did not exhibit carcinogenic activity, asphalt fume did. Coal tar also exhibited activity, which was attributed to a high concentration of benzo[a]pyrene (Belinky et al., 1988; Niemeier et al., 1988). Asphalt fume, however, does not contain any specific class of known carcinogens at a comparable level that might account for its carcinogenic activity. One conclusion arising from the NIOSH study was that the bioactivity of asphalt fume could be due to the presence of cocarcinogens or tumor promoters. In the two-stage model (initiation-promotion) of chemical carcinogenesis (Berenblum and Shubik, 1947; Boutwell, 1974; Friedewald and Rous, 1944), initiation is considered to be dependent on a chemical interacting with DNA and causing mutation (Miller and Miller, 1981). The mechanism of promotion is less clearly defined, partly because various biochemical responses are induced by tumor promoters (Hecker et al., 1982; Slaga et al., 1978). One such response is inhibition of intercellular communication (Bohrman et al., 1988; Elmore et al., 1987; Klaunig and Ruch, 1987; Trosko et al., 1980,1981; Tsushimoto et al., 1982; Yamasaki, 1987; Yotti et al., 1979). It is believed that inhibition of intercellular communication by a tumor promoter isolates an initiated or preneoplastic cell from growth regulatory signals of surrounding cells, which leads to development of neoplasia. In the present study, the V79 metabolic cooperation assay was used to evaluate the effect of asphalt fume condensate and five separate condensate fractions on intercellular communication. MATERIALS AND METHODS Asphalt Fume Fractions Asphalt fume was generated by heating (316°C) a type III asphalt produced by Exxon, Inc. (Roofing Products, Beacon Sales, Inc., Sommerville, Mass.). The type III asphalt was originally generated by distillation and air blowing of Arabian crude oil. The "parent" fume was collected in a series of cold traps, combined and fractionated into five fractions by preparative-scale high-performance liquid chromatography (Belinky et al., 1988). The fractions were dried and then reconstituted by mixing equal parts (w/v) of fraction and solvent. The solvent used a 50/50 mixture of acetone and cyclohexane, which is the same as that used in the mouse bioassay (Niemeier et al., 1988). The fume fractions were further diluted

ASPHALT FUME INHIBITS INTERCELLULAR COMMUNICATION

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in acetone so that 25- to 50-(i\ aliquots could be added to 5 ml of culture medium in 60-mm culture dishes to attain test concentrations. The solvent alone was nontoxic to V79 cells.

Journal of Toxicology and Environmental Health 1991.34:95-102.

V79 Cell Cultures The 6-thioguanine-sensitive (6TGS) cells were derived from Chinese hamster lung fibroblasts, and the 6-thioguanine-resistant (6TGr) cells were derived from x-ray-irradiated wild-type 6TGS cells. Both lines were kindly provided by Dr. J. E. Trosko of Michigan State University, East Lansing, Mich. Cells were maintained in modified Eagle's minimal essential medium (Gibco, Grand Island, N.Y.), containing 3% fetal bovine serum (Hyclone, Logan, Utah) without antibiotics or fungicides. Cultures were incubated at 37°C in a humidified 5% CO2 air atmosphere. V79 Metabolic Cooperation Assay The V79 metabolic cooperation assay is dependent on the transfer, via gap junctions, of the toxic phosphorylated metabolite of 6thioguanine from wild-type 6TGS cells to 6TGr cells. The 6TGr cells are unable to metabolize 6-thioguanine to a toxic substrate and survive to form colonies when intercellular communication is inhibited. The protocol used here has been previously described (Bohrman et al., 1988; Jone et al., 1987) and will be discussed only briefly. The "metabolic cooperation" between the sensitive and resistant V79 cells was assessed by coculturing 100 6TGr cells with 4 x 105 6TGS cells in 5 ml of medium in 60mm dishes. Asphalt fume solutions were added 4 h after plating 6TGr and 6TGS cells, and 6-thioguanine was added 15 min later to a final concentration of 10 fig/m\. After 3 d, medium was replaced with fresh medium containing only 6-thioguanine but no asphalt fume. Three to 4 d later cells were washed with phosphate-buffered saline, then fixed and stained with 1% crystal violet. A statistically significant increase over the number of treated 6TGr colonies surviving relative to control 6TGr colonies was considered indicative of inhibition of intercellular communication. Cytotoxicity Assays Three preliminary cytotoxicity assays were performed in order to establish a concentration range for the metabolic cooperation assay so that the maximum concentration tested would be marginally toxic. A fourth cytotoxicity assay was performed in parallel with the metabolic cooperation assay. A significant reduction in colonies in treated cultures relative to control cultures was considered a cytotoxic response.

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Statistical Analysis A one-tailed Dunnett's test (p < .01) was used to compare the number of colonies per dish in cocultures treated with the test fractions to the number of colonies per dish in control cocultures treated only with solvent. RESULTS

Journal of Toxicology and Environmental Health 1991.34:95-102.

Asphalt Fume Fractions The five fractions obtained from the parent fume were analyzed by gas chromatography/mass spectroscopy. The mass spectra enabled the characterization of the compounds present in each fraction. Families of chemical compounds in the five fractions are summarized in Table 1. Cytotoxicity Cytotoxic concentrations were not obtained for the parent mixture because insolubility limited the maximum concentration that could be tested. Although the parent asphalt fume condensate did not significantly reduce colony formation in the cytotoxicity assay, a trend was obtained suggesting that the fume was slightly cytotoxic at the maximum concentrations tested (Table 2). A similar solubility problem was evident with fraction A, and none of the concentrations tested were cytotoxic. TABLE 1. Composition of Fractions by Chemical Class as Identified by Gas Chromotography/Mass Spectroscopy Fraction

Composition

A

C9 to C35 alkanes Alkylated benzenes Alkylated naphthalenes

B

Alkylated benzothiophenes Alkylated dibenzothiophenes Alkylated benzonaphthothiophenes Alkylated phenanthrenes C6 to C26 olefins

C

Alkylated phenylethanones C2 to C11 alkylated dihydrofuranones

D

Alkylated phenols Alkylated ketones

E

C6 to C22 alkylated ketones Alkylated naphthols and phenols Note. Reprinted from Belinky et al. (1988).

ASPHALT FUME INHIBITS INTERCELLULAR COMMUNICATION

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TABLE 2. Cytotoxicity of Asphalt Fume Condensates in V79 Cells Fractions Concentration (/ig/ml)

Journal of Toxicology and Environmental Health 1991.34:95-102.

0.0 1.0 2.5 5.0 7.5

10.0 15.0 20.0 25.0 40.0 50.0

Parent

A

97 96 95 90 91 87

78

± 7

115

± 6

95 ± 11

82 80

± 9 ± 8

114 109

± 3 ± 11

80

± 7

108

78

± 8

78

± 9

± + + ± ± ±

10 14 8 12 6 14

B

C

92

9

E

D

100 + 14 92 ± 12

74 82 74 73

± ± ± ±

10 9 9 5

62 ± 7 62 ± 7 60 + 10 56 ± 6

± 8

90 ± 12

66

± 11

47 + 10 34 ± 7*

± 11*

67 ± 7*

7

± 4*

± 3*

2 ± 2*

Note. Values are means ± SD of the number of colonies in at least 10 cultures. Cytotoxicity was determined on 100 6TCr cells plated in each culture. An asterisk indicates that the mean number of surviving colonies is significantly less (p < .01) than the number of colonies surviving at 0 fig/ml of test fraction.

Fractions B, C., D, and E were all cytotoxic at concentrations between 15 and 20 jig/ml. Inhibition of Metabolic Cooperation The parent compound and all of the fractions, with the exception of fraction A, inhibited intercellular communication in a concentrationdependent fashion (Table 3). Fraction A inhibited metabolic cooperation only at the maximum concentration tested. Fractions B, C., D, and E inhibited metabolic cooperation at concentrations of 10 /xg/ml or less. Fraction D was the most potent and inhibited metabolic cooperation at 2.5 fig/ml

DISCUSSION The present results demonstrate that asphalt fume fractions significantly inhibit intercellular communication in V79 cells. The fractions exhibiting the greatest activity (D, E) were those containing alkylated ketones, alkylated naphthols, and phenols. The least effective fraction (A) contained the long-chain alkanes and the alkylated benzenes and alkylated naphthalenes. Interestingly, the long-chain alkanes n-dodecane and 1-phenyldodecane have been reported to be tumor promoters (Bingham and Falk, 1969; Bingham and Nord, 1977), yet we previously found that they did not inhibit metabolic cooperation in the V79 assay (Bohrman et al., 1988).

M. TORAASON ETAL.

100

TABLE 3. Inhibition of Intercellular Communication in V79 Cells by Asphalt Fume Condensates Fractions Concentratioi (jig/ml)

Journal of Toxicology and Environmental Health 1991.34:95-102.

0.0 1.0 2.5 5.0 7.5

10.0 15.0 20.0 25.0 40.0 50.0

Parent

A

19 + 4 19 ± 5 23 ± 6

12

± 3

13

±

4

14 ± 2

17 12

± 4 ± 4

12 18

± ±

3 6

14

± 3

23

±

39

±

27 ± 6 32 ± 7* 36 ± 7*

B

C

D

E

24 ± 5 26 + 4

19 25 32 41

± + ± ±

5 5 6* 7*

6*

64 ± 8*

99

± 12*

6*

118 ± 9*

112

± 11*

13 15 21 37

± 2 ± 4 ± 4 ± 10*

76 + 7* 89 + 6*

17 + 5 40 + 8*

112 + 14*

20 + 4*

Note. Values are means ± SD of the number of colonies in at least 10 cultures. Metabolic cooperation was determined between 100 6TCr cells cocultured with 4 x 105 6TGS cells. An asterisk indicates that the mean number of 6TGr colonies surviving because of impaired intercellular communication with 6TGS cells is significantly greater (p < .01) than the number of colonies surviving at 0 /tg/ml of test fraction.

With the exception of fraction D, there was a relatively close association between concentrations of asphalt condensate that inhibited intercellular communication and concentrations that were cytotoxic. This association indicates that the observed increase in 6TGr cells in the metabolic cooperation assay may not be due entirely to inhibition of gap junctional intercellular communication. At certain concentrations of asphalt fume, there appears to be a paradoxical outcome. For example, fraction B at 40 /ig/ml (Table 2) prevented the growth of nearly all 6TGr cells in the cytotoxicity assay, but about 40% of 6TGr cells survived in the metabolic cooperation assay (Table 3). The difference in susceptibility is due partly to the fact that the cytotoxicity assay does not reproduce exactly the conditions in the metabolic cooperation assay. In addition, nonpolar compounds such as the asphalt fumes would tend to move out of solution and partition in the cell membrane. In the cytotoxicity assay where cell density is low, there would be more fume per cell and the potential for greater toxicity, which is exactly what was observed. The reduced toxicity in the metabolic cooperation assay would allow colony formation of 6TGr cells at fume concentrations that prevented 6TGr colony formation in the cytotoxicity assay—that is, of course, if intercellular communication is disrupted and the toxic metabolite of 6-thioguanine is not transferred via gap junctions from 6TGS cells to 6TGr cells. It is not apparent from the present results if gap junctional function was impaired, if the number of junctions was reduced, or if communication was blocked because of reduced cell contact. It may well be that the toxicity

Journal of Toxicology and Environmental Health 1991.34:95-102.

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of the fume condensates caused retraction of cells and increased survival of 6TGr cells because of reduced physical contact with 6TGS cells. Although the mechanism is not evident, the effects of asphalt fume condensates are comparable to the effects of cigarette smoke condensates in V79 cells (Hartman and Rosen, 1983). Concentrations of cigarette condensate that inhibited metabolic cooperation ranged from 1 to 50 /ig/ml, and paralleled cytotoxic concentrations much like the asphalt fume. Recently, the effects of cigarette smoke condensate on metabolic cooperation have been confirmed with dye coupling experiments performed with tracheal epithelial cells (Rutten et al., 1988). In addition, the inhibition of dye coupling and metabolic cooperation correlates with skin tumor-promoting activity of cigarette smoke condensate (Heck et al., 1990). Therefore, if the similarity between cigarette smoke and asphalt fume condensates goes beyond inhibition of metabolic cooperation in V79 cells, the carcinogenicity of the asphalt fume condensate may be due in part to the presence of tumor promoters acting through inhibition of intercellular communication. However, inhibitors of intercellular communication, like tumor promoters, are species and organ specific (Klaunig and Ruch, 1987; Saez et al., 1987). Before impaired gap junctional communication can be considered a factor in the skin carcinogenesis of asphalt fume, additional investigation into the inhibition of intercellular communication in rodent or human skin keratinocytes is necessary.

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Freidewald, W. F., and Rous, P. 1944. The initiating and promoting elements in tumor production. J. Exp. Med. 80:101-126. Hartman, T. G., and Rosen, J. D. 1983. Inhibition of metabolic cooperation by cigarette smoke condensate and its fractions in V-79 Chinese hamster lung fibroblasts. Proc. Natl. Acad. Sci. USA 80:5305-5309. Heck, J. D., Wenk, M. L., and Bennett, M. B. 1990. Mouse skin painting bioassay of cigarette smoke condensates for promoting activity in ICR Swiss and SENCAR mice. Toxicologist 10:234. Hecker, E., Fusenig, N. E., Kunz, W., Marks, F., and Thielman, H. W. 1982. Cocarcinogenesis and Biological Effects of Tumor Promoters, Carcinogenesis a Comprehensive Survey, vol. 7. New York: Raven Press. IARC. 1984. Polynuclear aromatic hydrocarbons part 4: Bitumens, coal-tars and derived products, shale-oils and soots. IARC Monogr. Bval. Carcinogen. Risk Chem. Hum., 35. Lyon, France. Jone, C., Erickson, L., Trosko, J. E., and Chang, C. C. 1987. Effect of biological toxins on gapjunctional intercellular communication in Chinese hamster V79 cells. Cell Biol. Toxicol. 3:1-15. Klaunig, J. E., and Ruch, R. J. 1987. Strain and species effects on the inhibition of hepatocyte intercellular communication by liver tumor promoters. Cancer Lett. 33:137-150. Miller, R. E., and Miller, J. A. 1981. Mechanism of chemical carcinogenesis. Cancer 47:1055-1064. Niemeier, R. W., Thayer, P. S., Menzies, K. T., Von Thuna, P., Moss, C. E., and Burg, J. 1988. A comparison of the skin carcinogenicity of roofing asphalt and coal tar pitch fumes. In Polynuclear Aromatic Hydrocarbons, eds. M. Cooke and A. J. Dennis, pp. 609-647. Columbus, Ohio: Battelle Press. Rutten, A. A. J. J. L., Jongen, W. M. F., de Hann, L. H. J., Hendrikson, E. G. J., and Koeman, J. H. 1988. Effect of retinol and cigarette-smoke condensate on dye-coupling intercellular communication between hamster tracheal epithelial cells. Carcinogenesis 9:315-320. Saez, J. C., Bennett, M. V. L., and Spray, D. C. 1987. Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes. Science 236:967-969. Slaga, T. J., Sivak, A., and Boutwell, R. K. 1978. Mechanisms of Tumor Promotion and Cocarcinogenesis. Carcinogenesis a Comprehensive Survey, vol. 2. New York: Raven Press. Trosko, J. E., Dawson, B., Yotti, L. P., and Chang, C.-C. 1980. Saccharin may act as a tumor-promoter by inhibiting metabolic cooperation between cells. Nature 285:109-110. Trosko, J. E., Yotti, L. P., Dawson, B., and Chang, C.-C. 1981. In vitro assay for tumor promoters. In Short Term Tests for Chemical Carcinogens, ed. H. Stich, pp. 420-427. New York: SpringerVerlag. Tsushimoto, C., Trosko, J. E., Chang, C.-C.,and Aust, S. D. 1982. Inhibition of metabolic cooperation in Chinese hamster V-79 cells in culture by various polybrominated biphenyl (PBB) congeners. Carcinogenesis 3:181-185. Wallcave, L., Garcia, H., Feldman, R., Lijinsky, W., and Shubik, P. 1971. Skin tumorigenesis in mice by petroleum asphalts and coal-tar pitches of known polynuclear aromatic hydrocarbon content. Toxicol. Appl. Pharmacol. 18:41-52. Yamasaki, H. 1987. The role of cell-to-cell communication in tumor promotion. Banbury Report 25, Nongenotoxic Mechanisms of Carcinogenesis, pp. 297-309. Cold Springs Harbor, N.Y.: Cold Springs Harbor Laboratory. Yotti, L. P., Chang, C.-C.,and Trosko, J. E. 1979. Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206:1089-1091. Received October 19, 1990 Accepted March 18, 1991

Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates.

Asphalt fume condensate is a skin carcinogen in mice, yet this complex mixture contains relatively low levels of known carcinogenic initiators. Conseq...
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