Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of alkane tumor‐promoting agents on chemically induced mutagenesis in cultured v79 Chinese hamster cells George R. Lankas , C. Stuart Baxter & Robert T. Christian To cite this article: George R. Lankas , C. Stuart Baxter & Robert T. Christian (1978) Effect of alkane tumor‐promoting agents on chemically induced mutagenesis in cultured v79 Chinese hamster cells, Journal of Toxicology and Environmental Health, 4:1, 37-41, DOI: 10.1080/15287397809529642 To link to this article: http://dx.doi.org/10.1080/15287397809529642
Published online: 20 Oct 2009.
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Date: 07 November 2015, At: 05:57
EFFECT OF ALKANE TUMOR-PROMOTING AGENTS ON CHEMICALLY INDUCED MUTAGENESIS IN CULTURED V79 CHINESE HAMSTER CELLS George R. Lankas, C. Stuart Baxter, Robert T. Christian Department of Environmental Health, University
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of Cincinnati Medical School, Cincinnati, Ohio
Linear alkanes of specific chain length between 6 and 16 carbon atoms, an aryl derivative of dodecane, and a phorbol diester were tested in a cell culture system for relative ability to enhance mutagenesis induced by a chemical carcinogen, methylazoxymethanol acetate (MAM). Mutation frequencies at the ouabain-resistance locus were measured. Results indicated an excellent correlation between the relative activities of the above compounds in enhancing mutagenesis in the in vitro culture system and their tumor-promoting activities in mouse skin. None of the compounds tested showed mutagenic activity per se, further lending support to the theory that promoters act via derepression of latent carcinogen-induced damage to the genome.
INTRODUCTION In industrial and ambient environments a number of agents have been identified that, although noncarcinogenic per se, are capable of enhancing the carcinogenic response of animals when administered subsequent but not prior to carcinogen exposure. The action of these agents, termed promoters, was first demonstrated, in the case of the naturally occurring phorbol diesters, in mouse skin (Berenblum, 1941). These experiments led to the hypothesis of a two-stage mechanism of cancer induction, whereby carcinogen treatment was proposed to first induce irreversible damage (i.e., mutations) in the cell genome. Below a certain concentration of the carcinogen, a change in cell phenotype to malignancy did not occur, but could subsequently be effected by exposure to either higher carcinogen concentrations or a promoter, which caused "expression" of the latent damage and thus neoplastic transformation. A direct inference that can be made from the above theory is that promoters of carcinogenesis should also enhance carcinogen-induced For valuable advice and discussion we would like to thank Dr. Eula Bingham, Department of Environmental Health, University of Cincinnati Medical Center and Occupational Safety and Health Administration. This work was supported by a Chemical Industry Institute of Toxicology Fellowship and by National Institutes of Health grants ES 00159 and ES 00127. Requests for reprints should be sent to Stuart Baxter, Department of Environmental Health, University of Cincinnati Medical School, Cincinnati, Ohio 45267. 37 Journal of Toxicology and Environmental Health, 4:37-41,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0401-0037$2.25
38
G. R. LANKASETAL.
Downloaded by [Universite Laval] at 05:57 07 November 2015
mutagenesis in eukaryotic cells. In corroboration of this, we and others have found that phorbol and its tetradecanoyl acetate diester (TPA) enhanced carcinogen-induced mutagenesis of mammalian cells in culture (Lankas et al., 1977; Trosko et al., 1977). These findings suggested a relatively inexpensive and rapid means of identifying promoters of environmental importance, one avoiding the expense, time, and complexity involved in traditional experiments in mouse skin. In this paper we demonstrate the potential of our system in detecting and correlating the relative activities of linear alkanes with even chain lengths of from 6 to 16 carbon atoms. These hydrocarbons are present in cigarette smoke, human sebum, and petroleum distillate. The members from decane to hexadecane have been shown to have promoting activity in mouse skin and have been implicated in enhancement of carcinogenesis in humans (Bingham and Horton, 1966; Sice, 1966; Van Duuren and Goldschmidt, 1976). MATERIALS AND METHODS V79 Chinese hamster cells were grown in Eagle's minimum essential medium supplemented with 1.5 X vitamins and amino acids and 10% fetal calf serum (Gibco, Grand Island, New York). For routine culture the cells were grown in 75-cm2 plastic tissue culture bottles (Corning Glass Works, Corning, New York) without antibiotics in an atmosphere of 95% air and 5% CO 2 - For the mutagenesis assay 2 X 10 s cells were seeded in 100-mm plastic petri dishes (Lux Scientific Corp., Newbury Park, California) in 10 ml growth medium supplemented with 100 units penicillin and 100 jug/ml streptomycin (Gibco, Grand Island, New York). After 2-4 hr, to allow for cell attachment, methylazoxymethanol acetate (MAM) (Aldrich Chemical Co., Milwaukee, Wisconsin), dissolved in medium without serum, was added to the plates to a final concentration of 24 jug/ml. After 24 h of exposure the medium in the plates was removed and replaced by fresh medium plus the chemical being tested for promoting activity. /7-Decane, A7-dodecane, A7-tetradecane, 1-phenyldodecane, and nhexadecane were gifts from E. Bingham of our department. /7-Hexane was certified grade (Fisher Scientific Co., Pittsburgh, Pennsylvania). The above chemicals were mixed with acetone (20 /xl/ml) to give a 0.2% v/v solution. TPA (Consolidated Midland Corp., Brewster, New York) was dissolved in acetone to a concentration of 1 mg/ml. Aliquots of these solutions were added to the cultures after removal of the mutagen. The plates were gently swirled to ensure an even distribution of the chemical. Ouabain-resistant mutants were selected by adding growth medium containing 1 m/W ouabain (Sigma Chemical Co., St. Louis, Missouri) 48-72 h after dosing with the MAM. The alkanes were again added so that exposure to the promoter continued throughout ouabain selection. After 2 wk the plates were washed, fixed with ethanol, and stained with Giemsa and the number of mutant colonies was determined. Replicates of 10 plates per point were made.
EFFECTS OF ALKANES ON MUTAGENESIS
39
Concurrent with the mutagenesis assay, toxicity assays were performed to determine the number of surviving cells after the various chemical treatments. Cells (1.5 X 10 2 ) were seeded in 60-mm plastic culture dishes (Corning Glass Works, Corning, New York) in 2 ml medium. Treatments were identical to those in the mutagenesis experiments, with the exception that no ouabain was used and surviving colonies were counted after 8-9 d of incubation. Replicates of four plates per point were done to determine the cloning efficiency.
Downloaded by [Universite Laval] at 05:57 07 November 2015
RESULTS The effects of /7-hexane, /7-decane, A7-dodecane, fl-tetradecane, and rt-hexadecane on the frequency of mutations induced by MAM are presented in Table 1. In every case the alkanes were present at 0.12-m/W equimolar concentrations. It is apparent that at the concentration employed, no alkane was toxic and the MAM-induced toxicity was not affected. No alkane showed mutagenic activity per se. Of the alkanes tested, statistically significant enhancement of the MAM-induced mutation frequency was observed with /7-decane, /7-dodecane and A7-tetradecane, but not with A7-hexane or /7-hexadecane. The results are in remarkably good agreement with the relative promoting activities reported for these agents
TABLE 1. Effect of Linear Alkanes on Mutagenesis in V79 Cells
Treatment 0 Control Acetone (0.1%) n-Hexane n-Decane n-Dodecane n-Tetradecane n-Hexadecane MAM MAM + 0.1% acetone MAM + n-hexane MAM + n-decane MAM +n-dodecane MAM + n-tetradecane MAM + /7-hexadecane
Survival (% of control) 100 92 97 103 110 105 91 28 28 25 23 28 28 27
"Alkanes added at a concentration of 0.12 mM. "Average + 95% confidence interval (Mest). C NS, not significant. d
p < 0.05.
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of alkane tumor‐promoting agents on chemically induced mutagenesis in cultured v79 Chinese hamster cells George R. Lankas , C. Stuart Baxter & Robert T. Christian To cite this article: George R. Lankas , C. Stuart Baxter & Robert T. Christian (1978) Effect of alkane tumor‐promoting agents on chemically induced mutagenesis in cultured v79 Chinese hamster cells, Journal of Toxicology and Environmental Health, 4:1, 37-41, DOI: 10.1080/15287397809529642 To link to this article: http://dx.doi.org/10.1080/15287397809529642
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 8
View related articles
Citing articles: 17 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh19 Download by: [Universite Laval]
Date: 07 November 2015, At: 05:57
EFFECT OF ALKANE TUMOR-PROMOTING AGENTS ON CHEMICALLY INDUCED MUTAGENESIS IN CULTURED V79 CHINESE HAMSTER CELLS George R. Lankas, C. Stuart Baxter, Robert T. Christian Department of Environmental Health, University
Downloaded by [Universite Laval] at 05:57 07 November 2015
of Cincinnati Medical School, Cincinnati, Ohio
Linear alkanes of specific chain length between 6 and 16 carbon atoms, an aryl derivative of dodecane, and a phorbol diester were tested in a cell culture system for relative ability to enhance mutagenesis induced by a chemical carcinogen, methylazoxymethanol acetate (MAM). Mutation frequencies at the ouabain-resistance locus were measured. Results indicated an excellent correlation between the relative activities of the above compounds in enhancing mutagenesis in the in vitro culture system and their tumor-promoting activities in mouse skin. None of the compounds tested showed mutagenic activity per se, further lending support to the theory that promoters act via derepression of latent carcinogen-induced damage to the genome.
INTRODUCTION In industrial and ambient environments a number of agents have been identified that, although noncarcinogenic per se, are capable of enhancing the carcinogenic response of animals when administered subsequent but not prior to carcinogen exposure. The action of these agents, termed promoters, was first demonstrated, in the case of the naturally occurring phorbol diesters, in mouse skin (Berenblum, 1941). These experiments led to the hypothesis of a two-stage mechanism of cancer induction, whereby carcinogen treatment was proposed to first induce irreversible damage (i.e., mutations) in the cell genome. Below a certain concentration of the carcinogen, a change in cell phenotype to malignancy did not occur, but could subsequently be effected by exposure to either higher carcinogen concentrations or a promoter, which caused "expression" of the latent damage and thus neoplastic transformation. A direct inference that can be made from the above theory is that promoters of carcinogenesis should also enhance carcinogen-induced For valuable advice and discussion we would like to thank Dr. Eula Bingham, Department of Environmental Health, University of Cincinnati Medical Center and Occupational Safety and Health Administration. This work was supported by a Chemical Industry Institute of Toxicology Fellowship and by National Institutes of Health grants ES 00159 and ES 00127. Requests for reprints should be sent to Stuart Baxter, Department of Environmental Health, University of Cincinnati Medical School, Cincinnati, Ohio 45267. 37 Journal of Toxicology and Environmental Health, 4:37-41,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0401-0037$2.25
38
G. R. LANKASETAL.
Downloaded by [Universite Laval] at 05:57 07 November 2015
mutagenesis in eukaryotic cells. In corroboration of this, we and others have found that phorbol and its tetradecanoyl acetate diester (TPA) enhanced carcinogen-induced mutagenesis of mammalian cells in culture (Lankas et al., 1977; Trosko et al., 1977). These findings suggested a relatively inexpensive and rapid means of identifying promoters of environmental importance, one avoiding the expense, time, and complexity involved in traditional experiments in mouse skin. In this paper we demonstrate the potential of our system in detecting and correlating the relative activities of linear alkanes with even chain lengths of from 6 to 16 carbon atoms. These hydrocarbons are present in cigarette smoke, human sebum, and petroleum distillate. The members from decane to hexadecane have been shown to have promoting activity in mouse skin and have been implicated in enhancement of carcinogenesis in humans (Bingham and Horton, 1966; Sice, 1966; Van Duuren and Goldschmidt, 1976). MATERIALS AND METHODS V79 Chinese hamster cells were grown in Eagle's minimum essential medium supplemented with 1.5 X vitamins and amino acids and 10% fetal calf serum (Gibco, Grand Island, New York). For routine culture the cells were grown in 75-cm2 plastic tissue culture bottles (Corning Glass Works, Corning, New York) without antibiotics in an atmosphere of 95% air and 5% CO 2 - For the mutagenesis assay 2 X 10 s cells were seeded in 100-mm plastic petri dishes (Lux Scientific Corp., Newbury Park, California) in 10 ml growth medium supplemented with 100 units penicillin and 100 jug/ml streptomycin (Gibco, Grand Island, New York). After 2-4 hr, to allow for cell attachment, methylazoxymethanol acetate (MAM) (Aldrich Chemical Co., Milwaukee, Wisconsin), dissolved in medium without serum, was added to the plates to a final concentration of 24 jug/ml. After 24 h of exposure the medium in the plates was removed and replaced by fresh medium plus the chemical being tested for promoting activity. /7-Decane, A7-dodecane, A7-tetradecane, 1-phenyldodecane, and nhexadecane were gifts from E. Bingham of our department. /7-Hexane was certified grade (Fisher Scientific Co., Pittsburgh, Pennsylvania). The above chemicals were mixed with acetone (20 /xl/ml) to give a 0.2% v/v solution. TPA (Consolidated Midland Corp., Brewster, New York) was dissolved in acetone to a concentration of 1 mg/ml. Aliquots of these solutions were added to the cultures after removal of the mutagen. The plates were gently swirled to ensure an even distribution of the chemical. Ouabain-resistant mutants were selected by adding growth medium containing 1 m/W ouabain (Sigma Chemical Co., St. Louis, Missouri) 48-72 h after dosing with the MAM. The alkanes were again added so that exposure to the promoter continued throughout ouabain selection. After 2 wk the plates were washed, fixed with ethanol, and stained with Giemsa and the number of mutant colonies was determined. Replicates of 10 plates per point were made.
EFFECTS OF ALKANES ON MUTAGENESIS
39
Concurrent with the mutagenesis assay, toxicity assays were performed to determine the number of surviving cells after the various chemical treatments. Cells (1.5 X 10 2 ) were seeded in 60-mm plastic culture dishes (Corning Glass Works, Corning, New York) in 2 ml medium. Treatments were identical to those in the mutagenesis experiments, with the exception that no ouabain was used and surviving colonies were counted after 8-9 d of incubation. Replicates of four plates per point were done to determine the cloning efficiency.
Downloaded by [Universite Laval] at 05:57 07 November 2015
RESULTS The effects of /7-hexane, /7-decane, A7-dodecane, fl-tetradecane, and rt-hexadecane on the frequency of mutations induced by MAM are presented in Table 1. In every case the alkanes were present at 0.12-m/W equimolar concentrations. It is apparent that at the concentration employed, no alkane was toxic and the MAM-induced toxicity was not affected. No alkane showed mutagenic activity per se. Of the alkanes tested, statistically significant enhancement of the MAM-induced mutation frequency was observed with /7-decane, /7-dodecane and A7-tetradecane, but not with A7-hexane or /7-hexadecane. The results are in remarkably good agreement with the relative promoting activities reported for these agents
TABLE 1. Effect of Linear Alkanes on Mutagenesis in V79 Cells
Treatment 0 Control Acetone (0.1%) n-Hexane n-Decane n-Dodecane n-Tetradecane n-Hexadecane MAM MAM + 0.1% acetone MAM + n-hexane MAM + n-decane MAM +n-dodecane MAM + n-tetradecane MAM + /7-hexadecane
Survival (% of control) 100 92 97 103 110 105 91 28 28 25 23 28 28 27
"Alkanes added at a concentration of 0.12 mM. "Average + 95% confidence interval (Mest). C NS, not significant. d
p < 0.05.
