Skip to main content
SpringerLink
Log in

Metabolism and binding of benzo[a]pyrene in randomly-proliferating, confluent and s-phase human skin fibroblasts

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-3H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BaP:

benzo[a]pyrene

BaP-4,5-diol:

trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene

BaP-7,8-diol:

trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene

Bap-9,10-diol:

trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene

CM:

complete medium

HNF:

human neonatal foreskin

HPLC:

high pressure liquid chromatography

PAH:

polycyclic aromatic hydrocarbon

PDL:

population doubling

RP:

randomly proliferating

References

  • BAIRD, W.M. and DIAMOND, L. (1978). Metabolism and DNA binding of 23 polycyclic aromatic hydrocarbons by human diploid fibroblasts. Int. J. Cancer 22:189–195.

    Google Scholar 

  • COHEN, G.M., HAWS, S.M., MOORE, B.P. and BRIDGES, J.W. (1976). Benzo(a)pyren-3-yl hydrogen sulfate, a major ethyl acetate-extractable metabolite of benzo(a)pyrene in human, hamster, and rat lung cultures. Biochem. Pharmacol. 25:2561–2570.

    Google Scholar 

  • COOPER, C.S., GROVER, P.L. and SIMS, P. (1983). The metabolism and activation of benzo[a]pyrene. In: Progress in Drug Metabolism. (J.W. Bridges and L.F. Chasseaud, eds.), Vol. 7, pp. 295–396. John Wiley and Sons, Ltd., Chichester.

    Google Scholar 

  • CUNNINGHAM, M.J. (1985). The induction and inhibition of benzo(a)pyrene metabolism in human epidermal keratinocytes and dermal fibroblasts. Ph.D. Thesis, pp. 1–161.

  • EKELMAN, K.B. and MILO, G.E. (1978). Cellular uptake, transport, and macromolecular binding of benzo(a)pyrene and 7,12-dimethylbenz(a)-anthracene by human cells in vitro. Cancer Res. 38:3026–3032.

    Google Scholar 

  • FURANO, A.V. (1971). A very rapid method for washing large numbers of precipitates of proteins and nucleic acids. Anal. Biochem. 43:639–640.

    Google Scholar 

  • GELBOIN, H.V. (1980). Benzo(a)pyrene metabolism, activation, and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes. Physiol. Rev. 60:1107–1166.

    Google Scholar 

  • HUBERMAN, E. and SACHS, L. (1973). Metabolism of the carcinogenic hydrocarbon benzo(a)pyrene in human fibroblast and epithelial cells. Int. J. Cancer 11:412–418.

    Google Scholar 

  • INBASEKARAN, M.N., WITIAK, D.T., BARONE, K. and LOPER, J.C. (1980). Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz(a)-anthracene. J. Med. Chem. 23:278–281.

    Google Scholar 

  • KUROKI, T. and HEIDELBERGER, C. (1971). The binding of polycyclic aromatic hydrocarbons to the DNA, RNA and protein of transformable cells in culture. Cancer Res. 31:2168–2176.

    Google Scholar 

  • KUROKI, T., HOSOMI, J., MUNAKATA, K., ONIZUKA, T., TERAUCHI, M. and NEMOTO, N. (1982). Metabolism of benzo(a)pyrene in epidermal keratinocytes and dermal fibroblasts of humans and mice with reference to variation among species, individuals, and cell types. Cancer Res. 42:1859–1865.

    Google Scholar 

  • MAHER, V.M. and McCORMICK, J.J. (1984). Role of DNA lesions and excision repair in carcinogen-induced mutagenesis and transformation in human cells. In: Biochemical Basis of Chemical Carcinogenesis. (H.G.R. Jung, M. Kramer, H. Marquardt and F. Oesch, eds.), pp. 143–158. Raven Press, New York.

    Google Scholar 

  • MILO, G.E., BLAKESLEE, J., YOHN, D.S. and DiPAOLO, J.A. (1978a). Biochemical activation of aryl hydrocarbon hydroxylase activity, cellular distribution of polynuclear hydrocarbon metabolites, and DNA damage by polynuclear hydrocarbon products in human cells in vitro. Cancer Res. 38:1638–1644.

    Google Scholar 

  • MILO, G.E. and DiPAOLO, J.A. (1978b). Neoplastic transformation of human diploid cells in vitro after chemical carcinogen treatment. Nature 275:130–132.

    Google Scholar 

  • MILO, G.E. and DiPAOLO, J.A. (1980). Presentation of human cells with extrinsic signals to induce chemical carcinogenesis. Int. J. Cancer 26:805–812.

    Google Scholar 

  • MILO, G.E., OLDHAM, J.W., ZIMMERMAN, R., HATCH, G.G. and WEISBRODE, S.A. (1981). Characterization of human cells transformed by chemical and physical carcinogens in vitro. In Vitro 17: 719–729.

    Google Scholar 

  • MILO, G.E. and CASTO, B.C. (1986). Conditions for transformation of human fibroblast cells: An overview. Cancer Lett. 31:1–13.

    Google Scholar 

  • PHILIPS, D.H. (1983). Fifty years of benzo[a]pyrene. Nature 303:468–475.

    Google Scholar 

  • REIGNER, D., McMICHAEL, T., BERNO, J. and MILO, G.E. (1976). Processing of human tissue to establish primary cultures in vitro. Tissue Culture Association Manual. 2:273–276.

    Google Scholar 

  • RUDIGER, H.W., MARXEN, J., KOHL, F-V., MELDERIS, H. and WICHERT, P.V. (1979). Metabolism and formation of DNA adducts of benzo(a)pyrene in human diploid fibroblasts. Cancer Res. 39:1083–1088.

    Google Scholar 

  • SELKIRK, J.K., NIKBAKHT, A. and STONER, G.D. (1983). Comparative metabolism and macromolecular binding of benzo(a)pyrene in explant cultures of human bladder, skin, bronchus and esophagus from eight individuals. Cancer Lett. 18: 11–19.

    Google Scholar 

  • SIMS, P. and GROVER, P.L. (1974). Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. Cancer Res. 20:165–274.

    Google Scholar 

  • SIMS, P. (1980). The metabolic activation of chemical carcinogens. Br. Med. Bull. 36:11–18.

    Google Scholar 

  • STEVENS, C.W., BOUCK, N., BURGESS, J.A. and FAHL, W.E. (1985). Benzo(a)pyrene diol-epoxides: Different mutagenic efficiency in human and bacterial cells. Mutat. Res. 152:5–14.

    Google Scholar 

  • TEJWANI, R., NESNOW, S. and MILO, G.E. (1980a). Analysis of intracellular distribution and binding of benzo(a)pyrene in human diploid fibroblasts. Cancer Lett. 10:57–65.

    Google Scholar 

  • TEJWANI, R., TREWYN, R.W. and MILO, G.E. (1980b). Benzo(a)pyrene metabolism by transformable human skin fibroblasts. Fed. Proc. 39:2091.

    Google Scholar 

  • TONG, C., FAZIO, M. and WILLIAMS, G.M. (1980). Cell cycle-specific mutagenesis at the hypoxanthine phosphoribosyltransferase locus in adult rat liver epithelial cells. Proc. Natl. Acad. Sci. USA. 77:7377–7379.

    Google Scholar 

  • WOHLER, W., BERTRAM, C.R., SCHURER, C.C. and RUDIGER, H.W. (1977). Benzopyrene metabolism in human diploid fibroblasts. Hum. Hered. 27:222.

    Google Scholar 

  • WOHLER, W., BERTRAM, C.R., SCHURER, C.C. and RUDIGER, H.W. (1978). Benzopyrene metabolism in human diploid fibroblasts. Mongr. Hum. Genet. 10:165–170.

    Google Scholar 

  • YANG, S.K., McCOURT, D.W., ROLLER, P.P. and GELBOIN, H.V. (1976). Enzymatic conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene through a single enantiomer of r-t,t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. 73:2594–2598.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Urology, Stanford University Medical Center, Stanford, CA

    Mary Jane Cunningham

  2. The Ohio State University Comprehensive Cancer Center and the Department of Physiological Chemistry, 410 West 12th Avenue, Columbus, OH

    Ponnamma Kurian

Authors
  1. Mary Jane Cunningham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ponnamma Kurian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. George E. Milo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cunningham, M.J., Kurian, P. & Milo, G.E. Metabolism and binding of benzo[a]pyrene in randomly-proliferating, confluent and s-phase human skin fibroblasts. Cell Biol Toxicol 5, 155–168 (1989). https://doi.org/10.1007/BF00122650

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00122650

Key words

Search

Navigation