Skip to main content

Advertisement

SpringerLink
Log in

Interactive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoids on proliferation and differentiation in cultured human keratinocytes: quantification of cross-linked envelope formation

  • Original Investigation
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Dioxins are potent inducers of chloracne in humans. This skin aberration can be interpreted as an altered differentiation pattern of acinar sebaceous base cells and a change in the rate of terminal differentiation of the keratinocytes. We measured this rate induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in primary cultures of human keratinocytes. As parameters for differentiation, we quantified the 35S-methionine incorporation into cross-linked envelopes (revealing the total CLE biomass), as well as the number of microscopically visible CLEs. It was shown that TCDD is a very potent inducer of both CLE biomass and number with a half-maximal effect concentration (EC50) of 1.4 nM. CLE biomass was maximally increased 10-fold and the number of cells in culture producing a CLE was increased from 15% in control cultures to maximally 75% of the cells in TCDD-treated cultures. Both effects were Ca2+-dependent and increased with elevated cell density, being optimal in post-confluent cultures. Retinoic acid dose-dependently decreased the effect of 10−8 M TCDD, 10−6 M having a nearly complete antagonistic action. This interaction of retionic acid with TCDD-induced differentiation was non-competitive. Retinol was equally potent as an antagonist of the TCDD-induced elevation of CLE formation as compared with retinoic acid. Retinyl palmitate and etretinate were not very effective as TCDD antagonists. Supplementation of hydrocortisone suppressed the TCDD-induced keratinocyte differentiation. It was concluded that CLE biomass quantification provides a reliable and sensitive parameter for keratinocyte differentiation. In this in vitro system it is shown that TCDD strongly induces a switch from proliferation to terminal differentiation and that this effect can be antagonized effectively by retinoic acid and retinol.

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.

Similar content being viewed by others

References

  • Allen JG, Bloxham DP (1989) The pharmacology and pharmacokinetics of the retinoids. Pharmac Ther 40: 1–27

    Article  CAS  Google Scholar 

  • Blumenberg M, Connolly DM, Freedberg IM (1992) Regulation of keratin gene expression: The role of the nuclear receptors for retinoic acid, thyroid hormone, and vitamin-D3. J Invest Dermatol 98: S42-S49

    Article  Google Scholar 

  • Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81: 33s-40s

    Article  PubMed  CAS  Google Scholar 

  • Boyce ST, Ham RG (1985) Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum-free media. J Tissue Cult Meth 9: 83–93

    Article  Google Scholar 

  • Brouwer A, Blaner WS, Kukler A, Van de Berg KJ (1988a) Study on the mechanism of interference of 3,4,3′,4′-tetrachlorobiphenyl with the plasma retinol-binding proteins in rodents. Chem-Biol Interact 68: 203–217

    Article  PubMed  CAS  Google Scholar 

  • Brouwer A, Kulker A, Van de Berg KJ (1988b) Alterations in retinoid concentrations in several extrahepatic organs of rats by 3,4,3′,4′-tetrachlorobiphenyl. Toxicology 50: 317–330

    Article  PubMed  CAS  Google Scholar 

  • Brouwer A, Hakansson H, Kukler A, Van de Berg KJ, Ahlborg UG (1989a) Marked alterations in retinoid homeostasis of Sprague-Dawley rats induced by a single i.p. dose of 10 μg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology 58: 267–283

    Article  PubMed  CAS  Google Scholar 

  • Brouwer A, Reijnders PJH, Koeman JH (1989b) Polychlorinated biphenyl (PCB)-contaminated fish induces vitamin A and thyroid hormone deficiency in the common seal (Phoca vitulina). Aquatic Toxicol 15: 99–106

    Article  CAS  Google Scholar 

  • Cline PR, Rice RH (1983) Modulation of involucrin and envelope competence in human keratinocytes by hydrocortisone, retinyl acetate, and growth arrest. Cancer Res 43: 3203–3207

    PubMed  CAS  Google Scholar 

  • Crow KD (1981) Chloracne and its potential clinical implications. Clin Exp Dermatol 6: 243–257

    Article  PubMed  CAS  Google Scholar 

  • Fuchs E, Green H (1987) Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell 25: 617–625

    Article  Google Scholar 

  • Greenlee WF, Dold KM, Osborne R (1985) Actions of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) on human epidermal keratinocytes in culture. In Vitro Cell Dev Biol 21: 509–512

    Article  PubMed  CAS  Google Scholar 

  • Greenlee WF, Osborne R, Dold KM, Hudson LG, Young MJ, Toscano Jr WA (1987) Altered regulation of epidermal cell proliferation and differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In: Hodgson E, Bend JR, Philpot RM (eds) Reviews in biochemical toxicology vol. 8. Elsevier, New York, pp 1–35

    Google Scholar 

  • Hennings HD, Michael D, Cheng C, Steinert P, Holbrook L, Yuspa SH (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19: 245–254

    Article  PubMed  CAS  Google Scholar 

  • Hennings H, Kruszewski FH, Yuspa SH, Tucker RW (1989) Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes. Carcinogen 10: 777–780

    Article  CAS  Google Scholar 

  • Hough-Monroe L, Milstone LM (1991) Quantitation of cross-linked protein: an alternative to counting cornified envelopes as an index of keratinocyte differentiation. Anal Biochem 199: 25–28

    Article  PubMed  CAS  Google Scholar 

  • Hudson DL, Weiland KL, Dooley TP, Simon M, Watt FM (1992) Characterisation of eight monoclonal antibodies to involucrin. Hybridoma 11: 367–379

    PubMed  CAS  Google Scholar 

  • Jetten AM, Shirley JE (1986) Characterization of transglutaminase activity in rat tracheal epithelial cells; regulation by retinoids. J Biol Chem 261: 15091–15101

    Google Scholar 

  • Johnson ES (1992) Human exposure to 2,3,7,8-TCDD and risk of cancer. Crit. Rev. Toxicol 21: 451–463

    Article  CAS  Google Scholar 

  • Kimbrough RD (1984) SKin lesions in animals and humans: a brief overview. In: Poland A, Kimbrough RD (eds) Banbury report vol. 18, Biological mechanisms of dioxin action. Meeting, Cold Spring Harbor, N.Y., USA, pp 357–363

  • King I, Mella SL, Sartorelli AC (1986) A sensitive method to quantify the terminal differentiation of cultured epidermal cells. Exp Cell Res 167: 252–256

    Article  PubMed  CAS  Google Scholar 

  • Kociba RJ, Schwetz BA (1982) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Drug Metab Rev 13: 387–406

    Article  PubMed  CAS  Google Scholar 

  • Landers JP, Bunce NJ (1991) The Ah receptor and the mechanism of dioxin toxicity. Biochem J 276: 273–287

    PubMed  CAS  Google Scholar 

  • Michel S, Courseaux A, Miquel C, Bernardon J-M, Schmidt R, Shroot B, Thacher S, Reichert R (1991) Determination of retinoid activity by an enzyme-linked immunosorbent assay. Anal Biochem 192: 232–236

    Article  PubMed  CAS  Google Scholar 

  • Osborne R, Dold KM, Greenlee WF (1987) Evidence that 2,3,7,8-tetrachlorodibenzo p-dioxin and thyroid hormones act through different mechanisms in human keratinocytes. Toxicol Appl. Pharmacol 90: 522–531

    Article  PubMed  CAS  Google Scholar 

  • Plewig G (1970) Lokalbehandlung der Chloracne (Halowaxacne) mit Vitamin A-säure. Der Hautarzt 21: 465–470

    CAS  Google Scholar 

  • Poland AP, Glover E, Kende AS (1976) Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is the receptor for the induction of aryl hydrocarbon hydroxylase. J Biol Chem 251: 4936–4946

    PubMed  CAS  Google Scholar 

  • Ponec M, Kempenaar JA, De Kloet ER (1981) Corticoids and cultured human epidermal keratinocytes: specific intracellular binding and clinical efficacy. J Invest Dermatol 76: 211–214

    Article  PubMed  CAS  Google Scholar 

  • Ponec M, Weerheim A, Kempenaar J, Boonstra J (1988) Proliferation and differentiation of human squamous carcinoma cell lines and normal keratinocytes: effects of epidermal growth factor, retinoids, and hydrocortisone. In Vitro Cell Dev Biol 24: 764–770

    Article  PubMed  CAS  Google Scholar 

  • Puhvel SM, Conner MJ, Sakamoto M (1991) Vitamin A deficiency and the induction of cutaneous toxicity in murine skin by TCDD. Toxicol Appl. Pharmacol 107: 106–116

    Article  PubMed  CAS  Google Scholar 

  • Regnier M, Eustache J, Shroot B, Darmon M (1989) Bioassays for retinoic acid-like substances using cultured human keratinocytes. Skin Pharmacol 2: 1–9

    Article  PubMed  CAS  Google Scholar 

  • Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6: 331–344

    Article  PubMed  CAS  Google Scholar 

  • Rice RH, Cline PR (1984) Opposing effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone on growth and differentiation of cultured malignant human kerationcytes. Carcinogen 5: 367–371

    Article  CAS  Google Scholar 

  • Rice RH, Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: Activation of the cross-linking by calcium ions. Cell 18: 681–694

    Article  PubMed  CAS  Google Scholar 

  • Rice RH, Cline PR, Coe EL (1983) Mutually antagonistic effects of hydrocortisone and retinyl acetate on envelope competence in cultured malignant human keratinocytes. J Invest Dermatol 81: 176s-178s

    Article  PubMed  CAS  Google Scholar 

  • Rosenthal DS, Griffiths CEM, Yuspa SH, Roop DR, Voorhees JJ (1992) Acute or chronic topical retinoic acid treatment of human skin in vivo alters the expression of epidermal transglutaminase, loricrin, involucrin, filagrin, and keratins 6 and 13 but not keratins 1, 10, and 14. J Invest Dermatol 98: 343–350

    Article  PubMed  CAS  Google Scholar 

  • Rubin AL, Rice RH (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons suppress retinoid-induced tissue transglutaminase in SCC-4 cultured human squamous carcinoma cells. Carcinogen 9: 1067–1070

    Article  CAS  Google Scholar 

  • Safe SH (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annv Rev Pharmacol Toxicol 26: 371–399

    Article  CAS  Google Scholar 

  • Safe S (1989) Polychlorinated biphenyls (PCBs): mutagenicity and carcinogenicity. Mutat Res 220: 31–47

    PubMed  CAS  Google Scholar 

  • Safe S (1990) Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit Rev Toxicol 21: 51–88

    Article  PubMed  CAS  Google Scholar 

  • Serre G, Mils V, Haftek M, Vincent C, Croute F, Réano A, Ouhayoun J-P, Bettinger S, Soleilhavoup J-P (1991) Identification of late differentiation antigens of human cornified epithelia, expressed in re-organized desmosomes and bound to cross-linked envelope. J Invest Dermatol 97: 1061–1072

    Article  PubMed  CAS  Google Scholar 

  • Siegenthaler G, Saurat J-H, Ponec M (1990) Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes. Biochem J 268: 371–378

    PubMed  CAS  Google Scholar 

  • Stewart AF, Reik A, Schütz G (1991) A simpler and better method to cleave chromatin with DNasel for hypersensitive site analyses. Nucl Acid Res 19: 3157

    Article  CAS  Google Scholar 

  • Suskind RR (1985) Chloracne, “the hallmark of dioxin intoxication”. Scand J Work Environ Health 11: 165–171

    PubMed  CAS  Google Scholar 

  • Thacher SM, Rice RH (1985) Keratinocyte-specific trans-glutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell 40: 685–695

    Article  PubMed  CAS  Google Scholar 

  • Thunberg T (1984) Effect of TCDD on vitamin A and its relation to TCDD-toxicity. In: Poland A, Kimbrough RD (eds) Banbury report vol. 18, Biological mechanisms of dioxin action. Meeting, Cold Spring Harbor, N.Y., USA, pp 333–343

  • Tindall JP (1985) Chloracne and chloracnegens. J Am Acad Dermatol 13: 539–558

    Article  PubMed  CAS  Google Scholar 

  • Tong P, Mayes D, Wheeler L (1986) Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes. Biochem Biophys Res Commun 138: 483–488

    Article  PubMed  CAS  Google Scholar 

  • Tsao MC, Walthall BJ, Ham RG (1982) Clonal growth of normal human epidermal keratinocytes in a defined medium. J Cell Physiol 110: 219–229

    Article  PubMed  CAS  Google Scholar 

  • Urabe H, Asahi M (1985) Past and current dermatological status of Yusho patients. Environ Health Perspect 59: 11–15

    Article  PubMed  CAS  Google Scholar 

  • Vahlquist A, Törmä A (1988) Retinoids and keratinization: current concepts. Int. J Dermatol 27: 81–95

    Article  PubMed  CAS  Google Scholar 

  • Van Pelt FNAM, Hassing IGAM, Stelling MA, Seinen W, Blaauboer BJ (1992) Induction of terminal differentiation in cultured human keratinocytes by polychlorinated aromatic hydrocarbons as meansured by cell size analysis. Toxicol Appl Pharmacol 113: 240–245

    Article  PubMed  Google Scholar 

  • Violette SM, King I, Sartorelli AC (1989) Antagonistic effects of retinoic acid and hydrocortisone on the terminal differentiation of human squamous carcinoma cells. J Invest Dermatol 93: 165–168

    Article  PubMed  CAS  Google Scholar 

  • Watt FM, Green H (1982) Stratification and terminal differentiation of cultured epidermal cells. Nature 295: 434–436

    Article  PubMed  CAS  Google Scholar 

  • Yuspa SH, Ben T, Steiner P (1982) Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells. J Biol Chem 257: 9906–9908

    PubMed  CAS  Google Scholar 

  • Zile MH (1992) Vitamin-A homeostasis endangered by environmental pollutants. Proc Soc Exp Biol Med 201: 141–153

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research Institute of Toxicology, Utrecht University, PO Box 80.176, NL-3508 TD, Utrecht, The Netherlands

    Jos A. M. Berkers, Ine Hassing & Bas J. Blaauboer

  2. Department of Toxicology, Wageningen Agricultural University, PO Box 8000, NL-6700 EA, Wageningen, The Netherlands

    Bert Spenkelink & Abraham Brouwer

Authors
  1. Jos A. M. Berkers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ine Hassing
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abraham Brouwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bas J. Blaauboer
    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

Berkers, J.A.M., Hassing, I., Spenkelink, B. et al. Interactive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoids on proliferation and differentiation in cultured human keratinocytes: quantification of cross-linked envelope formation. Arch Toxicol 69, 368–378 (1995). https://doi.org/10.1007/s002040050186

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s002040050186

Key words

Search

Navigation