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An ex vivo model of the rat trachea to study the effect of inhalable toxic compounds

Research in Experimental Medicine volume 196, Article number: 195 (1996) Cite this article

Abstract

Different cell culture and organ systems are used to evaluate the physiological responses of the airways to the effects of carcinogenic [e.g., benzo(a)pyrene] and anticarcinogenic (e.g., retinoids) compounds on cellular growth and differentiation. However, in contrast to in vivo conditions dissociated epithelial cells or tracheal ring cultures are covered with medium. Therefore, we developed an ex vivo perfusion model enabling evaluation of morphology and metabolism of different compounds under near-physiological conditions. The trachea was surrounded with culture medium and perfused with air by means of a small animal respirator. To test the viability of the system under various experimental conditions tracheal probes were incubated with either retinoids (retinol 10−5 mol/l; retinyl palmitate 10−5 mol/l) or benzo(a)pyrene (10−7 mol/l) for up to 7 days. At the end of the incubation period metabolites in the trachea and in the medium were measured by means of high-performance liquid chromatography. Samples were examined by light microscopy, and by scanning and transmission electron microscopy for cell morphology. Glycoconjugate expression was assessed by lectin histochemistry. Specimens incubated in a retinoid-supplemented medium revealed no alterations in the distribution of cell types and characteristics of the epithelial layer compared with tracheal biopsies assessed immediately after removal from the animals. Glycoconjugate patterns especially remained intact. Histological changes after incubation with benzo(a)pyrene resembled in vivo morphology of vitamin A-deficient rats. An important advantage of this in vitro model compared with common cell or organ cultures is the preservation of the original phenotype and environment of the tracheobronchial surface. In addition, carcinogenic substances, such as benzo(a)pyrene, can easily be applied by airway or through the medium.

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References

  1. Ammigan N, Nair UJ, Lalitha VS, Bhide SV (1991) Carcinogenicity studies of pyrolysed tobacco products in vitamin A-deficient Sprague-Dawley rats. J Cancer Res Clin Oncol 117:50–54

    PubMed  Article  CAS  Google Scholar 

  2. Biesalski HK (1990) Separation of retinyl esters and their geometric isomers by isocratic adsorption high-performance liquid chromatography. Methods Enzymol 189:181–189

    PubMed  CAS  Article  Google Scholar 

  3. Biesalski HK, Gabbert HE, Gerharz CD, Engers R, Brodda K (1990) Uptake and metab of retinoic acid induces inhibition of cell growth: a study in a rat rhabdomyosarcoma cell line (BA-HAN-1C) using nonlinear theoretical models. Int J Vit Nutr Res 60:6–17

    Google Scholar 

  4. Biesalski HK, Stofft E (1992) Biochemical, morphological, and functional aspects of systemic and local vitamin A deficiency in the respiratory tract. Ann NY Acad Sci 669:325–331

    PubMed  CAS  Article  Google Scholar 

  5. Biesalski HK, Weiser H (1990) Microdetermination of retinyl esters in guinea pig tissues under different vitamin A-status conditions. J Micronutr Anal 7:97–116

    CAS  Google Scholar 

  6. Bonanni F, De Luca L (1974) Vitamin A-dependent fucose-glycopeptide from rat tracheal epithelium. Biochim Biophys Acta 343:632–637

    PubMed  CAS  Google Scholar 

  7. Bullock GR (1984) The current status of fixation for electron microscopy: a review. J Microscopy 133:1–15

    CAS  Google Scholar 

  8. Chopra DP, Cooney RA, Taylor GW (1990) Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster. Cell Tissue Kinet 23:575–586

    PubMed  CAS  Google Scholar 

  9. Chopra DP, Joiakim AP (1991) Differences in lectin binding in squamous metaplasia induced by benzo(a)pyrene and vitamin A deficiency in hamster tracheal explants. In Vitro Cell Dev Biol 27A:223–228

    PubMed  CAS  Google Scholar 

  10. Chopra DP, Joiakim AP (1991) Alterations in sugar residues in squamous metaplasia in hamster tracheal explants induced by benzo(a)pyrene and its reversal by retinoic acid. In Vitro Cell Dev Biol 27A:229–233

    PubMed  CAS  Google Scholar 

  11. Chopra DP, Klinger MM, Sullivan JK (1989) Effects of vitamin A on growth and differentiation of human tracheobronchial epithelial cell cultures in serum-free medium. J Cell Science 93:133–142

    PubMed  CAS  Google Scholar 

  12. Clark AB, Randell SH, Nettesheim P, Gray TE, Bagnell B, Ostrowski LE (1995) Regulation of ciliated cell differentiation in cultures of rat tracheal epithelial cells. Am J Respir Cell Mol Biol 12:329–338

    PubMed  CAS  Google Scholar 

  13. Cooper HS, Farano P, Coapman RA (1987) Peanut lectin binding sites in colons of patients with colitis ulcerosa. Arch Pathol Lab Med 111:270–275

    PubMed  CAS  Google Scholar 

  14. Couch MJ, Pauli BU, Weinstein RS, Coon JS (1988) Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid. J Cell Biochem 37:187–197

    Article  Google Scholar 

  15. Davies PJ, Basilion JP, Chiocca EA, Johnson J, Poddar S, Stein JP (1988) Retinoids as generalized regulators of cellular growth and differentiation. Am J Med Sci 296:164–170

    PubMed  Article  CAS  Google Scholar 

  16. DeLuca LM (1991) Retinoids and their receptors in differentiation, embryogenesis, and neoplasia. FASEB J 5:2924–2933

    CAS  Google Scholar 

  17. Denning MF, Verma AK (1994) The mechanism of the inhibition of squamous differentiation of rat tracheal 2C5 cells by retinoic acid. Carcinogenesis 15:503–507

    PubMed  Article  CAS  Google Scholar 

  18. Edes TE (1991) Exposure to the carcinogen benzo(a)pyrene depletes tissue vitamin A. Nutr Cancer 15:159–166

    PubMed  CAS  Article  Google Scholar 

  19. Edes TE, Gysbers DS (1993) Carcinogen-induced tissue vitamin A depletion. Potential protective advantages of beta-carotene. Ann NY Acad Sci 686:203–212

    PubMed  Article  CAS  Google Scholar 

  20. Forstner G (1995) Signal transduction, packaging and secretion of mucins. Annu Rev Physiol 57:585–605

    PubMed  CAS  Google Scholar 

  21. Gerlach T, Biesalski HK, Weiser H, Haeussermann B, Baessler KH (1989) Vitamin A in parenteral nutrition: uptake and distribution of retinyl esters after intravenous application. Am J Clin Nutr 50:1029–1038

    PubMed  CAS  Google Scholar 

  22. Gross EA, Mellick PW, Kari FW, Miller FJ, Morgan KT (1994) Histopathology and cell replication responses in the respiratory tract of rats and mice exposed by inhalation to glutaraldehyde for up to 13 weeks. Fundam Appl Toxicol 23:348–362

    PubMed  Article  CAS  Google Scholar 

  23. Gudas LJ, Sporn MB, Roberts AB (1994) Cellular biology and biochemistry of the retinoids. In: Sporn MB, Roberts AB, Goodman DS (eds) The retinoids: biology, chemistry and medicine. Raven Press. New York, pp 443–520

    Google Scholar 

  24. Hayat MA (1974) Principles and techniques of scanning electron microscopy: biological applications, vol 1. Van Nostrand Reinhold, New York

    Google Scholar 

  25. Hilfer SR, Schneck SL, Brown JW (1986) The effect of culture conditions on cytodifferentiation of fetal mouse lung respiratory passageways. Exp Lung Res 10:115–136

    PubMed  CAS  Google Scholar 

  26. Honda T, Schulte BA, Spicer SS (1989) Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells. Histochem J 21:241–247

    PubMed  Article  CAS  Google Scholar 

  27. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577–580

    PubMed  CAS  Google Scholar 

  28. Izzotti A, Balansky R, Scatolini L, Rovida A, De-Flora S (1995) Inhibition byN-acetylcysteine of carcinogen DNA adducts in the tracheal epithelium of rats exposed to cigarette smoke. Carcinogenesis 16:669–672

    PubMed  Article  CAS  Google Scholar 

  29. Jetten AM, Rearich JI, Smits HL (1986) Regulation of differentiation of airway epithelial cells by retinoids. Biochem Soc Trans 14:930–933

    PubMed  CAS  Google Scholar 

  30. Jetten AM, Vollberg TM, Nervi C (1992) Hyperplasia and squamous metaplasia in the tracheobronchial epithelium: alterations in the balance of growth and differentiation factors. In: Newell GR, Hong WK (eds) The biology and prevention of aerodigestive tract cancers. Plenum Press, New York, pp 89–93

    Google Scholar 

  31. Kaartinen L, Nettesheim P, Adler KB, Randell SH (1993) Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol Anim 29A:481–492

    PubMed  CAS  Google Scholar 

  32. Karnovsky MJ (1965) Formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137/A 270

    Google Scholar 

  33. Kelsen SG, Johnson RA, Mest S, Stauber Z, Zhou S, Aksoy M, Hilfer SR (1993) Explant culture of rabbit tracheobronchial epithelium: structure and prostaglandin metabolism. Am J Respir Cell Mol Biol 8:472–479

    PubMed  CAS  Google Scholar 

  34. Lotan R, Lotan D, Meromsky L (1984) Correlation of retinoic acid-enhanced sialyltransferase activity and glycosylation of specific cell surface sialoglycoproteins with growth inhibition in a murine melanoma cell system. Cancer Res 44:5805–5512

    PubMed  CAS  Google Scholar 

  35. Manna B, Ashbaugh P, Bhattacharyya SN (1995) Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture. Inflammation 19:489–502

    PubMed  Article  CAS  Google Scholar 

  36. Mass MJ, Kaufman DG (1983) A comparison between the activation of benzo(a)pyrene in organ cultures and microsomes from the tracheal epithelium of rats and hamsters. Carcinogenesis 4:297–303

    PubMed  Article  CAS  Google Scholar 

  37. McDowell EM, Keenan KP, Huang M (1984) Effects of vitamin A deprivation on hamster tracheal epithelium. A quantitative morphologic study. Virchows Arch B Cell Pathol Incl Mol Pathol 45:197–219

    PubMed  CAS  Google Scholar 

  38. McManus JFA (1948) Histological and histochemical uses of periodic acid. Stain Technol 23:99–108

    CAS  PubMed  Google Scholar 

  39. Peters BP, Goldstein IJ (1979) The use of fluorescein-conjugatedBandeiraea simplicifolia B4-isolectin as a histochemical reagent for the detection of α-D-galactopyranosyl groups. Exp Cell Res 120:321–334

    PubMed  Article  CAS  Google Scholar 

  40. Pinnock CB, Douglas RM, Badcock NR (1986) Vitamin A status in children who are prone to respiratory tract infections. Aust Pediatr 22:95–99

    CAS  Google Scholar 

  41. Plotkin GM, Wolf G (1980) Vitamin A and galactosyltransferase of tracheal epithelium. Biochim Biophys Acta 615:94–102

    PubMed  CAS  Google Scholar 

  42. Reen W, Nolan E, Turner C (1985) Expression of tracheal differentiated functions in serum-free hormone-supplemented medium. J Cell Physiol 125:167–181

    Article  Google Scholar 

  43. Reviere R, Schneider S, Woolbright K (1995) Associations between disease and occupation: hypotheses generated from the National Mortality Followback Survey. Am J Ind Med 27:195–205

    PubMed  CAS  Google Scholar 

  44. Richardson KC, Jarret L, Finke EH (1960) Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol 35:313–316

    PubMed  CAS  Google Scholar 

  45. Saunders NA, Bernacki SH, Vollberg TM, Jetten AM (1993) Regulation of transglutaminase type I expression in squamous differentiating rabbit tracheal epithelial cells and human epidermal keratinocytes: effects of retinoic acid and phorbol esters. Mol Endocrinol 7:387–398

    PubMed  Article  CAS  Google Scholar 

  46. Scott JE, Dorling J (1965) Differential staining of acid glycosaminoglycans/mucopolysaccharides by alcian blue in salt solution. Histochemie 5:191–212

    Article  Google Scholar 

  47. Serra R, Pelton RW, Moses HL (1994) TGF beta 1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures. Development 120:2153–2161

    PubMed  CAS  Google Scholar 

  48. Shiba M, Ohiwa T, Klein-Szanto AJP (1984) Lectin-binding sites in preneoplastic and neoplastic lesions of human and rodent respiratory tract. J Natl Cancer Inst 72:43–51

    PubMed  CAS  Google Scholar 

  49. Sommer A, Tarwotjo I, Djunaedi E, West KP Jr, Loeden AA, Tilden R, Mele L (1986) Impact of vitamin A supplementation on childhood mortality. A randomised controlled community trial. Lancet I:1169–1173

    Article  Google Scholar 

  50. Spicer SS, Schulte BA (1992) Diversity of cell glycoconjugates shown histochemically, a perspective. J Histochem Cytochem 40:1–38

    PubMed  CAS  Google Scholar 

  51. Stofft E, Biesalski HK, Niederauer U, Zschäbitz A, Weiser H (1992) Morphological changes in the tracheal epithelium of guinea pigs in conditions of “acute” vitamin A deficiency. Int J Vit Nutr Res 62:143–142

    Google Scholar 

  52. Stoward PJ, Spicer SS, Miller RL (1980) Histochemical reactivity of peanut lectin-horseradish peroxidase conjugate. J Histochem Cytochem 28:979–990

    PubMed  CAS  Google Scholar 

  53. Strum JM, Latham PS, Schmidt ML, McDowell EM (1985) Vitamin A deprivation in hamsters. Correlations between tracheal epithelial morphology and serum/tissue levels of vitamin A. Virchows Arch B Cell Pathol Incl Mol Pathol 50:43–57

    PubMed  CAS  Article  Google Scholar 

  54. Wolterbeek AP, Roggeband R, van Moorsel CJ, Baan RA, Koeman JH, Feron VJ, Rutten AA (1995) Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA repair activities in hamster tracheal epithelium in organ culture. Cancer Lett 91:205–214

    PubMed  Article  CAS  Google Scholar 

  55. Zeitlin PL, Loughlin GM, Guggino WB (1988) Ion transport in cultured fetal and adult rabbit tracheal epithelia. Am J Physiol 254:C691–698

    PubMed  CAS  Google Scholar 

  56. Zschäbitz A, Weiser H, Stofft E, Gabius HJ, Biesalski HK (1996) Modulation of the glycoconjugate expression in the tracheobronchial epithelium during sustained hypovitaminosis A. Histol Histopathol 11:395–405

    PubMed  Google Scholar 

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Affiliations

  1. Institut für Biologische Chemie und Ernährungswissenschaft der Universität Stuttgart-Hohenheim, Fruhwirthstrasse 12, D-70593, Germany

    H. K. Biesalski & L. Engel

  2. Institut für Anatomie der Johannes Gutenberg-Universität Mainz, D-55099, Mainz, Germany

    E. Stofft & A. Zschäbitz

Authors
  1. H. K. Biesalski
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  2. L. Engel
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  3. E. Stofft
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  4. A. Zschäbitz
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Biesalski, H.K., Engel, L., Stofft, E. et al. An ex vivo model of the rat trachea to study the effect of inhalable toxic compounds. Res. Exp. Med. 196, 195 (1996). https://doi.org/10.1007/BF02576842

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  • DOI: https://doi.org/10.1007/BF02576842

Key words

  • Benzo(a)pyrene
  • Morphology
  • Organ culture
  • Rat trachea
  • Vitamin A