Skip to main content
SpringerLink
Log in

Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay

  • Molecular Medicine
  • Published:
The clinical investigator Aims and scope Submit manuscript

Abstract

The detection of DNA single-strand breaks (SSB) in human mononucleated white blood cells (MWBC) using a modified version of the nick translation assay is presented. This assay allows rapid and sensitive examination of SSB using only 5 ml heparinized blood for an eightfold determination. The assay was standardized by incubation of MBWC in vitro with N-methyl-N′-nitro-N-nitro-soguanidine (MNNG), a known genotoxic agent. In vitro incubation of MWBC with MNNG induced a dose-dependent increase in DNA-SSB at doses between 5 and 500 μM MNNG. The detection limit for the assay was 5 μM MNNG. To assess the suitability of this assay to detect SSB in vivo a controlled study was performed in which volunteer smokers (n = 5), nonsmokers (n = 5) exposed to environmental tobacco smoke (ETS), and nonsmoker controls (n = 5) were compared. The study lasted 4 experimental days, 2 control and 2 exposure days. On control days (days 1 and 3) smokers and nonsmokers sat in an unventilated 45 m3 room for 8 h. On the exposure days (days 2 and 4) each of the five smokers smoked 24 cigarettes in 8 h, while the five nonsmokers were exposed to the ETS generated by the smoking volunteers. High exposure to tobacco smoke was confirmed by dosimetry of carboxyhemoglobin (COHb), plasma nicotine and cotinine levels. Blood was drawn before and after each exposure on all 4 experimental days for determination of DNA-SSB in lymphocytes immediately after isolation of blood cells. COHb, plasma nicotine, and cotinine levels were considerably increased in both smokers and nonsmokers exposed to ETS on days 2 and 4. DNA-SSB were detected in all volunteers with intra-and interindividual day to day and morning to evening variations. After smoking, SSB increased on day 2 and on day 4 in smokers. In nonsmokers exposed to ETS no exposure-related variation in SSB levels was found. We conclude that the modified nick translation assay is sensitive enough to detect SSB induced in vivo by exposure to a genotoxic agent and could therefore be used in biological monitoring at the workplace.

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

COHb:

carboxyhemoglobin

DMEM:

Dulbecco's modification of Eagle's minimal essential medium

SSB:

single-strand break

ETS:

environmental tobacco smoke

MNNG:

N-methyl-N′-nitro-N-nitrosoguanidine

MWBC:

mononucleated white blood cells

NT:

nick translation

References

  1. Anai H, Maehara Y, Sugimachi K (1988) In situ nick translation method reveals DNA strand scission in HeLa cells following heat treatment. Cancer Lett 40:33–38

    Google Scholar 

  2. Boyum A (1977) Separation of lymphocytes, lymphocyte subgroups and monocytes. A review. Lymphology 10:71–76

    Google Scholar 

  3. Conner JM, Oldaker G, Murphy JJ (1990) Method for assessing the contribution of environmental tobacco smoke to respirable suspended particles in indoor air. Environ Technol Lett 11:189–196

    Google Scholar 

  4. Dunn BP, Vedal S, San RHC, Kwan W-F, Nelems B, Enarson DA, Stich HF(1991) DNA adducts in bronchial biopsies. Int J Cancer 48:485–492

    Google Scholar 

  5. Everson RB, Randerath E, Santella RM, Cefalo RC, Avitts TA, Randerath K (1986) Detection of smoking-related covalent DNA adducts in human placenta. Science 231:54–57

    Google Scholar 

  6. Fenech M, Morley AA (1985) Solutions to the kinetic problem in the micronucleus assay. Cytobios 43:233–246

    Google Scholar 

  7. Feyerabend C, Russell MAH (1990) A rapid gas-liquid chromatographic method for the determination of cotinine and nicotine in biological fluids. J Pharm Pharmacol 42:450–452

    Google Scholar 

  8. Holz O, Krause T, Scherer G, Schmidt-Preuß U, Rüdiger HW (1990) 32P-postlabelling analysis of DNA adducts in monocytes of smokers and passive smokers. Int Arch Occup Environ Health 62:299–303

    Google Scholar 

  9. International Agency for Research on Cancer (1986) Monographs on the evaluation of the carcinogenic risk of chemicals to humans, vol. 38, tobacco smoking. IARC, Lyon

    Google Scholar 

  10. Langone J, Gjika HB, Van Vunakis H (1973) Nicotine and its metabolites: radioimmunoassay for nicotine and cotinine. Biochemistry 12:5025–5030

    Google Scholar 

  11. Kohn KW (1991) Principles and practice of DNA filter elution. Pharmacol Ther 49:55–77

    Google Scholar 

  12. Kohn KW, Ewig RA (1973) Alkaline elution analysis, a new approach to the study of DNA single strand interruptions in cells. Cancer Res 33:1849–1853

    Google Scholar 

  13. Kohn KW, Friedman CA, Ewig RAG, Igbal ZM (1974) DNA chain growth during replication of asynchronous L1210 cells. Alkaline elution of large DNA segments from cells lysed on filters. Biochemistry 13:4134–4139

    Google Scholar 

  14. Lett JT, Klucis ES, Sun C (1970) On the size of the DNA in mammalian chromosome: structural subunits. Biophys J 10:272–292

    Google Scholar 

  15. Libbus BL, Illenye SA, Craighead JE (1989) Induction of DNA strand breaks in cultured rat embryo cells by crocidolite asbestos as assessed by nick translation. Cancer Res 49:5713–5718

    Google Scholar 

  16. Manoharan K, Kinder D, Banerjee MR (1987) DMBA induced DNA damage in mammary epithelial cells in vitro measured by a nick translation assay. Cancer Biochem Biophys 9:127–132

    Google Scholar 

  17. Masuck TM, Taylor AR, Lough J (1990) Arabynosyl-cytosine-induced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation. J Cell Physiol 144:12–17

    Google Scholar 

  18. Nakayama T, Kaneko M, Kodama M, Nagata C (1985) Cigarette smoke induces DNA single-strand breaks in human cells. Nature 314:462–464

    Google Scholar 

  19. Noll H (1967) Characterization of macromolecules by constant velocity sedimentation. Nature 215:360

    Google Scholar 

  20. Nose K, Okamoto H, (1983) Detection of carcinogen-induced DNA breaks by nick translation in permeable cells. Biochem Biophys Res Commun 111:383–389

    Google Scholar 

  21. Ogden MW (1989) Gaschromatographic determination of nicotine in environmental tobacco smoke: collaborative study. J Assoc Off Anal Chem 72:1002–1006

    Google Scholar 

  22. Ogden MW, Maiolo K (1990) Comparison of GC and LC for determining solanesol in environmental tobacco smoke. LC/GC 10:459–462

    Google Scholar 

  23. Panigrahi GB, Walker IG (1991) The reaction of acetyl-4-hydroxyaminoquinoline 1-oxide with DNA: quantitation of single-strand break formation and hyper-reactivity of DNA termini. Carcinogenesis 12:963–967

    Google Scholar 

  24. Rüdiger HW, Ehlert U, Roser M, Reimers U, Lehnert G (1988) Erfahrungen mit einem modifizierten Mikronukleustest zur Erfassung genotoxischer Risiken. Verh Dtsch Ges Arbeitsmed 28:113–116

    Google Scholar 

  25. Sarto F, Faccioli MC, Cominato I, Levis AG (1985) Aging and smoking increases the frequence of sister chromatid exchanges (SCE) in man. Mutat Res 144:183–187

    Google Scholar 

  26. Saul RL, Ames BN (1986) Background levels of DNA damage in the population. In: Simic M, Grossmann L, Upton A (eds), Mechanisms of DNA damage and repair. Plenum, New York. pp 529–535

    Google Scholar 

  27. Savela K, Hemminki K (1991) DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabeling assay. Carcinogenesis 12:503–508

    Google Scholar 

  28. Snyder RD (1986) Evaluation of putative inhibitors of DNA excision repair in cultured human cells by the rapid nick translation assay. Mutat Res 173:279–286

    Google Scholar 

  29. Snyder RD, Matheson DW (1985) Nick translation — a new assay for monitoring DNA damage and repair in cultured human fibroblasts. Environ Mutagen 7:267–279

    Google Scholar 

  30. Turner S, Cyr L, Gross AR (1992) The measurement of environmental tobacco smoke in 585 office environments. Environ Int 18:19–28

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Abteilung Arbeitsmedizin, Klinik für Innere Medizin IV, Universität Wien, Währinger Gürtel 18-20, A-1090, Wien, Austria

    M. Einhaus, O. Holz, R. Meißner, T. Krause, K. Warncke, I. Held & H. W. Rüdiger

  2. Analytisch-biologisches Forschungslabor Prof. Dr. med. F. Adlkofer, Goethestrasse 20, D-80336, München, Germany

    G. Scherer, A. R. Tricker & F. Adlkofer

Authors
  1. M. Einhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. O. Holz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. Meißner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. Krause
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K. Warncke
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. I. Held
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. Scherer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. R. Tricker
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. F. Adlkofer
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. H. W. Rüdiger
    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

Einhaus, M., Holz, O., Meißner, R. et al. Determination of DNA single-strand breaks in lymphocytes of smokers and nonsmokers exposed to environmental tobacco smoke using the nick translation assay. Clin Investig 72, 930–936 (1994). https://doi.org/10.1007/BF00190755

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00190755

Key words

Search

Navigation