DNA damage by gliotoxin from Aspergillus fumigatus. An occupational and environmental propagule

Adduct detection as measured by 32P DNA radiolabelling and two-dimensional thin-layer chromatography

Michelle C. Golden, Sae J. Hahm, R. E. Elessar, S. Saksonov, Jacob J. Steinberg

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Gliotoxin is produced by the fungus Aspergillus fumigatus. Aspergillus is widespread in the environment and this ubiquitous nature results in disease and co-carcinogenesis to be distributed world-wide. Gliotoxin contains an epipolythiodioxopiperazine (ETP) ring that is believed to be involved in redox reactions. The reactive oxygen species produced interact with DNA to form hydroxylated and other altered DNA products. To measure DNA adduct formation, we used 32P radiolabelling and, after enzymatic DNA digestion, separated adducts in two dimensions using thin-layer chromatography (2D-TLC), with ultimate autoradiography and densitometry. HeLa DNA was incubated with 0.1 mmol l-1 and 0.3 mmol l-1 of gliotoxin (and necessary redox agents) for 1 and 20 h. We found an increase in 6-hydro-5,6- dihydroxythymidine (thymine glycol) monophosphate [d(TG)MP] from 0.0% to 30.4%, an increase in 8-hydroxy-2'-deoxyguanidine monophosphate [8(OH)dGMP] from 0.0% to 4.2%, an increase in deoxynucleotide diphosphate (dNDP) from zero adducts to six DNA adducts, as well as an increase of other as yet unidentified adducts. Also, time exposure may have a greater effect than concentration based on a 20-h incubation with 0.3 mmol l-1 gliotoxin that completely obliterates the pyrimidines deoxythymidine 3'-monophosphate (dTMP) and deoxycytidine 3'-monophosphate (dCMP).

Original languageEnglish (US)
Pages (from-to)97-104
Number of pages8
JournalMycoses
Volume41
Issue number3-4
StatePublished - 1998

Fingerprint

Gliotoxin
radiolabeling
Aspergillus fumigatus
Thin Layer Chromatography
thin layer chromatography
DNA damage
DNA Damage
DNA adducts
DNA Adducts
DNA
Oxidation-Reduction
Deoxycytidine Monophosphate
redox reactions
Pyrimidines
thymine
glycols
densitometry
Densitometry
Diphosphates
pyrimidines

Keywords

  • Adduct
  • Aspergillus fumigatus
  • DNA damage
  • Free radical
  • Gliotoxin
  • Mycotoxin

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Dermatology
  • Microbiology

Cite this

DNA damage by gliotoxin from Aspergillus fumigatus. An occupational and environmental propagule : Adduct detection as measured by 32P DNA radiolabelling and two-dimensional thin-layer chromatography. / Golden, Michelle C.; Hahm, Sae J.; Elessar, R. E.; Saksonov, S.; Steinberg, Jacob J.

In: Mycoses, Vol. 41, No. 3-4, 1998, p. 97-104.

Research output: Contribution to journalArticle

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abstract = "Gliotoxin is produced by the fungus Aspergillus fumigatus. Aspergillus is widespread in the environment and this ubiquitous nature results in disease and co-carcinogenesis to be distributed world-wide. Gliotoxin contains an epipolythiodioxopiperazine (ETP) ring that is believed to be involved in redox reactions. The reactive oxygen species produced interact with DNA to form hydroxylated and other altered DNA products. To measure DNA adduct formation, we used 32P radiolabelling and, after enzymatic DNA digestion, separated adducts in two dimensions using thin-layer chromatography (2D-TLC), with ultimate autoradiography and densitometry. HeLa DNA was incubated with 0.1 mmol l-1 and 0.3 mmol l-1 of gliotoxin (and necessary redox agents) for 1 and 20 h. We found an increase in 6-hydro-5,6- dihydroxythymidine (thymine glycol) monophosphate [d(TG)MP] from 0.0{\%} to 30.4{\%}, an increase in 8-hydroxy-2'-deoxyguanidine monophosphate [8(OH)dGMP] from 0.0{\%} to 4.2{\%}, an increase in deoxynucleotide diphosphate (dNDP) from zero adducts to six DNA adducts, as well as an increase of other as yet unidentified adducts. Also, time exposure may have a greater effect than concentration based on a 20-h incubation with 0.3 mmol l-1 gliotoxin that completely obliterates the pyrimidines deoxythymidine 3'-monophosphate (dTMP) and deoxycytidine 3'-monophosphate (dCMP).",
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