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

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).

Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

“…In this specific case, the toxins were not associated with spores or mycelium but rather were absorbed into the materials themselves. The concentrations of gliotoxin known to lower ciliary beating frequency or cause DNA adduct formation were above 0.2 and 32.6 g/ml, respectively (1,6). These can be compared to the levels detected in the experimentally mold-damaged building materials reported here, from 1 to 40 ng of gliotoxin per cm 2 depending on the building material tested.…”

“…These reactive oxygen species or other radicals generated through redox cycling are also capable of causing DNA damage as demonstrated elsewhere in in vitro studies (3,5,6). In this study, we found that bacterial DNA repair systems appear to The mechanism of colonization is still poorly understood, but the ability of culture filtrates of A. fumigatus to decrease ciliary beating frequency (gliotoxin has been identified as a cilioinhibitory factor) and to damage human respiratory epithelium in vitro (R. Amitani, A. Sato, Y. Matsui, A. Niimi, K. Yamada, E. Tanaka, T. Murayama, K. Maeda, and F. Kuze, American Thoracic Society meeting, Am.…”

Evaluation of DNA adduction of AZT in peripheral blood leukocytes of HIV-infected individuals by 32P-post-labeling thin-layer chromatography: a feasibility study

“…We developed 32 P-post-labeling 2-D TLC assay to detect radioactivity labeled nucleic acid components quantitatively and qualitatively [20]. This assay has been successfully reported to measure 2 -deoxyuridine-3 -monophosphate (dUMP) adduct [21], 5-bromo-2 -deoxiuridine (BUDR) adduct [22], and gliotoxin induced adduct [23] in tested DNA samples. This technique is sensitive enough to detect one adduct per 10 5 nucleotides.…”

Nucleic Acids and Their Derivatives

“…One can use as much specific chemical characterization as possible (58) but ultimately rely on fingerprint analysis. Many published figures demonstrate examples of fingerprint chromatograms (55,56,59).…”