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).
Adriamycin (ADM, or doxorubicin hydrochloride) is an effective antineoplastic agent whose use is restricted by its well-described, dose-dependent cardiotoxicity. This study measures ADM DNA adduct formation by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thinlayer chromatography (2D-TLC), and quantitating the adducts with autoradiography and densitometry. Thirty-six male Sprague-Dawley rats are randomized to receive ADM at varying intraperitoneal (i.p.) injection concentrations: 0.9% saline i.p. controls, 4 mg/kg ADM i.p., and 6 mg/kg ADM i.p. Myocardial and pulmonary tissues are harvested 48 hours after i.p. injection for autoradiographic and histopathologic analyses. The results indicate differences in the amount and type of adduct formation as a function of ADM concentration. Increased partial depurination of dGMP and dAMP occurs with increasing ADM concentration at equal incubation times. This depurination correlates with the emergence of new adducts HM-dUMP, 8-OH-dGMP, HM-dCMP, and Me-dCMP. The quantification of these adducts can potentially represent an early marker of ADM cardiotoxicity and thereby optimize the efficacy of individual chemotherapy regimens while minimizing adverse effects.
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