We recently reported the preferential accumulation of 8-hydroxydeoxyguanosine (8OHdG) adducts in cardiac mitochondrial DNA (mtDNA) following acute intoxication of rats with doxorubicin (C.M. Palmeira et al., Biochim. Biophys. Acta, 1321 (1997) 101-106). The concentration of 8OHdG adducts decreased to control values within 2 weeks. Since conventional antineoplastic therapy entails repeated administration of small doses of doxorubicin, it was of interest to characterize the kinetics for the accumulation and repair of 8OHdG adducts in the various DNA fractions. Weekly injections of doxorubicin (2 mg/kg, i.p.) to adult male Sprague-Dawley rats caused a cumulative dose-dependent increase in the concentration of 8OHdG adducts in both mtDNA and nuclear DNA (nDNA) from heart and liver. Following six weekly injections, the concentration of 8OHdG in cardiac mtDNA was 50% higher than liver mtDNA and twice that of cardiac nDNA. In contrast to the rapid repair of 8OHdG observed during the first days following an acute intoxicating dose of doxorubicin, the concentration of 8OHdG adducts remained constant between 1 and 5 weeks following the last injection. This was true for all DNA fractions examined. The cardioselective accumulation and persistence of 8OHdG adducts to mtDNA is consistent with the implication of mitochondrial dysfunction in the cumulative and irreversible cardiotoxicity observed clinically in patients receiving doxorubicin cancer chemotherapy.

II (Prognostic).

The purpose of this investigation was to determine whether acute doxorubicin intoxication causes a preferential accumulation of 8-hydroxydeoxyguanosine (8OHdG) adducts to mitochondrial DNA (mtDNA) as opposed to nuclear DNA (nDNA), particularly in cardiac tissue. Adult male rats received a single i.p. bolus of doxorubicin (15 mg/kg) and were killed 1-14 days later. Acute intoxication with doxorubicin caused a 2-fold greater increase in 8OHdG adducts to mtDNA compared to nDNA, the concentration of adducts to both nDNA and mtDNA being 20%-40% greater for heart as opposed to liver. For both tissues, the relative abundance of adducts was highest at the earliest time-point examined (24 h) and decreased to control values by 2 weeks. The temporal dilution of 8OHdG adducts was not the result of cell hyperplasia and was only partially due to amplification of the mitochondrial genome, most probably via an increase in DNA copy number rather than a stimulation of mitochondrial biogenesis.

Level III.

Epidemiologic study, level III.

A method based on liquid chromatography (LC) combined with electrospray ionization tandem mass spectrometry for the analysis of the frequency of formation of 8-hydroxydeoxyguanosine (80HdGuo), a biomarker of oxidative damage to DNA, has been developed. This analytical technique has the advantage over gas chromatography combined with mass spectrometry of not requiring analyte derivatization, and over LC combined with ultraviolet and electrochemical detection of being chemospecific. The method was evaluated by determining increased frequency of formation of 80HdGuo in male Sprague-Dawley rats given a single intraperitoneal dose of Adriamycin compared to control rats. Detection of one oxidized deoxyguanosine per 5 x 10(5) deoxyguanosines was readily achieved.

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.

No abstract