Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry.

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.

Effect of 3,4-dichloroaniline on the early life stages of the zebrafish (Brachydanio rerio): results of a comparative laboratory study.

An early life-stage (ELS) test was conducted with the zebrafish and 3,4-dichloroaniline (3,4-DCA) in eight laboratories. Based on the results from all eight laboratories, an LOEC of 200 micrograms 3,4-dichloroaniline/liter applies for the early life stages of the zebrafish. Effects observed were reduction of the survival rate and malformations. If the 100 micrograms/liter concentration additionally tested by one laboratory is included in the assessment, an LOEC of 100 micrograms 3,4-DCA/liter is obtained for the survival rate and increase in length. The NOEC is 20 micrograms/liter. The present results of a comparative laboratory study with the zebrafish show that a 42-day ELS test can be conducted with this species of fish, and affords meaningful results.