Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded ΦX174 DNA

Johannes M S van Maanen, M. Vincent M Lafleur, Dennis R A Mans, Eric van den Akker, Cor de Ruiter, Peter R. Kootstra, David Pappie, John de Vries, Jan Retèl, Herbert M. Pinedo

Research output: Contribution to journalArticlepeer-review

Abstract

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded ΦX174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7-9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active ΦX174 DNA. The mean T37-values for inactivation of ss and RF ΦX174 DNA by 2.2 × 10-4M catechol at 37° and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss ΦX174 DNA with a mean T37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T37-value for inactivation of ss ΦX174 DNA by 1.8 × 10-4 M ortho-quinone at 37° and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss ΦX174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss ΦX174 DNA with a mean T37-value of 175 min. The rate of inactivation of RF ΦX174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss ΦX174 DNA: T37 = 49 min. When using excision repair deficient E.coli mutants (uvrA- or uvrC-), a higher inactivation of RF ΦX174 DNA was found: T37 = 29 min for uvrA- E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair. Neutral and alkaline sucrose gradient centrifugation of ss ΦX174 DNA incubated with the ortho-quinone at pH 4.0 revealed the absence of DNA-breakage, suggesting that introduction of strand breaks and alkali-labile sites does not play a role in the inactivation of DNA by the ortho-quinone. Adduct-formation probably plays a role in the inactivation of DNA by this metabolite.

Original languageEnglish (US)
Pages (from-to)3579-3589
Number of pages11
JournalBiochemical Pharmacology
Volume37
Issue number19
DOIs
StatePublished - Oct 1 1988
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology

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