Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded phi X174 DNA.

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 phi X174 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 phi X174 DNA. The mean T37-values for inactivation of ss and RF phi X174 DNA by 2.2 x 10(-4)M catechol at 37 degrees 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 phi X174 DNA with a mean T37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T37-value for inactivation of ss phi X174 DNA by 1.8 x 10(-4) M ortho-quinone at 37 degrees and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss phi X174 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 phi X174 DNA with a mean T37-value of 175 min. The rate of inactivation of RF phi X174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss phi X174 DNA: T37 = 49 min. When using excision repair deficient E. coli mutants (uvrA- or uvrC-), a higher inactivation of RF phi X174 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Multi-residue screening of a minimum package of anabolic steroids in urine with GC-MS.

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most abundant ion. Results of validation experiments are presented. The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.

Thyroid peroxidase: kinetics, pH optima and substrate dependency.

The oxidation of iodide, guaiacol and 2,2'-azino-di[3-ethyl-benzthiazoline-(6)-sulphonic acid] and the iodination of tyrosyl residues in bovine serum albumin, catalysed by partly purified thyroid peroxidase, were studied. The enzyme showed pH optima with all electron donors. With the exception of guaiacol, the position of the pH optima depended upon both the electron donor and hydrogen peroxide concentrations. With increased hydrogen peroxide concentrations the optima shifted to lower pH, and with increased iodide concentration to higher pH. For monoiodotyrosine (MIT) formation in bovine serum albumin the position of the pH optimum was also dependent on the hydrogen peroxide concentrations. The position of the pH optimum of the oxidation of guaiacol was pH 9 and independent of substrate and hydrogen peroxide concentrations. It is obvious from these findings that iodination reactions must be studied under well-defined conditions.

Free radical formation from the antineoplastic agent VP 16-213.

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 M NaCl at 20 degrees C, was 257 +/- 4 s. The signal recorded on incubation with HRP/H2O2 or MPO/H2O2 did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.

The role of metabolic activation by cytochrome P-450 in covalent binding of VP 16-213 to rat liver and HeLa cell microsomal proteins.

Covalent binding of 3H-labeled VP 16-213 to rat liver and HeLa cell microsomal proteins was studied in vitro. Metabolic activation by cytochrome P-450 was found to play a role in the covalent binding of VP 16-213 to rat liver microsomal proteins, as shown by the need of NADPH cofactor, the increased binding after phenobarbital pretreatment and the inhibition by SFK-525A. Addition of ascorbic acid or alpha-phenyl-N-tert. butylnitrone to the incubation mixture depressed covalent binding by about 85%, suggesting that formation of a reactive metabolite from the phenolic structure may be involved in the binding process. VP 16-213 did not inhibit aminopyrine N-demethylase at the concentration used in the binding experiments (17 microM), indicating that metabolism of its methylenedioxy group does not play a role in binding to microsomal proteins. HeLa cell microsomes were found to possess aminopyrine N-demethylase activity. Covalent binding of radiolabeled VP 16-213 to HeLa cell microsomes decreased by about 64% if NADPH was omitted.

Inactivation of phi X174 DNA by the ortho-quinone derivative or its reduction product of the antitumor agent VP 16-213.

Biologically active phi X174 DNA is inactivated by the ortho-quinone derivative of the antitumor agent VP 16-213, but not by VP 16-213 itself, VP 16-213 phenoxy radical or aqueous decomposition product(s) of the ortho-quinone. Reduction of the ortho-quinone by cytochrome P-450 reductase and NADPH results in deactivation of the ortho-quinone towards anti-phi X174 DNA activity. However, compared with the parent compound VP 16-213, reduction of the ortho-quinone results in substantial damage towards DNA.