New model system for testing effects of flavonoids on doxorubicin-related formation of hydroxyl radicals.

Doxorubicin belongs to anthracycline cytotoxic drugs and it is widely used as a major therapeutic agent in the treatment of various types of tumors. However,its therapeutic use is limited by the development of myelosuppression and cardiotoxicity after a specific cumulative dose is reached. The aim of this study was to investigate the effect of flavonoids, either natural or synthetic on doxorubicin-mediated formation of oxidative stress implicated in doxorubicin toxicity. Doxorubicin caused a concentration-dependent increase in the formation of hydroxyl radicals in minipig liver microsomes used as an in-vitro model system. When bacterial membranes heterologously expressing human NADPH cytochrome-P450 oxidoreductase were incubated with doxorubicin, formation of the superoxide radical under aerobic conditions and the doxorubicin­semiquinone radical under anaerobic conditions was detected. Forty different flavonoids were tested for their potency to prevent NADPH-induced or Fe2+-induced peroxidation of lipids in the microsomal system. According to the results, seven flavonoids were selected for evaluation of their potency to inhibit doxorubicin-dependent formation of hydroxyl radicals assessed by electron spin resonance. Myricetin, fisetin, and kaempferol were found to produce a significant protective effect against hydroxyl radicals in the minipig liver microsomal system. In conclusion, this study shows the use of a novel cost-effective in-vitro model system for preselection of antioxidants for testing of their protective effects against toxicity of anthracyclines and potentially other oxidative stress-inducing chemicals.

The effect of flavonoid derivatives on doxorubicin transport and metabolism.

This study investigated the effect of naturally occurring flavonoids and synthetic aurone derivatives on the formation of cardiotoxic doxorubicinol and transport of doxorubicin in breast cancer cells. Quercetin significantly inhibited the formation of doxorubicinol. Quercetin and aurones did not significantly affect transport of [14C]doxorubicin in human resistant breast cancer cells. In conclusion, quercetin should be further tested for its potency to decrease doxorubicin-mediated toxicity.

Cytochrome P450 destruction by benzene metabolites 1,4-benzoquinone and 1,4-hydroquinone and the formation of hydroxyl radicals in minipig liver microsomes.

Reactive metabolites of benzene 1,4-benzoquinone and 1,4-hydroquinone exert their toxic effects through covalent and/or oxidative damage to DNA and proteins. Since minipigs have been proposed as a suitable model species in toxicological and pharmacological research, the aim of this study was to explore mechanisms by which catechol, 1,4-hydroquinone and 1,4-benzoquinone destroy cytochrome P450 (P450) and induce oxidative stress in minipig liver microsomes. Our second goal was to assess the usefulness of minipig liver microsomes as a model system for the testing of the production of oxidative stress by clinically relevant quinone-containing compounds, e.g. anthracyclines. Of the three benzene metabolites tested, the highest P450 destruction was caused by 1,4-benzoquinone. This destructive effect did not correlate with the production of hydroxyl radicals as measured by ESR spin trapping which was the highest in samples containing 1,4-hydroquinone. Our results confirm previous findings that 1,4-benzoquinone exerts its effect mainly by direct attack on macromolecules while 1,4-hydroquinone rather stimulates the production of reactive oxygen species. Doxorubicin stimulated the production of hydroxyl radicals and the destruction of P450 similarly as 1,4-hydroquinone. Minipig liver microsomes should be further tested as a possibly suitable model system for the testing of potential modulators of the toxicity of doxorubicin.

Carbonyl reduction of the potential cytostatic drugs benfluron and 3,9-dimethoxybenfluron in human in vitro.

Benfluron (B, [5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is a potential benzo[c]fluorene antineoplastic agent with high activity against a broad spectrum of experimental tumors in vitro and in vivo. The structure of B has been modified to repress its rapid deactivation through carbonyl reduction on C7. 3,9-Dimethoxybenfluron (D, [3,9-dimethoxy-5-(2-N-oxo-2-N',N"-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene]) is one of the B derivatives developed. The present paper was designed to compare the C7 carbonyl reduction of B and D in microsomes, cytosol and hepatocytes from human liver. Two purified human enzymes, microsomal 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1) and cytosolic carbonyl reductase, were tested if they are responsible for B and D carbonyl reduction in the respective fractions. Indeed, carbonyl reduction of D in comparison to that of B was 4 and 6-10 times less extensive in human liver microsomes and cytosol, respectively. Moreover, about 10-20 times higher amounts of dihydro B than dihydro D were detected in primary culture of human hepatocytes. 11beta-HSD 1 was shown to be able to reduce B and D. For this enzyme, about 10 times higher rates of carbonyl reduction were observed for B than for D. Likewise, CR participates in B and D carbonyl reduction, although smaller amounts of both reduced metabolites were detected. In summary, carbonyl reduction of D was significantly less extensive than that of B in all in vitro experiments. This lower rate of D inactivation was especially pronounced in hepatocytes which represent a close to in vivo situation. Our results clearly demonstrate that dimethoxy substitution protects the carbonyl group of the benzo[c]fluorene moiety against the deactivation by microsomal and cytosolic reductases. Detailed knowledge on the participating enzymes may serve as a basis for the co-application of specific inhibitors in chemotherapy to further improve the pharmacokinetics of benzo[c]fluorene derivatives.