Aeroplysinin-1 (1) and the structurally related dienone 2 were cytotoxic to Ehrlich ascites tumor (EAT) cells and HeLa tumor cells in the microculture tetrazolium (MTT) and clonogenic assays. Both compounds are bromotyrosine derivatives, isolated from the marine spong Aplysina aerophoba. As the effective concentrations in the MTT assay were lower than in the clonogenic assay, 1 and 2 are able to cause growth inhibition as well as actual cell death in these cell lines. With an IC50 value of 8.2 microM (MTT assay, 2-h incubation, EAT cells), 1 was the more toxic compound. When the cells were depleted of glutathione by pretreatment with buthionine sulfoximine, they were significantly more sensitive toward 1 and 2 in the MTT assay. A dose-enhancement factor as high as 11.8 was found in EAT cells after 2-h incubation with 2. Using electron paramagnetic resonance we were able to measure free radical formation of 1 and 2, yielding the semiquinone structures 3 and 4, respectively, in a culture medium with tumor cells. It is concluded that free radicals are, at least in part, responsible for the cytotoxicity of 1 and 2. This conclusion is in line with expectations derived from the chemical structures of both compounds.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thinlayer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)-and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.

Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Their structures are suggested to be (4R)and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, andrat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4hydroxyochratoxin A.

Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively.