The algal hepatoxoxin okadaic acid is a substrate for human cytochromes CYP3A4 and CYP3A5

Abstract

The hepatotoxin okadaic acid (OA) was incubated with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Both CYP3A4 and CYP3A5 converted OA to a mixture of the same four metabolites, but incubation with CYP3A4 resulted in higher levels of conversion. Michaelis-Menten parameters, K(m) (73.4 microM) and V(max) (7.23 nmol of metabolitesnmol(-1)min(-1)) for CYP3A4 were calculated by analyzing double-reciprocal plots. LC-MS(n) analysis and chemical interconversion indicate that metabolites 2 and 3 are the 11S-hydroxy and 11R-hydroxy okadaic acid respectively, while metabolite 4 is 11-oxo okadaic acid. LC-MS(n) analysis of metabolite 1 shows a molecular ion which corresponds to an addition of 16 amu to OA, also suggesting hydroxylation, but the specific site has not been identified. The same four metabolites were produced upon incubation of okadaic acid with pooled human liver microsomes. This transformation could be completely inhibited with ketokonazole, and inhibitor of the CYP3A family of enzymes. The metabolites were determined to be only slightly less potent inhibitors of serine threonine protein phosphatase 2A (PP2A) when compared to OA. As PP2A is the principle molecular target for OA, these oxidative transformations may not effectively detoxify OA.

Figure 1

Structure of OA and metabolites and proposed fragmentation pathways.

Figure 2

Total ion chromatogram (TIC) of metabolic products of OA incubated with CYP3A4. (a) 0 h (b) 30 min. (c) Total ion chromatogram (TIC) of metabolic products of OA incubated with HLM. The mass range was from m/z 800 - 850.

Figure 3

(a) Metabolic profile of 50 μM OA incubated with 500 pmol CYP3A4 in 0.5 ml final volume. (b) Double-reciprocal plot (1/V vs 1/S)

Figure 4

Typical negative ESI MS/MS spectra of OA (a), metabolite 1 (b), 3 (c) and 4 (d).

Figure 5

MS³ spectra of fragment A from metabolite 2 (a), 3 (b) and 4 (c) in negative ESI mode.

Figure 6

Proposed MS³ fragmentation pathways of m/z 271 from metabolites 2 and 3 in negative ESI mode.

Figure 7

The dose-response inhibitory activity of OA and its metabolites on PP2A .