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 μM) and V max (7.23 nmol of metabolites nmol -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.

Proteomic profile in Perna viridis after exposed to Prorocentrum lima, a dinoflagellate producing DSP toxins

“…Peters et al (1998) demonstrated the presence of five different types of CYP including CYP1A, 2B, 2E, 3A, and 4A in M. galloprovincialis, all involved in detoxification of environmental pollutants. Guo et al (2010) reported that OA could be metabolized by human recombinant cytochrome CYP3A4 to produce four oxidized products. However, to date, little is known about the metabolism of DSP toxins in bivalves, although the involvement of detoxifying enzymes, hydrolyzing enzymes, as well as antioxidant enzymes, has been suggested (Manfrin et al, 2012;Vidal et al, 2014).…”

Gene expression profiling of human liver carcinoma (HepG2) cells exposed to the marine toxin okadaic acid

“…These recoveries are consistent with those observed from spiked media at similar concentrations. The poor recovery for the lowest concentration sample (HPLC-MS analysis of the original 6.2 nM sample was determined to be 2.4 nM) sample is more likely due to losses of OA at the lower concentration during the extraction process rather than metabolism of OA as none of the 4 previously characterized metabolites (Guo et al 2010) were detected in any of the samples.…”

“…Guo et al (2010) demonstrated that among 9 human CYP tested CYP3A4 and 3A5 metabolize OA to 4 metabolites. In order to ensure that all observed effects on HepG2 cells might be attributed to OA and not its metabolites, CYP 3A4 or 3A5 activity was assayed by incubation with testosterone.…”

“…Although Hashizume et al (2009) demonstrated an increased tolerance to OA in HepG2 cells transformed to express CYP1A2. Guo et al (2010) noted that among 9 human CYP tested including CYP1A2, only CYP3A4 and 3A5 metabolize OA. Therefore it is likely that OA did not induce CYP expression in HepG2 cells and OA toxicity in HepG2 cells may be attributed entirely to OA itself and not to its metabolites.…”

“…The combined extracts weredried under a stream of N 2 and reconstituted in either 1 ml (highest 3 concentration samples) or 200 μl (control and lowest 2 concentration samples) of methanol. HPLC-MS analysis was carried out on a Finnigan Surveyor series HPLC system (Thermo Fisher Scientific Inc., Waltham, MA) using a 5 μm C18, 250 mm × 4.6 mm Discovery column (Supelco, Bellefonte, PA) coupled to a Finnigan LCQ Deca XP MAX mass ion-trap spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) as described previously (Guo et al 2010). Ten μl of extract was injected.…”