In vitro phase I metabolism of vinclozolin by human liver microsomes

Abstract

1. Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes.

2. Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The K_{M app} for co-eluted M4/DTMBA and M7 was 24.2 ± 5.6 and 116.0 ± 52.6 μM, the V_{Max app} was 0.280 ± 0.015 and 0.180 ± 0.060 nmoles/min/mg protein, and the CL_{int app} was 11.5 and 1.5 mL/min/g protein, respectively. The K_i for M7 was 133.2 ± 63.9 μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP.

3. DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.

Keywords:

• Vinclozolin
• cytochrome P450
• antiandrogen
• biomarkers
• xenobiotic metabolism

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Marycarmen Cruz-Hurtado was a recipient of a scholarship from Consejo Nacional de Ciencia y Tecnología-México (Conacyt) (Number 421556). This work was supported by Conacyt (Grant number 168384).