Characterized in Vitro Metabolism Kinetics of Alkyl Organophosphate Esters in Fish Liver and Intestinal Microsomes

Abstract

Tris(2-butoxyethyl) phosphate (TBOEP) and tris( n-butyl) phosphate (TNBP) are the most commonly used alkyl organophosphate esters (alkyl-OPEs), and they increasingly accumulate in organisms and create potential health hazards. This study examined the metabolism of TNBP and TBOEP in Carassius carassius liver and intestinal microsomes and the production of their corresponding monohydroxylated and dealkylated metabolites. After 140 min of incubation with fish liver microsomes, the rapid depletion of TNBP and TBOEP were both best fitted to the Michaelis-Menten model (at administrated concentrations ranging from 0.5 to 200 μM), with a CL (intrinsic clearance) of 3.1 and 3.9 μL·min·mg protein, respectively. But no significant ( P > 0.05) biotransformation was observed for these compounds in intestinal microsomes at any administrated concentrations. In fish liver microsomes assay, bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) and bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate (3-OH-TBOEP) were the most abundant metabolites of TBOEP, and dibutyl-3-hydroxybutyl phosphate (3-OH-TNBP) was the predominant metabolite of TNBP. Similarly, the apparent V values (maximum metabolic rate) of BBOEHEP and 3-OH-TNBP were also respectively highest among those of other metabolites. Further inhibition studies were conducted to identify the specific cytochrome P450 (CYP450) isozymes involved in the metabolism of TNBP and TBOEP in liver microsomes. It was confirmed that CYP3A4 and CYP1A were the significant CYP450 isoforms catalyzing the metabolism of TNBP and TBOEP in fish liver microsomes. Overall, this study emphasized the importance of hydroxylated metabolites as biomarkers for alkyl-OPEs exposure, and further research is needed to validate the in vivo formation and toxicological implications of these metabolites.

Metabolism and fate of organophosphate ester (OPE) contaminants in rainbow trout (Oncorhynchus mykiss) using in vitro lab-based studies

“…In mammalian and avian species, rapid metabolism provided an explanation for the phenomenon of high OPE concentrations in environmental matrices with low OPE tissue residue levels. The degree to which fish are able to metabolize OPEs varies with species and as such, it is possible that compounds not metabolized well by rainbow trout are being transformed lower on the food chain or by other metabolically active tissues such as the intestine or gills (Hou et al, 2018(Hou et al, , 2019Lindström-Seppä et al, 1981;Sasaki et al, 1981;. Rapid metabolism may play a lesser role in the fate of aryl and chlorinated OPEs in a piscine context, leading to a potential to bioaccumulate.…”

“…varied for each OPE, though all were 1 -2 orders of magnitude less than VMAX values for TCIPP metabolized in human liver microsomes (1,470 ± 110 pmol/min/mg protein) as well as TBOEP and TNBP metabolized in crucian carp liver microsomes (462.9 ± 56.8 pmol/min/mg protein and 586.9 ± 179.5 pmol/min/mg protein, respectively) (Greaves et al, 2016b;Hou et al, 2018;Van den Eede et al, 2015, 2016a (Su et al, 2015b). Hydroxylated metabolites were also identified for TNBP in rat liver microsomes as TNBP-OH, which was further metabolized to TNBP-(OH)2 and DNBP (Sasaki et al, 1984).…”

“…The selected OPEs have been consistently detected in the environment and to a lesser degree in biota, making them an environmentally relevant choice for study. Additionally, previous in vitro studies with these OPEs in polar bear, ringed seal, herring gull, and crucian carp allow for cross-taxa comparison between avian, mammalian, and piscine models (Greaves et al, 2016b;Hou et al, 2018;Strobel et al, 2018a) Given the procedural changes from Chu et al (2011) to maintain optimal temperature conditions for fish microsomes, TBOEP was used in the positive control instead of TDCIPP for each batch. TBOEP was consistently depleted completely in the positive controls during the 100 minute incubation period.…”

“…enzymes (Hou et al, 2018;Lindström-Seppä et al, 1981;Van den Eede et al, 2013a). In addition to this, recent studies have reported depletion of TPHP and formation of DPHP in the absence of NADPH (Strobel et al, 2018a;Van den Eede et al, 2013a).…”

“…Previous studies have identified extrahepatic metabolic activities in both the gills and intestine of rainbow trout, and this latter metabolism can limit the bioavailability of compounds ingested orally (Kleinow et al, 1998;Lindström-Seppä et al, 1981;Van Veld et al, 1988). Hou et al (2018) used intestinal microsomes to investigate in vitro metabolism of TNBP and TBOEP in crucian carp. They detected no significant metabolism of both TNBP and TBOEP, which they suggested was due to lower levels of CYP450 enzymes in intestinal microsomes.…”