A microtiter‐plate‐based cytochrome P450 3A activity assay in fish cell lines

Abstract

Abstract-Enzymes belonging to the cytochrome P450 3A (CYP3A) subfamily play an important role in the metabolism of endogenous substances and xenobiotics, including pharmaceuticals. Xenobiotics can alter CYP3A expression and activity, and therefore, changes in CYP3A activity may serve as a biomarker of xenobiotic exposure. To determine changes in CYP3A enzyme activity for environmental risk assessment of xenobiotics including pharmaceuticals, high-throughput assays are needed, but these are missing for fish cells to date. Here, we report on the development of a fluorescent-based CYP3A high-throughput assay for four fish cell lines cultivated in 96-well plates based on 7-benzyloxy-4-trifluoromethylcoumarin as a CYP3A substrate. We show that human CYP3A substrate BFC is catalyzed by fish CYP3A enzymes to a fluorescent product. Its formation is dependent on cell numbers and incubation time. Furthermore, we demonstrate that with this new CYP3A assay induction and inhibition of enzyme activity by pharmaceuticals can be determined. This new cell-based assay is suitable for detection of alteration in CYP3A enzyme activity in largescale experiments for screening of pharmaceuticals occurring in the environment.

Transcriptional and post-transcriptional response of drug-metabolizing enzymes to PAHs contamination in red mullet (Mullus barbatus, Linnaeus, 1758): A field study

“…Nevertheless the good positive correlation between BFCOD, cyp1a gene transcription and dealkylating activities underlines the need for further investigation of genes encoding this activity and indicates BFCOD as a possible general marker of CYP induction by PAHs, in this species. A cyp3a induction model using primary mullet hepatocytes would be needed to establish whether cyp3a catalytic assays are reliable markers of PAHs contamination, as suggested in other species (Christen et al, 2009), and to investigate the relationship between cyp3a and BFCOD.…”

“…Response of cyp1a towards PAHs has been extensively studied and is known to be regulated by the aryl hydrocarbon receptor (AhR) (Stegeman and Hahn, 1994;Hahn, 1998). On the other hand, while cyp3a is known to be involved in the metabolism of several drugs (Hasselberg et al, 2008;Christen et al, 2009), its role and regulation in fish is still matter of debate. Few studies have directly addressed its involvement in the response to environmental contaminants, and field data are lacking altogether.…”

“…Moreover, species-specific differences in substrate specificity towards different compounds, including b-naphthoflavone, were recently reported (Smith and Wilson, 2010). Enzymatic activities generally associated with cyp3a include benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) (Hasselberg et al, 2008;Christen et al, 2009), testosterone hydroxylase (James et al, 2005), aminopyrine N-demethylase and erythromycin N-demethylase (Vaccaro et al, 2007), with some contradictory results with regards to their modulation in response to xenobiotics (Li et al, 2008).…”

Comparison of three fluorescent CYP3A substrates in two vertebrate models: pig and Atlantic salmon

“…In fish, BFC is believed to be the specific substrate for CYP3A (Christen et al, 2009). Our study showed that BFCOD activity was inhibited by KTZ in a non-competitive manner in salmon (Figure 3).…”

Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CYP3A enzyme activity

“…Catalytic activities of CYP3A have been determined in different fish by use of different substrates [16,21,22]. It was found that CYP3A enzymatic activity in fish can easily be determined by application of a modified human CYP3A activity assay, in which the conversion of a substrate by CYP3A to a fluorescent product is determined [14,15,[22][23][24][25][26].…”