Biotransformation of Benzo[a]pyrene by Three Rainbow Trout (Onchorhynchus mykiss) Cell Lines and Extrapolation To Derive a Fish Bioconcentration Factor

Abstract

Permanent fish cell lines constitute a promising complement or substitute for fish in the environmental risk assessment of chemicals. We demonstrate the potential of a set of cell lines originating from rainbow trout ( Oncorhynchus mykiss) to aid in the prediction of chemical bioaccumulation in fish, using benzo[ a]pyrene (BaP) as a model chemical. We selected three cell lines from different tissues to more fully account for whole-body biotransformation in vivo: the RTL-W1 cell line, representing the liver as major site of biotransformation, and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines, as important environment-organism interfaces, which likely influence chemical uptake. All three cell lines were found to effectively biotransform BaP. However, rates of in vitro clearance differed, with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with α-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A catalytic activity, and the progression of cellular toxicity upon prolonged BaP exposure revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell lines. Application of the cell-line-derived in vitro clearance rates into a physiology-based toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to 920 reported for rainbow trout in vivo.

Co-exposure to polystyrene plastic beads and polycyclic aromatic hydrocarbon contaminants in fish gill (RTgill-W1) and intestinal (RTgutGC) epithelial cells derived from rainbow trout (Oncorhynchus mykiss)

“…dG- N 2 -BPDE). Although BaP metabolite formation was not determined in RTgill-W1 and RTgutGC cells after BaP exposure in the present study, RTgutGC cells have previously been shown to metabolise BaP (Stadnicka-Michalak et al, 2018), and the detection of BaP-derived DNA adducts (i.e. dG- N 2 -BPDE) (Fig.…”

“…Stadnicka-Michalak et al (2018) observed similar moderate (~10%) effects on cytotoxicity (which was not significant in their study), in both these cell lines after BaP exposure for 48 h. Cytotoxicity of these pollutants is often associated with biotransformation which produces reactive metabolites and/or free-radicals (Imanikia et al, 2016; Banni et al, 2017; White et al, 2017; Jarvis et al, 2018; Reed et al, 2018). CYP1A1 enzyme activity in the RTgill-W1 cell line, as measured by ethoxyresorufin O-deethylase (EROD) activity, has been reported to be low and is not significantly induced by BaP (Stadnicka-Michalak et al, 2018), which may explain the low cytotoxicity to this PAH (present study) and extracts from oil contaminated sediment (Amaeze et al, 2015). To our knowledge, the cytotoxicity of 3-NBA has not been ascertained in RTgill-W1 and RTgutGC cell lines previously.…”

“…It has been recently subjected to an international round robin test which demonstrated to be robust and to show inter-laboratory reproducibility (Lillicrap et al, 2016). More recently the fish intestinal RTgutGC cell line (Kawano et al, 2011) has been used to evaluate the risk posed by novel pollutants (Langan et al, 2018; Stadnicka-Michalak et al, 2018). Thus, this cell line offers another in vitro model with the opportunity to reduce the numbers of fish used in regulatory procedures.…”

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity

“…One could argue that losses may also stem from biotransformation of the chemicals by the cells. Indeed, in a study to explore biotransformation for bioaccumulation prediction in fish, RTgill-W1 cells were shown to biotransform benzo(a)pyrene, albeit in the presence of 5% FBS in the exposure medium (Stadnicka-Michalak et al , 2018a). To what extent RTgill-W1 cells are capable of biotransformation within 24 h of exposure in L-15/ex is not yet known, though the relatively short exposure time combined with the simple nature of the medium suggests that biotransformation might, at minimum, be slowed.…”

<i>In vitro</i> metabolism of pesticides and industrial chemicals in fish

“…46) The basic EROD activity and its induction by BaP are highest in RTL-W1, and the metabolic activity in the depletion of BaP decreases in the order of RTL-W1>RT gut GC>RT gill -W1. 47,48) Both the epifluorescence observation of treated cells and the GC-MS analysis of cell extracts clearly indicated the uptake of BaP by the RT gut GC cells. 49)…”