In vitro-in vivo, cross-life stage and inter-species extrapolation of the biotransformation and uptake of benzo[a]pyrene in two fish species using toxicokinetic models
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Toxicokinetic (TK) models are in silico tools that are used to assess the uptake, biotransformation, and elimination of environmental contaminants. Such models can be used in ecological risk assessment (ERA) and in research to evaluate chemical bioaccumulation. This information is especially useful for fish, which are an important taxonomic group used in ERA. Presently, the TK models developed for fish are accurate for neutral organic chemicals; however, the models do not commonly consider chemicals that are actively biotransformed. Furthermore, most TK models for aquatic organisms focus strictly on the adult life stage, while few are explicitly developed for early-life stages (ELS). Thus, the overall objective of this thesis was to develop life-stage specific TK models for the rapidly biotransformed model chemical, benzo[a]pyrene (B[a]P), in two physiologically distinct species of fishes, i.e., the fathead minnow (Pimephales promelas) and the white sturgeon (Acipenser transmontanus). The first study (Chapter 2) focused on integrating biotransformation of B[a]P into life stage-specific TK models for a standard laboratory fish model, the fathead minnow. Whole-body embryos, and adult liver and gall bladder (bile) were collected from fish aqueously exposed to B[a]P. The respective tissues were analyzed for the activity of phase I and phase II enzymes to assess biotransformation capacity, and B[a]P metabolites were measured to act as a model validation data set. The biotransformation rate of B[a]P was determined using measurements of in vitro intrinsic clearance and implemented into a multi-compartment adult model using in vitro-in vivo extrapolation. For the embryo-larval stage, whole-body biotransformation was allometrically scaled from the calculations of adult biotransformation and directly implemented into a one-compartment embryo-larval TK model. No difference in phase I or II activity was observed with exposure to increasing concentrations to aqueous B[a]P; however, a significant increase in B[a]P metabolites was observed in both life stages. Both models showed good predictive power with model predictions within one order of magnitude of measured values. The second study (Chapter 3) focused on developing life-stage specific TK models for white sturgeon. Similar to chapter 2, whole-body embryos were sampled from white sturgeon larvae aqueously exposed to B[a]P for assessment of phase I and II activity, and for measurements of whole-body B[a]P metabolites. In contrast to chapter 2, however, whole-body biotransformation of B[a]P could not be scaled from the calculated value of sub-adult B[a]P biotransformation, and therefore, was internally calibrated. The calibrated value of whole-body biotransformation was implemented into a one-compartment embryo-larval TK model and used to make predictions of the internal concentrations of B[a]P metabolites. Due to logistical and ethical reasons (white sturgeon are an endangered species in Canada), an aqueous B[a]P exposure with the adult life stage could not be conducted. Instead, an experimental data set of four chemicals was compiled from previously conducted sub-adult sturgeon bioaccumulation studies found in the scientific literature to be used as a model validation data set. A multi-compartment TK model was parameterized using direct measurements of wet mass, tissue volume, tissue lipid fraction, tissue water content and cardiac output, and literature values of oxygen consumption, from sub-adult white sturgeon, and used to make predictions of the internal concentrations of organic contaminants in the sub-adult life stage. The model results showed that both models could accurately predict the bioaccumulation of organic contaminants within one order of magnitude. In chapter 4, the life-stage and species specific models were used to characterize the differences in the uptake and biotransformation of B[a]P between life-stages and species. In the fathead minnow, the results of the life-stage analysis showed that the embryo-larval life-stage accumulated a greater abundance of parent B[a]P and B[a]P metabolites; however, the accumulation was slower than what was observed in the adults. The results of the life-stage analysis in white sturgeon showed a similar uptake of parent B[a]P between life stages, but a larger abundance of B[a]P metabolites was predicted in the sub-adult life stage. In both species, the results suggest that the embryo-larval stage might have a lesser capacity to generate the enzymes required for biotransformation B[a]P. The comparative species analysis showed that in both the embryo-larval and sub-adult/adult life stages, white sturgeon showed substantially slower biotransformation of B[a]P. Accordingly, in both life stages, a lesser abundance of metabolites was predicted/measured in the white sturgeon. It was concluded that the embryo-larval life stage of white sturgeon would be comparatively more susceptible to the effects of aryl hydrocarbon receptor binding, as a result of the slower rates of biotransformation, while the sub-adult/adult life stage of the fathead minnow would be comparatively more susceptible to the genotoxic effects associated with B[a]P exposure, as a result of the faster rates of biotransformation. Overall, the models developed in this thesis contribute to the increasing scope of applications in which TK models can be used in ERA and research by providing a basis in which cross-life stage and interspecies extrapolation can be conducted for the bioaccumulation of actively biotransformed chemicals.
DegreeMaster of Science (M.Sc.)
SupervisorBrinkmann, Markus; Hecker, Markus
CommitteeWeber, Lynn; Jones, Paul; Laprairie, Robert
Copyright DateJanuary 2021
in vitro-in vivo extrapolation