Towards High Throughput Determination of Biotransformation Rates of Chemical Mixtures Using Isolated Perfused Trout Livers


In the field of environmental risk assessment, aquatic contaminants are typically characterized by their persistence, bioaccumulation potential, and toxicity (PBT). Bioconcentration factor (BCF) is the most common metric for evaluating bioaccumulation potential and is frequently estimated using Quantitative-Structure-Activity-Relationships (QSARs) and other in silico tools. These methods can estimate BCF based upon the physicochemical characteristics of a chemical and the toxicokinetic parameters of a model organism for chemicals which do not undergo biotransformation. For chemicals which are actively biotransformed, whole animal in vivo exposures have classically been required to determine BCF for regulatory acceptance. Recently, in vitro substrate depletion assays have been adopted as an alternative to in vivo testing due to concerns of cost and ethics. The results of these assays have been met with doubt due to uncertainties involved with in vitro-in vivo extrapolation (IVIVE) methods required to bridge the gap between target organs of biotransformation such as the liver, and whole-organism outcomes. The overall objective of this thesis was to validate an IVIVE approach for estimating biotransformation in rainbow trout (Oncorhynchus mykiss) by using the isolated perfused liver method, which represents an intermediate between in vitro and in vivo. The first study (Chapter 2) involved comparing direct measurements of hepatic clearance in the isolated perfused liver with in vitro determinations of clearance which were previously published as part of a collaborative international trial for the regulatory adoption of in vitro substrate depletion assays. This comparison was performed for the model biotransformation substrate chemicals pyrene, phenanthrene, 4-n-nonylphenol, deltamethrin, and methoxychlor. The hepatic clearance rate of these chemicals was determined in isolated perfused livers by measuring the difference between chemical concentration as it entered and exited the liver, giving a measure of chemical elimination due to biotransformation. Experiments were performed over a period of several hours to validate physiological performance, and measurements of glucose efflux and pH were used to confirm aerobic respiration and thus metabolic activity. In addition, the influence of protein binding on hepatic clearance was investigated by varying the concentration of bovine serum albumin (BSA) in perfusates spiked with chemical. Measured clearances were in good agreement with in vitro substrate depletion models coupled with an IVIVE approach which takes protein binding into account. Overall, this study indicated that uncertainty associated with current IVIVE models is likely due to extrahepatic biotransformation, variability in BCF test designs, and inaccuracies in partitioning estimates or other kinetic processes such as uptake across the gills, rather than the reliability of in vitro test methodologies. The second study (Chapter 3) focused on expanding the domain of applicability of the isolated perfused liver method following validation in Chapter 2 as well as concurrent research investigating mixture experiments in ionizable organic compounds (IOCs). One of the primary concerns about bioaccumulation assessment methods regardless of in silico, in vitro, or in vivo approaches is the limited number of chemicals which have been successfully tested. To achieve the throughput needed by modern chemical risk assessment frameworks, a mixture experiment was performed using the isolated perfused liver model. This study used a chemical mixture obtained from the United States Environmental Protection Agency (EPA) as part of the EPA’s non-targeted analysis collaborative trial (ENTACT). This mixture contained over 500 chemicals, and the study utilized advanced high-resolution-mass-spectrometry (HRMS) methods to detect individual compounds in mixture at the very low concentrations required for mixture experiments. Hepatic clearance was determined for 20 substances in this mixture simultaneously, representing diverse classes of chemicals including pharmaceuticals, pesticides, and industrial chemicals. This study demonstrated that the isolated perfused liver method can be a valuable tool for bioaccumulation screening, and validated the performance of this model for diverse groups of chemicals. Chapter 4 discusses the present state of bioaccumulation assessment with regard to biotransformed chemicals, focusing on the role of the isolated perfused liver method and the goals of validation and demonstration set out by this thesis. Many studies have focused on which factors involved in IVIVE drive the discrepancies between BCFs determined by in vivo exposures with those predicted by IVIVE approaches. The difference between chemical freely available for biotransformation in the systemic circulation in vivo versus in vitro has been identified as a source of uncertainty. This ratio has typically been assumed to be identical, as this assumption whilst mechanistically impossible leads to a more reliable prediction of BCF. In Chapter 2, it was demonstrated that the influence of protein binding on hepatic clearance can be incorporated into an IVIVE model to increase reliability, indicating that prior assumptions were inappropriate. Concerns over limited domain of applicability were addressed in Chapter 3 as well as in concurrent work involving IOCs, focusing on the high-throughput nature of these experiments in which six fish were used to screen 20 chemicals. In summary, the isolated perfused liver model advanced in this thesis work has contributed to the field of bioaccumulation assessment by validating presently adopted in vitro assays. Furthermore, this work has expanded the chemical scope of methods developed to achieve high-throughput predictions of biotransformation, serving as the basis for incorporation into reliable IVIVE models

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Last time updated on 07/02/2024

This paper was published in University of Saskatchewan Research Archive.

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