Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Variability in fish bioconcentration factors: Influences of study design and consequences for regulation.

The fish bioconcentration factor (BCF) is an important aspect within bioaccumulation assessments. Several factors have been suggested to influence BCF values - including species, developmental stage, mixture exposure, and calculation method. However, their exact contribution to variance in BCF values is unknown. Within this study we assessed the relative impact of these test characteristics on BCF values and analyzed the reproducibility of aquatic exposure bioconcentration tests. Linear mixed effects analyses were performed on a newly develop database to investigate the relationship between the response variable (i.e. lipid normalized log BCF values) and several test characteristics as fixed effects. Lower BCF values were observed for substances that were simultaneously applied with high molecular weight polycyclic aromatic hydrocarbons compared to single substance exposure (with an average difference of -0.81 log BCF). Also, lower BCFs upon kinetic determination were observed compared to steady-state BCFs (log BCF -0.27), and lower BCFs for species from the Ostariophysi subcohort level (log BCF -0.17 to -0.15). In addition, data analysis showed high variation within BCF values for single substances (average SD = log BCF 0.21), which questions the robustness of the current bioaccumulation assessments. For example, the 95% confidence range of a BCF value of 2500 ranges from 953 ('not-bioaccumulative') to 6561 ('very bioaccumulative'). Our results show that the use of one single BCF leads to a high uncertainty in bioaccumulation assessments. We strongly recommend that within future bioconcentration studies, the used experimental design and test conditions are described in detail and justified to support solid interpretation.

Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment.

In vitro substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log K ow chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate K M and V max values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between K M and chemical log K ow. Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous in vitro work with high log K ow compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true in vivo activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized in vivo testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.

Review of existing terrestrial bioaccumulation models and terrestrial bioaccumulation modeling needs for organic chemicals.

Protocols for terrestrial bioaccumulation assessments are far less-developed than for aquatic systems. This article reviews modeling approaches that can be used to assess the terrestrial bioaccumulation potential of commercial organic chemicals. Models exist for plant, invertebrate, mammal, and avian species and for entire terrestrial food webs, including some that consider spatial factors. Limitations and gaps in terrestrial bioaccumulation modeling include the lack of QSARs for biotransformation and dietary assimilation efficiencies for terrestrial species; the lack of models and QSARs for important terrestrial species such as insects, amphibians and reptiles; the lack of standardized testing protocols for plants with limited development of plant models; and the limited chemical domain of existing bioaccumulation models and QSARs (e.g., primarily applicable to nonionic organic chemicals). There is an urgent need for high-quality field data sets for validating models and assessing their performance. There is a need to improve coordination among laboratory, field, and modeling efforts on bioaccumulative substances in order to improve the state of the science for challenging substances.

Review of laboratory-based terrestrial bioaccumulation assessment approaches for organic chemicals: Current status and future possibilities.

In the last decade, interest has been renewed in approaches for the assessment of the bioaccumulation potential of chemicals, principally driven by the need to evaluate large numbers of chemicals as part of new chemical legislation, while reducing vertebrate test organism use called for in animal welfare legislation. This renewed interest has inspired research activities and advances in bioaccumulation science for neutral organic chemicals in aquatic environments. In January 2013, ILSI Health and Environmental Sciences Institute convened experts to identify the state of the science and existing shortcomings in terrestrial bioaccumulation assessment of neutral organic chemicals. Potential modifications to existing laboratory methods were identified, including areas in which new laboratory approaches or test methods could be developed to address terrestrial bioaccumulation. The utility of "non-ecotoxicity" data (e.g., mammalian laboratory data) was also discussed. The highlights of the workshop discussions are presented along with potential modifications in laboratory approaches and new test guidelines that could be used for assessing the bioaccumulation of chemicals in terrestrial organisms.