In vitro and in vivo metabolic stability of various fragrance materials and insect repellent in rainbow trout (Oncorhynchus mykiss).

Commercial fragrances consist of several thousand natural and synthetic substances formulated in complex combinations. These ingredients are frequently blended at very low concentrations but they are typically lipophilic and a few of them (e.g., synthetic musks) have been detected in aquatic systems, albeit at low concentrations. Few fragrances have guideline in vivo data on bioaccumulation, so in silico modeling has been widely used to estimate bioconcentration factors (BCFs). This study used in vitro metabolism assays with trout S9 cell fractions and cryopreserved hepatocytes to improve estimates of fish BCFs and to test published methods for extrapolating in vitro metabolic rate data to whole fish and corresponding BCFs. These estimates for several chemicals were compared with new in vivo bioconcentration measurements and previously published data on fragrances and the insect repellent, DEET. In total, 17 (20 including isomers) fragrance chemicals (abalyn, amberwood, amboryl acetate, bisabolene, cedroxide, coniferan, elemol, givescone, maritima, precyclemone B, polysantol, sandela, sanjinol, santalex, timberol and vernaldehyde) and DEET were metabolized at various rates. Only three materials tested did not appear to undergo enzymatic degradation (caryophyllene oxide, galaxolide and ketone patchouli). Even relatively slow rates of metabolism had a large influence on bioconcentration estimates. This work adds valuable information to the evolving body of work supporting the use of in vitro determinations of hepatic clearance to improve modeled predictions of bioaccumulation. It can also be used directly to help prioritize testing of potential bioaccumulative chemicals or serve as a more economical method for screening these chemicals.

In vitro evaluation of the metabolic stability of nine fragrance chemicals in trout and human hepatocytes.

In vitro metabolic stability of nine fragrance chemicals: p-tolyl acetate, cashmeran, ethylene brassylate, celestolide, galaxolide, traseolide, ambretone, tonalide and pentadecanolide, was evaluated in trout and human hepatocytes. The compounds were incubated with trout hepatocytes at 12°C and human hepatocytes at 37°C. Quantification of compound disappearance with time was performed using gas chromatography/mass spectrometry. in vivo hepatic intrinsic clearance values were calculated from the in vitro data. Significant metabolism was observed with trout hepatocytes for five of the nine fragrance chemicals, while all nine were metabolized significantly with human hepatocytes. Previously published models were used to examine expected bioaccumulation and persistence in whole organisms. Calculated half-lives due to metabolism of the nine chemicals are significantly shorter for humans than trout: <1 hour and <1 day, respectively. For all chemicals with demonstrated hepatic metabolism, the models indicate a lack of accumulation. For those where metabolism was demonstrated in trout, calculated bioconcentration factors would not be classified as bioaccumulative under prevailing regulatory systems.

A Comparison of In Vitro Metabolic Clearance of Various Regulatory Fish Species Using Hepatic S9 Fractions.

Bioaccumulation predictions can be substantially improved by combining in vitro metabolic rate measurements derived from rainbow trout hepatocytes and/or hepatic S9 fractions with quantitative structure-activity relationship (QSAR) modeling approaches. Compared with in vivo testing guidelines Organisation for Economic Co-operation and Development (OECD) 305 and Office of Chemical Safety and Pollution Prevention (OCSPP; an office of the US Environmental Protection Agency) 850.1730, the recently adopted OECD test guidelines 319A and 319B are in vitro approaches that have the potential to provide a time- and cost-efficient, humane solution, reducing animal use while addressing uncertainties in bioaccumulation across species. The present study compares the hepatic clearance of the S9 subcellular fraction of rainbow trout, bluegill, common carp, fathead minnow, and largemouth bass, discerning potential differences in metabolism between different warm- and cold-water species. With refinements to the in vitro metabolic S9 assay for high-throughput analysis, we measured in vitro clearance rates of seven chemicals crossing multiple classes of chemistry and modes of action. We confirmed that data from rainbow trout liver S9 fraction metabolic rates can be utilized to predict rainbow trout bioconcentration factors using an in vitro to in vivo extrapolation model, as intended in the OECD 319B applicability domain per the bioaccumulation prediction. Also, we determined that OECD 319B can be applied to other species, modified according to their habitat, adaptations to feeding behavior, and environmental conditions (e.g., temperature). Once toxicokinetics for each species is better understood and appropriate models are developed, this method can be an excellent tool to determine hepatic clearance and potential bioaccumulation across species. The present study could be leveraged prior to or in place of initiating in vivo bioconcentration studies, thus optimizing selection of appropriate fish species. Environ Toxicol Chem 2024;43:1390-1405. © 2024 SETAC.

Reduced biotransformation of polycyclic aromatic hydrocarbons (PAHs) in pollution-adapted Gulf killifish (Fundulus grandis).

Anthropogenic pollution represents a significant source of selection, potentially leading to the emergence of evolutionary adaptations in chronically exposed organisms. A recent example of this scenario corresponds to Gulf killifish (Fundulus grandis) populations inhabiting the Houston Ship Channel (HSC), Texas, USA, which have been documented to have adapted to this heavily contaminated environment. Although not fully elucidated, one particularly important aspect of their adaptation involves the reduced inducibility of the aryl hydrocarbon receptor (AhR) and, potentially, the alteration of major biotransformation pathways. In the present study, we employed a modified Organization for Economic Cooperation and Development (OECD) 319-B test guideline to explore population and sex-related differences in the hepatic biotransformation of six polycyclic aromatic hydrocarbons (PAHs) in F. grandis populations with different exposure histories. Pollution-adapted F. grandis showed significantly lower hepatic clearance of PAHs than non-adapted fish, especially for high molecular weight PAHs (chrysene, benzo[k]fluoranthene, and benzo[a]pyrene), with pollution-adapted females presenting the lowest clearance. The characterization of different phase I biotransformation enzymes revealed that the basal activity of CYP1A, fundamental in the biotransformation of PAHs, was significantly lower in pollution-adapted fish, especially in females, which showed the lowest activity. Contrarily, basal CYP2C9-like activity was significantly higher in pollution-adapted fish. These results demonstrate the importance of exposure and evolutionary histories in shaping organisms' responses to pollution and provide significant evidence of sex-specific biotransformation differences in F. grandis populations.

In vitro-in vivo biotransformation and phase I metabolite profiling of benzo[a]pyrene in Gulf killifish (Fundulus grandis) populations with different exposure histories.

Chronic exposure to pollution may lead populations to display evolutionary adaptations associated with cellular and physiological mechanisms of defense against xenobiotics. This could result in differences in the way individuals of the same species, but inhabiting different areas, cope with chemical exposure. In the present study, we explore two Gulf killifish (Fundulus grandis) populations with different exposure histories for potential differences in the biotransformation of benzo[a]pyrene (BaP), and conduct a comparative evaluation of in vitro and in vivo approaches to describe the applicability of new approach methodologies (NAMs) for biotransformation assessments. Pollution-adapted and non-adapted F. grandis were subjected to intraperitoneal (IP) injections of BaP in time-course exposures, prior to measurements of CYP biotransformation activity, BaP liver concentrations, and the identification and quantification of phase I metabolites. Additionally, substrate depletion bioassays using liver S9 fractions were employed for measurements of intrinsic hepatic clearance and to evaluate the production of metabolites in vitro. Pollution-adapted F. grandis presented significantly lower CYP1A activity and intrinsic clearance rates that were 3 to 4 times lower than non-adapted fish. The metabolite profiling of BaP showed the presence of 1­hydroxy-benzo[a]pyrene in both the in vitro and in vivo approaches but with no significant population differences. Contrarily, 9­hydroxy-benzo[a]pyrene and benzo[a]pyrene-4,5-dihydrodiol, only identified through the in vivo approach, presented higher concentrations in the bile of pollution-adapted fish relative to non-adapted individuals. These observations further the understanding of the evolutionary adaptation of F. grandis inhabiting heavily polluted environments in the Houston Ship Channel, TX, USA, and highlight the need to consider the evolutionary history of populations of interest during the implementation of NAMs.

Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals.

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.

Drug interaction potential of toremifene and N-desmethyltoremifene with multiple cytochrome P450 isoforms.

Toremifene is an effective agent for the treatment of breast cancer in postmenopausal women and is being evaluated for its ability to prevent bone fractures in men with prostate cancer taking androgen deprivation therapy. Due to the potential for drug-drug interactions, the ability of toremifene and its primary circulating metabolite N-desmethyltoremifene (NDMT) to inhibit nine human cytochrome P450 (CYP) enzymes was determined using human liver microsomes. Induction of CYP1A2 and 3A4 by toremifene was also investigated in human hepatocytes. Toremifene did not significantly inhibit CYP1A2 or 2D6. However, toremifene is a competitive inhibitor of CYP3A4, non-competitive inhibitor of CYP2A6, 2C8, 2C9, 2C19 and 2E1 and mixed-type inhibitor of CYP2B6. CYP inhibition by NDMT was similar in magnitude to toremifene. Toremifene did not induce CYP1A2 but increased CYP3A4 monooxygenase activity and gene expression in drug-exposed human primary hepatocytes. Although clinical doses of toremifene produce steady state exposures to toremifene and NDMT that may be sufficient to cause pharmacokinetic drug-drug interactions with other drugs metabolised by CYP2B6, CYP2C8, CYP3A4, CYP2C9 and CYP2C19, these data indicate that toremifene is unlikely to play a role in clinical drug-drug interactions with substrate drugs of CYP1A2 and CYP2D6.

Reliability of In Vitro Methods Used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial.

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intralaboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, whereas interlaboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO, INT; l/d/kg fish), both assays yielded similar levels of activity (<4-fold difference for all chemicals). Hepatic clearance rates (CLH; l/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss).

Trout provide a relatively easy source of hepatocytes that can be cryopreserved and used for a range of applications including toxicity testing and determination of intrinsic clearance. Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess metabolic stability of xenobiotics in fish by means of a substrate depletion approach. Variations on these methods, troubleshooting tips, and directions for use of extrapolation factors to express results in terms of in vivo intrinsic clearance are included. These protocols have been developed for rainbow trout, but can be adapted to other fish species with appropriate considerations.

Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction.

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account.

Cleavage of recombinant proenkephalin and blockade mutants by prohormone convertases 1 and 2: an in vitro specificity study.

Proenkephalin (PE) derived-peptides are thought to be generated predominantly through endoproteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). In order to compare cleavage site preferences of these convertases, we studied the processing of recombinant wild-type rat PE and of two mutant PEs by recombinant purified mouse PC1 and PC2. Western blot analyses of timed digestions showed that both mouse PC1 and PC2 were able to produce a variety of large and intermediate sized-peptides from wild-type PE as well as from the precursors mutated at initial blockade sites. PC2 exhibited a broader specificity against PE than PC1, generating a much greater number of peptide products. Mass spectrometric identification of cleavage products showed that PC2 appeared to be the principal enzyme involved in the generation of smaller active opioids. Both enzymes were able to cleave various KR- and KK-containing sites, but PC2 was also able to cleave efficiently at an RR-V site and a KK-M site not cleaved by PC1, suggesting the exclusion of large aliphatic residues at the P1' position in PC1 cleavage. Alternative cleavage sites were readily chosen by convertases in blockade mutants, confirming in vivo results that cleavages do not follow an obligatory order. Furthermore, glycosylated PE was less efficiently processed by PC2, indicating that glycosylation may serve as a mechanism to hinder processing.

Potential for retroposition by old Alu subfamilies.

Alu elements sharing sequence characteristics of the "old" subfamilies are thought to currently be retrotranspositionally inactive. We analyzed one of these old subfamilies of Alu elements, Sx, for sequence conservation relative to the consensus and the length of the "A-tail" as parameters to define the presence of potential Alu Sx source genes in the human genome. Sequence identity to the left half or the right half of the Alu Sx consensus sequence was evaluated for 4424 complete elements obtained from the human genome draft sequence. A small subset of Alu Sx left halves were found to be more conserved than any of the Alu Sx right halves. Selection for promoter function in active elements may explain the slightly higher conservation of the left half. In order to determine whether this sequence identity was the result of recent activity, or simply sequence conservation for older elements, PCR amplification of some of the loci containing Sx elements with conserved left/right halves from different primate genomes was carried out. Several of these Sx Alus were found to have amplified at a later evolutionary period (<35 mya) than expected based on previous studies of Sx elements. Analysis of "A-tail" length, a feature correlated with current retroposition activity, varied between Alu Sx element loci in different primates, where the length increased in specific Alu elements in the human genome. The presence of few conserved Alu Sx elements and the dynamic expansion/contraction of the A-tail suggests that some of these older subfamilies may still be active at very low levels or in a few individuals.