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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

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.

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 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 (

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.

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.

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.

The contribution of the prohormone-processing enzyme PC2 to the proteolytic maturation of proenkephalin was examined in three sets of studies. In the first, the processing of this precursor was compared in PC2-rich (Rin5f) and PC2-lacking (AtT-20) cell lines expressing proenkephalin by virtue of stable transfection. These studies showed that the time frame for processing of this precursor is cell line specific, with AtT-20 cells processing proenkephalin to peptide B much more rapidly than Rin cells. However, the latter cell line processed proenkephalin much more extensively, i.e., produced a greater proportion of the penta- to octapeptide enkephalins. The involvement of PC2 in these later processing events was analyzed by examining the processing of proenkephalin in PC2-overexpressing AtT-20 cell lines. These experiments yielded a processing profile similar to that observed for Rin cells, although the time frame of initial processing was similar to that found in AtT-20 cells. To confirm the physiological involvement of proenkephalin in the production of the small opioid peptides, we generated a Rin cell line in which the production of PC2 was impaired due to stable expression of antisense mRNA to this enzyme. These experiments provided conclusive evidence that the generation of Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu, but not the larger enkephalin-containing peptides, is mediated by PC2. Taken together, our data support the idea that PC2 is physiologically capable of mediating only the later processing steps of neuropeptide precursors. PC2 thus appears to be the primary enzyme responsible for the generation of bioactive opioid peptide species from proenkephalin.

In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 x 10(4) M-1 s-1 (Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp+ ++; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2, 4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s-1 (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s-1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s-1 (

Prohormones are known to be processed at various cleavage sites in a defined temporal order, suggesting the possibility of sequential unfolding of processing sites. In order to investigate whether sequential processing at predefined sites is in fact required for proper processing, site-directed mutagenesis was performed to block known initial cleavage sites within proenkephalin. Pulse-chase/immunoprecipitation experiments were employed to analyze the fate of mutant and native proenkephalins in stably transfected AtT-20 cells. While processing did not occur at blockaded sites, surprisingly, overall processing of mutant proenkephalins proceeded efficiently, and alternative sites were chosen. When compared with native proenkephalin, processing of mutant proenkephalins occurred more slowly at early stages and more quickly at later stages. Experiments employing endoglycosidase H indicated that the early slow processing of mutant proenkephalins may be due to delays in intracellular transport. Metabolic labeling studies showed that more efficient production of bioactive opioids occurred in all processing site blockade mutants examined; these results were confirmed using several different radioimmunoassays of stored peptide products. We conclude that efficient processing of prohormone precursors does not require a specific temporal order of processing events. The fact that mutant proenkephalins were more fully processed than native proenkephalin may provide a route for more efficient production of opioid peptides in applications for chronic pain treatment.

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.

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.

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.

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 (