Richard C. Kolanczyk

Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques

An in vitro male rainbow trout liver slice assay has been developed for long-term incubation of precision-cut slices for the detection of vitellogenin (VTG) protein induction in response to xenobiotic chemicals. The assay was optimized to allow 72 h of incubation of slices to maximize detection of VTG, while maintaining slice viability. Two methods of incubation frequently used with rat liver slices were compared: (1) slices were submerged in media (11 degrees C) and cultured in 12-well plates (PL) with continuous shaking; or (2) slices were floated onto titanium screens, placed into glass vials, and held under dynamic organ culture (DOC) conditions (11 degrees C). Slices (200 µm) in modified L-15 media were exposed to 1.0 µM 17beta-estradiol (E2) or diethylstilbestrol (DES). Protein from media and slice was sampled for Western blot analysis, using a polyclonal antibody to detect appearance of VTG protein. Maximum VTG was seen at 72 h, with detectable protein at 24 and 48 h in slices and media following PL incubation. In contrast, slices incubated in DOC showed little detectable VTG above background levels after 72 h. This difference was not attributable to protein loss to vial or plate surfaces. Standard viability assays did not reveal any differences between slices incubated in PL or DOC. However, histopathological examination revealed earlier and more severe vacuolization in slices incubated in DOC. Significantly more E2 uptake and conversion to water-soluble metabolites was noted in PL, compared with DOC, as well as more production of VTG in response to DES and E2, correlated with less histologic change. The in vitro assay described allows tissue-level assessment of estrogenicity in aquatic organisms, and will be useful for assessing not only comparative species receptor binding and transactivation, but also the role of tissue-specific activation factors in the estrogenic response of fish.

Effects of anesthesia (tricaine methanesulfonate, MS222) on liver biotransformation in rainbow trout (Oncorhynchus mykiss)

The effect of tricaine methanesulfonate (MS222) on rainbow trout liver biotransformation rates was investigated with a microsomal model; an in vitro preparation that can be employed with or without the use of an anaesthetic. Two experimental sets of rainbow trout microsomes were tested; one representing in vivo or surgical tricaine exposures and the other representing in vitro tissue/organ collection tricaine exposures. Microsomal incubations were performed on these two experimental groups with phenol as substrate to assess the effects of tricaine on Phase I (ring-hydroxylation) and II (glucuronidation) liver biotransformation by monitoring production of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG). The use of a 2-h 100 mg/l exposure of tricaine for surgical anesthesia with or without 24-h recovery did not significantly (P< or =0.05) affect rates of phenol (Phase I and II) biotransformation rates; nor, did the 5-min 300 mg/l tricaine exposure for isolated organ/tissue collection significantly (P< or =0.05) affect phenol (Phase I and II) biotransformation rates. There were also no significant statistical differences (P< or =0.05) in P450 protein levels, or 7-ethoxyresorufin-O-deethylase (EROD) activity in these microsomal assays between any of the tricaine treated rainbow trout and controls.

Biotransformation of 4-methoxyphenol in rainbow trout (Oncorhynchus mykiss) hepatic microsomes

Rainbow trout liver microsomes were used to study the O-demethylation and ring hydroxylation of 4-methoxyphenol (4-MP) (4-hydroxyanisole) at 11 and 25°C by directly measuring the production of the primary metabolite hydroquinone (HQ), 4-methoxycatechol (4-MCAT), and additional metabolites. An HPLC method with integrated ultraviolet (UV) and electrochemical detection (ECD) was developed for metabolite identification and quantification at low concentrations. Sample handling with appropriate buffers, solvents, low temperature and light prevented loss of extremely labile metabolites. Saturation kinetics for the production of HQ via O-demethylation of 4-MP (0.66‐40 mM) was never achieved, with substrate solubility being the limiting factor. The linear rate of HQ formation at 11°C was 22.092.2 (coefficient9S.E., r 2 0.91) pmol min 1 per mg protein per mM substrate, and at 25°C was 34.091.3 (r 2 0.99) pmol min 1 per mg protein per mM substrate. The second major microsomal metabolite 4-MCAT was also identified, with linear rates of ring hydroxylation determined to be 19.09 1.6 (r 2 0.94) and 17.290.5 (r 2 0.99) pmol min 1 per mg protein per mM substrate at 11 and 25°C, respectively. Unlike HQ production, the rate of 4-MCAT production was found to be similar at the two temperatures when linear formation rates were corrected for the effect of temperature on substrate and product solubility at 11°C. Measurement of ‘freely dissolved’ fraction was essential to the accurate determination of ring hydroxylation and O-demethylation reaction rates in rainbow trout microsomes incubated at physiological temperature. Experimental conditions were shown to affect dissolved 4-MP and HQ at 11°C (verified using microdialysis) while not altering substrate and product levels at 25°C. Small but detectable levels of 1,4-benzoquinone were observed in 4-MP microsomal incubations. 1,2,4-Trihydroxybenzene was also detected, with possible routes of production through hydroxylation of HQ or O-demethylation of 4-MCAT. A metabolic scheme for bioactivation of 4-MP is proposed and the significance of observed metabolic conversions in rainbow trout microsomes discussed in relation to aquatic toxicity of 4-MP.

Effects-based chemical category approach for prioritization of low affinity estrogenic chemicals

Regulatory agencies are charged with addressing the endocrine disrupting potential of large numbers of chemicals for which there is often little or no data on which to make decisions. Prioritizing the chemicals of greatest concern for further screening for potential hazard to humans and wildlife is an initial step in the process. This paper presents the collection of in vitro data using assays optimized to detect low affinity estrogen receptor (ER) binding chemicals and the use of that data to build effects-based chemical categories following QSAR approaches and principles pioneered by Gilman Veith and colleagues for application to environmental regulatory challenges. Effects-based chemical categories were built using these QSAR principles focused on the types of chemicals in the specific regulatory domain of concern, i.e. non-steroidal industrial chemicals, and based upon a mechanistic hypothesis of how these non-steroidal chemicals of seemingly dissimilar structure to 17ß-estradiol (E2) could interact with the ER via two distinct binding types. Chemicals were also tested to solubility thereby minimizing false negatives and providing confidence in determination of chemicals as inactive. The high-quality data collected in this manner were used to build an ER expert system for chemical prioritization described in a companion article in this journal.

MetaPath: an electronic knowledge base for collating, exchanging and analyzing case studies of xenobiotic metabolism.

The MetaPath knowledge base was developed for the purpose of archiving, sharing and analyzing experimental data on metabolism, metabolic pathways and crucial supporting metadata. The MetaPath system grew out of the need to compile and organize the results of metabolism studies into a systematic database to facilitate data comparisons and evaluations. Specialized MetaPath data evaluation tools facilitate the review of pesticide metabolism data submitted for regulatory risk assessments as well as exchange of results of complex analyses used in regulation and research. Customized screen editors called Composers were developed to automate data entry into MetaPath while also streamlining the production of agency specific study summaries such as the Data Evaluation Records (DER) used by the US EPA Office of Pesticide Programs. Efforts are underway through an Organization for Economic Co-operation and Development (OECD) work group to extend the use of DER Composers as harmonized templates for rat metabolism, livestock residue, plant residue and environmental degradation studies.

Rate and capacity of hepatic microsomal ring-hydroxylation of phenol to hydroquinone and catechol in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol at 11 and 25 degrees C by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and catechol (CAT). An HPLC method with integrated ultraviolet and electrochemical detection was used for metabolite identification and quantification at low (pmol) formation rates found in fish. The Michaelis-Menten saturation kinetics for the production of HQ and CAT over a range of phenol concentrations were determined at trout physiological pH. The apparent Kms for the production of HQ and CAT at 11 degrees C were 14+/-1 and 10+/-1 mM, respectively, with Vmaxs of 552+/-71 and 161+/-15 pmol/min per mg protein. The kinetic parameters for HQ and CAT at 25 degrees C were 22+/-1 and 32+/-3 mM (Km) and 1752+/-175 and 940+/-73 pmol/min per mg protein (Vmax), respectively. The calculated increase in metabolic rate per 10 degrees C temperature rise (Q(10)) was 2.28 for HQ and 3.53 for CAT production. These experiments assess the potential for metabolic bioactivation in fish through direct quantification of putative reactive metabolites at the low, but toxicologically significant, chemical concentrations found in aquatic organisms. This work initiates a series of studies to compare activation pathway, rate, and capacity across fish species, providing a basis for development of biologically-based dose response models in diverse species.

An in vivo microdialysis method for the qualitative analysis of hepatic phase I metabolites of phenol in rainbow trout (Oncorhynchus mykiss).

Development of reliable and accurate methodologies for determination of xenobiotic hepatic biotransformation rate and capacity parameters is important to the derivation of precise physiologically-based toxicokinetic (PB-TK) models. Biotransformation data incorporated into PB-TK models has, for the most part, depended on in vitro techniques designed to mimic the in vivo environment; however, data from direct in vitro/in vivo comparisons is limited. In this investigation we describe for the first time a method using in vivo microdialysis (MD) to qualitatively assess hepatic xenobiotic biotransformation of phenol in an unanesthetized fish. MD probes were surgically implanted into the livers of adult rainbow trout which were subsequently confined to respirometer-metabolism chambers. Phenol (1-300 mM) was delivered directly to the liver via the MD probe at a perfusion rate of 1 microl min(-1) which consistently resulted in a relative delivery of 77-85% of the phenol in the perfusate to the tissue over a 3 day experimental time frame. Location of the probe within the liver was also shown to have no effect on the delivery of phenol or on the type or quantity of phase I metabolites formed. Production of hydroquinone (HQ) and catechol (CAT), the primary phase I metabolites of phenol, was monitored through direct sampling of the hepatic extracellular fluid space via the MD probe. HQ and CAT production increased with increasing time of perfusion and with increasing concentration of phenol delivered to the liver. In the future, data obtained through in vivo MD will be useful in resolving uncertainties in biotransformation rate and capacity parameters, which are central to fish PB-TK modeling of chemical disposition.

A rule-based expert system for chemical prioritization using effects-based chemical categories

A rule-based expert system (ES) was developed to predict chemical binding to the estrogen receptor (ER) patterned on the research approaches championed by Gilman Veith to whom this article and journal issue are dedicated. The ERES was built to be mechanistically transparent and meet the needs of a specific application, i.e. predict for all chemicals within two well-defined inventories (industrial chemicals used as pesticide inerts and antimicrobial pesticides). These chemicals all lack structural features associated with high affinity binders and thus any binding should be low affinity. Similar to the high-quality fathead minnow database upon which Veith QSARs were built, the ERES was derived from what has been termed gold standard data, systematically collected in assays optimized to detect even low affinity binding and maximizing confidence in the negatives determinations. The resultant logic-based decision tree ERES, determined to be a robust model, contains seven major nodes with multiple effects-based chemicals categories within each. Predicted results are presented in the context of empirical data within local chemical structural groups facilitating informed decision-making. Even using optimized detection assays, the ERES applied to two inventories of >600 chemicals resulted in only ~5% of the chemicals predicted to bind ER.

A comparison of fish pesticide metabolic pathways with those of the rat and goat

ABSTRACT Ecological risk assessments are often limited in their ability to consider metabolic transformations for fish species due to a lack of data. When these types of evaluations are attempted they are often based on parent chemical only, or by assuming similarity to available mammalian metabolic pathways. The metabolism maps for five pesticides (fluazinam, halauxifen‐methyl, kresoxim‐methyl, mandestrobin, and tolclofos‐methyl) were compared across three species. A rapid and transparent process, utilizing a database of systematically collected information for rat, goat, and fish (bluegill or rainbow trout), and using data evaluation tools in the previously described metabolism pathway software system MetaPath, is presented. The approach demonstrates how comparisons of metabolic maps across species are aided by considering the sample matrix in which metabolites were quantified for each species, differences in analytical methods used to identify metabolites in each study, and the relative amounts of metabolites quantified. By incorporating these considerations, more extensive rat and goat metabolism maps were found to be useful predictors of the more limited metabolism of the five pesticides in fish. HighlightsA systematic approach for species comparison of metabolism studies is shown.Major metabolites from fish tissue were represented well in the rat and goat studies.Sample matrix is an important consideration in the fish, rat and goat studies.