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.

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.

The use of metabolising systems for in vitro testing of endocrine disruptors.

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

Discriminating redox cycling and arylation pathways of reactive chemical toxicity in trout hepatocytes.

The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl-1,4-naphthoquinone (MNQ), 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. More than 70% of cells died in 3 h when exposed to BQ or NQ; 50% died in 7 h when exposed to MNQ, with no mortality compared to controls after 7 h DMONQ exposure. A suite of biochemical parameters was assessed for ability to discriminate these reactivity pathways in fish. Rapid depletion of glutathione (GSH) with appearance of glutathione disulfide (GSSG) and increased dichlorofluoroscein fluorescence were used as indicators of redox cycling, noted with DMONQ, MNQ, and NQ. Depletion of GSH with no GSSG accumulation, and loss of free protein thiol (PrSH) groups (nonreducible) indicated direct arylation by BQ. All toxicants rapidly oxidized NADH, with changes in NADPH noted later (BQ, NQ, MNQ) or not at all (DMONQ). Biochemical measures including cellular energy status, cytotoxicity, and measures of reactive oxygen species, along with the key parameters of GSH and PrSH redox status, allowed differentiation of responses associated with lethality. Chemicals that arylate were more potent than redox cyclers. Toxic pathway discrimination is needed to group chemicals for potency predictions and identification of structural parameters associated with distinct types of reactive toxicity, a necessary step for development of mechanistically based quantitative structure-activity relationships (QSARs) to predict chemical toxic potential. The commonality of reactivity mechanisms between rodents and fish was also demonstrated, a step essential for species extrapolations.

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.

Effect of bovine serum albumin on the extent of ortho rearrangement of N-(sulfooxy)-2-fluorenylacetamide and of enzymatically activated N-hydroxy-2-fluorenylacetamide and on the binding of reactive esters to nucleic acids.

We have investigated the effect of the bovine serum albumin (BSA)-catalyzed ortho rearrangement of synthetic and enzymatically generated N-(sulfooxy)-2-fluorenylacetamide (NSF) to the O-sulfate esters on the binding of NSF to transfer ribonucleic acid (tRNA) and to deoxyribonucleic acid (DNA). Binding of synthetic NSF to tRNA and DNA decreased approximately 90 and 70%, respectively, in the presence of BSA. Under these conditions, the ortho rearrangement, a minor reaction in the absence of BSA, was nearly quantitative. The decrease of adduct formation to nucleic acids was not attributable to the competitive binding of NSF to BSA. Binding of NSF, generated by cytosolic sulfonation of the arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to tRNA, was diminished approximately 97% in the presence of BSA while the ortho rearrangement of the sulfonated substrate increased from less than 0.5% to approximately 50%. Adduct formation of DNA with N-hydroxy-2-fluorenylacetamide, activated by enzymatic sulfonation, was inhibited approximately 90% in the presence of BSA. In these experiments, the catalytic effect of BSA on the ortho rearrangement of enzymatically sulfonated N-hydroxy-2-fluorenylacetamide was of the same order as observed in the experiments with tRNA. The data obtained on the covalent interaction of DNA with enzymatically activated N-hydroxy-2-fluorenylacetamide indicate that, in addition to NSF, another electrophilic species accounts for binding of activated N-hydroxy-2-fluorenylacetamide to DNA. The data support the view that the reactive electrophile is N-acetoxy-2-fluorenamine, resulting from the N,O-transacetylation of N-hydroxy-2-fluorenylacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)

The catalytic effect of bovine serum albumin on the ortho rearrangement of the potential ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated enzymatically from N-hydroxy-2-(acetylamino)fluorene and evidence for substrate specificity of the enzymatic sulfonation of arylhydroxamic acids.

This investigation examines the catalytic effect of bovine serum albumin on the ortho rearrangement of the possible ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated from N-hydroxy-2-(acetylamino)fluorene by the sulfotransferase(s) in the cytosol of rat liver. With various preparations of cytosol, 55-75% of the substrate, N-hydroxy-2-(acetylamino)-fluorene, was found to rearrange to the nonmutagenic and noncarcinogenic o-(sulfooxy) esters, 1- and 3-(sulfooxy)-2-(acetylamino)fluorene, in the presence of bovine serum albumin, while less than 1% of the substrate rearranged in its absence. In presence of bovine serum albumin the cytosolic reduction of N-(sulfooxy)-2-(acetylamino)fluorene to 2-(acetylamino)fluorene decreased by 60-90% and its solvolytic degradation to 4-hydroxy-2-(acetylamino)fluorene by 80-90%. The covalent interaction of enzymatically generated N-(sulfooxy)-2-(acetylamino)fluorene with the nucleophilic acceptors, N-acetyl-L-methionine and guanosine, was lowered by greater than 90% by addition of bovine serum albumin. These measurements indicated that the albumin-catalyzed ortho rearrangement controls the rates of concurrent metabolic and degradative reactions of N-(sulfooxy)-2-(acetylamino)fluorene. The results are in agreement with previous findings of a catalytic effect of serum albumin on the ortho rearrangement of synthetic N-(sulfooxy)-2-(acetylamino)fluorene. In contrast to its catalytic effect on the formation of o-(sulfooxy) esters from N-(sulfooxy)-2-(acetylamino)fluorene, bovine serum albumin had no effect on the formation of o-(acetylamino)fluorenols. To assess the substrate specificity of bovine serum albumin, its effect on the rearrangement of N-hydroxy-2-(benzoylamino)fluorene, a carcinogenic analogue of N-hydroxy-2-(acetylamino)fluorene, was analyzed under conditions of cytosolic sulfonation.(ABSTRACT TRUNCATED AT 250 WORDS)