A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.

Assessing for interactions among chemicals in a mixture involves the comparison of actual mixture responses to those predicted under the assumption of zero interaction (additivity), based on individual chemical dose-response data. However, current statistical methods do not adequately account for differences in the shapes of the dose-response curves of the individual mixture components, as occurs with mixtures of full and partial receptor agonists. We present here a novel extension of current methods, which overcomes some of these limitations. Flexible single chemical concentration-effect curves combined with a common background parameter are used to describe an additivity surface along each axis. The predicted mixture response under the assumption of additivity is based on the constraint of Berenbaum's definition of additivity. Iterative algorithms are used to estimate mean responses at observed mixture combinations using only single chemical parameters. A full model allowing for different maximum response levels, different thresholds, and different slope parameters for each mixture component is compared to a reduced model under the assumption of additivity. A likelihood-ratio test is used to test the hypothesis of additivity by utilizing the full and reduced model predictions. This approach is useful for mixtures of chemicals with threshold regions and whose component chemicals exhibit differing response maxima (e.g., mixtures of full and partial agonists). The methods are illustrated with a combination of six chemicals in an estrogen receptor-alpha (ER-alpha) reporter gene assay.

The public and scientific concern that chemicals present in the human diet and the environment and their ability to disrupt the normal hormonal milieu in humans and wildlife have become a high-profile international issue. In 1998, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) convened by the Environmental Protection Agency (EPA) recommended a tiered testing approach for the evaluation of estrogen, androgen, and thyroid-related effects of some 87,000 commercial chemicals and environmental contaminants. The function of this committee concluded with its final report, and the further implementation of the recommended testing strategy has now been carried forward with the assistance of the Endocrine Disruptor Methods Validation Subcommittee. The function of this body is to provide advice to the EPA on scientific and technical issues related specifically to the conduct of studies required for the validation of assays proposed by the EDSTAC as part of the tiered screening program. The EDSTAC recommended and alternative screening batteries encompass four in vitro mammalian assays. The current methodologies and validation status of the proposed in vitro EDSTAC assays are discussed and consist of estrogen/androgen receptor binding, estrogen/androgen gene transactivation, and minced testis, and one alternate (placental aromatase) in vitro screening assay.

Most studies investigating interactions among endocrine-active chemicals have been limited to binary mixtures. This study reports on the preliminary evaluation an in vitro MCF-7 cell ER-alpha reporter gene system, coupled with a statistical methodology adapted for assessing interactions within ternary (3-chemical) mixtures. Two mixtures were initially chosen for assessment of the in vitro system's ability to detect additivity (mixture A) as well as greater-than-additive (mixture B) responses. Mixture A was composed of 17beta-estradiol (E2), ethinyl estradiol, and diethylstilbestrol and served as a control for additivity, whereas mixture B (E2, epidermal growth factor, insulin-like growth factor-I) was selected to model greater-than-additive interactions based on previous in vitro studies. After generating complete dose-response curves for each chemical, ternary mixtures were then tested in a full factorial design (4 concentrations per chemical, 64 treatment groups). A response surface was estimated using a nonlinear mixed model, and the observed responses were statistically analyzed for departures from the responses expected under the assumption of additivity. Mixture A exhibited additivity in vitro when the chemicals were present at concentrations in the linear range of their individual dose-response curves. For mixture B, in vitro analysis resulted in the additivity hypothesis being rejected (p < 0.001) because of a greater-than-additive interaction, as expected. A limited in vivo evaluation of mixture A was performed in the immature mouse uterotrophic assay (27 treatment groups), which agreed with the in vitro assessment of no significant departure from additivity ( p = 0.903). These findings demonstrate the ability of this in vitro methodology to detect additive, greater-than-additive, and less-than-additive interactions within ternary mixtures, which now allows for the assessment of environmentally relevant mixtures.

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