Mechanism of trifluralin-induced thyroid tumors in rats.

Trifluralin, an herbicide, has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats. This study was designed to determine the mechanism of thyroid hyperactivity after trifluralin exposure. A group of 15 male Fischer 344 rats were exposed to trifluralin-fortified (6500 ppm) diet for 2 weeks. The time weighted average daily intake of trifluralin was 441+/-77 mg/kg/day. Ten rats of the group were sacrificed and the sera analyzed for T3, T4, and TSH levels. The livers were also analyzed for selected T4-specific UGT gene expression and total UGT enzyme activity. In the trifluralin treated rats, the serum T3 and T4 levels decreased by 17% and 90%, respectively and TSH increased by 37% more than the control rats. Trifluralin-induced total hepatic UGT enzymes (2.4-fold) and mRNA expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). For the remaining 5 rats in the group, bile was collected for 2 h and analyzed for free and conjugated T3 and T4. The total amount of T4 in bile more than doubled in trifluralin treated rats. Trifluralin treatment increased bile flow, caused a 3.2-fold increase in biliary elimination of conjugated T4 and 63% increase in conjugated T3. Based on these data, the decrease in total serum T3 and T4 levels in the trifluralin treated rats was due to enhanced peripheral metabolism and an increase in bile flow that results in a compensatory increase in TSH synthesis and secretion. The increased levels of TSH with chronic exposure to trifluralin would exert a continuous stimulation of the thyroid gland leading to cellular hypertrophy and proliferation predisposing to the development of follicular cell tumors in rats.

Analysis of the interaction of phytoestrogens and synthetic chemicals: an in vitro/in vivo comparison.

In the evaluation of chemical mixture toxicity, it is desirable to develop an evaluation paradigm which incorporates some critical attributes of real world exposures, particularly low dose levels, larger numbers of chemicals, and chemicals from synthetic and natural sources. This study evaluated the impact of low level exposure to a mixture of six synthetic chemicals (SC) under conditions of co-exposure to various levels of plant-derived phytoestrogen (PE) compounds. Estrogenic activity was evaluated using an in vitro human estrogen receptor (ER) transcriptional activation assay and an in vivo immature rat uterotrophic assay. Initially, dose-response curves were characterized for each of the six SCs (methoxyclor, o,p-DDT, octylphenol, bisphenol A, beta-hexachlorocyclohexane, 2,3-bis(4-hydroxyphenyl)-propionitrile) in each of the assays. The six SCs were then combined at equipotent ratios and tested at 5-6 dose levels spanning from very low, sub-threshold levels, to a dose in which every chemical in the mixture was at its individual estrogenic response threshold. The SC mixtures also were tested in the absence or presence of 5-6 different levels of PEs, for a total of 36 (in vitro) or 25 (in vivo) treatment groups. Both in vitro and in vivo, low concentrations of the SC mixture failed to increase estrogenic responses relative to those induced by PEs alone. However, significant increases in response occurred when each chemical in the SC mixture was near or above its individual response threshold. In vitro, interactions between high-doses of SCs and PEs were greater than additive, whereas mixtures of SCs in the absence of PEs interacted in a less than additive fashion. In vivo, the SC and PE mixture responses were consistent with additivity. These data illustrate a novel approach for incorporating key attributes of real world exposures in chemical mixture toxicity assessments, and suggest that chemical mixture toxicity is likely to be of concern only when the mixture components are near or above their individual response thresholds. However, these data suggest that extrapolation from in vitro assays to in vivo mixture effects should be approached with caution.

A comparison of in vitro and in vivo EDSTAC test battery results for detecting antiandrogenic activity.

The androgen receptor (AR) transactivation, binding, and Hershberger assays are being developed for large-scale screening of chemicals for endocrine activity. The goal of this study was to evaluate the correlation between in vitro and in vivo antiandrogenicity assays using a variety of compounds (p,p'-DDE, flutamide (FLUT), spironolactone, procymidone, RU486, methoxychlor (MXC), benzo(a)pyrene (BAP), and selected metabolites). For the AR transactivation assay, AR(+) LNCaP prostate carcinoma cells were transfected with an inducible luciferase reporter construct (pGudLuc7ARE) and exposed for 24 h to test materials (< or = 10 microM) in the presence and absence of 1 nM of the AR agonist R-1881. Each of these materials, including the hydroxlated metabolites of BAP and MXC, produced significant antiandrogenic activity in vitro as evidenced by their inhibition of the response to R-1881. Similarly, in vitro AR binding experiments using the recombinant ligand-binding domain (LBD) of the human AR and fluorescence polarization (FP) methodology yielded IC50s comparable to that of testosterone for RU486 and 9-OH-BAP. Other parent compounds and metabolites exhibited lesser binding affinity. In vivo antiandrogenic activity was evaluated with the Hershberger assay, wherein castrated male CD rats were dosed by gavage for 10 days with (mg/kg per day): MXC (10, 50, 100, and 200), BAP (1, 10, 50, and 100), RU486 (1, 5, 10, and 25), and FLUT (10) in the presence of 0.4 mg/kg per day (sc) of testosterone propionate (TP). Neither BAP nor MXC produced significant decreases in accessory sex tissue (AST) weights relative to TP control. However, 200 MXC resulted in a significant decrease in body weight and 100 BAP significantly increased absolute and relative liver weights. RU486 (25) produced significant decreases in ventral prostate, seminal vesicle, and Cowper's gland weights without affecting body weight. FLUT (10) decreased all AST weights measured. The antiandrogenic activities of the remaining materials (p,p'-DDE, spironolactone, and procymidone) have been demonstrated in previous Hershberger assays. These data indicate the importance of including in vivo results in assessing the endocrine activity of test materials and further stress the importance of a weight of evidence approach in assessing endocrine activity of test materials.

Mode of mutagenic action for the biocide Bioban CS-1246 in mouse lymphoma cells and implications for its in vivo mutagenic potential.

The biocidal agent, BIOBAN CS-1246 (7-ethyl bicyclooxazolidine, CAS# 7747-35-5, CS-1246) induced a concentration-dependent mutagenic response in mouse lymphoma (L5178Y TK+/-) cells both with and without the addition of S9 metabolic activation. Previous data indicating the ability of CS-1246 to hydrolyze in aqueous media to generate formaldehyde (FA), led us to investigate the potential role of FA in the CS-1246-induced mutagenic response in the mouse lymphoma assay (MLA). To accomplish this, the MLA on CS-1246 was repeated in the presence of a metabolizing system (formaldehyde dehydrogenase/NAD+), which was shown to successfully inhibit the mutagenic response of formaldehyde in this assay system. Significantly, the observed mutagenicity of CS-1246 was completely abrogated when the cultures were supplemented with formaldehyde dehydrogenase/NAD+, suggesting that the positive MLA response was attributable to the generation of FA in situ. These results demonstrate that in vitro mutagenicity of CS-1246 in the MLA is most likely associated with FA. Negative results from two in vivo assays for genotoxicity were consistent with the known activity of FA in these assays. In the mouse bone marrow micronucleus (MNT), there were no significant increases in micronucleated polychromatic erythrocytes (with evaluation of 2000/animal), after treatment with 0.5, 1, and 2 g/kg/day CS-1246 (6/dose group) for 2 consecutive days and sacrifice 24 h later. Furthermore, in the unscheduled DNA synthesis (UDS) study, male F344 rats (5 /dose group), given a single oral gavage (0, 1, and 2 g/kg) and evaluated at two time points (2-4 and 14-15 h post dosing), did not elicit an UDS response, indicating a lack of DNA reactivity in vivo. Based on the negative in vivo findings, it can be inferred that the FA detoxification mechanisms that exist in intact organisms prevent the likelihood of generating FA at levels capable of causing genotoxicity following exposure to CS-1246 at low, environmentally relevant concentrations. The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.

In vitro models in endocrine disruptor screening.

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.

A novel flexible approach for evaluating fixed ratio mixtures of full and partial agonists.

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.

An approach for assessing estrogen receptor-mediated interactions in mixtures of three chemicals: a pilot study.

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.

Benzo(a)pyrene, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, alters cell adhesion proteins in human uterine RL95-2 cells.

This study compared the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two aryl hydrocarbon receptor agonists, on cell attachment and adherens junction proteins in RL95-2 human uterine endometrial cells. Exposure to 10 microM BaP significantly decreased cell attachment to Matrigel, whereas 10 nM TCDD had no effect. Immunocytochemistry and Western immunoblot analysis showed that BaP, but not TCDD, produced a marked loss of plasma membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries. BaP-treated cells exhibited significant decreases in beta-catenin and cadherin protein levels, while vinculin levels remained unchanged relative to control. In contrast, TCDD treatment had no effect on the levels of beta-catenin, cadherin, or vinculin. Further studies using the fluorescein labeled peptide phalloidin showed the presence of continuous subcortical actin filaments in control cells, whereas BaP-treated cells had subcortical actin aggregates. Thus, in contrast to TCDD, BaP produces a loss of cell attachment involving decreased localization of molecules important for cell-cell interactions in RL95-2 cells.

Assessment of interactions of diverse ternary mixtures in an estrogen receptor-alpha reporter assay.

This study used an MCF-7 cell based ER-alpha reporter gene assay to assess chemical interactions within the following ternary mixtures: (1) three synthetic pesticides, methoxychlor (MXC), o,p-DDT, and dieldrin; (2) three polyaromatic hydrocarbons, benzo[a]pyrene (BAP), 1,2-benzanthracene (BENZ), and chrysene (CHRY); and (3) an endogenous estrogen, [17beta-estradiol, (E(2))]; a phytoestrogen, genistein (GEN); and a synthetic estrogen, o,p-DDT. A full factorial design in which four concentrations of each chemical were assessed in all possible combinations (64 treatment groups) was utilized. In addition, mixtures were tested in both a low range (concentrations near the individual chemical response thresholds) and a high range ( approximately 2-10x higher) experiment. A response surface was estimated using a nonlinear mixed model, and the cumulative response in each mixture was evaluated for departure from additivity. The mixture of E(2), GEN, and DDT exhibited antagonistic interactions (p < 0.001) in both concentration ranges. However, specific interactions between E(2)/GEN and E(2)/DDT differed between the low and high range concentrations. The BAP/BENZ/CHRY mixture did not depart significantly from additivity (p = 0.66) in either concentration range, although response levels were generally low. The MXC/DDT/dieldrin mixture did not depart significantly from additivity in either the high (p = 0.065), or low dose range (p = 0.506), with generally minimal responses dominated by MXC and DDT. This methodology has allowed for a rigorous statistical evaluation of potential departures from additive interactions in endocrine active mixtures. In no case was a significantly greater-than-additive (synergistic) interaction observed.