Grantley D. Charles

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.

An in vitro screening paradigm for extracts of whole foods for detection of potential toxicants.

The application of organic, conventional and biotechnology techniques can alter the intrinsic levels of natural toxicants in crop foods and methods are needed to screen for unexpected changes in toxicant levels. We evaluated crude, aqueous preparations of 37 foods purchased from a local market in a battery of four in vitro mammalian toxicity screens. The foods were evaluated in one or more of the following tests: (1) cytotoxicity (37 foods) and (2) chromosomal aberration test (nine foods), both in Chinese hamster ovary cells, (3) limb bud micromass assay (nine foods) using 11-day old CD-1 mouse embryos and (4) estrogenicity (MCF-7 cells transfected with estrogen receptor and lucerifase reporter constructs, 12 foods). IC50s for cellular proliferation ranged from < 1% (v/v, garlic) to > 10% (v/v, 18 foods), the maximal concentration tested. Five of nine preparations (soybeans, broccoli, garlic, snow peas and corn) were clastogenic and two (soybeans and snow peas) inhibited chrondrogenesis in the limb bud micromass assay. Five of nine preparations (soybeans, snow peas, cumin, asparagus and bean sprouts) produced significant estrogenic responses. Overall, the 12 foods evaluated in two or more of the tests showed different patterns of response. These preliminary data indicate that screening for potential toxicants is possible with fast, relatively inexpensive in vitro tests. These in vitro tests, while potentially useful to detect unexpected toxicants in plants that may signal the need for further evaluation, are not directly useful to predict human or animal risk from eating these plants.

Incorporation of S-9 activation into an ER-α transactivation assay☆

We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-alpha transactivation assay. 17beta-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded consistently in the assay with average EC(50) values of 9.6 x 10(-11) M and 1.2 x 10(-5) M, respectively. In the presence of a 0.1% S-9 fraction, the EC(50) for E2 was increased to 1.4 x 10(-9) M and that for MXC decreased to 4.9 x 10(-7) M, with both compounds demonstrating increased secondary metabolite formation as evidenced by HPLC analysis. Consistent with these data, metabolites of E2 and MXC exhibited decreased and increased potencies, respectively, in the assay system relative to the parent molecules. S-9 was compatible with the MCF-7 reporter assay and has the potential to enhance detection of proestrogenic materials.

Investigations of Oxidative Stress, Antioxidant Response, and Protein Binding in Chlorpyrifos Exposed Rat Neuronal PC12 Cells

ABSTRACT Chlorpyrifos (CPF) is a widely used organophosphate insecticide. In addition to its known properties of cholinesterase inhibition, the production of reactive oxygen species (ROS) has been suggested as a possible toxic mechanism. To investigate CPF-generated ROS, rat neuronal PC12 cells were exposed to CPF concentrations of 0 to 5000 μg/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 μM of the antioxidant Trolox for 0 to 5 h. Paraquat served as a positive control for ROS. The fluorescent probe 2,7-dichlorodihydro-fluorescein and the MTS assay were used to measure ROS and cytotoxicity, respectively. Examinations into CPF-albumin binding were also conducted. CPF was not strongly cytotoxic to PC12 cells, causing only mild cytotoxicity at 5000 μg/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 μg/mL, and at 1 to 5 h at 5000 μg/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 μg/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 μg/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at ≥500 μg/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.

Optimization of an estrogen receptor-α transcriptional activation assay for testing a diverse spectrum of chemicals

Estrogen receptor (ER) transactivation assays were initially designed to study endogenous mechanisms of steroid hormone action, but more recently have been used to assess industrial chemicals for potential estrogenic activity. Given the diverse spectrum of physicochemical properties of these chemicals, we examined the effects of pH (a weak organic and strong inorganic acid and base), hyperosmolality (NaCl, mannitol) and two different vehicles (DMSO, Triton X‐100) on responses to estradiol‐17β (E2) in an ER transactivation assay. MCF‐7 human breast cancer cells were transiently transfected with a chimeric estrogen receptor (Gal4‐HEG0) and a Gal4‐regulated luciferase reporter gene (17m5‐G‐Luc), treated with E2 under various test conditions, and then assessed for ER‐mediated luciferase activity. Maximal E2‐induced reporter activity was observed at pH 7.8 (pre‐incubation), but was markedly reduced at pH ⩽7.5 or ⩾8.0 (P < 0.001), even though there was no evidence of cytotoxicity. Hyperosmolality induced by addition of mannitol (⩾25 mM) resulted in significant decreases in overall assay responsiveness, whereas NaCl (⩾80 mM) decreased the sensitivity of the assay by increasing the no‐observed‐effect level for E2 compared to control cultures (330 mOsm). The maximal DMSO concentration that resulted in consistently high E2‐induced reporter activity was 0.1%, whereas concentrations of Triton X‐100 above 1 ppm inhibited E2‐induced reporter responses and were cytotoxic above 10 ppm. These results indicate that various physicochemical factors have the potential to confound assay data if not kept within predefined operational limits. Copyright

Analysis of resulting data from estrogen receptor reporter gene assays

Screening methods for detecting single chemicals and chemical mixtures that have endocrine effects are of increasing importance. One such assay used by many laboratories to screen for potential estrogenicity is the estrogen receptor (ER) transcriptional activation assay, also known as an ER “reporter gene assay.” When applied in screening situations or to mechanistic studies, the assay appears to be quite reliable in giving a qualitative indication of a compound’s potential to activate ER-regulated target genes. Reporter gene assays also are advantageous for more complex applications, such as the analysis of chemical mixtures, but such applications present some challenges for statistical analysis. In particular, the need to transiently transfect cells with DNA constructs each time a reporter gene experiment is run may be a major factor contributing to a significant amount of interexperiment variability in strictly reproducing concentration-effect curves. This article reports on the use of nonlinear mixed models to account for the different sources of variability in analyzing such data. A population-averaged model is selected for use in the analysis of mixtures of chemicals. The models are illustrated with data from an ER reporter gene assay used to analyze tertiary mixtures of chemicals expected to exhibit additivity or synergy based on prior scientific reports. The models yielded the expected conclusions, and thus, validated the statistical approach.