Interspecies differences in susceptibility to perturbation of thyroid homeostasis: a case study with perchlorate.

Despite many physiological similarities, humans and rats exhibit notably different susceptibilities to thyroid perturbation. Considerable research has recently been conducted on the thyroid-active chemical perchlorate, a chemical of emerging environmental and regulatory interest. While the data indicate humans and rats exhibit similar dose-response relationships in terms of acute inhibition of thyroidal iodide uptake, the two species appear to exhibit notable differences in terms of thyroid hormone response, the toxicologically significant consequence of iodide uptake inhibition. We analyzed dose-response data for changes in serum T(3), T(4), and TSH levels from studies in humans, rats, mice, and rabbits. We found that thyroid homeostasis in the rat appears to be strikingly more sensitive to perchlorate than any of the other species. Rats exhibited an increase in serum TSH at 0.1mg/kg-day whereas other species remained unresponsive even at doses of 10mg/kg-day. Less pronounced but consistent effects were seen with serum T(3) and T(4). These cross-species comparisons provide strong evidence that data obtained from rat studies should be critically evaluated for their relevance to humans. If rat data are used to develop toxicity criteria for perchlorate, we propose that this is an instance where an inter-species uncertainty factor less than one is supportable. DISCLOSURE STATEMENT: One of the authors (BDB) has been hired by Lockheed Martin Corporation as an expert in litigation involving perchlorate. A portion of the initial research presented in this paper was conducted in conjunction with her role in that matter.

Procedures for health risk assessment in Europe.

This report compares cancer classification systems, health risk assessment approaches, and procedures used for establishing occupational exposure limits (OELs), in various European countries and scientific organizations. The objectives were to highlight and compare key aspects of these processes and to identify the basis for differences in cancer classifications and OELs between various scientific organizations and countries. Differences in cancer classification exist in part due to differences in the ultimate purpose of classification and to the relative importance of different types of data (i.e., animal vs human data, mechanistic data, and data from benign vs malignant tumors). In general, the groups surveyed tend to agree on classification of chemicals with good evidence of carcinogenicity in humans, and agree less on classification of chemicals with positive evidence in animals and inadequate or limited evidence in humans. Most entities surveyed distinguish between genotoxic and nongenotoxic chemicals when conducting risk assessments. Although the risk assessment approach used for nongenotoxic chemicals is fairly similar among groups, risk assessment approaches for genotoxic carcinogens vary widely. In addition to risk assessment approaches, other factors which can affect OELs include selection of the critical effect, use of health-based vs technology-based exposure limits, and consideration of technological feasibility and socioeconomic factors.

Effects of O6-benzylguanine on growth and differentiation of P19 embryonic carcinoma cells treated with alkylating agents.

The purpose of this study was to evaluate the impact of modulating the repair of O6-alkylguanine adducts on the developmental toxicity of alkylating agents. Alkylating agents that have been shown to induce developmental toxicity following either in vitro or in vivo exposure were chosen for this investigation, and include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). P19 cells are pluripotent murine embryonic carcinoma cells that can be induced by all trans retinoic acid (RA) to differentiate into cells that are biochemically and morphologically very similar to cells of the central nervous system. These cells are useful for studying the ability of chemicals to affect neuronal viability and differentiation. Neuronally differentiating P19 cells were pretreated with O6-benzylguanine (O6-Bg), a potent and specific inhibitor of the O6-alkylguanine-DNA-alkyltransferase (AT) protein that repairs lesions at the O6-position of guanine. In previous studies using micromass rat embryo midbrain cells, O6-Bg greatly potentiated the ability of MNU but not ENU to inhibit differentiation, and did not significantly alter the effects of either MNU or ENU on viability. In the P19 cells, we found that AT inhibition potentiated the effects of MMS, MNU, and EMS to inhibit both viability and differentiation. Additionally, AT inhibition had a much greater effect on toxicity of the methylating agents, as compared to the ethylating agents. These results suggest that O6-alkylguanine adducts can inhibit both viability and differentiation in P19 cells treated with alkylating agents.

Evaluation of P19 cells for studying mechanisms of developmental toxicity: application to four direct-acting alkylating agents.

P19 cells are pluripotent murine embryonic carcinoma (EC) cells that can be induced by all-trans-retinoic acid (RA) to differentiate into cells that are biochemically and morphologically similar to cells of the central nervous system. We have established these cells as a reproducible cell system to evaluate potential effects of agents disrupting neuronal differentiation. The viability of P19 cells was assessed using a neutral red assay. Uptake of [3H]-gamma-amino butyric acid ([3H]GABA) was assessed as a marker of neuronal differentiation. We observed significant increases in [3H]GABA over time, corresponding with the appearance of cells with neuronal morphologies. 2,4-Diaminobutyric acid, a specific inhibitor of high affinity neuronal GABA uptake, reduced [3H]GABA uptake by approximately 75%. Additionally, [3H]GABA uptake in cells treated with dimethylsulfoxide (DMSO), which differentiate into mesodermal derivatives, was approximately 25% of uptake observed in RA-exposed, neuronally differentiated P19 cells. The morphology of P19 cell cultures correlated with [3H]GABA uptake: high [3H]GABA uptake was only observed in cultures with distinct neuronal morphologies. These results suggest that [3H]GABA uptake is a good indicator of neuronal differentiation in P19 cells. The responsiveness of P19 cells to developmental toxicants was assessed by comparing effects in P19 cells with effects observed previously in primary cultures of differentiating rat embryo midbrain (CNS) cells. Alkylating agents chosen for this investigation include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). The rank order of potency of these alkylating agents in the CNS cells was MMS > MNU > ENU > EMS. With the exception of ENU, concentrations that caused effects on growth and differentiation in the P19 cells were very comparable to those causing similar effects in CNS cell cultures. Our results with P19 cells suggest that this EC cell line may also be a useful in vitro cell system for studying mechanisms of developmental toxicity, with the advantages of being an established cell line.

Toxicity of four alkylating agents on in vitro rat embryo differentiation and development.

The relative developmental toxicity of four direct acting, alkylating agents was determined in primary cultures of differentiating rat embryo midbrain (CNS) and limb bud (LB) cells and compared with that observed in the rat whole embryo postimplantation culture system. The alkylating agents tested include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents have been shown to produce developmental toxicity following either in vitro or in vivo exposure. Viability for both CNS and LB was assessed by a neutral red dye assay. Differentiation of CNS cells was assessed by hematoxylin staining of neurons; differentiation of LB cells was assessed by Alcian blue staining of extracellular proteoglycans. Relative potencies of these compounds in the cell culture system were not the same as those observed in the embryo culture system. Whereas rank order of potency in the cell culture system, for viability and differentiation, was MMS > MNU > ENU > EMS, rank order in the embryo culture system, for embryo lethality and malformations, was MNU > ENU > MMS > EMS. Effective concentrations for cell culture viability and differentiation by MNU and ENU in cell culture were about three to nine times higher than comparable values previously reported for embryos, while effective concentrations for MMS and EMS were two to seven times lower than those observed in the embryos. Differences in potency between the two culture systems may be related to differences in formation and repair of DNA adducts, as well as differences in culture conditions.