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Final Report: Improving Methods for Identifying Noncancer Risks Application of Cell Kinetic Models for Methylmercury Risk Assessment
EPA Grant Number: R825358Title: Improving Methods for Identifying Noncancer Risks Application of Cell Kinetic Models for Methylmercury Risk Assessment
Investigators: Faustman, Elaine , Leroux, Brian
Institution: University of Washington
EPA Project Officer: Reese, David H.
Project Period: October 1, 1996 through September 30, 1999
Project Amount: $390,827
RFA: Exploratory Research - Human Health (1996)
Research Category: Health Effects
Description:
Objective:The overall goal of the project was to improve procedures for estimating the risk of developmental toxicity in humans. The attainment of this goal requires the development of biologically based dose-response models for noncancer risks that can be accurately and appropriately extrapolated from test species to humans and estimate risks at low, environmentally relevant doses. The models currently used to evaluate risk in humans from exposure to potential toxic agents do not account for the complexities of events occurring during organogenesis nor do they adequately incorporate mechanistic difference information, concentration nor time considerations. Investigators at the University of Washington Department of Environmental Health focused on the development of biologically-based mathematical models to describe the dynamic process of organogenesis, based on modeling of cell kinetics using branching processes and nonlinear mixed effects regression models. Biological experiments were performed for model developmental toxicants (e.g., methylmercury [MeHg]) to provide detailed molecular and cellular information that was incorporated into the models. MeHg is a naturally occurring organometal that is of concern because of the large population exposed through fish consumption and because epidemiologic studies implicate even low levels, such as those expected among subsistence fish consumers, with adverse neurobehavioral development. The information collected included in vivo and in vitro data on timing, concentration, and rates of dynamic cell processes such as changes in cell cycle genes in differentiation and migration as well as growth and replication rates. As this information is both species and age specific, incorporation of such biologically based information improves our ability to accurately model species and age dependent differences in susceptibility.
The original grant proposal included a timeline that identified eight activities to be performed during the 3-year grant period:
- Design of in vitro and in vivo experimental plan
- In vitro experiments with methylmercury
- Identification of cell cycle changes
- Characterization of cell cycle gene expression
- Initiation of methylmercury study in vivo
- Simulation testing of model variables to assess model sensitivity
- Comparison of in vivo and in vitro methylmercury results with model predictions
- Final biological experiments defined and conducted.
In Vitro Experiments With Methylmercury and Identification of Cell Cycle Changes
MeHg Effects on Embryo CNS Cells In Vitro. To study the developmental toxicity of MeHg, we investigated the effects of MeHg on cell viability, differentiation and cell cycling using primary rat embryo central nervous system (CNS) cells. As with other regions of the developing CNS, midbrain cell proliferation appears to be sensitive to the effects of in utero MeHg exposure. The flow cytometric results demonstrate a MeHg-induced inhibition of cell cycling in both S and G2/M, and suggest an effect in G1; these results are consistent with previous in vivo and in vitro investigations. In addition, cell cycle progression rates appear to be the most sensitive indicator of adverse cell cycle effects following MeHg exposure. Cell cycle inhibition, as measured by progression through one round of cell division, demonstrates that MeHg-induced inhibition occurs by1 µM MeHg, however, changes in cell cycle distribution are not clearly evident at levels below 2 µM, and indicates that MeHg may affect cell cycle progression without significant effects on the distribution of cells in each cell cycle phase. These data support the hypothesis that MeHg selects against cycling cells, that multiple mechanisms can result in cell cycle inhibition, and that the mechanisms underlying cell cycle inhibition change as a function of exposure concentration.
By comparing the cytotoxicity and cell cycle inhibition elicited by MeHg with that induced by colchicine, it is observed that MeHg was approximately 80-fold less potent on a molar basis than colchicine in inducing mitotic arrest in G2/M. Furthermore, at similar levels of cytotoxicity, colchicine caused approximately 5-fold more cell cycle arrest in G2/M. These results suggest that the cytotoxicity elicited by MeHg cannot be ascribed solely to cell cycle arrest in G2/M alone, and provide evidence that the low neuronal cell counts observed in the brains of infants and animals exposed to MeHg in utero are likely to result from a combination of cell cycle inhibition (which may involve G1, S, and G2/M) and cell death.
Characterization of Cell Cycle Gene Expression. To understand the possible underlying mechanism of MeHg-induced cell cycle inhibition, we initiated a study of the effect of MeHg on genes known to be involved in the cell cycle control and growth arrest. We began by evaluating the constitutive expression level of Gadd genes (Gadd45 and Gadd153) during cell proliferation and commitment to differentiation. The Gadd genes are involved in growth arrest in response to stress and DNA damage, and act at the G1/S checkpoint where we have observed cell cycle inhibition following MeHg exposure. Gadd45 mRNA expression decreased with neuronal differentiation in CNS cells and increased during LB differentiation. Gadd153 mRNA expression remained low throughout CNS differentiation, but, in contrast to CNS, was markedly expressed during LB differentiation. These observations may indicate a differential role for these genes in regulating the cell cycle during differentiation in these two cell populations. This and other studies in our laboratory have shown differences in the cell proliferation kinetics of these two cell populations and the role of Gadd genes in defining these relationships is under investigation in other studies. To evaluate the effect of MeHg on the expression of Gadd 45 and Gadd 153, total RNA was isolated from cells treated with MeHg (0-2 µM, 24 hours). Preliminary findings show a concentration-related increase in the amount of Gadd45 and Gadd153 expression in the MeHg-exposed cells compared to the untreated cells. Differences in the induction profiles of these two genes has been previously observed, however to our knowledge, this is first evaluation of the effects of MeHg on these genes. Because peak induction for Gadd 45 has been reported to be maximal 4 hours after exposure to an inducer, our measurement of Gadd 45 expression at 24 hours post-exposure may under-represent induction.
In Vivo Studies of MeHg
MeHg and cell cycle regulatory gene expression. To examine whether the induction of cell cycle regulatory gene expression can occur following chronic MeHg exposure in vivo, a collaborative effort with Dr. Terrance Kavanagh was initiated as he had already initiated an in vivo study to evaluate the effects of MeHg on developmental immunology. Female mice were exposed to MeHg 4 weeks prior to conception. Gestation Day 12 and Day 16 embryos were collected, and midbrain and LB were isolated for gene expression studies. For comparison, RNA from exposed adult female mice was isolated and analyzed. While analysis of embryonic data is still in progress, our data indicate that genes relevant for cell cycle control, Gadd45, Gadd153, and p21, are induced in various tissues of adult female mice following 4 weeks of MeHg exposure.
To determine whether the induction of Gadd genes in response to MeHg exposure was via a known stress response pathway, the cell cycle regulatory gene, p21, was targeted for additional investigation. Exposure to MeHg elicits a dose-dependent activation of p21 gene expression in LB cells, however, this induction is not observed in CNS cells. Because we have previously observed a differential expression of p21 and cell cycle kinetics between CNS and LB cells during the time course of differentiation, p21 induction may well be dependent on the state of cell cycle and differentiation. Thus, our studies strongly suggest the need for careful dose and time evaluations to determine the involvement of these cell cycle regulatory genes in MeHg developmental toxicity.
In addition to its effects on gene expression, MeHg has been shown to alter intracellular redox status in the primary CNS cells. To address the relationship between MeHg and oxidative stress, primary CNS cells were evaluated for intracellular glutathione. These cells were also pre-incubated with N-acetyl cysteine (NAC), a glutathione (GSH) enhancer, for 24 hours prior to a subsequent 24-hour exposure to MeHg. The purpose of these studies were two-fold. First, we were interested in determining if the vulnerability of the developing CNS to MeHg lies in its intracellular GSH content. The intracellular GSH content and the activity of _Bglutamyl cycsteine synthetase (GCS) were determined with and without MeHg exposure in primary cultures of rat embryonic CNS cells. In addition, the effect of GSH modulation on MeHg-induced cytotoxicity was determined. Second, we characterized the mechanism of GCS regulation, initially by studying the GCS heavy chain subunit (GCS-HC). Primary embryonic limb bud cells were used as a reference cell type for comparing the response of CNS cells. The results indicate that constitutive intracellular GSH content, GCS activity, and GCS-HC mRNA and protein levels of CNS cells were approximately ten-, two-, five-, and ten-fold higher, respectively, than those in limb bud cells of the same gestational period. A dose-dependent increase in GSH levels and GCS activity was observed in CNS and limb bud cells following 1 and 2 µM MeHg exposure for 20 hours. Further characterization of GCS up-regulation in CNS cells showed that the increase in GCS activity following MeHg exposure, unlike limb tissues, was not accompanied by an elevation of GCS-HC mRNA and protein levels. Pretreatment of cells with N-acetylcysteine led to a significant increase in intracellular GSH, while L-buthionine- (S,R)-sulfoximine (BSO) pretreatment resulted in decreased GSH levels, however neither pretreatment had a significant impact on MeHg-induced cytotoxicity in either cell type. Our results suggest that although oxidative stress may mediate aspects of MeHg toxicity, disruption of GSH homeostasis alone is not responsible for the sensitivity of embryonic CNS cells to MeHg.
Role of p21 or p53 Genotype in Mediating MeHg=s Effects on Neuron Cell Cycle. Because our research has revealed MeHg induced changes in cell cycle inhibition and has revealed changes in several key cell cycle regulatory genes, we were interested in determining if these genes were essential for MeHg induced cell cycle inhibition. To accomplish this goal we isolated fibroblasts from p21 or p53 transgenic mouse embryos at day 14 of gestation. Cells of different genotypes (wild type, heterozygous and null) relative to p21 and p53 were cultured. Early passage (4-6) cells were treated with 0,2,4 and 6 µM MeHg for 24 hours. Colchicine, a known mitotic inhibitor, was used as positive control. Changes in cell cycle distribution after continuous MeHg treatment was analyzed by DNA content-based flow cytometry using DAPI fluorescent staining and was assessed in the cells of different genotypes following MeHg treatment.
Involvement of p21WAF1/CIP1 in Methylmercury-Induced Cell Cycle Inhibition. To evaluate the role of p21, a cell cycle protein involved in the G1 and G2 phase checkpoints, in the cell cycle inhibition induced by MeHg we utilized primary mouse embryonic fibroblasts (MEFs) of different p21 genotypes (wild type, heterozygous and null). Using these cultures we observed that MeHg induced an increase in proportion of cells in G2/M at 2 and 4 µM MeHg irrespective of p21 genotype (p"0.05). Effects of MeHg on cell cycle progression were also evaluated using BrdU-Hoechst flow cytometric analysis. Inhibition of cell cycling was observed in all p21 genotypes after continuous exposure to MeHg for 24 and 48 hours. p21 null (-/-) cells reached the second-round G1 at a higher fraction compared to the wild type (+/+) and heterozygous (+/-) cells (p"0.05). This study confirms previous observations that MeHg inhibits cell cycle progression through delayed G2-M transition and that the G2/M accumulation induced by MeHg is independent of p21. However, MeHg mediated inhibition of cell cycle as assessed by evaluating the proportion of cells reaching the second round G1 was dependent on p21 genotype.
Role of p53 Genotype in Methylmercury-Induced Cell Cycle Inhibition. To evaluate the role that p53 cell regulatory protein had on MeHg induced cell cycle inhibition, primary mouse fibroblasts of different p53 genotypes (wild type; heterozygous and null) were treated with MeHg and proportion of cells in each of the cell cycle phases was evaluated. Dose dependent increases in proportion of cells in G2/M phase was seen and was consistent with our previous observations. The overall level of MeHg mediated increases in G2/M was dependent upon p53 genotype status at low concentrations of MeHg (2 and 4 µm concentrations that produced minimal effects on cell viability) however all genotypes responded to MeHg. Additional studies are planned to confirm proportion of cells in G2/M using cell cycle dependent cyclin proteins, as they can distinguish true G2/M cells from non-disjunctional products.
Development and Simulation of Biologically-Based Mathematical Models and Comparison of In Vivo and In Vitro MeHg predictions. Because of our limited knowledge regarding the controls governing cell growth, this field of study relies heavily on the use of mathematical models to test hypotheses, to provide a framework for synthesizing all available information, and to examine the relative impacts on cell proliferation from environmental or intrinsic factors, and the downstream effects of proliferative alternations on morphogenesis and behavior.
The mathematical models developed in this study are based on the biological processes observed in laboratory studies. These models, known as biologically based dose-response (BBDR) models, can provide a basis for defining the dose-response relationship for subtle responses associated with lower, environmentally relevant doses. Investigators were interested in looking at fetal rat development after maternal exposure to methylmercury at varying doses.
A preliminary model of MeHg toxicokinetics (TK) was developed for the rat and linked to a prior toxicodynamic (TD) model developed by Leroux, et al. The TK model examines different stages of fetal growth and considers physical changes in the fetus such as fetal blood available and organ weights. In the TD model, changes in the population of committed fetal neural cells are estimated based on the observed effects of MeHg on rates of cellular death, proliferation and differentiation in vivo. By linking the TD and TK models investigators were able to refine estimates of methylmercury concentrations in the developing fetus and to examine cell cycle changes and cell death rates leading to possible physical and behavioral alterations or alterations in neural development..
The preliminary model has shown an adequate fit to experimental TK data. Analysis of the results of the linked TK/TD model revealed that fetal rat exposure did not increase at higher maternal doses. This surprised researchers and revealed that further refinement of the model parameters is needed. The differences could be related to maternal metabolism or inter-species differences. Two papers that describe this aspect of the study are in-process (Lewandowski, et al, see Appendix).
Conclusions:Journal Articles on this Report: 3 Displayed | Download in RIS Format
| Other project views: | All 27 publications | 4 publications in selected types | All 3 journal articles |
| Type | Citation | ||
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Faustman EM, Lewandowski TA, Ponce RA, Bartell SM. Biologically based dose-response models for developmental toxicants: lessons from methylmercury. Inhalation Toxicology 1999;11(6-7):559-572. |
R825358 (Final) R825173 (1999) R831709 (2005) |
not available |
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Ou YC, Thompson SA, Ponce RA, Shroeder J, Kavanagh TJ, Faustman EM. Induction of the cell cycle regulatory gene, p21 (Waf1, Cip1) following methylmercury exposure in vitro and in vivo. Toxicology and Applied Pharmacology 1999;157:203-212. |
R825358 (Final) R831709 (2005) |
not available |
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Ou YC, White CC, Krejsa, CM, Ponce RA, Kavanagh TJ, Faustman EM. The role of intracellular glutathione in methylmercury-induced toxicity in embryonic neuronal cells. Neurotoxicology, Volume 20, Issue 5, October 1999, Pages 793-804. |
R825358 (Final) R831709 (2005) |
not available |
non-cancer, development, modeling, risk assessment, rodent. , Toxics, Water, Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Risk Assessments, genetic susceptability, Health Risk Assessment, Mercury, Environmental Chemistry, National Recommended Water Quality, heavy metals, risk assessment, age, dose-response models, methylmercury, developmental toxicity, mixed effects regression model, interindividual variability, dose response, age dependent response, exposure, cell kinetic models, health risks, human exposure, neurotoxicity, mercury cycling
Progress and Final Reports:
Original Abstract