Complex chemical mixtures may be released into the environment from a variety of sources including hazardous waste sites. Components of chemical mixtures and their metabolites may be genotoxic leading to cancer and heritable gene mutations. Chemical analysis alone does not always provide the most accurate information from which to estimate the risk of adverse effects associated with exposure to mixtures. Current methods to estimate the human health risk for complex mixtures assume additive effects of the components. Although it is assumed that this approach is protective of human and ecological health, it is also recognized that chemical mixtures may induce a variety of interactions including potentiation, synergism, and antagonism. A combined testing protocol, using chemical analysis coupled with a battery of in vitro, in vivo, and in situ bioassays, provides the most accurate information from which to estimate risk. Such a combined testing protocol provides information to describe the major organic and inorganic constituents, as well as the pharmacokinetics and potential interactions of chemical mixtures. This research was conducted to investigate the potential genotoxic effects of complex chemical mixtures of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated aromatics (PCA) using microbial bioassays (Salmonella/microsome assay and the E. coli prophage induction assay), the 32P-postlabeling assay in mice, and in situ measurements of genotoxicity using flow cytometry. Samples of environmental media and wildlife tissues were collected from four National Priority List Superfund sites within the United States. In general, chemical analysis was not always predictive of mixture toxicity. Although biodegradation reduced the concentration of total and carcinogenic PAHs in soils and groundwater, the genotoxicity of extracts from environmental media did not display a corresponding reduction. Mixtures of polychlorinated biphenyls (PCBs) extracted from sediments were found to inhibit the genotoxicity of PAH mixtures when administered dermally to rodents. This inhibition exhibited a dose-response relationship, with the adduct frequency reduced at increasing doses of sediment extract. Finally, PAH concentrations in environmental media and tissues were found to correlate with DNA damage in wildlife receptors. An integrated approach, combining in vitro and in vivo methods to characterize genotoxicity provides more accurate information from which to estimate uptake and risk associated with exposure to complex mixtures and should be considered in both the human and ecological risk assessment process.

Bone is a dynamic tissue, where bone forming osteoblasts and bone resorbing osteoclasts maintain homeostasis. Research into bone toxicology has largely focused on pharmaceutical side effects adversely affecting bone development. However, many environmental toxicants can regulate bone homeostasis. Recently, the nuclear receptor peroxisome proliferator activated receptor gamma (PPARγ) has emerged as an important target of environmental toxicants. PPARγ dimerizes with the retinoid-X receptor alpha (RXRα), is a central transcription factor in adipogenesis, and in bone can transdifferentiate osteoblasts into adipocytes by suppressing osteogenic pathways. The central hypothesis of this dissertation is that environmental chemicals can adversely affect bone homeostasis by activating nuclear receptors in bone cells – particularly osteoblasts and osteoclasts – to perturb cellular differentiation and function. Three study aims were developed to test and refine this hypothesis. First, a set of structurally diverse environmental PPARγ agonists were individually applied to mouse primary bone marrow mesenchymal stromal cell cultures undergoing osteogenic differentiation. In vitro PPARγ ligand treatment suppressed osteogenesis and stimulated adipogenesis. Organotin compounds (tributyltin, triphenyltin) in particular more efficaciously suppressed osteogenesis. The second aim characterized the effects of in vivo tributyltin exposure on bone microarchitecture in female C57Bl/6 mice. Tributyltin exposure resulted in a thinner cortical bone, but significantly increased trabecular mineralization. Further analyses suggested that tributyltin did not suppress osteoclast numbers but rather changed osteoclast function, minimally attenuating the resorptive function and enhancing their ability to generate osteogenesis-stimulating factors. Furthermore, tributyltin activated not only PPARγ, but also RXR and liver X receptors. The third aim established the utility of Generalized Concentration Addition in modeling PPARγ activation by mixtures of full and partial PPARγ agonists. A complex mixture of multiple phthalate compounds activated an in vitro PPARγ reporter assay, and the individual dose-responses of each compound were used to construct modeled responses. The comparisons of empirical data and model predictions supported the use of Generalized Concentration Addition in modeling a complex mixture of environmental PPARγ agonists. Together, these studies support and establish important toxicological mechanisms related to PPARγ and RXRα activation in different aspects of bone biology and provide a basis for studying mixture effects of PPARγ agonists.

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The three insecticides of interest were lindane (an organochlorine), malathion (an organophosphate) and piperonyl butoxide (PBO; a synergist). Based on minimum cytotoxicity (> LC25), the following concentrations were chosen for the pesticide mixture studies: 70μM lindane (Lind), 50μM malathion (Mal) and 55μM PBO. In the AlamarBlue cytotoxicity assay, individual pesticide and mixtures of malathion/PBO (MP) and malathion/lindane (ML) prompted cytotoxicity with varying intensities (Mal 18.8%, Lind 20.4%, PBO 23.5%, ML 53.6% and MP 64.9%). Cytopathological analysis revealed apoptotic features in treated cells and the DNA Ladder Assay confirmed the presence of DNA fragments. The specific mode of cell death was examined via the 7-aminoactinomycin D (7-AAD) Staining Assay. Apoptosis was detected in each treatment (Mal 6.5%, Lind 12.0%, PBO 13.2%, ML 19.3% and MP 23.4%). Furthermore, 7-AAD staining in combination with fluorescent-labeled monoclonal antibodies, PE-CD45RB/220 and FITC-CD90, was performed. B-cells were more susceptible to Mal and PBO treatments than were T-cells. The pro-oxidant activity of the pesticides was monitored via the Dichlorofluorescin Diacetate assay. Exposure to pesticides for 15 minutes increased H2O2 production above the controls, Mal 21.1%; Lind 10.8%; PBO 25.9%; ML 26.8%; MP 37.8%. The activities of antioxidant enzymes, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were altered by these treatments. GR was significantly reduced for the pesticide mixtures only (control: 51.7; Mal: 48.2; Lind: 50; PBO: 52.3; ML: 40.5; MP: 42 Units/mg). GSH-Px activity was severely reduced for all the pesticide treatments (control: 44.9; Mal: 30.2; Lind: 30.6; PBO: 32.4; ML: 21.1; MP: 21.1 Units/mg). These results indicate that exposure to these pesticide and pesticide mixtures induces apoptosis and oxidative stress. / Master of Science