This thesis examines the toxicity of the herbicide glyphosate (two formulations ¡V technical grade and Roundup Biactive RB) and the insecticide chlorpyrifos CPF to a model freshwater food chain of a producer and consumer. The importance of studying the toxicity of low (environmentally realistic) concentrations of pesticides to non-target organisms is highlighted. An extensive literature review on the toxicity of glyphosate and chlorpyrifos to aquatic organisms is provided. The requirements for the maintenance of algal (Chlorella vulgaris, Chlorella pyrenoidosa and Pseudokirchneriella subcapitata) and Daphnia carinata cultures are discussed. The effects of two formulations of the herbicide glyphosate (technical grade and Roundup Biactive„µ) and the insecticide chlorpyrifos on the growth of Chlorella pyrenoidosa and Pseudokirchneriella subcapitata were studied, and the EC50 values determined. Hormesis was observed when P. subcapitata was exposed to concentrations of Roundup equal to 7% and 4% of its EC50 respectively. When exposed to chlorpyrifos concentrations 0.3-5 ƒÝg/L, hormesis was observed for both algal species with a maximum at 0.06% of EC50. The effects of sublethal concentrations of chlorpyrifos on population characteristics of Daphnia carinata were investigated in multiple-generation toxicity testing using individual culture. Exposure to chlorpyrifos affected survival and fecundity of animals in the first generation. In the second generation the most affected endpoint was time to the first brood with an indication of hormesis. LC50 tests were then conducted using animals of the third generation from each of the exposures in individual tests. Results of testing the third generation showed a constant significant decline in LC50 in the order of control daphnids through to ¡¥0.1 LC50¡¦ pre-exposed daphnids. The same experimental protocol was used in testing of glyphosate (technical grade and Roundup Biactive). Glyphosate was tested in two different media: sea salt solution and M4 medium, while Roundup Biactive was tested in M4 medium. Results indicated that glyphosate and Roundup Biactive had low toxicity to Daphnia. Hormesis was evident in sea salt medium exposures in the first and second generations of daphnids with glyphosate. When exposed to glyphosate and Roundup Biactive in M4 medium animals showed no indication of hormesis. It is hypothesized that glyphosate may have compensated for the lack of microelements in the sea salt medium, and possible mechanisms discussed.The modifying effect of glyphosate on the toxicity of cadmium to Daphnia carinata was studied using the same experimental design. Low concentrations of Roundup Biactive reduced the toxicity of cadmium, and the performance of daphnia was enhanced in terms of animal size, survival, fecundity, and the rate of natural increase in both generations in the presence of glyphosate. However when the third generation was tested for their sensitivity to Cd in the 48-h LC50 experiments there was no difference between RB-free and RB-spiked treatments in pair wise comparisons, indicating that no adaptation mechanisms were involved in the enhancement. The implications of these observed effects for environmental freshwater food chains subjected to pesticide exposure are discussed and recommendations on modifying pesticide use are provided.

This project developed methodology for in vitro toxicity assessment of contaminated sites using the Promega?? MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay performed on human cells (HepG2 and Skin fibroblasts). The project included the development of a method for extracting contaminants from soil based on leaching and centrifugation. A number of solvents and surfactants were assessed for their suitability as extracting agents. The Zwitterionic surfactant CHAPS ({3[(3-Cholamidopropyl) dimethylammonio] propanesulphonic acid}), which is an irritant in vivo, was found suitable for in vitro toxicity assessment applications. CHAPS was found to be the least toxic surfactant in vitro when tested on skin fibroblasts (NOEC: 1800??577 ppm, IC50: 4000??577 ppm) and HepG2 cells (NOEC: 833??289 ppm, IC50: 5300??287 ppm). The chosen surfactant was used in three different methods for extraction of Toluene and Xylene spiked in 2 g and 10g soil. The combination comprising of 0.1% (s/w) CHAPS and cosolvent 1% (w/w) Isopropanol, at their respective NOEC (No Observed Effective Concentration) toxicity values, showed good recovery of the nonpolar organic compounds in comparison to the recovery by 0.1% CHAPS and 0.5% CHAPS. The study found additive interactions to be the most common form of toxicity for 16 concentration combinations of Formaldehyde (polar), Toluene and Xylene (nonpolar) when compared to predicted toxicity (R2=0.943, P&lt0.0001). When assessing the in vitro toxicity of unknown (blind) contaminated soil samples, the Hazard Index (HI) predicted from the chemical analyses results showed a relatively good correlation (R2&gt0.7062, n=26) when compared to the experimental toxicity results on HepG2 cells. Furthermore, the comparison of Australian Health Investigation Levels (HIL) with in vitro toxicity testing gave similar correlation (R2&gt0.6882, n=26) on HepG2 cells. The overall project suggests the potential application of the zwitterionic surfactant (CHAPS) in sampling contaminants from soils in an in vitro toxicity assessment. This study demonstrates the application of in vitro toxicity assessment using human cells for the prediction of toxic risk as a sentinel to human toxicity from a contaminated site.

Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

Nanoparticles (particles less than 100 nm in at least one dimension) exhibit chemical properties that differ from their bulk counterparts. Furthermore, they exhibit increased potential for systemic toxicities due to their deposition deep within pulmonary tissue upon inhalation. Thus, standard regulatory assays alone may not always be appropriate for evaluation of their full spectrum of toxicity. Systems biology (e.g., the study of molecular processes to describe a system as a whole) has emerged as a powerful platform proposed to provide insight in potential hazard, mode of action and human disease relevance. This work makes use of systems biology to characterize carbon black nanoparticle-induced toxicities in pulmonary and extra-pulmonary tissues (i.e., liver and heart) in mice over dose and time. This includes investigations of gene expression profiles, microRNA expression profiles, tissue-specific phenotypes and plasma proteins. The data are discussed in the context of potential use in human health risk assessment. In general, the work provides an example of how toxicogenomics can be used to support human health risk assessment.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

The long-term natural history of ovarian reserve after adjuvant chemotherapy for breast cancer has been poorly described. We recruited 52 breast cancer survivors treated with adjuvant chemotherapy before 40 years of age who remained premenopausal after chemotherapy treatment. Twenty (38.5%) were more than five years out from treatment. Ovarian reserve estimates were compared with a control group. Anti-Müllerian hormone (AMH), follicle stimulating hormone and luteinizing hormone demonstrated significant differences consistent with reduced ovarian reserve in breast cancer survivors. Mean AMH was 6.65 pmol/l in survivors compared to 17.43 in controls (p < 0.001). Attained age and age at the time of treatment were correlated with AMH levels in breast cancer survivors. Conclusion: Ovarian reserve is significantly reduced in young breast cancer survivors. Age is the major predictor of AMH level in survivors. A 35 year old breast cancer survivor has an AMH level similar to a 45 year old control.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

The long-term natural history of ovarian reserve after adjuvant chemotherapy for breast cancer has been poorly described. We recruited 52 breast cancer survivors treated with adjuvant chemotherapy before 40 years of age who remained premenopausal after chemotherapy treatment. Twenty (38.5%) were more than five years out from treatment. Ovarian reserve estimates were compared with a control group. Anti-Müllerian hormone (AMH), follicle stimulating hormone and luteinizing hormone demonstrated significant differences consistent with reduced ovarian reserve in breast cancer survivors. Mean AMH was 6.65 pmol/l in survivors compared to 17.43 in controls (p < 0.001). Attained age and age at the time of treatment were correlated with AMH levels in breast cancer survivors. Conclusion: Ovarian reserve is significantly reduced in young breast cancer survivors. Age is the major predictor of AMH level in survivors. A 35 year old breast cancer survivor has an AMH level similar to a 45 year old control.