Chemical exposure of the skin has become a route of entry of some chemicals into the body and has come under major review in recent times. This research aims to develop a method of estimating skin exposure that is both reliable and non-prohibitive in cost. This involved the design and testing of skin patches adaptable for monitoring skin exposure to chemicals using several different types of absorbents which could be easily worn against skin. The final design of the patch used either activated charcoal or tenax as the absorbing medium. The patches were then desorbed with a solvent in order to analyse the chemicals. The results of the study showed that many skin exposures do not have a direct relationship with inhalation exposures, which is important because currently there are no estimates of the levels of skin exposures that may have potential long term health effects. The patch has proved successful for detecting the presence and determining the amount of chemicals that come in contact with the skin. Charcoal patches have the widest application, but are not suitable for all situations and tenax should be used on these occasions. / Doctor of Philosophy (PhD)

This study assessed the consequences of a single binge exposure to ethanol on postnatal day (PN) 4 in the Purkinje cell population of the rat cerebellar vermis. The acute temporal pattern of apoptosis and the long-term permanent deficit in Purkinje cells across the entire vermis and on a lobular basis was investigated. It also investigated the lobular based Purkinje cell vulnerability to ethanol-induced apoptosis. On PN4 Sprague-Dawley rat pups from timed matings were randomly assigned to either an alcohol exposed (AE; 4.5g/kg, 10.2% (v/v) as 2 doses 2 hours apart) via intragastric intubation, or sham intubated control (IC) group. Immunolabelling methods were used to detect active capase-3 in cerebellar vermal Purkinje cells hourly from 1 to 12 hours and 14, 16, 20, and 24 hours, following the first intubation. Semi-quantitative methods were used to determine the ratio of active caspase-3, a marker of apoptosis, labelled Purkinje cells per total Purkinje cells within the entire cerebellar vermis and on a lobular basis. The onset of caspase-3 activation occurred 3-4 hours following the initial ethanol exposure and peaked 10 hours post-ethanol, the magnitude of increase in active caspase-3 varied across lobules. To determine the long-term consequences a second cohort of pups was treated as per above and the total number of Purkinje cells in the adult (PN60) cerebellar vermis, as well as in lobules I/II, VII and IX, was determined using the optical disector method combined with the Cavalieri technique. A significant deficit was found in the entire vermis (21%), as well as lobules I/II (48%), VII (25%) and IX (38%). A third cohort of suckle control (SC) pups was used to investigate the lobular based vulnerability to apoptosis in normal cerebellar vermal Purkinje cells on PN4. The levels of Bcl-2 and Bax mRNA expression were investigated using laser capture microdissection (LCM) to isolate the Purkinje cell lobular based populations combined with quantitative RTPCR (qPCR) technique. This method enabled the determination of comparative levels of mRNA expression attributable to the Purkinje cell population in identified lobules. Results indicated that Bcl-2 is expressed in cerebellar Purkinje cells and does not vary across the lobules. However, Bax expression could not be confirmed. This thesis demonstrates the effects of a moderate ethanol binge on cerebellar vermal Purkinje cells on PN4. Findings demonstrate that apoptosis is initiated rapidly and results in the permanent loss of Purkinje cells, the degree of which is lobular dependent and greater than seen in the vermis as a whole. Thus significant cell loss can occur in discrete regions which may have specific discrete impairments in cerebellar function. Importantly for the human population, this study indicates that a single moderate alcohol binge is sufficient to cause significant permanent neuronal loss in the developing fetus, the onset of which occurs rapidly, and may result in specific brain dysfunctions.

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.

The toxic effects of degrading the chlorinated hydrocarbons trichloroethylene (TCE), chloroform (CF) and cis-1,2-dichloroethylene (cis-1,2-DCE) were studied in the bacterium Nitrosomonas europaea. N europaea is an ammonia-oxidizing bacterium that obtains all of its energy from the oxidation of ammonia to nitrite. This metabolic process involves two enzymes, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). AMO has a broad substrate range and is also capable of oxidizing TCE, CF, and cis-1,2-DCE. Effects of degrading these chlorinated compounds on both AMO and HAO were studied. Cells were inactivated with known inhibitors of both AMO (light) and HAO (hydrogen peroxide) to provide comparison studies. Oxidation of the three chlorinated hydrocarbons did not always result in similar toxic effects to the cells. Whole cell studies indicated that oxidation of TCE and CF resulted in a loss of both NH������- and N���H���- dependent 0��� uptake rates, while in vitro studies indicated that at lower concentrations of both TCE (���0.05 mM) and CF (���0.10 mM) neither AMO or HAO appear to be the primary sites of inactivation. The oxidation of cis-1,2-DCE appeared to specifically inactivate AMO both in in vivo and in vitro assays. N europaea cells were also pretreated with the AMO inhibitor acetylene and incubated with the chlorinated hydrocarbons. Results of both whole cell 0��� uptake rates and the in vitro HAO assay confirms the hypothesis that the chlorinated hydrocarbons must be turned over in order to produce a toxic effect in N. europaea cells. / Graduation date: 1999

Graduation date: 1996

Graduation date: 1996

Graduation date: 1995

Graduation date: 1994

The basis for the species difference between B6C3F1 mice (susceptible) and Fischer 344 rats (resistant) to 4- vinylcyclohexene (VcH)-induced ovarian tumorigenicity was investigated. Greater than 95% of a single oral 400 mg/kg dose of [¹⁴C]VCH was eliminated in 48 hr by mice and rats. Approximately 50-60% of the administered dose was excreted in the urine, while the remaining 30-40% of the dose was expired as organically soluble radioactivity. VCH-treated mice had dramatically higher blood concentrations of the VCH metabolite VCH-1,2-epoxide compared to VCH-treated rats. Furthermore, mouse hepatic microsomes catalyzed the conversion of VCH to VCH-1,2-epoxide at greater rates than rat hepatic microsomes. The destruction of oocytes was used as an index of ovarian toxicity to compare the potency of VCH and VCH epoxides in the mouse and rat. VCH markedly reduced the number of small oocytes in mice while no detectable change in oocyte number occurred in rats. Epoxide metabolites of VCH destroyed oocytes in both species at lower doses than VCH. Inhibition of VCH epoxidation reduced VCH-1,2-epoxide blood levels and partially protected mice from VCH-induced ovarian toxicity. Thus, the conversion of VCH to epoxides and the subsequent destruction of oocytes are critical steps in the induction of ovarian tumors by VCH. Rats may be resistant because the amount of VCH converted to epoxides is insufficient to destroy oocytes. The biochemical basis for the species difference in the rate of VCH epoxidation by hepatic microsomes from mice and rats was investigated. studies using inducers and inhibitors of certain cytochrome(s) P450 showed that hepatic microsomes of female mice perform VCH epoxidation at greater rates than rats because of the constitutive expression of P450 IIA and lIB forms. Hepatic microsomes of human females perform VCH epoxidation at lower rates than rats. This suggests that if the rate of epoxidation of VCH by the liver is the most important factor determining susceptibility to VCH toxicity then the rat may better model the response of humans exposed to VCH than mice.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences