4-vinylcyclohexene diepoxide (VCD) is an environmental xenobiotic formed as a by-product in the manufacture of rubber and therefore potential human exposure is likely. VCD destroys half of the small pre-antral (25-100 μm) follicles in ovaries of rats following 15 days of dosing. The overall goal of this research, was to determine the mode and earliest time for identification of follicular destruction and examine the specificity of the response for 25-100 μm follicles. The particular involvement of protein synthesis and gene expression in this ovotoxic response was also examined. After daily dosing with VCD (80 mg/kg), the rate of protein synthesis in 25-100 μm follicles was inhibited following 3, 6, and 10 hr of in vitro incubation with VCD; whereas, the inhibition in the rate of protein synthesis at 3 hr in 25-100 μm follicles from untreated animals was reversed at 6 and 10 hr. Furthermore, follicular viability was compromised to a greater extent in 25-100 μm follicles from dosed versus untreated animals. Following 10 days of daily dosing with VCD, there was an increase in random DNA fragmentation in 25-100 μm follicles; however, there was not a reduction in the numbers of primordial and primary (25-100 μm) follicles. Morphological analysis showed changes characteristic of an apoptotic-like form of cell death in oocytes and granulosa cells of primordial and primary follicles 4 hr following 10 days of daily dosing. There was an increase in levels of mRNA for bax, manganese superoxide dismutase (MnSOD) and microsomal epoxide hydrolase (mEH) in 25-100 μm follicles following 10 days dosing with VCD, but the increase was not observed in large pre-antral (100-250 μm) follicles or liver. However, decreases in levels of mRNA for bax in liver and mEH in 100-250 μm follicles were observed. These results suggest that repeated dosing makes 25-100 μm follicles more susceptible to VCD-induced cellular changes and that VCD-induces an apoptotic-like form of cell death which is mediated through changes in levels of expression of genes associated with death of the follicular cells.

The overall objective of this dissertation was to estimate the in vivo hepatic clearance (CL(H)) of compounds using in vitro methods of drug metabolism. The isolated perfused rat liver, precision-cut liver slices and hepatocyte were used to estimate in vitro CL(H) and compared to in vivo CL(H) Two compounds, benzoic acid and tolbutamide were chosen as model compounds. An isolated perfused rat liver (IPRL) apparatus was developed to measure hepatic extraction ratio. Three compounds, antipyrine, ethanol and lidocaine were used to characterize the apparatus. The ability of the IPRL to utilize oxygen was also investigated. Antipyrine extraction ratio was independent of perfusate flow rate, while the extraction of ethanol and lidocaine were flow-dependent. The extraction ratios of benzoic acid and tolbutamide were determined. The CL(H) of benzoic acid and tolbutamide was 4.43 ± 0.84 mL/min and 1.52 ± 0.59 mL/min, respectively. The intrinsic clearance of benzoic acid and tolbutamide was determined in precision-cut liver slices and scaled to the whole liver using total protein. The CL(H) of benzoic acid in rat liver slices was 2.13 ± 0.71 mL/min, while the intrinsic clearance in humans was 270 mL/min. The CL(H) of tolbutamide in rat and human liver slices was 0.019 mL/min and 3.16 mL/min, respectively. The intrinsic clearance of benzoic acid and tolbutamide was determined in rat hepatocytes and scaled to the whole liver using total number of cells in the rat liver. The CL(H) of benzoic acid and tolbutamide in rat hepatocytes was 3.55 ± 1.29 mL/min and 0.57 ± 0.16 mL/min, respectively. The CL(H) of benzoic acid and tolbutamide on intravenous dosing in the rat was 8.02 ± 1.01mL/min and 0.49 ± 0.06 mL/min, respectively. Precision cut liver slices under-estimated the CL(H) of benzoic acid and tolbutamide. The under-estimation is probably due to the inability of the drug to permeate the liver slice over the short time course of the initial rate experiments. The CL(H) of benzoic acid and tolbutamide in rats was better predicted by the IPRL and hepatocytes.

Toxicity of arsenic species is dependent on chemical oxidation state. Inorganic arsenic in the trivalent state, arsenite or As(III), is more biologically active than pentavalent arsenic, arsenate or As(V), and is more toxic by most measures. As(V), however, is more stable and prevalent in the environment. One consequence of environmental exposure is peripheral vascular disease, which is primarily due to vascular changes, but toxicity to the erythrocyte has not been evaluated. To understand toxicity and the implications of arsenic oxidation state, human erythrocytes were utilized to model the uptake, biotransformation (metabolism) and toxicity of sodium arsenate, As(V). It was first established that biotransformation, both in vivo and in vitro, would not be limited by uptake of As(V) into the cell. Evidence suggested that reduction was accomplished by at least two separate pathways. All reductive metabolism was dependent on the presence of reduced thiols including both non-protein thiols (glutathione; GSH) and protein thiols (ProSH). These pathways are: (1) chemically mediated reduction by GSH and (2) protein mediated reduction. It was established that the protein-dependent pathway required a reduced protein thiol and also required the presence of GSH. This points to reduction through a redox coupling to form a protein mixed disulfide (ProSSG). Toxicity to the erythrocyte was evaluated by determining total cell death, morphologic changes and effects on the energy cofactor adenosine triphosphate (ATP). Based on these three parameters, the erythrocyte was more susceptible to As(V) and not As(III) as other tissues are. The morphologic effects on the cell were also consistent with ATP depletion. These changes were characterized by formation of morphologically altered cells that are unable to deform in circulation effectively and occlude the microcirculation. This could contribute to vascular tissue damage associated with arsenic-induced circulatory disorders. In summary, the erythrocyte is able to take in As(V) which is detrimental to the ability of the cell to perform its intended function. Biotransformation to As(III) would therefore be a detoxifying event, and understanding the factors involved in biotransformation will help to understand human susceptibility to arsenic-induced vascular disease.

Recent intervention trials reported that smokers given dietary β-carotene supplementation were at increased risk of lung cancer and overall mortality. These results were unexpected based on previous observational epidemiology, which suggested β-carotene would decrease the onset of lung cancer in these individuals. The mechanism by which β-carotene supplementation results in increased lung cancer among smokers has yet to be defined. However, one proposed explanation is that β-carotene acts as a prooxidant in lungs exposed to cigarette smoke and exacerbates oxidative damage. This project has examined the consequences resulting from interactions of cigarette smoke with β-carotene in model systems. A novel β-carotene oxidation product, 4-nitro-β-carotene has been identified from smoke oxidation of beta-carotene in solution. This product has been isolated by both reverse-phase and cyano-column HPLC, and characterized by UV-vis spectroscopy, APCI-LC-MS, and NMR spectrometry. This class of products includes cis and all-trans isomers of 4-nitro-β-carotene. A new method for measuring primary products of lipid peroxidation has been developed. This method is specific and sensitive for the determination of 9- and 13-hydroxy fatty acid methyl esters of octadecadienoic acid as pentafluorobenzoyl esters by GC-ECD. These compounds are derived from the first stable products of linoleic acid oxidation. Using the method discussed above, the interactions between β-carotene and cigarette smoke were examined in a liposomal model system. DLPC liposomes showed little differences in oxidative damage with or without β-carotene incorporation. This result was consistent with gas-phase or whole smoke exposures. The effect of β-carotene on the oxidation of other antioxidants was also examined. Both the lipid soluble antioxidant α-tocopherol and the water soluble antioxidant ascorbate showed lower oxidation rates due to smoke exposure in the presence of β-carotene than without. These data indicate that β-carotene does not have prooxidant effects in this system. β-carotene is oxidized by cigarette smoke in model systems. 4-Nitro-β-carotene is one of several classes of products resulting from cigarette smoke oxidation of β-carotene. β-Carotene incorporation did not increase oxidation of lipid or other antioxidants upon smoke exposure. It is unlikely a prooxidant effect of β-carotene is responsible for increased lung cancer observed in recent intervention trials.

Methyleugenol is a component of essential oils and is found in many foods and consumer products. Structurally similar to safrole, a known rodent carcinogen, methyleugenol has been shown to be a potent rodent hepatocarcinogen in a National Toxicology Program two-year bioassay. The mechanism by which methyleugenol causes tumors is suspected to mimic that of safrole. However, it is unknown if methyleugenol produces metabolites analogous to the proximate carcinogens of safrole. The hypothesis of this study is that methyleugenol is a genotoxicant after metabolic activation. The data presented show that methyleugenol is rapidly excreted from the body of rodents after oral and IV dose, primarily in the urine. Metabolites found in the urine include hydroxylated derivatives that are subsequently conjugated. The same basic pattern of metabolites is produced in isolated hepatocytes and microsomes from both rodents and humans. Mice produce more of these metabolites and at a faster rate than do rat or human derived systems. Among the metabolites identified in microsomes is 1 '-hydroxymethyleugenol, the analogous metabolite to the proximate carcinogen of safrole. Results of the Unscheduled DNA synthesis assay in hepatocytes show that methyleugenol is positive (genotoxic) in both rats and mice but possibly much less so in human derived hepatocytes, and that genotoxicity can be blocked by preventing sulfation in rodent hepatoctyes. Thus methyleugenol is capable of forming the proximate carcinogenic molecule (1' - hydroxymethyleugenol) and the subsequent sulfation of that molecule may lead to the moiety responsible for DNA damage and potentially eventual tumorigenesis.

Accidental exposure to arsine (AsH₃) is possible in the semiconductor industry, metal mining, painting and herbicide preparation. First symptoms include intravascular hemolysis and dark red urine (hematuria), followed by abdominal pain, jaundice, and anemia. Exposure to AsH₃ is fatal in up to 25% of the reported human cases, usually caused by acute oliguric renal failure. The mechanism of AsH₃ toxicity in the kidney is unknown and was studied in vitro using established cell lines, primary cells, and isolated kidney. The hypothesis was that AsH₃ cause renal toxicity by its conversion to arsenite (AsIII). Renal cells were more susceptible to As(III) cytotoxic effects on ion homeostasis and cell integrity, but AsH₃ showed oxidative stress-like toxicity. Red blood cells were only susceptible to direct AsH₃ cytotoxicity. Hepatocytes, chosen because liver is also affected by AsH₃, were susceptible to both arsenicals. It was established that AsH₃ produce tissue specific toxicity. The toxicity of the AsH₃-produced hemolysate was also investigated. The complete hemolysate was toxic and this toxicity was associated with the soluble hemolytic products. AsH₃-induced nephrotoxicity was also studied in the isolated rat kidney. Unmetabolized AsH₃ was more toxic than hemolytic products in this system. Damage was found in the glomeruli, tubular epithelial cells, and vascular peritubular capillaries. Finally, the total amount of arsenicals produced by AsH₃ oxidation in the rat kidney and liver homogenates was determined. As(III) was formed four times as much compared to As(V) in the kidney. By comparison, the liver metabolized less than half of the arsenite formed by the kidney. In summary, in vitro systems were used to model tissue selectivity for AsH₃ toxicity and to investigate AsH₃ renal cytotoxicity. Red blood cells and hepatocytes were susceptible to unmetabolized AsH₃. AsH₃ was required to form As(III) to produce renal toxicity. The soluble hemolytic products produced by AsH₃ also contributed to the in vitro renal toxicity. Renal dysfunction produced by AsH₃ exposure (the cause for mortality), is caused by a combination of AsH₃-produced oxidative-stress toxicity and by cell integrity damage produced by As(III) formed from AsH₃ oxidation, and delivered to the kidney as soluble toxicants in the hemolysate.

Multiple myeloma is a B-cell malignancy characterized by the latent accumulation in bone marrow of monoclonal plasma cells with a low proliferative index and an extended life span. Interleukin-6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the Signal Transducer and Activator of Transcription (STAT) proteins. We report that one STAT family member Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line, U266. U266 cells are shown to constitutively overexpress Bcl-XL protein when compared to the IL-6 independent, Stat3 negative, human myeloma cell line 8226. Comparison of the two cell lines reveals that U266 cells are inherently resistant to Fas-mediated apoptosis and cytotoxic drugs when compared to 8226 cells. Inhibition of the IL-6/Jak2/Stat3 pathway in U266 cells by the Jak kinase inhibitor, AG490, or dominant negative Stat3 expression construct (Stat3/3) results in downregulation of Bcl-XL expression, and enhanced sensitivity to Fas-mediated apoptosis. Treatment of myeloma patient specimens with AG490 was sufficient to inhibit Stat3 phosphorylation and activation of Stat3. In addition, AG490 downregulated the expression of Bcl-XL mRNA and protein as determined by RNase protection assay (RPA) and flow cytometry, respectively. We also found that ectopic overexpression of Bcl-XL rescued Fas-induced apoptosis in U266 cells following inhibition of the Jak2/Stat3 pathway. Furthermore, enforced overexpression of Bcl-XL increased the resistance of U266 cells to chemotherapeutic drugs. However, instead of sensitizing U266 cells to drug-induced apoptosis, the JAK inhibitor AG490 antagonized drug-induced apoptosis. The inhibition of drug-induced apoptosis by AG490 was due to inhibition of cyclin D1 expression resulting in cell cycle arrest. These studies demonstrate that IL-6 induced activation of the Jak2/Stat3 pathway controls the expression of several genes that regulate cell proliferation and survival. Two of these genes are Bcl-XL and cyclin D1. Blocking Stat3 activation enhanced sensitivity of U266 myeloma cells to Fas-mediated apoptosis but reduced the efficacy of cell-cycle dependent cytotoxic drugs.

The primary focus of this research is to develop an in vitro method that uses an appropriate formulation:blood ratio and contact time to evaluate the degree of hemolysis occurring after an intravenous injection. The effects of both the formulation composition and the formulation:blood contact time on hemolysis are given in Chapters I-IV. Since hemolysis is shown to increase as either the formulation:blood ratio and/or the contact time increases for various pharmaceutical vehicles, a small formulation:blood ratio and short contact time must be selected to determine the degree of hemolysis occurring as the formulation is rapidly diluted by the blood after an intravenous injection. Using a formulation:blood ratio of 0.1 and a contact time of 1 second, a dynamic method has been developed to evaluate intravascular hemolysis. The ability of this method to accurately evaluate hemolysis occurring after an injection is determined by comparing hemolysis data generated with this dynamic method to in vivo hemolysis data obtained from the literature. The results of this comparison are given in chapter V. The in vitro hemolysis data are shown to be in agreement with in vivo hemolysis data.

Photochemical and antioxidant reactions of α-tocopherol (α-TH, vitamin E) were studied by monitoring the fate of α-TH in UVB irradiated liposomes, solution, and mouse skin. α-TH was rapidly depleted in UVB irradiated mouse skin and in vitro systems. Oxidative damage, assessed by monitoring lipid peroxidation, was suppressed in UVH-irradiated liposomes until α-TH was depleted to 20% of initial levels. In all three systems, products previously identified as marker products for peroxyl radical scavenging by α-TH were observed, including α-tocopherolquinone (α-TQ), α-tocopherolquinone 2,3-epoxide (α-TQE 1), α-tocopherolquinone 5,6-epoxide (α-TQE 2) and several 8a-(hydroperoxy) epoxytocopherones. In addition, a product resulting from photochemical consumption of α-tocopherol, a spirodimer, was identified by HPLC and HPLC-MS. These studies provide the first evidence of the fate of vitamin E in UVB irradiated in vitro and in vivo systems. We have also assessed the hydrolysis of the vitamin E ester α-tocopherol acetate (α-TAc) to the active antioxidant α-TH in mouse epidermis and in supernatant from epidermal homogenates. Topically administered α-tocopherol prevents UVB photocarcinogenesis in C3W mice, whereas α-tocopherol acetate does not. Hydrolysis in skin was monitored in mice treated topically with deuterium labeled α-TAc (d₃-α-TAc). α-TAc was hydrolyzed to α-TH in mouse skin, and this effect was enhanced by prior UVB treatment. We hypothesized that prior UVB exposure may increase hydrolysis of α-TAc by increasing epidermal esterase activity. Non specific esterase (NSE) activity was measured in the 2000 x g supernatant from epidermis of unirradiated and irradiated mice. α-Napthyl acetate (α-NA), a NSE substrate, was converted to α-napthol (α-NOH) in supernatants from unirradiated mice. Hydrolysis of α-TAc to α-TH also occurred in supernatant from unirradiated mice, and hydrolysis of both α-NA and α-TH was enhanced in supernatants from UVB-irradiated mice. These data indicate that NSE activity was increased by UVB in the skin, that α-TAc is converted to α-TH in the homogenate fraction containing NSE, and that UVB exposure modulates the metabolism of α-TAc to α-TH in vivo.

During the past decade precision-cut tissue slices have begun to be utilized for toxicity and metabolism studies. These studies have primarily involved a single organ type. In this study, a new preparation of rat renal cortical slices was validated and used to investigate the toxicity of chlorotrifluoroethylene and its cysteine and glutathione conjugates. An additional level of complexity was added by utilizing a sequential incubation system in which rat renal cortical slices were directly incubated in the medium from liver slice incubations. Once the new renal slice preparation and sequential incubation system had been validated, these new methods were used to study the mechanism of toxicity of chlorotrifluoroethylene and it metabolites. The hypothesis being tested in these studies is that sequential biotransformation in the liver and the kidney is required for CTFE nephrotoxicity. In these studies I developed a sequential incubation system with precision-cut rat liver slices as the drug activating system and renal cortical slices as the target tissue for toxicity. Utilizing the sequential incubation system, I found that first incubating liver slices with CTFE and then transferring kidney slices to this liver slice incubation medium causes toxicity in the kidney slices. I also found that this toxicity correlates well with the toxicity observed with kidney slice incubations with the cysteine and glutathione conjugates of CTFE. By incubating slices with inhibitors of the various enzymes in the proposed metabolic pathway of CTFE, it was determined that glutathione conjugation in the liver and subsequent degradation by gamma-glutamyltranspeptidase are important steps in toxicity of CTFE. Although previous research with inhibitors of β-lyase have indicated that β-lyase is an essential enzyme in the bioactivation of CTFE, inhibition of the pyridoxal phosphate cofactor of this enzyme in renal slices did not reduce the toxicity of conjugates of CTFE. There was no reduction in toxicity when dipeptidases were inhibited when transport via the organic anion transporter or neutral amino acid transporter were inhibited. These data indicate that the glutathione conjugate of CTFE is formed in the liver and that the subsequent metabolism of this glutathione conjugate in the kidney is required for nephrotoxicity.