4-Vinylcyclohexene (VCH) is an intermediate in the production of rubber and styrene. Following repeated exposure, VCH depletes the small pre-antral follicles of B6C3F1 mice. Interestingly, the female rat is resistant to VCH-induced ovotoxicity, which can be partially attributed to a reduced ability in forming the toxic metabolites (VCH-1,2-epoxide, VCH-7-8-epoxide, and ultimately, 4-vinyl-1-cyclohexene diepoxide [VCD]), as compared to mice. Current studies have focused on species dependent bioactivation of VCH, stereochemical considerations in VCH bioactivation, and the role of cytochrome P450 (CYP), particularly following repeated exposure to VCH. Hepatic microsomes isolated from mice pretreated with VCH converted VCH to epoxides to a greater extent than control mice or rats, or VCH-pretreated rats. In microsomes from both control animals and those pretreated with VCH, (R)-VCH formed more VCH-1,2-epoxide while (S)-VCH formed more VCH-7,8 epoxide, demonstrating enantioselectivity in metabolism. Rodents were also dosed with equitoxic doses of VCH, VCH-1,2-epoxide, or [VCD]), for 10d and investigated for CYP expression. VCH or VCH-1,2-epoxide pretreatments were effective in increasing total CYP levels in the mouse only. Of the multiple specific CYP isoforms tested, expression of only CYP2A and CYP213 were induced following VCH and VCH-1,2-epoxide pretreatment in the mouse. Neither CYP2A nor CYP2B were induced in the rat. While CYP2E1 plays a role in metabolism of the structurally related 1,3-butadiene, current studies demonstrate that these pretreatments caused no increases in CYP2E1 protein levels/activity. Those data, combined with the data showing no differences in epoxidation of VCH or its monoepoxides in CYP2E1-deficient mouse hepatic microsomes compared to those of mice that do have CYP2E1, indicated that CYP2E1 is not an important isoform in the species-specific bioactivation of VCH. These results have interesting correlations with human protein metabolism studies. Of several human hepatic CYP isoforms tested, CYP2E1 and CYP2B6 were the only isoforms that significantly catalyzed the epoxidation of VCH. In conclusion, CYP2A and CYP2B are critical isoforms in species-dependent VCH bioactivation, and the rat is resistant to VCH ovotoxicity, at least in part, because CYP induction does not occur following repeated exposure to VCH.

Women are born with a finite number of primordial follicles, the smallest follicles in the ovary. Once these follicles are destroyed, they cannot be replaced and after extensive loss, ovarian failure (menopause) can occur. The industrial chemical 4-vinylcyclohexene diepoxide (VCD) induces depletion of these follicles and causes premature ovarian failure in rats. VCD-induced ovotoxicity has been found to accelerate a natural process in the ovary, atresia, which occurs via apoptosis. The mechanism(s) by which VCD enhances follicular atresia are unknown; however, it has been shown to alter the expression of several genes and proteins associated with apoptosis. While downstream signaling events of VCD are becoming identified, the early signaling events of this pathway have not yet been determined, but may involve a receptor-mediated cascade. Therefore, these studies tested the hypothesis that VCD-induced ovotoxicity involves a nuclear receptor-mediated pathway that leads to increased atresia. Concurrent treatment of rats with VCD and estradiol selectively protected primary follicles from loss by an estrogen receptor-mediated mechanism via a reduction of caspase-3-induced apoptosis. VCD does not alter ER number, affinity, circulating estradiol levels, or directly bind ERbeta. Concurrent dosing of rats with VCD and an AhR antagonist prevented primordial and primary follicle loss via a reduction in caspase-3-induced apoptosis. Repeated dosing with VCD was shown to up-regulate expression of AhR mRNA; however, VCD did not alter expression of AhR-mediated genes glutathione-S-transferase Ya1 or Ya2 nor CYP 1A1 protein. AhR-deficient mice were still susceptible to VCD-induced follicle loss. Repeated dosing with VCD reduced Heat Shock Protein (HSP) 90 expression in small primary follicles. Analogs of the ER and AhR did not alter HSP90 protein, nor did a loss of HSP90 function induce follicle loss or potentiate VCD-induced follicle depletion. While the ER, AhR, and HSP90 are all co-localized in the oocyte nucleus of primordial and primary follicles, no evidence was seen to support that these proteins are interacting. Taken together, the ER is able to prevent VCD-induced ovotoxicity in primary follicles, the AhR is not required for VCD-induced follicle loss, and HSP90 does not appear to play a central role in follicle depletion caused by VCD.

4-Vinylcyclohexene (VCH) is released during rubber manufacture as an environmental contaminant. It causes destruction of ovarian follicles and ovarian cancer in mice. To determine its long term effects on the female reproductive system, female B6C3F1 mice were administered sesame seed oil or VCH (800 mg/kg) ip daily for 30 days. Mice were killed when in diestrus at 30, 60, 120, 240, or 360 days after VCH administration. Ovaries and uteri were weighed and ovaries prepared for histology. Primary and secondary follicles were counted and FSH determined by RIA. The number of estrous cycles differed from controls in the 30 day group. Uterine weight differed in the 30, 60, and 360 day groups. Primary follicles, secondary follicles and ovarian weight decreased in the VCH groups. FSH increased in the 240 and 360 day VCH groups. These studies indicate that VCH causes a dramatic and permanent reduction in ovarian follicles with a reduction in ovarian weight the most obvious indicator of follicle destruction.

Mineral hydrocarbons (MHC) produce hepatic microgranulomas and granulomas following repeated administration to female Fischer-344 (F-344) rats. Female Sprague-Dawley (S-D) rats are less sensitive to these MHC-induced hepatic effects. Comparative studies were conducted to characterize the pharmacokinetics and disposition of a representative C26 MHC, [1-14C]1-eicosanylcyclohexane ([14C]EICO), in these two rat strains. Following a single oral dose of MHC, F-344 rats had a higher blood Cmax of [14C]EICO, a longer time to Cmax, and a greater blood AUC compared to S-D rats. Fecal excretion was the major route of elimination of parent [ 14C]EICO for both rat strains. Both rat strains eliminated 11% of the dose in the urine by 96 h. However, S-D rats eliminated the majority of [14C]EICO-metabolites in the urine by 16 h, while F-344 rats excreted [14C] equivalents in a time-dependent manner. At 96 h, 3% of the radioactive dose was recovered in livers of F-344 rats, but only 0.1% in S-D rats. Parent [14C]EICO was determined to be retained in livers of F-344 rats. Only metabolites of [14C]EICO were present in urine, the two major being 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid. Inhibition of hepatic P450 activity using ABT prior to MHC administration resulted in a significant increase in [14C]EICO retention in livers of F-344 rats at 96 h. Pretreatment with clofibrate, a CYP4A inducer, decreased the amount of [14C]EICO retained in livers of F-344 rats at 96 h. Dietary exposure from MHC for 2 weeks resulted in an increased amount of MHC retained in livers of F-344 rats, whereas little was retained in treated S-D rats. The majority of MHC was retained in hepatocytes, whereas little was detected in Kupffer cells (KC). This was evidenced following transmission electron microscopy analysis of liver sections of F-344 rats. Multiple MHC droplets were observed in hepatocytes, while few droplets were detected in KC. Results from these studies suggest that inherent strain-differences in systemic exposure, rates of elimination, and hepatic retention of MHC exist following MHC exposure. The CYP4 enzyme family plays a role in the metabolism, clearance, and retention of [14C]EICO, while the phagocytic activity of KC is less significant after a single oral dose of MHC. In addition, repeated exposure of MHC alters the morphology of hepatocytes of F-344 rats but not S-D rats. These differences just described suggest that F-344 rats are inherently predisposed to the observed MHC-induced hepatic effects. (Abstract shortened by UMI.)

MEKK4 is a recently identified serine/threonine kinase. Upon initiation of this study, MEKK4 had been shown to phosphorylate MKK6, an upstream kinase of the stress activated p38 MAP kinase, although no agonist or directly upstream activating kinase had been reported. To identify proteins that interact with MEKK4, a recombinant form of the protein was used as bait to bind interacting proteins from mammalian cell extracts. Using this method the calcium binding protein, annexin II, was identified as an interacting partner with MEKK4. The Far-Western immunoblot was another approach used to examine interacting proteins and revealed that a 120 kiloDalton protein interacts with MEKK4. It was then postulated and subsequently confirmed that this 120 kiloDalton protein was the calcium regulated tyrosine kinase, Pyk2. Studies proceeded to examine the interaction between Pyk2 and MEKK4 and revealed that MEKK4 was an angiotensin II-induced, extracellular calcium-dependent substrate for Pyk2 kinase activity. It was subsequently observed that MEKK4 functioned in the reported pathway of angiotensin II enhancing cyclooxygenase II transcription. Again using recombinant MEKK4 as bait, the MAP kinase, ERK1, was also found to interact with MEKK4. Additional studies demonstrated that ERK1 was not a substrate for MEKK4. Instead, the Pyk2-interacting tyrosine phosphatase, SHP-2, was found to bind to and regulate MEKK4 tyrosine phosphorylation. As studies by other groups had detailed SHP-2 as an important regulator for ERK activation, experiments shifted to examining a role for MEKK4 in scaffolding SHP-2 regulation of ERK activation. Studies confirmed that the SH2 domains of SHP-2 were important in regulating the subset of the total cellular ERK population bound to MEKK4. Collectively these results demonstrate a dual function for MEKK4; as an active kinase that functions upstream of the p38 MAP kinase in the Pyk2 pathway; and as a scaffold for SHP-2 regulation of ERK activity. This study then defines two novel, potentially overlapping, cellular roles for MEKK4 in regulating the cardiovascular response to angiotensin II.

The biogeochemical cycle of arsenic (As) is very complex. The current practice of measuring total arsenic concentrations, especially in soil environments, ignores any differences in arsenic form or bioavailability, and therefore misrepresents the risk of arsenic to human health. Biotic and abiotic transformations of arsenic change its mobility, availability, and toxicity. A key reaction is arsenite (As(III)) oxidation to arsenate (As(V)), a form that is less bioavailable and fifty times less toxic. The role of microorganisms may be key to understanding As(III) oxidation rates in soils. This research studies how As(III) oxidation rates may be altered when soil microorganisms have had previous arsenic exposure. My primary hypothesis is: Previous exposure to arsenic affects the soil microbial population, allowing it to more quickly oxidize, and therefore detoxify, additional arsenite challenges. Microcosms of soils with different histories of arsenic contamination were amended with As(III). As(III) oxidation rates were compared by monitoring the bioavailable (water-extractable) arsenic concentrations using high performance liquid chromatography--hydride generation--atomic fluorescence spectrometry. Although As(III) oxidation occurred in all biologically active soils, reaction rates were positively correlated with previous As exposure, even with fewer and less diverse microorganisms in the chronically contaminated soils. 16SrDNA denaturing gradient gel electrophoresis analysis detected an emerging band with sequence homology to Thiomonas sp. NO115 after the highest contaminated soil was given the highest amendments. Bacterial isolates from that soil also showed different As transformation abilities, even when under the same selection pressures. To determine if faster As(III) oxidation rates could be induced in an arsenic-naive soil, control soils were given multiple doses of As(III). Changes in As(III) oxidation rate were dependent on both the number of previous doses and the challenge dose. When a soil inoculated with microorganisms extracted from a pre-exposed soil was challenged with 500ppm As(III), As(III) disappearance was more similar to an adapted soil, showing that it was the change in the microbial population that was responsible for the change in the As(III) oxidation rate. By studying how microbial populations adapt to environmental stressors like As(III), we may better understand not only the microorganisms, but also the biogeochemistry of arsenic.

Ultraviolet exposure causes epidermal damage including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions has been associated with mutagenesis and probably contributes to development of nonmelanoma skin cancer. Topically applied α-tocopherol previously has been shown to prevent UVB induced damage and skin tumor induction in mice. Thus, α-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. This dissertation explored possible mechanisms of photoprotection exerted by α-tocopherol. The UVB dose penetrating the epidermis was measured by monitoring the formation of UV induced DNA photodamage. Thymine dimer levels in DNA were analyzed by capillary gas chromatography with electron capture detection. Topical application of α-tocopherol prior to UVB exposure afforded significant protection against thymine dimer formation in mouse epidermis. Other vitamin E compounds and sunscreen compounds were less potent than α-tocopherol in their ability to reduce dimer formation. Multiple factors such as UVB absorbance and penetration of compounds into the skin could affect the photoprotective abilities of these compounds. The log integrated absorbance in the UVB was highly related to inhibition of thymine dimer formation in vitro, but not in vivo, where tocopherol derivatives conferred superior protection against UVB induced DNA photodamage despite having lower UVB absorbances. α-Tocopherol conferred the greatest photoprotection. DMSO was used as the vehicle to enhance penetration of the sunscreening agents. DMSO markedly increased the ability of commercial sunscreen compounds to inhibit dimer formation, but only modestly increased the photoprotective ability of the tocopherol homologues, suggesting that the tocopherol compounds penetrate the skin well regardless of vehicle. Cellular uptake and distribution of tocopherols was measured in an in vitro system. α-Tocopherol rapidly associated with cultured mouse keratinocytes and was transferred to the nucleus of the cells. The time course of α-tocopherol uptake corresponded temporally with DNA photoprotection in these cells. δ-Tocopherol and γ-tocopherol also afforded protection against DNA photodamage. γ-Tocopherol was taken up with similar kinetics as α-tocopherol, whereas δ-tocopherol accumulated to a lesser extent. These studies contributed evidence for the photoprotective actions of α-tocopherol and provided a strong rationale for the use of α-tocopherol in sunscreen products.

Studies presented in this dissertation determined biochemical mechanisms underlying the modulation of chemical-induced liver injury by retinol and GdC₃ The first objective was to determine the role of inflammatory cells in the retinol potentiation of CCl₄-induced liver injury. Plasma alanine aminotranferase activities and histological analysis of liver sections both illustrated significant potentiation of CCl₄ hepatotoxicity by a single dose of retinol. The mechanism for this potentiation involves priming of Kupffer cells (KC) (i.e. by enhancing their response to toxic stimuli) as established by chemical inhibitors of KC, isolated KC, and immunohistochemical analysis of liver sections. Additional studies estimated the effect of retinol on non-inflammatory processes (i.e. cytochrome P450 (P450) activity). While total P450 content was not increased, the activity and concentration of CYP 2E1 were both significantly elevated following treatment with a single dose of retinol. These findings suggest that a single pretreatment with retinol potentiates CCl₄ hepatotoxicity by multiple mechanisms that involve increased biotransformation and inflammatory cell activities. Based on the findings with retinol, another immunomodulating agent, GdCl₃ might also alter the activity of hepatic biotransforming enzymes. Having established that GdCl₃ inhibits the activity of KC, the purpose of these studies was to determine the effect of GdCl₃ on the content and activity of hepatic P450. GdCl₃ treatment reduced total hepatic microsomal P450 as well as aniline hydroxylase activity by 30% in male and 20% in female rats. Hepatocytes isolated from rats pretreated with GdCl₃ were less susceptible to toxicity caused by CCl₄ but not cadmium, a hepatotoxic chemical not bioactivated by P450. Thus GdCl₃-mediated protection from toxicity in vivo might involve decreased biotransformation and inflammatory cell activities. Data presented in this dissertation suggest that, in addition to altering the inflammatory response to toxicants, retinol and GdCl₃ may modulate liver injury by altering the P450-mediated bioactivation of chemicals. Considering the multiple effects described here for each of these compounds, investigators should be cautious in the interpretation of data utilizing retinol or GdCl₃ to implicate KC as the sole contributor to toxicological mechanisms. This is especially important in models of chemical-induced injury in which bioactivation is a key feature.

Cocaine is a psychomotor stimulant which is widely abused. To understand the behavior of cocaine, the disposition kinetics of the compound were characterized using the rat as an animal model. A sensitive HPLC assay was developed to quantitate cocaine and metabolites (cocaethylene, norcocaine, benzoylecgonine, and benzoyl-norecgonine) in plasma and urine samples. Since ecgonine methyl ester has insufficient UV absorptivity, the quantitation of this compound in blood and urine was performed by a GC/NPD method. The effects of dose and route of administration on the disposition kinetics of cocaine were studied in male Sprague-Dawley rats. The total systemic clearance of cocaine following i.v. and s.c. administration are dose-independent. The clearance of cocaine (CL/F) following i.p. administration is dose-dependent. Cocaine absorption following subcutaneous injection was slow but complete (F = 1.0). The extraction ratio of cocaine in the lung (1-F) is about 0.18. The oral bioavailability of cocaine (F = 0.05) was low compared to i.p. administration (F = 0.35-0.63). Various properties of cocaine in the blood (blood clearance, blood to plasma ratio and plasma protein binding) were characterized using blood from the experimental animals and humans (male and female). Cocaine degradation in blood was dose-independent. In rat, cocaine degradation in blood was slow and due to the non-enzymatic degradation. In humans, cocaine is metabolized by a cholinesterase enzyme and this reaction can be inhibited by NaF. Ethanol had no influence on cocaine degradation either in human or rat blood. Cocaine blood to plasma ratio was dose-independent and was not influenced by NaF and ethanol. Plasma protein binding of cocaine in the rat was independent of concentration but depended upon plasma pH. Gender and time of the menstrual cycle had no influence on cocaine degradation, blood to plasma ratio or plasma protein binding in humans. A simple device has been modified for serial venous blood sampling which permits the simultaneous measurement of locomotor activity in the freely moving rat. The relationship between the locomotor activity following a single short i.v. infusion of cocaine and cocaine plasma concentrations can be adequately described by the Sigmoid-Emax model or by the same model coupled with an effect compartment.

The studies presented in this dissertation were designed to test the hypothesis that embryonic N-acetyltransferase (NAT) acetylates 4-aminobiphenyl (4ABP), potentially affecting embryonic aromatic amine toxicity. NAT1 and NAT2 mRNAs were detected in C57BL6/J mice in gestational day (GD) 10 embryo/placental tissue, GD 15 embryo and GD 15 placenta tissue, and GD 18 extrahepatic embryonic tissue. Only NAT2 mRNA was detected in GD 18 hepatic tissue. NAT1 was not found in GD 18 or neonatal day (ND) 3 liver. NAT activity was present at all three gestational time points where NAT mRNA was detected. 4ABP NAT activity increased as gestation advanced. Activity at ND 4 was 1.5 fold higher than GD 10 tissue and 1.2 fold higher than GD 15. Neonatal hepatic tissue showed very little difference between ND 2 and ND 4. Preliminary kinetic constants were determined for GD 18 through ND 4. The average Km was 74 muM and the average Vmax was 0.78 nmol/min/mg. Finally, in vivo studies were conducted to determine if there was embryonic exposure to 4ABP or 4AABP. The amount of 4ABP in embryonic or placental tissue remained constant over the three time periods tested. While the level of 4AABP in the placenta did not change during gestation, the amount of 4AABP detected in the GD 18 embryonic tissue increased significantly over the other time points. 5-15% of 4ABP and 20-30% of 4AABP maternal blood levels were detected in the embryonic/placental tissue. Less than 0.03% to 0.06% of the maternal dose was found to have been converted to 4AABP in embryonic tissue. In summary, (1) NAT1 and NAT2 mRNAs were found in embryonic tissue; (2) functional NAT protein was present in the embryo; and (3) 4ABP and 4AABP were found in the embryo following maternal exposure to 4ABP. This suggests that embryonic NAT may contribute to the developmental toxicity of aromatic amines.