Induction of hepatic microsomal hydroxylative enzymes is an important aspect of detoxication of fat-soluble toxicants. The magnitude of induction depends on numerous factors, such as the nature and dose of toxicant as well as dietary factors. Research was conducted on (1) endrin tolerance in rats, (2) preliminary comparisons of inductive effects of various organochlorine pesticides in rats to select compounds for further study in guinea pigs, (3) general effects of various dietary factors on induction, and (4) effects of ascorbic acid deficiency on induction of hepatic microsomal hydroxylative enzymes by organochlorine pesticides in guinea pigs. Measurements were made of body weight gain, feed consumption, liver weight, in vivo and in vitroenzyme activities, and body levels of pesticides and vitamins. Tolerance developed in rats fed 25 ppm endrin in the diet. There was severe intoxication during the first week but complete recovery of rate of body weight gain and feed consumption occurred during the second week, in spite of continued ingestion of the endrin-containing diet. Induction of endrin-degrading microsomal enzymes was proposed as the mechanism for tolerance. Pretreatment with the potent inducer, dieldrin, diminished the severity of endrin intoxication. However, pretreatment with another inducer, phenobarbital, afforded less protection in proportion to the extent of microsomal enzyme induction. Organochlorine pesticides, tested for their inductive capacity in rats, in decreasing order of effectiveness then fed as 25 ppm of the diet for 15 days, were heptachlor epoxide, dieldrin, endrin, 1,1-Bis-(p-chlorophenyl)-2,2,2-Trichloroethane (DDT), Ovex, gamma-chlordane, and lindane. Of these, DDT and dieldrin were compared over the range of 1 to 50 ppm of the diet. Dieldrin was a more potent inducer at all dietary levels. At low doses both compounds produced greater induction when measured by hexobarbital sleep time than by the in vitro enzyme procedures. At high doses, the in vitro 0-ethyl 0-p-nitrophenyl phenyl-phosphonothioate (EPN) detoxication was a more responsive measure. Dieldrin, DDT, and lindane were fed to guinea pigs at 25 ppm of the diet for 15 days, but only dieldrin stimulated a significant level of induction. DDT antagonism of dieldrin storage seen in rats by Street (Sci. 146:1580, 1964) did not occur in guinea pigs, but rather dieldrin antagonized DDT storage. Some general dietary factors affecting induction in rats were observed. A semipurified diet lowered the baseline microsomal enzyme activity but supported induction as effectively as a conventional diet. Vitamin A at very high dietary levels induced enzyme activity; this induction was apparently additive to that produced by 1 ppm dieldrin. Other fat-soluble Vitamins produced inconsistent responses. Ascorbic acid deficiency in guinea pigs impaired induction by dieldrin. Impairment was seen by the second day on the deficient diet. However, dieldrin was able to produce a small amount of induction at all stages of deficiency. In frank scurvy, induction by DDT and lindane was completely blocked, but there was a moderate level of induction by dieldrin. Maintenance of maximum induction was related to dietary rather than liver levels of ascorbic acid; 50 ppm ascorbic acid in the diet was grossly inadequate while 200 ppm supported about 80% of the induction produced by feeding 2000 ppm. It was concluded that (1) microsomal enzyme induction is important in resistance to organochlorine intoxication, (2) factors found in the normal diet can induce microsomal enzyme activity, (3) high dietary levels of ascorbic acid are necessary to support maximum induction, and (4) dieldrin is an inducer of such high potency that it can stimulate a limited amount of induction in spite of ascorbic acid deficiency.

Despite distinct dissimilarities, diverse cancers express several common pro-tumorigenic traits. We present here evidence that the pro-apoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated PKA activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide- or siRNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA and phospho-T155 dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a pro-cancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.

Within the last few decades, epidemiological evidence has linked exposure to air pollution, both its particles and its organic components, with cardiovascular disease (CVD) progression. CVD is a life long disease with the disruption of the endothelium being the inaugural event in this inflammatory process. The vascular endothelium is extremely susceptible to environmental insults given its tremendous surface area and that it is in constant contact with blood and components circulating within the blood, including xenobiotics. The endothelium is important as a barrier from blood constituents however, dysfunction of this barrier leads to the influx of lymphocytes and granulocytes that lead to the fatty build‐up characteristic of atherosclerosis. The studies presented in this dissertation tested the hypothesis that two unique environmental contaminants, alumina nanoparticles and benzo[a]pyrene (B[a]P), lead to increased endothelial cell dysfunction, characterized by increased adhesion molecule expression. Alumina nanoparticles induced vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and E‐selectin (ELAM‐1), as well as increased monocyte adhesion to activated endothelium. Polystyrene nanoparticles did not elicit this response. B[a]P induced ICAM‐1 expression, but only after toxification by aryl hydrocarbon receptor (AhR) controlled enzymes. Silencing of either AhR or the membrane microdomains called caveolae attenuated the B[a]P‐induced ICAM‐1 response. It was also shown that the induction of ICAM‐1 occurred by signaling through MEK, p‐38 MAPK, and activator protein‐1 (AP‐1). These data provide a novel mechanism by which air pollutants like B[a]P may cause increased atherosclerosis and describe a new toxicant, alumina nanoparticles, as a possible threat for the development of inflammatory diseases, such as atherosclerosis. Little is known about dietary interventions capable of alleviating xenobiotic‐induced toxicity. Nutrition is an obtainable and inexpensive means of possible preventative therapy. With this in mind, it was also hypothesized that plant polyphenols, such as flavonoids, can down‐regulate B[a]P‐induced ICAM‐1. Selective flavonoids, containing both a 4’ B‐ring hydroxyl substitution and a 2‐3 C‐ring double bond, protected against B[a]P‐induced ICAM‐1 activation, however this protection did not correlate with the flavonoid’s antioxidant capacity.

Par‐4 is a pro‐apoptotic tumor suppressor that is mutated, suppressed or inactivated in cancer. Par‐4 exploits components of the extrinsic pathway to cause apoptosis selectively of cancer cells. This study identified Par‐4 as an essential component of the apoptotic pathway induced by TRAIL, which selectively targets cancer cells. RNA interference‐mediated knockdown of Par‐4 rendered cancer cells unresponsive to TRAIL‐induced apoptosis. Cells with knocked‐down levels of Par‐4 were deficient in the activation of the apoptosis‐initiator caspase‐8 and the apoptosis‐effector caspase‐3 in response to TRAIL. Par‐4 was identified as a critical mediator of membrane translocation of caspase‐8 and the adapter protein FADD. Surprisingly, Par‐4 was also found to interact with caspase 8 in untreated cells, and was cleaved at the N‐terminus at aspartic acid residue 123 in response to TRAIL. This, along with another cleavage by caspase‐9 effectively generated a fragment containing the functional module of Par‐4, the SAC domain, which is sufficient for apoptosis of cancer cells. Moreover, TRAIL activated caspase‐8 was also found to be involved in nuclear translocation of Par‐4, a crucial step during apoptosis induction by Par‐4. Together, our findings suggest that Par‐ 4 is an essential downstream target of caspase‐8 that is activated by TRAIL signaling and that, in turn, activates caspase‐8 and the downstream apoptotic pathway in response to TRAIL.

The renin angiotensin system (RAS) exerts many diverse physiological functions throughout the body, mediated by its effector peptide, angiotensin II (AngII). AngII has been linked with a variety of different functions ranging from the initiation of severe vascular pathologies, such as atherosclerosis and abdominal aortic aneurysm (AAA), to mundane physiological processes of fluid homeostasis, vascular contraction, and regulation of blood pressure. To provide a potential link between these functions, an in-depth analysis of regional effects of AngII on aortic vasculature was performed. The studies presented in this dissertation tested the overall hypothesis of whether regional changes exist in the vasculature in response to angiotensin II (AngII). We first infused AngII into C57BL/6 animals and studied the aortic morphology in detail. On first glance, we detected a thickening throughout the aorta, with no overt changes from region to region. However, upon further analysis, it was demonstrated that there was a region-specific aortic arch hyperplasia, versus the hypertrophy in the remainder of the aorta. Through a series of experiments, this hyperplasia was linked to the redox-mediated protein Id3. Further analysis of the vasculature demonstrated AngII exerted aortic contractions which were limited to the infrarenal aorta. These contractions were mediated by the AT1b receptor subtype in the RAS. We also demonstrate that AngII leads to suprarenal specific formation of AAA, which can be attenuated by the deletion of specific innate immune mediator proteins, such as MyD88 and TLR4. Overall, these data suggest many region-specific roles for AngII in the aortic vasculature and provide many novel findings as to the cause of these effects.

ATP-binding cassette (ABC) transporters are transmembrane proteins that transport a wide variety of substrates across intra and extra-cellular membranes. A few examples of endo and xenobiotic substrates are metabolic products, lipids, sterols, and drugs. An important function of ABC transporters involved in export is to prevent intracellular the buildup of toxic products. Several ABC transporters have also been associated with drug resistance upon treatment with chemotherapeutic agents. P-glycoprotein (P-GP) and the multidrug resistant (MRP) transporters of the ABC C family are examples of transporters that confer chemo-resistance. We have studied two unique roles of ABC transporters in the liver and the heart. In the liver, maintenance of bile secretion is important during lactation to ensure proper absorption of nutrients for the offspring. Three main ABC transporters are involved in this process: ABCB11 (transports bile acids), ABCB4 (transporters phospholipids), and ABCG5/ABCG8 (transports cholesterol). In the rat, expression of ABCB11 remains the same as the size of the bile acid pool increases. However, the expression of ABCG5/ABCG8 is abolished, preventing excessive export and loss of cholesterol from the liver. The regulation of these transporters during lactation maintains the production of bile acids from cholesterol by decreasing export while preventing toxicity from bile acids by maintaining bile flow. Another protective role of ABC transporters is seen in oxidative stress-induced toxicity of cardiac tissue following treatment with Doxorubicin (DOX), a drug used in cancer treatment. Multidrug resistance protein 1 (Mrp1) can transport toxic products by conjugation with sulfate, glutathione (GSH) or glucuronide. In Mrp1-/- mice, DOX causes advanced cell damage through intracellular edema and increased apoptotic nuclei. However, P-glycoprotein expression increases upon DOX treatment, potentially compensating for the loss of Mrp1. Mrp1 can also transport GSH, GSH disulfide (GSSG), and products of oxidation, like GSH conjugates. In the absence of Mrp1, GSH levels are increased in the heart, providing protection against oxidative stress. Both of these examples in liver and heart show the diversity of ABC transporters and the role they play in preventing cell toxicity. These studies also provide insight into ways to prevent cell toxicity through manipulation of ABC transport proteins.

An individual’s naïve level of response (LR) to ethanol is predictive of their lifetime likelihood to abuse alcohol. LR is heavily genetically influenced, suggesting that the genes responsible for LR may also be central to the development of abuse disorders. Our laboratory uses the model organism C. elegans to investigate the genetic influences on responses to acute ethanol exposure. We recently found that changes in TAG levels can alter LR. From this result we investigated the role of long-chain polyunsaturated fatty acids (LC-PUFAs) as well enzymes involved in lipid modifications of proteins. We found that LC-PUFAs are necessary for acute functional tolerance and that supplementation of eicosapentaenoic acid is able to rescue AFT. We also identified mutations in several palmitoyltransferases, a thioesterase, and elongases that alter AFT. These novel results highlight the importance of fatty acids in the response to ethanol and suggest exciting new potential therapeutic targets.

Lung cancer is a particularly devastating disease, accounting for the most deaths among all cancer types in the United States. Despite a reduction in the country’s smoking rates, cigarette smoking remains the number one risk factor for lung cancer. Additionally arsenic exposure, which occurs primarily through contaminated drinking water in the U.S., is associated with increased lung cancer incidence. The nucleotide excision repair (NER) pathway is critical for maintenance of genomic fidelity, removing DNA lesions that could otherwise promote DNA mutations and drive carcinogenesis. Tobacco smoking introduces significant amounts of DNA damage and produces characteristic DNA mutations found in lung cancers of smokers, and arsenic increases lung cancer risk in smokers beyond the risk of smoking along. The contributions of these chemicals to DNA damage and cancer have been well documented, but few studies have examined their effects on DNA repair pathways, particularly the nucleotide excision repair (NER) pathway. Arsenic, while not directly mutagenic, promotes the carcinogenicity of other compounds including agents that produce DNA damage that is repaired by the NER pathway. In this dissertation I investigated the effects of cigarette smoke condensate (CSC, a whole-smoke tobacco surrogate) and arsenic on NER. I observed that CSC or arsenic treatment inhibited NER as measured by a slot-blot assay using UV-induced photolesions as model substrates to measure NER. The abundance of Xeroderma Pigmentosum complementation group C (XPC), a critical NER protein, was significantly reduced in all lines treated with either chemical, while XPA protein was unaffected. CSC and arsenic also affected RNA levels of certain NER genes. Finally, proteasome-regulated XPC turnover was affected by CSC and arsenic treatment, suggesting a potential mechanism for XPC protein inhibition. The observed impairment of NER by CSC is critically important in tobacco cancer etiology – CSC introduces DNA damage, some of which is repaired exclusively by NER, and CSC inhibits the NER pathway as well, providing a two-sided assault on cellular genetic fidelity. I then adapted the NER assay to measure repair in lymphocytes isolated from human subjects of a study investigating the high incidence of lung cancer in Appalachian Kentucky. I observed an age-dependent decline in NER efficiency that was modulated by subject smoking status and a reduced NER efficiency among current smokers in the lung cancer patient population compared to control subjects in the youngest age group, suggesting individual DNA repair capacity measured with this repair assay may be a biomarker for lung cancer susceptibility.

The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs and MutSβ recognizing 2-16-nt ID heteroduplexes. The goal of this dissertation research was to understand the mechanism underlying differential mismatch recognition by human MutSα and MutSβ and to characterize the unique functions of human MutSα and MutSβ in MMR. In this study, recombinant human MutSα and MutSβ were purified. Binding of the proteins to a T-G mispair and a 2-nt ID mispair was analyzed by gel-mobility assay; ATP/ADP binding was characterized using a UV cross-linking assay; ATPase activity was measured using an ATPase assay; MutSα amd MutSβ’s mismatch repair activity was evaluated using a reconstituted in vitro MMR assay. Our studies revealed that the preferential processing of base-base and ID heteroduplexes by MutSα and MutSβ respectively, is determined by the significant differences in the ATPase and ADP binding activities of MutSα and MutSβ, and the high ratio of MutSα:MutSβ in human cells. Our studies also demonstrated that MutSβ interacts similarly with a (CAG)n hairpin and a mismatch, and that excess MutSβ does not inhibit (CAG)n hairpin repair in vitro. These studies provide insight into the determinants of the differential DNA repair specificity of MutSα and MutSβ, the mechanism of mismatch repair initiation, and the mechanism of (CAG)n hairpin processing and repair, which plays a role in the etiology and progression of several human neurological diseases.

The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity.