No description available.

The median effect equation (Chou, 1976) is developed and presented as a biologically plausible model for quantitative risk assessment. First, the biological plausibility is established; second, necessary statistical development is accomplished; third, the median effect equation is applied to 9 datasets (3 real from the Carcinogenic Potency Database, Gold et al., 1984, and 6 theoretical datasets) to assess the models' performance and ability of fit a variety of dose-response curves; last, the median effect model is applied to the estimation and use of the TD$\sb{50}$. The median effect model is compared to the multistage model (Crump et al., 1976) and the two stage model (Moolgavkar and Knudson, 1981). Underlying assumptions are compared and contrasted using data on reversibility of steps, steady state assumption and the definition of "biological plausibility". To place median effect model results in perspective, RsD (risk specific doses) (10$\sp{-1}$-10$\sp{-6}$) are calculated and compared with those of the multistage model.

Metabolic homeostasis is controlled, in part, by a family of proteins called nuclear receptors through which lipophilic hormones and hormone-like molecules regulate gene expression. One such nuclear receptor is peroxisome proliferator activated receptor γ (PPARγ). Its activation is essential for white, brite (brown-in-white) and brown adipogenesis, adipocyte function, mature adipocyte maintenance, and insulin sensitivity. PPARγ activation regulates energy homeostasis by both promoting storage of excess energy as lipids in white adipocytes and stimulating energy dissipation in brite and brown adipocytes. Accumulation of white adipocytes significantly increases the risk of obesity and metabolic syndrome. On the other hand, brown and brite adipocytes potentially counteract metabolic disease-related symptoms. The adipocyte differentiation and function as well as insulin sensitizing activities of PPARγ are regulated separately through differential post-translational modifications and/or co-regulator recruitment, with ligands having distinct abilities to activate each of PPARγ’s functions. These provide mechanisms by which a ligand could induce adipogenesis without stimulating PPARγ’s health promoting functions (i.e. insulin sensitivity, energy dissipation). The central hypothesis of this dissertation is that compared to therapeutic PPARγ ligands (i.e. rosiglitazone), environmental PPARγ ligands will activate a distinct PPARγ transcriptional program that disrupts adipose and metabolic homeostasis. Two study aims were developed to test and refine this central hypothesis. The first aim identified genes and pathways that differentiate environmental PPARγ ligands from endogenous and therapeutic chemicals. In primary mouse bone marrow multipotent stromal cells and 3T3-L1 cells, the environmental PPARγ ligands tributyltin (TBT, an antifouling agent and plasticizer) and triphenyl phosphate (TPhP, an organophosphate flame retardant) induced transcriptomic profiles that were distinct from rosiglitazone. All ligands induced adipogenesis; yet, only rosiglitazone strongly enriched pathways related to brown fat differentiation and mitochondrial processes and induced brite adipocyte gene markers (Cidea, Elovl3, Ucp1). Using the transcriptional profiles from 3T3-L1 adipocytes differentiated in the presence of 76 different chemicals, a taxonomy was built to identify environmental chemicals as PPARγ-modifying chemicals distinct from known PPARγ-modifying therapeutics. The second aim investigated the role of phosphorylation of PPARγ in defining environmental ligand-induced changes in adipocyte differentiation and function. In differentiated 3T3-L1 cells, rosiglitazone and TPhP both induced adipogenesis through PPARγ, but only rosiglitazone enhanced mitochondrial biogenesis and mitochondrial respiration, which contribute to healthy energy expenditure. Rosiglitazone, but not TPhP, protected PPARγ from phosphorylation at Ser-273. However, in 3T3-L1 cells in which PPARγ cannot be phosphorylated, TPhP was able to induce mRNA expression of a suite of brite adipocyte genes. In male C57BL/6J mice fed either a low or high fat diet, TPhP caused a significant decrease in brite adipocyte gene expression (Elovl3, Ucp1) in mature adipocytes from inguinal adipose tissue. Together, these studies support our hypothesis that environmental PPARγ ligands (i.e. TBT and TPhP) skew adipocyte differentiation toward white adipogenesis at the expense of brite adipogenesis, potentially because of differential post-translational modification of PPARγ.

There are few examples of therapeutic treatments in chemically-induced toxicity compared to pretreatments that protect against chemical injury. Cystamine treatment 12 hours after carbon tetrachloride (CCl₄) was reported to have therapeutic effects on CCl₄-induced hepatic necrosis via an unknown mechanism. The objectives of this project were to develop. quantitative animal models to characterize cystamine treatment of chemically-induced hepatotoxicity and to use the models to investigate possible mechanisms of the therapeutic effect. Cystamine produced dose related therapeutic effects against both CCl₄ and galactosamine-induced hepatic necrosis in male Sprague-Dawley rats. The therapeutic effect on galactosamine-induced damage demonstrated that cystamine has therapeutic effects that are unrelated to inhibition of early biochemical events initiating damage. This was an important finding since cystamine pretreatment will prevent CCl₄-induced hepatic damage by inhibiting the bioactivation of CCl₄. Cystamine-induced hypothermia did not cause a delay in the appearance of maximal hepatic damage. Cystamine also did not stimulate hepatic protein synthesis in intoxicated rats. Although cystamine was reduced to cysteamine in the livers of galactosamine treated rats the hepatic sulfhydryl content was only transiently affected by cystamine. Cystamine did not reduce toxicant-induced hepatic calcium accumulation, despite the fact that the influx of extracellular calcium into toxicant damaged cells is considered by many to be an irreversible event causing cell death. Cystamine also did not alter subcellular calcium distribution in toxicant treated rats or enhance recovery of microsomal calcium sequestration in CCl₄ treated rats. Since cystamine is metabolized to cysteamine in vivo and cysteamine can chelate calcium the effect of chelating agents and cysteamine analogs on galactosamine-induced hepatic damage was tested. Therapeutic effects were observed for the calcium chelators EDTA and EGTA, agents with a chelating structure similar to cysteamine (ie. a free amine and a free sulfhydryl on adjacent carbons), or agents which may be metabolized t0 such structures. The results suggest that calcium chelation may be a mechanism of therapeutic action in chemically-induced hepatotoxicity. A reduction of free calcium concentration via chelation would explain reduced cytotoxic consequences of toxicant-induced hepatic calcium accumulation.

Pyrrolizidine alkaloids (PAs) produce a variety adverse hepatic effects, including acute toxicity, carcinogenicity and potent, persistent antimitotic effects. Additionally several have been evaluated as antineoplastic agents. PAs constitute significant health hazards to man and domestic animals. The mechanism(s) by which PAs induce these effects are not known. These studies were designed to test the hypothesis that some or all of the adverse hepatic effects and possibly the antineoplastic activity of PAs associate with or are mediated by a genotoxic interaction with cellular DNA. The first objective of the studies was to verify the in vivo gentoxicity of the PAs, in the process characterizing the type(s) of DNA damage induced. Hepatic DNA damage induced by the model PA monocrotaline (MCT) was assessed following i.p. administration to adult male Sprague-Dawley rats. DNA damage was characterized by the alkaline elution technique. MCT was found to induce both DNA-DNA interstrand and DNA-protein cross-links. No evidence was seen for the induction of DNA single-strand breaks, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. DNA-DNA interstrand cross-linking reached a maximum within 12 hr and thereafter decreased over a protracted period. By 96 hr post administration, the calculated cross-linking factor was no longer statistically different from zero (control). Further studies were performed to test the effects of agents known to modulate the formation/disposition of the proposed reactive intermediate and the toxic effects of the PAs. Consistent with its involvement in the mechanism of the toxicity of the PAs, genotoxicity was shown to modulate in the same direction and to similar degree as does the toxicity. Other PAs, or derivatives thereof, were evaluated in addition to MCT. Structural requirements for DNA cross-linking potential were shown to be similar to those required for the induction of toxic and antimitotic effects, again consistent with the involvement of DNA cross-linking in the mechanism of these effects. Indicine N-oxide however, an experimental antineoplastic agent, was shown not to mediate its cytotoxic effects via this mechanism.

No description available.

Forensic toxicology is a branch of science that involves the analysis of drugs and other substances in biological fluids and tissues such as blood, urine, and oral fluid to aid medical or legal investigation of death, poisoning, and drug use. Due to the various components of different matrices, efficient and effective sample preparation techniques are necessary for reliable and accurate analysis. Following sample clean-up, a sensitive, specific, and robust method is ideal for consistent detection, identification, and quantitation of analytes. With the rise of drug abuse, there is a growing need to develop a single method that can target multiple classes of drugs quickly and effectively. This study validated two different sample preparation techniques for the detection and quantitation of six drug classes comprised of twenty-three drugs and metabolites in oral fluid. The drug classes were as follows: amphetamines, local anesthetics, opioids, hallucinogens, antidepressants, and novel psychoactive substances (NPS). Amphetamines used were amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA). Local anesthetics contained benzoylecgonine (BZE), cocaine, and lidocaine. Opioids included codeine, methadone, morphine, 6-monoacetylmorphine (6-MAM), fentanyl, and oxycodone. Hallucinogens included lysergic acid diethylamide (LSD) and phencyclidine (PCP). Antidepressants were amitriptyline, citalopram, fluoxetine, and trazodone. Lastly, NPS included ethylone, α-pyrrolidinopentiophenone (α-PVP), and 2,5-dimethoxy-4-iodophenethylamine N-(2-methoxybenzyl) (25I-NBOMe). Supported liquid extraction (SLE) and solid phase extraction (SPE) were assessed followed by confirmatory analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Both methods were validated according to guidelines in the Standard Practices for Method Validation in Forensic Toxicology set by the American Academy of Forensic Science (AAFS) Standards Board (ASB). Parameters assessed include calibration model, bias, precision, limit of detection (LOD), limit of quantitation (LOQ), dilution integrity, ion suppression/enhancement, interference studies, and stability. Matrix recovery was added as another parameter. All calibration models were 0.99 or greater and all compounds were stable for at least 72 hours. Bias, precision, LOD, LOQ, dilution integrity, and interferences were similar between both methods. SLE yielded slightly better LOD and LOQ values. SLE had greater values of matrix recovery as well as lower levels of ionization suppression/enhancement. Overall, SLE was determined to be the better method of sample preparation for this panel of drugs in oral fluid. Not only did it yield higher values for several of the parameters assessed but it also was more efficient (1 hour versus 2 hours) while using less solvent.

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No description available.

No description available.