Browsing by Subject "Biomarkers"
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Item Development and testing of gene expression biomarkers for gonadal dysgenesis in conjunction with the US EPA endocrine disruptor screening program's Tier 2 larval amphibian growth and development assay(2014-04) Haselman, Jonathan ThomasThe Endocrine Disrupter Screening Program of US EPA has recently developed a Tier 2 testing guideline using model amphibian species Xenopus laevis. The Larval Amphibian Growth and Development Assay assesses a chemical's endocrine-related effects in vivo and generates concentration-response information for ecological risk-assessment. Currently, the assay relies on histopathological evaluations for identifying endocrine-related reproductive effects. However, histopathology can seldom define the chemical mode of action and is not easily interpreted in the context of risk assessment when the effects are minimal to moderate. This study explores the use of gene expression biomarkers in the gonad that could potentially inform a chemical's mode of action and detect adverse reproductive effects that are otherwise uncharacterized by histopathology. To identify candidate biomarkers, global expression was analyzed in differentiating ovary and testis tissues of Xenopus tropicalis. Genetic programs responsible for reproductive maturation in gonad tissues were characterized and provided a foundation from which specific genes could be selected proximal to a model chemical's known mode of action. Four genes were selected within the putative androgen molecular network to evaluate as biomarkers of the anti-androgen mode of action characteristic of a common fungicide, prochloraz. Following continuous exposure to prochloraz throughout embryonic, larval and juvenile development in Xenopus laevis, assessments of growth, liver and kidney pathology, and reproductive development were made. To evaluate the predictive capabilities of the candidate genes, one gonad was kept in situ for histopathological evaluations while the other was processed for mRNA analyses. Results indicate that prochloraz exposure caused metabolic toxicity in the liver and kidney; it caused testis degeneration coincident with the onset of androgen-mediated spermatogenesis and inhibited regression of Müllerian ducts. Two of the four candidate genes showed increases in expression at the high test concentration and appeared to be predictive of an anti-androgen-induced adaptive response. The behavior of these biomarkers stimulated valuable discussion and generated testable hypotheses to better understand the evolution of molecular mechanisms driving gonad development in a cross-species context. This study provides a model for expression-based biomarker development in endocrine tissues and offers direction toward enhanced ecological risk-assessment.Item DNA Modifications As Biomarkers For Precision Medicine In Patients Treated With Alkylating Drugs(2023) Guidolin, ValeriaDNA alkylating drugs have been used as frontline medications to treat cancer for decades. Their chemical reaction with DNA leads to the blockage of DNA replication, which impacts cell replication. While this impacts rapidly dividing cancerous cells, this process is not selective and results in highly variable and oftentimes severe side effects in patients undergoing alkylating-drug based therapies. This observation supports the need for the development of biomarkers able to identify patients who effectively respond to certain therapies and recognize those who instead will develop serious side effects. The use of DNA adducts as predictive biomarkers has been proposed by several studies and herein reviewed. This dissertation focuses on the development and application of analytical methods able to comprehensively screen DNA adducts produced by alkylating drugs. The first study of this dissertation evaluates busulfan reactivity with DNA. Busulfan can promptly react with DNA, therefore, taking advantage of our DNA adductomic approach, DNA adducts formed by reacting busulfan with calf-thymus DNA were characterized. Samples collected from 6 patients undergoing busulfan-based chemotherapy prior to allogeneic hematopoietic cell transplantation were analyzed for the presence of busulfan-derived DNA adducts. Among the 15 adducts detected in vitro, 12 were observed in the patient blood confirming the presence of a large profile of DNA adducts in vivo. Two of the detected adducts were structurally confirmed by comparison with synthetic standards and quantified in patients. Similarly, in the second study, an extensive profile of DNA adducts generated by cyclophosphamide was characterized. Cyclophosphamide is metabolically activated and converted to phosphoramide mustard and acrolein, which are responsible for its efficacy and toxicity. Our DNA adductomic method has been optimized and tailored to maximize the detection of cyclophosphamide-derived adducts. Furthermore, the use of 15N-bacterial DNA served as further confirmation for DNA adduct identification and structural elucidation. This investigation led to the detection of 40 DNA adducts in vitro and 20 DNA adducts in patients treated with cyclophosphamide. The last study focused on the synthesis of a cyclophosphamide-derived DNA adduct to be used for quantitation and as an internal standard for future studies. Departing from reported synthetic schemes, several different approaches were tested. This study is currently ongoing, but the reaction schemes tested allowed for a better understanding of dGuo-alkylation regioselectivity. Overall, the work described in this thesis set the stage for the evaluation of a relationship between busulfan and cyclophosphamide DNA adducts and therapy outcome to identify DNA adducts to be used to stratify patients and distinguish who will benefit from therapies from those who may experience severe adverse toxic outcomes.Item Elevated levels of 1-hydroxypyrene and NOe-Nitrosonornicotine in the urine of smokers with head and neck cancer: a matched control study(2014-04) Khariwala, Samir SureshBackground: Head and neck squamous cell carcinoma (HNSCC) is associated with tobacco use. Still, most smokers do not develop HNSCC. The mechanisms of varying susceptibility to HNSCC are poorly studied to date. Tobacco metabolite research provides insight regarding the innate metabolism and excretion of carcinogens. Methods: Smokers with HNSCC (cases) were compared to smokers without HNSCC (controls) in a matched cohort. The tobacco metabolites studied are: 1-hydroxypyrene (1-HOP), Nf-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Results: In 33 subjects, mean 1-HOP was 1.82 pmol/mg creatinine vs 1.08 pmol/mg creatinine (p=0.004) and mean NNN was 0.10 pmol/mg creatinine vs 0.04 pmol/mg creatinine (p=0.01) in cases and controls, respectively. NNAL did not differ between groups. Conclusions: Smokers with HNSCC have elevated urinary levels of 1-HOP and total NNN compared to matched controls suggesting an increased effective exposure to these carcinogens. Tobacco constituent metabolites may be useful in understanding tobacco-related carcinogenesis in HNSCC.Item The fatty acids - inflammation relationship across the lifecycle(2011-10) Wang, HuifenDietary fatty acid intake, reflected by the endogenous fatty acid profile, has been associated with the pathogenesis and progression of cardiovascular diseases (CVD). Additional evidence is needed about the specific roles of individual fatty acids in the pathogenesis of inflammation, which is closely linked to CVD risk factors and interwined with oxidative stress and hemostatic dysfunction. It is also important to explore such relationships across the lifecycle. This dissertation, which includes four manuscripts, investigates the relations between fatty acids, biomarkers of inflammation (and oxidative stress and hemostasis), and cardiovascular health among different age groups. Specifically, the influences of adiposity and a genetic variant on the fatty acid/inflammation relations were also explored. The first manuscript used data from a study of obesity, insulin resistance and CVD risk factors in adolescents. A cross-sectional analysis was conducted to examine whether overweight status modified the relations between serum phospholipid fatty acids from dairy fats (i.e. 15:0 and 17:0 fatty acids) and inflammation/oxidative stress among adolescents with a mean age of 15 years. Inverse associations were found between dairy fatty acids and three biomarkers of inflammation and oxidative stress among overweight adolescents, but not their normal weight counterparts. In additional analyses, we further examined the same study question on other fatty acids and observed similar effect modification of adiposity. Only in overweight adolescents, but not in normal adolescents, 18:0 and 20:3ù6 fatty acids were positively, while 20:4ù6 and 22:6ù3 fatty acids were inversely, related to inflammation/oxidative stress. The second manuscript examined whether the cross-sectional relations between dietary intakes of polyunsaturated fatty acids (PUFA) and inflammation differed by genetic variant, peroxisome proliferator-activated receptor gamma (PPARã) Pro12Ala polymorphism. A biracial cohort of middle-aged adults enrolled in the year-20 exam of the Coronary Artery Risk Development in Young Adults (CARDIA) study was studied. In women, higher dietary intakes of 20:4ù6, 20:5ù3 and 22:6ù3 fatty acids were related to lower levels of IL-6 (an inflammatory biomarker) among Ala allele carriers. In contrast, these PUFA/IL-6 relations were positive among male Ala carriers, and absent among female Pro homozygotes. Male Pro homozygotes who consumed more 20:5ù3 and 22:6ù3 fatty acids tended to have a lower IL-6 level. The last two manuscripts were both prospective studies using data from the Atherosclerosis Risk in Communities (ARIC) study, which enrolled middle-aged adults. This cohort has been followed since year 1987-89. Manuscript 3 examined the interactions between dietary fatty acid intake and inflammatory/hemostatic factors in relation to incident coronary heart disease (CHD) and ischemic stroke (IS). Dietary intakes of 18:2ù6 and 20:4ù6 fatty acids were found to modify the associations between serum albumin and incident CHD/IS. The prediction of low serum albumin level, a potential inflammatory biomarker, on incident CHD/IS was attenuated with increasing intake of 18:2ù6 fatty acid or decreasing intake of 20:4ù6 fatty acid. Manuscript 4 included 3,715 ARIC participants enrolled at the Minnesota field center who had plasma phospholipid fatty acid measurements. In manuscript 4, the focuse was to determine whether inflammation/hemostasis mediated the relation of phospholipid fatty acids with incident CHD and IS. Inflammation and hemostasis, represented by levels of factor VIIIc (VIIIc), white blood cell count (WBC) and fibrinogen, mediated the positive associations of 18:0 and 20:3ù6 fatty acids with incident CHD. A similar but less significant pattern was found for 16:1ù7 in relation to incident IS. Lower WBC, but not VIIIc or fibrinogen, partially explained the inverse relations of 17:0 and 20:4ù6 fatty acids with incident CHD. In conclusion, this dissertation documents the associations between diverse fatty acids, inflammation and the development of CVD among different age-groups. The findings have enhanced the understanding of the health effects of individual fatty acids and the underlying mechanisms of fatty acid-inflammation-CVD relations, which are useful for advising food manufacturers and guiding CVD prevention.Item Identifying Mechanisms And Biomarkers Predictive Of Efficacy Of Vaccines Against Opioid Use Disorders And Overdose(2022-08) Crouse, BethanyOpioid use disorders (OUD) and overdose are public health crises that are worsening despite the availability of approved pharmacotherapies. Active immunization with anti-opioid conjugate vaccines is a novel therapeutic strategy to treat OUD and prevent overdose. To date, clinical studies suggest that efficacy of anti-drug conjugate vaccines is limited to a subset of individuals who can produce optimal antibody responses. To increase positive treatment outcomes and clinical success, this research program investigated several complementary strategies to increase OUD vaccine efficacy. First, mechanisms of optimal anti-opioid vaccine response are investigated by elucidating the immunological mechanisms behind a previously established interleukin-4 (IL-4) mediated increase in vaccine efficacy. These studies found that depletion of IL-4 resulted in a Type I IL-4R mediated increase in germinal center formation and germinal center T cell response which leads to increased opioid-specific antibody secreting cells, and that vaccine efficacy is dependent on a balanced Th1/Th2 T cell response in mice. Next, these results provided a blueprint for next generation anti-fentanyl vaccine formulations incorporating novel adjuvants targeting toll-like receptors (TLRs). These data show that a TLR7/8 agonist adjuvant increases vaccine efficacy in rodent and porcine models of fentanyl misuse and overdose. Third, vaccine design and immunization paradigms were assessed to optimize the efficacy of a novel carfentanil vaccine alone and in combination with a lead fentanyl vaccine. Longer linker lengths and a co-administered bivalent immunization strategy were associated with increased vaccine efficacy. Then, environmental factors contributing to the immune response are investigated by testing whether changes in the gastrointestinal microbiome would affect vaccine efficacy and whether these specific changes in the microbiome could be utilized as biomarkers. These studies revealed that changes in the microbiome in specific pathogen free or immune-experienced rodents did not affect efficacy of anti-oxycodone or anti-fentanyl vaccines. Finally, exploratory studies were performed to identify putative biomarkers that may be predictive of anti-opioid vaccine response in preclinical and clinical investigations. These studies indicate that pre-immunization concentration of IL-4 is correlated with vaccine efficacy in genetically diverse mice, and that specific cytokines may be of interest as indicators of immune response in human patients. Overall, the findings outlined in this research program support the use of novel adjuvants and predictive biomarkers to increase clinical efficacy of vaccines to treat OUD and overdose.Item Mass Spectrometry Based Quantification of 1, 3-Butadiene Induced DNA Adducts: Potential Biomarkers of Cancer Risk(2014-11) Sangaraju, DewakarChemical carcinogenesis involves metabolic activation of carcinogens to electrophilic species which can react with important cellular biomolecules including DNA to form covalent adducts. Covalent carcinogen-DNA adducts which are not removed by DNA repair mechanisms can induce transforming mutations, ultimately leading to cancer. Hence, carcinogen-DNA adducts are deemed the ultimate biomarkers of carcinogen exposure, metabolic activation, and possibly of cancer risk. 1,3-Butadiene (BD) is a recognized human and animal carcinogen present in cigarette smoke, automobile exhaust, wood fires, and also in some occupational settings such as BD monomer and polymer plants. BD is metabolically activated by CYP2E1 to form three electrophilic epoxides: 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, EBD, and DEB can modify DNA bases to form covalent DNA adducts such as N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII), N7-(2, 3, 4-trihydroxybut-1-yl)-guanine (N7-THBG) and 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD). Although BD-DNA adducts had been successfully detected and quantified in tissues of laboratory animals exposed to relatively high concentrations of BD ( ≥ 6.25 ppm), they had not been previously quantified in humans, preventing their use as biomarkers of BD exposure, metabolic activation, and cancer risk. The main purpose of this research was to develop ultra-sensitive bioanalytical methodologies based on mass spectrometry to enable the detection and quantitation of BD-DNA adducts in animals treated with sup-ppm levels of BD and in exposed human populations. In Chapter 2 of the thesis, a novel nanoHPLC-nanoESI+-MS/MS method was developed for sensitive, accurate, and precise quantitation of BD-induced guanine-guanine cross-links (1,4-bis-(guan-7-yl)-2,3,-butanediol, bis-N7G-BD) in tissues of laboratory mice treated with low - sub-ppm concentrations of BD (0.5-1.5 ppm) which approximate human occupational exposure to BD (1 ppm). Bis-N7G-BD concentrations increased in a concentration-dependent manner in mouse liver DNA as a function of BD exposure. In Chapter 3 of this Thesis, we investigated DNA repair mechanisms responsible for bis-N7G-BD repair using isogenic Chinese hamster lung fibroblasts proficient or deficient in nucleotide excision repair (NER) and Fanconi Anemia (FA) repair pathways. We found that while both pathways contributed to bis-N7G-BD removal, FA pathway was most effective at alleviating the toxicity and replication blockage imposed by bis-N7G-BD cross-links. To enable BD-DNA adduct detection in humans, we developed an isotope dilution capillary HPLC-ESI+-HRMS/MS methodology for the most abundant BD-DNA adducts identified in vivo: N7-(2,3,4-trihydroxybut-1-yl)-guanine (N7-THBG) (Chapter 4). This method was successfully applied to quantify N7-THBG adducts in blood leukocyte DNA of smokers, nonsmokers, and occupationally exposed workers. In addition, we have developed an isotope dilution nanoLC/ESI+-HRMS3 methodology for the quantitation of BD-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human blood leukocyte DNA and human urine (Chapters 5 and 6). This method was applied to quantify EB-GII adducts in blood and urine of BD-exposed populations such as smokers, nonsmokers, and occupationally exposed workers. Overall, during the course of the studies described in this Thesis, we have developed a range of novel mass spectrometry-based quantitative methods which have excellent sensitivity, accuracy, and precision, and can be used for future human BD exposure biomonitoring studies. Furthermore, these methodologies are now being employed in epidemiological studies to identify any ethnic/racial differences in BD bioactivation and to help understand the origins of ethnic/racial differences in lung cancer risk in smokers.Item Peri-implantitis Prognosis Using Metabolomic Biomarkers in Peri-Implant Crevicular Fluid: A Longitudinal Study(2021-05) Alassy, HatemBackground: Peri-implant diseases, peri-implantitis (PI) and peri-implant mucositis (PIM), are highly prevalent in subjects with dental implants. Despite this prevalence, diagnosing peri-implant disease (PID) remains challenging because of lack of accuracy and precision of periodontal probing and dental radiographs. Furthermore, these diagnostic tools document history of disease rather than current disease activity. There is no current model to predict the progression of PID. Biomarkers are commonly used in medicine to objectively determine disease state, or responses to a therapeutic intervention. Biomarkers in peri-implant crevicular fluid (PICF) show promise in their diagnostic and prognostic value. Metabolomic analysis of PICF quantifies molecules associated with host and bacterial metabolism may reflect on pathophysiology of peri-implantitis. Un-targeted metabolomics allows the discovery of unknown biomarkers without bias to correlate them with peri-implantitis and its progression. Aim: We hypothesize that the simple metabolites in PICF are predictors of future peri-implantitis progression. We aim to define the unique set of metabolites in the PICF that establish a reliable method for early prediction of bone-loss progression in peri-implantitis. Methods: Clinical and radiographic examinations and PICF samples were collected from 130 implants in 71 subjects at baseline, 6, 12, 18 and 24 months. At baseline, 59 implants were healthy (bone loss < 2mm; PD 4mm), 33 implants had PI (bone loss ≥ 3mm; PD ≥ 6mm) and 38 other implants had bone loss ≥ 2mm and <3mm and PD 5mm. Radiographic bone level changes of 112 implants and relative metabolites in PICF samples were measured at each 6 months interval using proton nuclear magnetic resonance (H-NMR) spectroscopy. MetaboAnalyst 5.0 software correlated metabolite levels with radiographic bone changes of ≥ 1mm within a 6-month interval. Results: In the cross-sectional component at baseline, univariate ROC curve analysis demonstrated that the Cadaverine/Lysine signature was significantly correlated with peri-implantitis (AUC= 0.76; 95% CI 0.658-0.855, p< 0.000) versus healthy implants. While alpha ketoglutarate was significantly correlated with healthy implants (AUC= 0.706; 95% CI 0.593-0.819; p= 0.002). In the longitudinal component, the metabolite levels in PICF of untreated diseased implants that demonstrated progressive radiographic bone loss of ≥ 1mm within a 6-month interval (group A, n=6) were compared to the metabolites of healthy-non-progressing implants (group B, n=26) and to diseased-non-progressing implants (group C, n=8). Proline and 1-3-diaminopropane levels could predict future bone loss of ≥ 1mm (AUC= 0.917 and 0.854 respectively) whereas glucose and arginine levels could predict the absence of bone loss in group C and group B respectively (AUC= 0.896 and 0.801) although statistical significance was not reached for all 4 metabolites. Biotin and propionate levels were higher in group C compared group A and group B (ANOVA p< 0.001; AUC biotin= 0.889; AUC propionate= 0.87). Valine levels were higher in both group A and group C compared to group B (p= 0.002; AUC= 0.841). Conclusions: PICF metabolites identified using H-NMR spectroscopy mapped a specific metabolomic profile able to identify implants with peri-implantitis versus healthy implants with moderate accuracy. Furthermore, specific metabolites discriminated between progressive disease versus non-progressing disease status and health.Item The use of biomarkers to determine the severity of osteoarthritis in the tarsus of an older horse population(2017-12) Coppelman, ElizabethBackground: Osteoarthritis (OA) is a group of diseases of different causes that ultimately lead to synovitis, subchondral bone remodeling, and articular cartilage degeneration. OA commonly develops in the distal intertarsal (DIT) and tarsometatarsal (TMT) joints of performance horses. Currently, the most accurate method of identifying OA in these joints is a combination of thorough physical, lameness, and radiographic examinations. However, many horses may have pain attributed or localized to these joints with minimal radiographic changes present. A novel way to identify and classify the degree of OA is through the measurement of molecular biomarkers. Molecular biomarkers of OA reflect quantitative and dynamic variations associated with joint metabolism. Objectives: (1) define the direct and indirect biomarker concentrations in synovial fluid from the tibiotarsal, distal intertarsal, and tarsometatarsal joints in horses with varying degrees of tarsal OA, and (2) validate/refute that the biomarker concentrations in these joints increase with severity of OA in the distal joints as determined by a radiographs (all joints), MRI (PIT, DIT, TMT joints), arthroscopic evaluation/ gross pathology (TT joint), and (3) determine if biomarker concentrations in the TT synovial fluid (SF) can be used to evaluated OA severity in the DIT and TMT joints. Methods: A cohort study of 11 older horses (>8 years) with variable amounts of OA in the tarsal joints identified on radiographs were included. The TT joints were examined by arthroscopy/gross examination. The distal tarsal joints were examined by MRI. Biomarkers BAP, CPII, C2C, CTX II, CS846 were examined in the distal tarsal joints; additional biomarkers C1,2C, IL-1β, IL-6, IL-8, IL-10, and TNFα were examined in the TT joint. Various statistical analyses were used to determine association between imaging modalities and biomarkers to degrees of OA severity. Results: In the TT joint, C2C and IL-6 were the best biomarkers distinguishing OA severity. There was more pathology present in TT joints than could be seen on radiographs, suggesting that arthroscopic surgery is still the best method to evaluate TT joint OA. In the distal tarsal joints, radiographs were better at distinguishing OA and correlated to the corresponding MRIs, but underestimated the degree of SCB bone sclerosis, and number and size of osteophytes in many of the cases. MRI also provided information about cartilage damage and SCB hyperintensity. The severity of SCB sclerosis and presence of SCB hyperintensity on MRI was a good indicator for separating moderate/severe from mild OA. Of the biomarkers evaluated, the best at determining OA severity in the DIT joint were BAP, CPII, and C2C. These biomarkers also correlated to subchondral bone hyperintensity on MRI. In the TMT joint, CPII was the best biomarker to determine OA severity. No biomarker was identified in the SF in the TT joint capable of identifying OA in the distal tarsal joints. Conclusions: Biomarkers have the potential to be a valuable source of information about the OA disease process in the tarsal joints. SF from the joint of interest must be collected. Further research is needed with more horses. To the author’s knowledge, this is the first study examining biomarkers in an older horse population and hopes it provides a template for forthcoming research.