Thesis (Ph.D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references. / Quantification of adducts in human samples is a challenging task in analytical chemistry. Humans are typically exposed to low levels of a wide range of different carcinogens. As a result, adduct quantification requires both a highly sensitive and a highly selective method. This project has focused on the development of new laser-induced fluorescence (LIF) based methods for the quantification of adducts in human samples. Two new instruments were built for LIF analysis. A cryogenic LIF instrument using a pulsed tunable dye laser as the excitation source was developed to obtain low temperature fluorescence emission spectra. A second instrument using a LIF detector interfaced to a HPLC separation system was developed to provide a higher level of selectivity than the cryogenic LIF system. The LIF instruments were used to quantify adducts of benzo[a]pyrene-7,8-diol-9,10- epoxide (BPDE) in human lung tissue and human bronchial epithelial cell histones and DNA and in human serum albumin. The presence of BPDE adducts in human lung tissue and human bronchial epithelial cell histones and DNA was proven by cryogenic fluorescence line-narrowing (FLN) spectroscopy. (-)-anti-BPDE-serum albumin adducts were quantified by HPLC-LIF in plasma obtained from 63 healthy volunteers. The method was shown to have a coefficient of variability of 22% and a detection limit of 20 attomoles of BPDE adduct. Adducts were detected in 60 of 63 samples (95%) at an average level of 0.22 femtomoles of adduct per mg of albumin. The investigation of histones as possible long-term molecular dosimeters was another focus in this research. The close proximity of histones and DNA coupled with the lack of repair and long lifetime make histones very attractive candidates as molecular dosimeters. The kinetics of histone turnover can most likely be accurately modeled by cellular turnover rates in vivo. Histone adducts were detected in human lung tissue and human bronchial epithelial cells. Histones were also shown to form adducts with BPDE, aflatoxin B1 (AFB,) and nitrosomethylurea (NMU) in TK6 cells grown in vitro. Histone adducts of AFB 1 , NMU, and BPDE were shown to be stable over 5 cell divisions. / by Can Cemil Özbal. / Ph.D.

Thesis (Ph. D. in Pharmacology and Toxicology)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2001. / Includes bibliographical references (p. 131-153). / Heparin-like glycosaminoglycans (HLGAGs) are complex polysaccharides found both in extracellular matrix (ECM) and at the cell surface where they, in a sequence-specific manner, bind to and regulate the biological activity of numerous proteins. Due to a lack of effective tools to study structure-function relationship of these complex molecules, the role(s) of HLGAGs in cancer remains largely indirect and poorly understood. To study the role(s) of HLGAGs in cancer growth, metastasis and angiogenesis, tumor systems were studied using heparinases I and III which have distinct substrate specificities. Studies, focusing on the biochemistry of the heparinase active site, elucidated key interactions important for enzymatic activities, thus enabling the biological studies presented here. In vitro cell culture study with heparinases as tools clearly showed that cell surface HLGAGs are involved in regulating fundamental cellular activities including cell proliferation, invasion and adhesion. Significantly, heparinase I and III demonstrated distinct effect on these cellular activities with heparinase III inhibiting tumor cell proliferation, invasion and adhesion in selected model systems and heparinase I inhibiting these processes. Further in vivo studies in animal tumor models confirmed and expanded the distinct responses to heparinase I and III treatment. HLGAG fragments generated from heparinase treatment were found to be responsible for the effect of heparinase treatment in tumor models. Moreover, cell surface HLGAGs containing cryptic activatory and inhibitory sequence information were identified and characterized. In contrast to heparinase I- generated HLGAG fragments, heparinase III- generated HLGAG fragments were shown here to inhibit both primary tumor growth and secondary lung metastasis. / (cont.) The inhibition of tumor by heparinase III treatment was attributed to inhibition of specific signaling pathways such as FGF2 signaling. In an effort to develop a non-invasive and efficient delivery strategy for HLGAG-based therapeutics, HLGAGs were prepared as dry aerosol of defined characteristics for pulmonary inhalation. Pharmacokinetics study revealed efficient absorption for both formulated and unformulated dry aerosolized HLGAGs after pulmonary inhalation. Significantly, pulmonary inhalation of aerosolized HLGAGs was demonstrated to be effective in treating both local respiratory diseases including tumor metastasis and systemic thrombosis. / by Dongfang Liu. / Ph.D.in Pharmacology and Toxicology

Thesis (S.M.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references (leaves 46-51). / Bleomycin is known to cause double strand breaks in vitro. Little is known, however, about its mechanism of genotoxicity in vivo. One way to probe the mechanism of genotoxicity of a DNA damaging agent in vivo is to compare the relative sensitivities of a wild type Escherichia coli strain to a panel of isogenic repair deficient mutants. If the pathway defective in the mutant is known (e.g., base excision repair, alkyl transferase repair, nucleotide excision repair, and so on), the sensitivity of the mutant can reveal mechanistic insight into the mode of killing by the DNA damaging agent. In this study, mutants deficient in recombinational repair, specifically recF, recBCD, ruvABC, recG and recGruvC, were examined for sensitivity to bleomycin. This sensitivity was tested in both dividing and non-dividing cells in order to analyze the effect of cell division on the cytotoxicity of bleomycin. When non-dividing cells were treated, the recBCD and recGruvC mutants, demonstrated high sensitivity to bleomycin. The recF mutant, on the other hand, demonstrated no sensitivity. These results were consistent with the conclusion that bleomycin induces double strand breaks in vivo that are repaired by the recombinational repair double strand break pathway. It also suggests that no damage was induced by bleomycin that required repair by the daughter strand gap pathway. Examining the sensitivity of recombinational repair deficient mutants to bleomycin also gave new insights about the mechanism of recombinational repair. Both ruvABC and recG gene products resolve Holliday junctions; however, they are thought to work on separate recombinational pathways. In this study, although the recGruvC strain was highly sensitive to bleomycin, the ruvABC and the individual recG and ruvC strains were not. This result suggested redundancy in the functions of the RecG and RuvABC proteins. Dividing cells showed a marked increase in sensitivity to bleomycin as compared to non-dividing cells. In addition, the functional redundancy of recG and ruvABC mutants was no longer seen. The ruvABC strain demonstrated high sensitivity equal to that of the recGruvC and recBCD strains whereas the recG and ruvC strains were only slightly sensitive. Under these conditions of increased cytotoxicity, additional functions of the RuvAB enzymes became important for suppression of toxicity. This result suggested a change in the mechanism of bleomycin's genotoxicity. / by Sophie Currier. / S.M.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2002. / Vita. / Includes bibliographical references. / Aflatoxin B1 (AFB1) is a fungal metabolite that contaminates the food supply in certain areas of the world. It is produced by Aspergillusflavus and related fungi that grow on improperly stored foods such as corn, rice, and peanuts. Epidemiological studies have shown a correlation between exposure to AFBI and incidence of hepatocellular carcinoma (HCC). Mutations in p53 are observed in over 50% of the HCC samples studied, and a unique mutational hotspot occurs at the third position of codon 249 in this gene, yielding almost exclusively GC to TA transversions. It is of interest to evaluate the mutagenic properties of specific chemical structures of AFBI adducts in order to determine which of these may be responsible for the mutations that may play a role in the formation of HCC. The primary DNA adduct formed by the epoxide of AFB is the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFBI-N7-Gua) adduct, which can lead to two secondary lesions, an apurinic site or a ring opened formamidopyrimidine (FAPY) adduct, which itself has two rotameric forms. This study focuses on of the determination of how well cells tolerate each of the AFB1-FAPY rotamers and of the type and frequency of mutations caused by the persistent AFB I-FAPY adduct in a site specifically modified M13 viral vector transfected into E. coli. Four major results were concluded from this work. First, one of the rotamers of AFBI-FAPY is a strong block to DNA replication, even when bypass polymerases are employed by the cell. / (cont.) Second, the G to T mutation frequency of the AFB I-FAPY adduct is at least six fold greater than that observed for the AFBi-N7-Gua adduct. Third, a spectrum of mutations that is unique to the AFBI-FAPY adduct was observed. Fourth, cell strains expressing different bypass polymerases responded differently when challenged with the AFB1-FAPY and AFBI-N7-Gua DNA adducts. These results show that AFB I-FAPY is the most toxic and mutagenic species of aflatoxin adduct studied to date. / by Maryann E. Smela. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2001. / Includes bibliographical references (leaves 186-193). / Residents of small communities or neighborhoods often observe what appears to them to be an excessively high number of cancer cases. Some of these observations are brought to the attention of public health officials in the hope that an environmental cause within these communities may be discovered and subsequently eliminated. However, what appears to be a cluster of cancer cases locally may be a chance occurrence when viewed from the perspective of the entire state or region. The primary aim of this work was to obtain and analyze a data set of sufficient magnitude to provide a means to discover if distributions of cancer mortality rates among communities within a particular state, for any of the most common forms of cancer, were compatible with chance. To this end cancer mortality data for six of the largest states in the United States (California, Florida, Massachusetts, New York, Pennsylvania and Texas) were collected, converted into mortality rates and analyzed to discover if the variations among communities could be accounted for by chance alone. These data comprised one third of all recorded deaths in the period of approximately 1969 to 1998. The 21 most common forms of adult and 6 most common forms of pediatric cancers were organized to permit analyses within each of the 6 states with regard to age (0-19, 65-84 and >/= 85 years), gender and ethnicity (European American and Non-European American descent). Key to this work was the mode of statistical analysis. For each community an expected mortality rate and its expected distribution was defined by the average mortality rate for all communities within each state and the binomial distribution, respectively. These expected distributions were summed for all communities to define the expected chance distribution of community mortality rates for each state, cancer, gender, age cohort and ethnicity. This produced nearly 800 separate "chance" distributions. Each of these was compared to the corresponding observed distribution using the Kolmogorov-Smimov statistical test. This test was designed to discover statistically significant differences between any two distributions. Here it was used to determine which of the nearly 800 observed distributions could not be accounted for by chance alone. Of these comparisons, 16 were found to have observed distributions significantly different from the expected by chance distributions. All 16 had distributions that exhibited greater dispersion than expected by chance, and none had distributions with less dispersion than the chance expectation. / by Janice A. Vatland. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2001. / Includes bibliographical references (leaves 136-140). / Glycosaminoglycans are complex polysaccharides that exist at the cell/extracellular matrix interface. As such, these information-dense molecules are key regulators of extracellular signals. However, to date, there has been a lack of effective biochemical and analytical tools for the analysis of glycosaminoglycan structure and hence, there is very little understanding of exactly how glycosaminoglycan structure impinges on function. Development of such tools is especially important for a key subset of glycosaminoglycans, i.e., heparin/heparan sulfate-like glycosaminoglycans (HLGAGs). As a first step in the development of tools to study HLGAG structure, biochemical studies were completed on the heparinases, a family of three HLGAG-degrading enzymes from Flavobacterium heparinum. With heparinase I, it was found that calcium is a necessary cofactor for optimal activity and that two putative calcium coordinating motifs exist in heparinase I. With heparinase II, a single cysteine and three histidines were found to be critical for proper enzymatic function. Finally, with heparinase III, two histidines were found to be catalytically important. / (cont.) In addition to the development of the heparinases, sensitive analytical methodologies were developed to determine HLGAG structure. A novel mass spectrometric procedure was developed and used to define heparinase's II mechanism of action on defined oligosaccharide substrates. In addition, capillary electrophoresis, in combination with heparinase digestion, was used for the compositional analysis of HLGAG oligosaccharides. Coupling of these analytical techniques with a property-encoded nomenclature to handle the information density of HLGAGs enabled the development of a sequencing procedure for HLGAGs. These procedures were then used to probe a number of key biological processes that are mediated by HLGAGs. The sequencing approach developed herein was used to determine the structure of HLGAG oligosaccharides involved in hemostasis and viral binding. In addition, these approaches were used to probe HLGAG binding by clinically and biologically important signaling molecules, viz., fibroblast growth factor and endostatin. Together, these studies represent a foundation with which it possible to develop further HLGAG structure/function relationships, opening new avenues of research and making possible HLGAG-based therapeutics. / by Zachary Shriver. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2001. / Includes bibliographical references. / The overall aim of this project was to investigate various cellular responses and toxic effects of nitric oxide, NO' and in vitro and in vivo. Nitric oxide gives rise to a complex spectrum of reactive species in oxygenated solution. The complexity of nitric oxide's chemistry is recapitulated in its effects on cells. Exposure to nitric oxide can result in changes on many different levels in cells ranging from protein and DNA damage, to damage to organelles and changes in gene expression, and even in cell death. Many models to study nitric oxide have been developed and will be used to study various responses to nitric oxide and related species. Nitric oxide and peroxynitrite-induced cellular damage has been and continues to be studied extensively using in vitro systems. Two systems have been used in this project, a delivery system for NO' as well as a cell line, which produces NO'. A Silasticʾ membrane delivery system can be used to treat bacteria or cells to mimic in vivo exposure to nitric oxide. Mutations induced by nitric oxide in a set of Salmonella tester strains can be studied utilizing this delivery system. Activated RAW264.7 macrophage cells have been used as an in vitro model of nitric oxide production and cytotoxicity SJL/J mice bearing the transplantable lymphoma RcsX have been established as an in vivo model of nitric oxide production and toxicity. / (cont.) This in vivo mouse model can be used to test results found in vitro. Specifically, the relationship between nitric oxide production and prostaglandin synthesis and glutathione homeostasis can be explored. Glutathione was found to be induced by nitric oxide production in this model, and this increase was due to increases in y-glutamylcysteinyl synthetase activity. This thesis studied both the regulatory and toxic effects of nitric oxide, both endogenously produced and from exogenous sources. / by Teresa Leah Wright. / Ph.D.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2001. / Includes bibliographical references (p. 346-354). / Overall cancer mortality rates have risen from about 4% of all deaths in the early 20th century to about 25% of all deaths by the end of the century in the United States. To assess any potential hypotheses for this increase required knowledge of the mortality rate changes specific to each form of cancer, and the time points when these rates had changed. For this purpose, population and cancer mortality data of the U.S. were collected and organized to create age-specific mortality rates for each birth decade from the 1800s forward, delineated by the organ of incidence. Concurrently, cancer survival data were collected so as to correct for any effect of improved treatment on historical changes in cancer mortality rates. To analyze these data, a mathematical model for the three-stage process of carcinogenesis (initiation, promotion, and progression) was developed to estimate for each birth decade cohort the value of the fraction of the cohort at lifetime risk for that cancer, the value of the growth rate of the respective precancerous lesion, and the values for the mutation rates of normal and precancerous cells in the organ of incidence. This methodology permits the analysis of the potential historical effect of new chemical exposures during the last century on cancer mortality rates. These chemical exposures represent potential risk factors that determine the fraction of the population at risk of developing cancer (lifetime, primary risk factor), or that hasten death by cancer by altering either mutation or cell kinetic rates (accelerating, secondary risk factor.) / (cont.) COLON CANCER: Application of this model on the colon cancer mortality data resulted in the estimate that 42% of the population in the U.S. was at risk for developing colon cancer, independent of gender or race. More importantly, there was no significant historical change in the calculated fraction at risk for birthyear cohorts from 1860 to 1940, suggesting that the primary risk factors for colon cancer are not environmental. Although direct observation of in vivo mutation rates of colonic cells does not yet exist, the calculated rate for the first initiation mutation in the colon was interestingly found to be similar to the mutation rate observed for the hprt locus in human peripheral T-cells (-2.1 x 10-7 per cell year) and the spontaneous mutation rate of the hprt locus of human B-cells in culture. The estimate for initiation mutation rates increased no more than two-fold from the birthyear cohort of 1860 to the birthyear cohort of 1940, except for European American females for which calculated initiation mutation rates were historically invariant, but since the accuracy of primary data for mortality rates and survival rates cannot be ascertained, the apparent small differences might admittedly arise from unknown biases. Evaluation of the parameter of the growth rate of precancerous lesions showed no significant historical change on this parameter. Curiously, the calculated doubling rate of these lesions (-0.17-0.21) was found to be similar to the growth rate of children, suggesting that the required initiation events have the net effect of potentially reactivating pathways involved in child development. / (cont.) The predominant historical change in the observed mortality rates for colon cancer occurred only at old ages. ... / by Pablo Herrero Jimenez. / Sc.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2002. / Includes bibliographical references (leaves 176-200). / The study of human mutagenesis requires methods of measuring somatic mutations in normal human tissues and inherited mutations in human populations. Such methods should permit measurement of rare mutations in the presence of abundant wild-type copies and should be general to the human genome. A sensitivity of 2 x 10-6 for point mutations was recently achieved in human cells using a novel method of target isolation, constant denaturant capillary electrophoresis (CDCE), and high-fidelity polymerase chain reaction (hifi-PCR) (Li-Sucholeiki and Thilly, 2000). This method is applicable to 100-base pair (bp) DNA domains juxtaposed with a naturally occurring domain of a higher melting temperature, or a natural clamp. Such sequence domains represent about 9% of the human genome. To permit analysis of rare point mutations in the human genome more generally, this thesis developed a procedure in which a clamp can be ligated to any 100-bp sequence of interest. This procedure was combined with the previous method to create a new method of point mutational analysis that is not dependent on a naturally occurring clamp. To demonstrate the new method, a sequence with a natural clamp, a part of the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (cDNA-bp 223-318), was analyzed using both the natural and ligated clamps. A sensitivity of 2 x 10-5 in human cells was demonstrated using the ligated clamp as opposed to 5 x 10-6 using the natural clamp. / (cont.) The sensitivity of the new method using the ligated clamp was demonstrated to be limited by the fidelity of Pfu DNA polymerase used for PCR. The sequence of the ligated clamp accounted for the differences in sensitivity as a result of causing a decreased efficiency of mutant enrichment by CDCE. The new method can be applied to measure somatic mutations in normal human tissues, such as lung tissues, in which point mutations at fractions above 10-5 have been observed. This method can also detect predominant inherited mutations even for genes carrying recessive deleterious alleles in pooled samples derived from a large number of individuals. / by Andrea Seungsun Kim. / Ph.D.

Anthropogenic activities such as oil spills are major sources of polycyclic aromatic hydrocarbon (PAH) pollution in the environment. Bivalves such as C. virginica can accumulate high levels of PAHs in tissue due to a limited metabolic capacity for these compounds. Accordingly, bivalves have served as key biomonitoring species for contaminants and exposure to PAH through seafood consumption can also be an important risk to human health due to the toxic and carcinogenic potential of these compounds. For evaluating bivalve PAH levels, conventional analyses are limited due to extensive time and expense and unreliability. This work demonstrates the application of immunological techniques to overcome such limitations in conventional techniques and to explore PAH kinetics and partitioning mechanisms within oysters. Biosensor technology coupled with a PAH antibody was employed to rapidly and inexpensively screen PAH levels in adult oysters in an Elizabeth River watershed monitoring survey. Through a novel extension of a fundamental chemistry theory, PAH concentrations measured in oyster fluid by biosensor were used to predict tissue concentrations. Biosensor-derived predictions had a strong association with tissue concentrations measured by conventional chemical analysis. A strong association between the biosensor and tissue concentrations when compared against regulatory PAH thresholds and efficient mapping of PAH levels throughout the watershed, demonstrates the real-world value of the method. The biosensor was also employed in PAH kinetics studies. Oysters were exposed to crude oil water accommodated fractions (WAF) in the laboratory to explore the application of the biosensor in oil spill response; however, further work is needed to improve the precision of biosensor-derived predictions at non-steady state. In a field exposure study, PAH levels in cultured triploid and diploid oysters deployed at a PAH hotspot in the Elizabeth River were compared to wild oysters inhabiting the site. Differences in PAH kinetic trends were observed between oyster fluid and tissue. When combined with the observed differences in PAH levels in specific tissue types between transplanted and wild oysters, there is evidence that internal partitioning and tissue-specific kinetic rates may be important factors in determining the overall PAH body burden in an oyster and warrants further investigation to improve precision in future biomonitoring efforts. The fluorescently tagged PAH antibody was also employed in an immunohistochemical (IHC) technique to visualize complex PAH mixtures within oyster tissue. Oysters were collected throughout the laboratory WAF exposure, and the observed change in signal intensity in tissue followed a similar trend to measured PAH concentrations. In visualizing transplanted vs. wild oyster tissue, the trends in signal intensity supported the differences observed in tissue-specific PAH concentrations between groups. Overall, the biosensor shows promise as a tool to overcome current analytical challenges faced in environmental monitoring of biota. While further work is needed to understand the influence of chemical and biological factors on PAH kinetics and biosensor-derived tissue predictions, the unique analytical features of these technologies are valuable for addressing these mechanistic questions. When coupled with IHC, these immunologic techniques can provide new insight to address complexities in environmental pollution and health risk assessments that cannot be as feasibly and inexpensively answered by standard methods.