Current concepts concerning nicotine's central nervous system (CNS) mechanism(s) of action suggest that this drug is producing its effects via an interaction at nicotiniccholinergic receptors (nAChRs) which open a membrane cation channel. Following initial opening of the channel, nicotine appears to induce a rapid desensitization of the nAChRs, closing the channel and resulting in a cessation of nicotine's effects. Research presented here will provide evidence of this secondary desensitization process in vivo by demonstrating nicotine's ability to induce acute tolerance in the discriminative stimulus (DS) paradigm. The ability of nicotine to elicit DS control of behavior was significantly reduced via challenge doses of (800, 1200, and 1600 ugjkg, s.c.) of nicotine administered 60-180 minutes prior to the training dose (400 ugjkg, s.c.). Eight out of twenty rats demonstrated this phenomena, with time and dose varying, suggesting that these effect may be contingent upon the individual rat studied. It appears that we have found a means of investigating cellular mechanisms in vivo using operant behavior.

<p> This dissertation includes three separate but related studies performed in partial fulfillment of the requirements for the degree of Doctor of Public Health in Environmental and Occupational Health. The main goal this dissertation was to develop and assess quantitative relationships for predicting doses associated with chronic non-cancer toxicity levels in situations where there is an absence of chronic toxicity data, and to consider the applications of these findings to chemical substitution decisions. Data from National Toxicology Program (NTP) Technical Reports (TRs) (and where applicable Toxicity Reports), which detail the results of both short-term and chronic rodent toxicity tests, have been extracted and modeled using the Environmental Protection Agency&rsquo;s (EPA&rsquo;s) Benchmark Dose Software (BMDS). Best-fit minimum benchmark doses (BMDs) and benchmark dose lower limits (BMDL) were determined. Endpoints of interest included non-neoplastic lesions, final mean body weights and mean organ weights. All endpoints were identified by NTP Pathologists in the abstract of the TRs as either statistically or biologically significant. A total of 41 chemicals tested between 2000 and 2012 were included with over 1700 endpoints for short-term (13 week) and chronic (2 year) exposures. </p><p> Non-cancer endpoints were the focus of this research. Chronic rodent bioassays have been used by many methodologies in predicting the carcinogenic potential of chemicals in humans (1). However, there appears to be less emphasis on non-cancer endpoints. Further, it has been shown in the literature that there is little concordance in cancerous endpoints between humans and rodents (2). The first study, Quantitative Relationship of Non-Cancer Benchmark Doses in Short-Term and Chronic Rodent Bioassays (Chapter 2), investigated quantitative relationships between non-cancer chronic and short-term toxicity levels using best-fit modeling results and orthogonal regression techniques. The findings indicate that short-term toxicity studies reasonably provide a quantitative estimate of minimum (and median) chronic non-cancer BMDs and BMDLs. </p><p> The next study, <i>Assessing Implicit Assumptions</i> in Toxicity Testing Guidelines (Chapter 3) assessed the most sensitive species and species-sex combinations associated with the best-fit minimum BMDL10 for the 41 chemicals. The findings indicate that species and species-sex sensitivity for this group of chemicals is not uniform and that rats are significantly more sensitive than mice for non-cancerous outcomes. There are also indications that male rats may be more than the other species sex groups in certain instances. </p><p> The third and final study, <i>Comparing Human Health</i> Toxicity of Alternative Chemicals (Chapter 4), considered two pairs of target and alternative chemicals. A target is the chemical of concern and the <i>alternative </i> is the suggested substitution. The alternative chemical lacked chronic toxicity data, whereas the target had well studied non-cancer health effects. Using the quantitative relationships established in Chapter 2, Quantitative Relationship of <i>Non-Cancer Benchmark Doses in Short-Term and Chronic Rodent Bioassays,</i> chronic health effect levels were predicted for the alternative chemicals and compared to known points of departure (PODs) for the targets. The findings indicate some alternatives can lead to chemical exposures potentially more toxic than the target chemical.</p>

Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references (leaves 186-205). / by Xiaocheng Li-Suckoleiki. / Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references (leaves 63-69). / by Curtis Lee X. Glover. / S.M.

Thesis (S.M.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 1999. / Includes bibliographical references (leaves 57-62). / by Jose Angel Márquez, Jr. / S.M.

A sediment toxicity test using the freshwater oligochaetes Lumbriculus variegatus and Tubifex tubifex was performed. We evaluated acute and chronic toxicity affects of copper and cadmium on reproduction in both species and the bioaccumulation of both metals by L. variegatus using artificial sediment. L. variegatus bioconcentrated copper 22‐fold and cadmium 16‐fold after a 14‐day exposure to spiked artificial sediments with 0.02% organic content. The EC50 for T. tubifex varied depending upon endpoint from 2.7 to 2.8 mg/L for cadmium and from 8.4 to 8.9 mg/L for copper. The EC50 for L. variegatus was 2.2 mg/L for cadmium and 3.9 mg/L for copper. Based on these results, L. variegatus appears to be more sensitive to metal toxicity in artificial sediments than T. tubifex.

It has been suggested that the CB1 G-protein-coupled receptor is internalized following agonist binding and activation of the second messenger pathways. The process of desensitization and resensitization is intimately involved with receptor internalization. Phosphorylation alters tolerance to cannabinoids thus contributing to tolerance. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. These findings led us to look at which kinase(s) may be involved in cannabinoid tolerance. We therefore hypothesize that by preventing phosphorylation of the CB1 receptor, we may reverse tolerance. We evaluated our hypothesis by testing the role of several kinases in tolerance: protein kinase A (PKA), protein kinase C (PKC), protein kinase C (PKG). Beta Adrenergic Receptor Kinase (β-ARK), Phosphatidylinositol 3-kinase (PI3K) and the src family tyrosine kinase. We also looked at cAMP and cGMP analogs. We evaluated PKA using KT5720, a PKA inhibitor; PKC using bisindolylmaleimide I, HCI] (bis), a PKC inhibitor; PKG using KT5823, a PKG inhibitor; β-ARK using Low molecular weight heparin (LMWH), a β-ARK inhibitor; PI3K using LY294002, a PI3K inhibitor and PP1 a src family tyrosine kinase inhibitor. The cAMP analog was dibutyryl-cAMP and the cGMP analog was dibutyryl-cGMP. ICR mice were rendered tolerant to △9- tetrahydrocannabinol (△9-THC) by administering injections of 20mg/kg △9-THC s.c. every 12 hours for 6.5 days. The mice were subsequently challenged 24 hours later with an ED8O of △9-THC at 20μg/mouse (i.t.). Antinociception was measured by the tail-flick test, %MPE’s and ED5O’s were calculated. The PKG inhibitor, KT5823, showed no significant change in %MPE. The β-ARK inhibitor, LMWH, showed no significant change in the %MPE. The PI3K inhibitor, LY294002, showed no significant change in the %MPE. Inhibition of PKC, by bis had no effect on tolerance, but at a higher dose attenuated the antinociceptive effect of △9-THC in non-tolerant mice. PPl, the src family tyrosine kinase inhibitor, reversed tolerance. KT5720, the PKA inhibitor reversed △9- THC tolerance. These data support a role for PKA and tyrosine kinase in phosphorylation events in THC tolerant mice. (Supported by NIDA grants K02DA00186 and P5ODA05274).

The activity of protein kinase C (PK-C) has been implicated in regulating the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C activators, phorbol 12,13- dibutyrate, mezerein, and bryostatin 1, on the proliferation and lineage commitment of enriched CD34+ human myeloid progenitor cells stimulated by lL-3, GM-CSF, stem cell factor and the lL-3/GM-CSF hybrid cytokine plXY321. Coadministration of these PK-C activators with plateau concentrations of rlL-3 or rGM-CSF induced 100-150% increase in the number of day 14 CFU-GM.with a selective stimulation on neutrophil and macrophage lineages while inhibiting eosinophilic growth. plXY321 stimulated an equivalent number of CFU-GM, including a predominant eosinophilic component, when compared to the combination of saturating levels of GM-CSF and lL-3. Bryostatin 1, when coadministered with plXY321 (or with the combination of lL-3 and GM-CSF), selectively enhanced the growth of neutrophilic and monocytic lineages while inhibiting eosinophil development. The inhibition of eosinophil colonies by bryostatin 1 was not mimicked by the coadministration of rSCF, rG-CSF or rCSF-1 with plXY321. Furthermore, neutralizing antibodies to rG-CSF and rCSF-1 failed to block potentiation of neutrophil or macrophage colony formation stimulated by bryostatin 1 in conjunction with plXY321, suggesting that accessory cell effects are not solely responsible for this phenomenona. rSCF synergistically enhanced plXY321 induced colony formation by an average of 144% by selectively stimulating neutrophilic and eosinophilic growth. Coadministration of bryostatin 1 with rSCF and plXY321 further increased colony formation by an average of 81%. This combination selectively stimulated cells of the macrophage lineage, and inhibited eosinophil differentiation. However, bryostatin 1 inhibited erythroid (BFU-E) and erythroid/myeloid mixed (CFU-GEMM) colonies induced by plXY321 alone or in combination with rSCF. Together these results indicate that 1) PK-C activity is involved in the growth and lineage commitment of early and committed myeloid progenitor cells. 2) Pharmacologic manipulation of PK-C may regulate the growth and differentiation of those cells exposed to early hematopoietic growth factors. This study raises the possibility that pharmacologic intervention at PK-C,in conjunction with hematopoietic growth factors, might be useful in the ex vivo expansion of hematopoietic progenitor cells.

Previous reports have indicated a dichotomy in the actions of TCDD on humoral immunity, both in vitro and in vitro, in which enhancements and suppression have been identified. The latter effect has been correlated with induction of liver P4501A1 enzyme activity, a response which is regulated by the Ah-gene locus. Additionally, the primary alteration in suppression of antibody responses is in the differentiation of the B cell. Therefore, the current investigation was undertaken to determine the relationship between these dual actions of TCDD on humoral immunity as related to its direct actions on B lymphocyte function. Specific emphasis was placed on determining the potential role of the Ah-gene locus in the modulation of the B cell and in vitro antibody responses. Initial observations determined that: 1) the degree of suppression of in vitro antibody responses in B6C3F1 mouse (an Ah-high responder strain) splenocytes is dependent on both the lot, concentration, and type (i.e., fetal bovine versus newborn bovine) of serum used in culture; and 2) there is a similarity in the actions of TCDD and Staphylococcus aureus Cowan strain I (SAC); a polyclonal B cell activator. These observations prompted the study of the direct effects of TCDD on B cells in different stages of in vivo activation and in the presence of different serum environments for which SAC has been reported to have opposing actions. TCDD was found to increase background levels of proliferation and differentiation in resting B cells (Go); thereby suggesting that resting B cells are a primary target in enhancement of immune responses by TCDD. However, no effect was seen on either response when the cells were stimulated with lipopolysaccharide (LPS). In cycling B cells (G1), TCDD caused suppression of both background and LPS-stimulated proliferation and differentiation and demonstrated a serum dependency that paralleled its actions in whole lymphocyte antibody responses; thereby demonstrating that it is the cycling B cell that is the primary target in suppression of antibody responses by TCDD and that both proliferation and differentiation are affected. The modulatory role of serum was also determined in primary hepatocyte cultures using P4501Al enzyme induction as the endpoint. Sera which supported either no effect or enhancements in both whole lymphocyte and purified B cell responses did not support induction of P4501A1 activity above BSA, a protein control. Conversely, sera which supported a consistent and dose-related suppression of Mug antibody responses were found to enhance the induction of P4501A1 activity. More importantly, normal mouse serum was found to allow for the full expression of the Ah-dependent phenomena in vitro, where primary hepatocytes and splenocytes from Ah-high responder (B6C3F1) and Ah-low responder (DBA/2) mouse strains were affected by TCDD in a manner that parallels the effects seen in the two strains following in vivo exposures. The latter results using mouse sera are consistent with a role by the Ah-gene locus in the direct effects of TCDD on whole lymphocyte and purified B cell antibody responses. However, the results with other, more traditional sources of sera, indicate that the ultimate expression of the TCDD- induced responses can be modulated by serum factors, which are at present unidentified. Furthermore, the results of this investigation indicate that the susceptibility of the cell, as related to its stage in the cell cycle, can contribute to the complex effects that are seen in the alteration of antibody responses following TCDD exposure.

In response to growing concerns over wetland habitat loss and the associated impact on water resources, federal and state legislation has been enacted to protect vulnerable wetland habitats from the impacts of humans. In order to examine the efficacy of current coastal resource policy and its implementation, a study was conducted in Mount Pleasant, South Carolina, a coastal city of the Atlantic, focusing on the quantification of wetland habitat change over time within a specific area of interest. The study incorporated an assessment of the effects of escalating population pressures and subsequent urban development on local wetland habitats due to the inherent threat of habitat degradation resulting from negligent development practices. The research methodology included a series of stakeholder interviews conducted within the Mount Pleasant community in order to define the key players who shape coastal resource policy formation, implementation, and enforcement. Further, a spatial analysis examined land use change over time. A historical record of regional land use derived from remotely sensed satellite imagery enabled the measurement of land use change over time. The results of a change detection analysis indicate an acceleration of wetland habitat loss in the second decade chosen for analysis in spite of strengthened coastal resource regulations enacted within the same time period. These results support a need for improved regulatory enforcement strategies and utilization of conservation-driven development practices.