Introduction. Birth defects and low birth weight/preterm delivery are the leading causes of a infant mortality in the United States. However, their etiologies remain mostly unknown. There is evidence suggestive of a link between exposure to pesticides and reduced infant health. We sought to answer the question of whether spatial variation in the incidence of infant mortality in the general population is correlated with variation in agricultural pesticide use. Methods. We examined the variation during 1996--2001 among 48 U.S. states of rates of infant mortality due to birth defects, and rates of infant mortality due to low birth weight or preterm delivery. We also examined the incidence of 41 specific birth defects through 1989--2001 in 33 states. We related these variables to the rate of agricultural pesticide use per state. Results. After controlling for socio-economic and behavioural risk factors, among-state variation in the rate of pesticide use accounted for over one quarter of the variation in infant mortality rates due to birth defects (r2=0.29). We did not find a significant relationship between pesticide use and infant mortality due to low birth weight/preterm delivery. Conclusion. Our results support the hypothesis that adverse effects on infant health in the general population are associated with broad scale pesticide use. To overcome the inferential limitations of this study, further research using individual exposure and outcome data is needed.

Les méthodes actuelles d’analyse pour rechercher des xénobiotiques et/ou leurs métabolites dans les liquides biologiques peuvent dans certaines occasions manquer de sensibilité. Ainsi, il est souvent difficile de mettre en évidence les substances utilisées par des agresseurs pour commettre des actes délictuels sur leurs victimes (vols ou encore viols), parce que ces substances administrées en très faibles quantités ont généralement des demi-vies courtes, ou que les recherches de ces substances se font plusieurs jours après l’agression. De même dans le cadre de l’exposition aux polluants de l’environnement et particulièrement aux hydrocarbures aromatiques polycycliques, il est difficile de mettre en évidence l’importance de l’exposition de salariés ou encore de la population générale à ces composés. Dans le cadre de la soumission chimique, l’utilisation de la chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (MS / MS) permet d’obtenir une sensibilité suffisante notamment en mode MRM pour détecter des substances ou leurs métabolites dans les liquides biologiques de victimes. L’utilisation de la même technologie permet de rechercher les métabolites hydroxylés des hydrocarbures aromatiques polycycliques. Les objectifs du travail ont été de montrer l’applicabilité de la spectrométrie de masse dans deux domaines. Le premier se rapporte à la soumission chimique, domaine dans lequel après avoir rappelé, les définitions, la prévalence des cas de soumission chimique en Europe et dans les pays anglophones, la liste des substances psychoactives incriminées, les difficultés couramment rencontrées lors des investigations et les recommandations pour améliorer la détection des substances aux niveau des analyses toxicologiques, est rapporté un cas de soumission chimique par le diéthylamide de l'acide lysergique ou LSD, par mise en évidence de ce composé et de son métabolite principal par UPLC-MS/MS. Au cours du travail sur la mise en évidence des substances de la soumission chimique, nous avions préparé les extraits biologiques en mélangeant des extraits effectués à pH acide et alcalin et en ionisant les molécules en mode électrospray négatif et positif. Il est vite apparu que certaines molécules avaient une limite de détection trop élevée. Nous avons alors développé une méthode spécifique pour ces molécules afin d’améliorer leur détection. Le second domaine d’application a été la mise en évidence des métabolites hydroxylés des hydrocarbures aromatiques polycycliques. Dans un premiers temps, nous avons réalisé une revue générale de ces substances polluantes de l’environnement, puis nous avons validé par chromatographie liquide de haute performance couplée à la spectrométrie de masse une méthode qui permet d’identifier et de doser ces métabolites dans l’urine de sujets exposés ou non à ces HAP. / Les méthodes actuelles d’analyse pour rechercher des xénobiotiques et/ou leurs métabolites dans les liquides biologiques peuvent dans certaines occasions manquer de sensibilité. Ainsi, il est souvent difficile de mettre en évidence les substances utilisées par des agresseurs pour commettre des actes délictuels sur leurs victimes (vols ou encore viols), parce que ces substances administrées en très faibles quantités ont généralement des demi-vies courtes, ou que les recherches de ces substances se font plusieurs jours après l’agression. De même dans le cadre de l’exposition aux polluants de l’environnement et particulièrement aux hydrocarbures aromatiques polycycliques, il est difficile de mettre en évidence l’importance de l’exposition de salariés ou encore de la population générale à ces composés. Dans le cadre de la soumission chimique, l’utilisation de la chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (MS / MS) permet d’obtenir une sensibilité suffisante notamment en mode MRM pour détecter des substances ou leurs métabolites dans les liquides biologiques de victimes. L’utilisation de la même technologie permet de rechercher les métabolites hydroxylés des hydrocarbures aromatiques polycycliques. Les objectifs du travail ont été de montrer l’applicabilité de la spectrométrie de masse dans deux domaines. Le premier se rapporte à la soumission chimique, domaine dans lequel après avoir rappelé, les définitions, la prévalence des cas de soumission chimique en Europe et dans les pays anglophones, la liste des substances psychoactives incriminées, les difficultés couramment rencontrées lors des investigations et les recommandations pour améliorer la détection des substances aux niveau des analyses toxicologiques, est rapporté un cas de soumission chimique par le diéthylamide de l'acide lysergique ou LSD, par mise en évidence de ce composé et de son métabolite principal par UPLC-MS/MS. Au cours du travail sur la mise en évidence des substances de la soumission chimique, nous avions préparé les extraits biologiques en mélangeant des extraits effectués à pH acide et alcalin et en ionisant les molécules en mode électrospray négatif et positif. Il est vite apparu que certaines molécules avaient une limite de détection trop élevée. Nous avons alors développé une méthode spécifique pour ces molécules afin d’améliorer leur détection. Le second domaine d’application a été la mise en évidence des métabolites hydroxylés des hydrocarbures aromatiques polycycliques. Dans un premiers temps, nous avons réalisé une revue générale de ces substances polluantes de l’environnement, puis nous avons validé par chromatographie liquide de haute performance couplée à la spectrométrie de masse une méthode qui permet d’identifier et de doser ces métabolites dans l’urine de sujets exposés ou non à ces HAP

Antagonists have been crucial in the characterization of nicotine's pharmacology. Initial evidence for the existence of central nicotinic receptors was based on the fact that nicotine produced a number of behavioral effects that were antagonized by ganglionic blockers that crossed the blood-brain barrier, such as mecamylamine and pempidine. Although the mechanism of action of these compounds has been studied extensively in the periphery, little is known about their mechanisms of action in the brain. These compounds are thought to be noncompetitive antagonists due to the fact that they do not compete for agonist binding to brain homogenate in vitro. However, pharmacological evidence in support of noncompetitive antagonism is lacking. Dose-response curves for nicotine were determined in the presence of various doses of pempidine for depression of spontaneous activity and antinociception in mice. Pempidine was found to shift the dose-response curves for these effects of nicotine in a manner consistent with noncompetitive antagonism. A number of mecamylamine analogs were investigated for antagonism of these central effects of nicotine as well. These studies revealed that the N-, 2-, and 3-methyls were crucial for optimal efficacy and potency and suggests that these compounds possess a specific mechanism of action, possibly involving a receptor. Furthermore, the structure-activity relationships for the mecamylamine analogs were found to be different than that previously reported for the agonists, suggesting that they do not act at the same site. The binding of [3H]-L-nicotine and [3H]-pempidine was studied in vitro to mouse brain homogenate and in situ to rat brain slices. The in situ binding of [3H]-L-nicotine to rat brain slices was quantitated autoradiographically to discrete brain areas in the presence and absence of 1, 10 and 100 µM nicotine and pempidine. Pempidine did not effectively displace [3H]-L-nicotine binding. The studies with [3H]-pempidine failed to demonstrate saturable binding. The evaluation of the antagonism of nicotine by mecamylamine and pempidine presented in this thesis supports a noncompetitive action of these compounds in the brain. The shift in the dose-response curves for nicotine, the structure-activity relationship for mecamylamine analogs and the binding studies are consistent with this hypothesis. The noncompetitive nature of these compounds suggests that they do not compete for the binding site of the agonist, and that endogenous nicotinic antagonists may exist in the brain.

Arecoline and nicotine are two psychoactive cholinergic alkaloids. Arecoline is primarily a muscarinic agonist while nicotine, at low doses, is a nicotinic agonist. The experiments in this dissertation investigated two major areas: (1) the role of different factors in the development of tolerance to the behavioral effects of arecoline and nicotine, and (2) the possible mechanism and site of action of the discriminative stimulus (DS) effects of arecoline and nicotine. The role of dispositional and physiological factors comfiared to behavioral factors in the development of tolerance to the effects of arecoline and nicotine on operant behavior was assessed in Experiments I and II, respectively. In part one of Experiment I, rats were trained to respond (M1 a variable-interval 15 second (VI-15) schedule for milk reinforcement. Dose-effect relationships were assessed prior to and during chronic arecoline (1.74 mg/kg/day) treatment. After 21 days Of arecoline administration prior to the session, the dose-effect relationship for total responses was not shifted. However, the dose-effect relationship for total reinforcements was shifted to the right. In part two of Experiment I, rats were trained to respond on a fixed-ratio 20 (FR-ZG) schedule for milk reinforcement. Dose-effect relationships were assessed prior to and during chronic arecoline (0.87 mg/kg/day) administration. One group of rats received daily injections of arecoline prior to the seSsion and a second group received arecoline injections after the session. Daily administration of arecoline resulted in a greater shift to the right of the dose-effect relationship in the pre-session group compared to the post-session group. These data demonstrate the importance cflf behavioral factors in the development of tolerance to arecoline. In Experiment II, rats were trained to respond on a VI-15 second schedule of milk reinforcement. Dose-effect relationships were determined prior to and during chronic nicotine (2.28 mg/kg/day) administration. One group of rats received daily injections Of nicotine prior to the session, another group received nicotine injections after the session. After 36 days of chronic treatment, similar degrees of tolerance were observed in both groups, however the group receiving post-session nicotine developed tolerance at a faster rate. The data suggested that 21 complex interaction of nicotine and the experimental environment affected the rate of tolerance development. Experiment III characterized the DS effect of arecoline. Using a two-lever operant paradigm, rats were trained to discriminate arecoline from saline on a VI-12 second schedule of milk reinforcement. Rats could learn to discriminate 1.74 mg/kg arecoline from saline, but not 0.58 mg/kg from saline. Agonist and antagonist studies demonStrated that the DS effect of arecoline is mediated through central muscarinic receptors. In Experiment IV, the ability of physostigmine to interact fiith the DS effect of nicotine (1.14 mg/kg) and arecoline (1.74 mg/kg) was assessed. Physostigmine (0.125 mg/kg) pretreatment shifted the dose-effect relationship for arecoline to the left but did not affect that of nicotine. Physostigmine (0.25 mg/kg) almost completely generalized to the DS effect of arecoline but not to the DS effect of nicotine. These data suggest an interaction of endogenous acetylcholine with muscarinic receptors but not with nicotinic receptors. In Experiment V, the ability hf arecoline and nicotine injected directly into the dorsal hippocampus (DH) and mesencephalic reticular formation (MRF) to generalize to the DS effect of peripherally administered arecoline (1.74 mg/kg) and nicotine (1.14 mg/kg) was assessed; Nicotine injected into these sites generalized in a dose-related manner to nicotine. The MRF was slightly more sensitive than the DH. Arecoline injected into either site did not generalize to the DS effect of, peripherally administered arecoline. However, a decrease in response rates was observed.

The access of drugs to variofis sites of action in the body is impeded by a succession of membranes. Likewise, the removal of a drug or foreign substance from the body is similarly dependent on the ability of the substance under consideration to permeate biological barriers. A con-committant problem generally arises concerning the overall time course of drug action: that of drug storage through binding or similar processes. Schanker (196h) however, has stated that "Although the mechanisms of localization and those of membrane transfer are in many respects different problems, there are some instances in which they are inseparable parts of the same problem." Examples include phenomena whereby the binding of a substance to the cell membrane is required for transport (Rosenberg and Wilbrandt, 1955; Peters; 1960; Lacko and Burger, 1961; Schwartz and Matsui, 1967; Stein, 1967). From a functional standpoint, body membranes may be classified in three major categories (Schanker, 1962b); membranes several cell layers thick such as skin, those one cell layer thick such as the brush border epithelium of the intestine, and membranes less than one cell in thickness such as the cell membrane itself or the membranes of organelles (e.g. mitochondria). Thus, except for the barriers associated with subcellular structures, the cell membrane 6 (or plasma membrane as it is often called) may be considered the fundamental unit of body membranes in general.

It generally accepted that the critical target of ionizing radiation in cellular reproductive death is DNA. What is less known is what types of lesions are involved, how they are repaired and how various agents enhance, protect or modify them. One of the main reasons for these difficulties has been the lack of assays for molecular damage sensitive enough to measure yields at doses where significant numbers of cells survive. Recently however, various techniques have appeared that exhibit this sensitivity. One of these, Rydbergs’ double-label unwinding method, can routinely detect damage in the DNA of mammalian cells from doses as low as 0.1 Gy. We have used this technique to measure initial yields, repair kinetics and residual yields of damage in the dose range 1-6 Gy. We have found, in substantial agreement with measurements at higher doses, the existence of at least two distinct repair processes with half-lives of 7 minutes and 250 minutes respectively. We have also found that the distribution of breaks between the fast and slow processes can be altered by various conditions; for example: whether or not the irradiation was done in the presence of oxygen or nitrogen, post-treatment in hypertonic solution, incubation with ARA-A, (an inhibitor of DNA synthesis) or post-treatment in dimethyl sulphoxide. We have also found that the yield of unrepaired lesions (as defined by damage remaining after two hours incubation at 37°C) seems to include a quadratic variation with initial dose suggesting that among the slowly repairing breaks, there exist complex, combination-type lesions (which may include DNA double-strand breaks). Such studies, combined with results from other assays should help in understanding the nature of the lesions in DNA and the repair processes acting on them and will eventually aid in identifying the critical lesion(s) responsible for cell death. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Polycyclic aromatic hydrocarbons (PAHs) are environmental ovotoxicants commonly released in cigarette smoke and vehicle exhaust. PAHs have been shown to target small ovarian follicles (25-100 μm) after a single high dose. Once irreplaceable primordial follicles are depleted in the ovary, ovarian failure results. The occupational chemical, 4-vinylcyclohexene diepoxide (VCD), induces pre-mature ovarian failure in mice and rats. A direct comparison between the ovotoxicity caused by VCD and that of PAHs was made by calculating an initial ovotoxic index (OI = ED₅₀ x 15d) in ovarian follicles of mice and rats. There are two hypotheses to be tested, first is that oocyte destruction can be prevented with a single dose of VCD. Second, three constituents of cigarette smoke, PAHs: 3-methylchloranthrene (3-MC), 9,10-dimethylbenzanthracene (DMBA) and benzo[a]pyrene (BaP) after repeated low-dose exposure results in apoptosis similar to VCD. VCD is protective against the normal rate of atresia in primary ovarian follicles following exposure to a single dose of VCD, and the mRNA expression of the cell death gene bax paralleled the changes in atresia. Using the initial OI, repeated low dose exposures were shown to be more toxic (lower OI) in the ovary than that previously reported from a single high dose. Furthermore, the calculation of the initial OI demonstrated DMBA to be the most potent chemical tested by destroying primordial follicles at the lowest dose. PAHs were also found to target secondary follicles. In the present study, the PAHs were also found to be associated with apoptosis (increased bax mRNA - DMBA and 3-MC; decreased bcl-x(L) mRNA - DMBA), in mice ovarian follicles. Confocal microscopy was used to visualize proteins of gene products from the bcl-2 family Bax (cell death) and Bcl-x(L) (cell survival) in mice. Large primary follicles illustrated consistently increased ratios of Bax/Bcl-x(L) for the three chemicals tested. These results suggest that repeated low dose exposure make ovarian follicles more susceptible to PAHs-induced cellular changes. Taken together with the confocal data, DMBA may be working through a different pathway than VCD and 3-MC, or the ovotoxic effects of DMBA are so potent that the effects due to 3-MC and VCD appear lower, and that large primary follicles may be the critical follicle stage determining the cells fate. The results obtained in these experiments can predict the potential reproductive effects initiated by VCD and PAHs.

The toxicity of methanol, atrazine, and copper to Pseudokirchneriella subcapitata (Korshikov) Hindák historically referred to as Selenastrum capricornutum Printz were determined following 96 hrs growth in a modified Goram's growth media. Methanol and atrazine inhibited fluorescence readings in the cultures by 50% (IC50) at concentrations of 2% and 82 µg/l respectively. These toxicity values compared favorably to other published reports. The IC50 for copper was 160 µg/l which is substantially higher than reported values. This is understandable because of the high chelating capacity of Goram's media. The use of stereologically derived relative volume in the chloroplasts, mitochondria, lipid bodies, phosphate bodies, and nucleus was investigated to determine if it could be used as a sensitive endpoint in toxicity tests. The volume fractions for the chloroplasts and mitochondria were normally distributed in control cells while the nuclei, phosphate bodies, and lipid bodies were not. The chloroplasts were the most dominate organelle occupying a mean relative volume of 46% and mitochondria occupied a mean relative volume of 3%. The nucleus and phosphate bodies occupied a median relative volume of 7% and 2% respectively. The lipid bodies were rare in section profile and no meaningful median relative volume could be calculated. Up to the 82nd percentile of sectioned profiles contained no recognizable lipid bodies. The use of relative volume was not a sensitive endpoint for use in toxicity tests. No significant differences in relative volume could be detected in the nucleus or phosphate bodies following any treatment. Limited differences were detected in the mitochondria, chloroplasts, and lipid bodies. The only significant differences that appear to be biologically significant occurred in methanol treated cells where an increase in the lipid bodies' relative volume was apparently concentration dependent. Significant differences in the relative volume of mitochondria and chloroplasts do not appear to be biologically significant.

The functional aspects of specific mutations, R30K, S291G, and I392T, found associated with AChE gene of OP- and carbamate-resistant Colorado potato beetle (CPB) were determined using recombinant AChEs. From the previous studies, azinphosmethyl resistant CPB strain (AZ-R) possesses S291G and R30K mutations and their AChE was 16-fold less sensitive to azinphosmethyl than that of insecticide-susceptible (SS) strain, in terms of biomolecular rate constant (ki). A carbamate-resistant strain (BERTS) showed high resistance to carbofuran (12-fold), but was relatively less resistant to azinphosmethyl (1.35-fold) than SS CPB. The AZ-R strain was 2.6-fold more resistant to carbofuran and 8-fold more resistant to azinphosmethyl than SS CPB, respectively, in a discriminating dose bioassay. A substrain of BERTS, BERTS-R, possessing only the S291 G mutation, elicited high resistance to carbofuran and moderate resistance to azinphosmethyl. The BERTS-S substrain, possessing S291G and I392T mutations, was susceptible to both azinphosmethyl and carbofuran. In this study, enzymatic properties of altered AChEs from CPB were examined using recombinant AChEs obtained from baculovirus expression system. The S291G mutation increased hydrolysis activity of larger substrates (e.g. BTC) and increased sensitivity to inhibition by larger inhibitors (e.g. paraoxon, DFP, and N-propyl carbofuran) in the altered recombinant AChEs. The R30K mutation caused further increasement of hydrolysis activity of larger substrates and the sensitivity to inhibition by larger inhibitors in combination with the S291G mutation. The 1392T mutation compensated the effect of S291G. Thus, the altered recombinant AChE with both S291G and I392T mutations elicited a substrate specificity and inhibitory properties more similar to the susceptible form of ACNE without mutations. Two rapid molecular diagnostic systems were successfully developed for monitoring of OP and pyrethroid resistances by determining resistant allele frequencies. These techniques were used to genotyping pyrethroid resistance in 16 field populations of CPB by detection of the kdr mutation. The predicted resistant levels of populations by genotyping were closely correlated to the esfenvalerate bioassay results by the rank-correlation coefficient of a non-parametric rank correlation test for independence. These two DNA based diagnostic procedures are reliable, accurate and affordable techniques for monitoring field population of CPB for resistance.

There is great concern over human exposure following the application of pesticides for the management of turf environments. This concern is expected and germane given the level and frequency of pesticide use, the extent of activities and time spent on turfgrass, and the exposure potential for infants, children, and adults alike. Much effort has been expended in the determination of applicator exposure issues and the means to mitigate problematic exposure situations before and during application of pesticides. However, there are potential exposure concerns for all who enter turfgrass areas following pesticide applications. The present research emphasizes dosimetry (measuring pesticide residues on cotton suits, gloves, and air samplers worn by golfers) and biomonitoring (measuring pesticide metabolites in urine of golfers) in conjunction with environmental monitoring to determine transfer and penetration factors. The direct and simultaneous determination of dosimetry and biomonitoring data provides a novel and complete database on how much pesticide is transferred from the treated turf to the golfer during the play of a round of golf, where transfer takes place, and subsequently how much pesticide is actually absorbed. Dermal exposure was determined to be the dominant exposure pathway to golfers. Exposure estimates based on a 1 h re-entry interval following full-course and full-rate applications of chlorpyrifos, carbaryl, and cyfluthrin are substantially below current US EPA acute Reference Dose (Rfd) values, indicating safe exposures. These already low exposures were successfully mitigated using several management strategies.