Michael D. Martin

Lorcaserin, a Novel Selective Human 5-Hydroxytryptamine2C Agonist: in Vitro and in Vivo Pharmacological Characterization

5-Hydroxytryptamine (5-HT)2C receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT2C full agonist. Lorcaserin bound to human and rat 5-HT2C receptors with high affinity (Ki = 15 ± 1 nM, 29 ± 7 nM, respectively), and it was a full agonist for the human 5-HT2C receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT2A and 5-HT2B receptors, respectively. Lorcaserin was also highly selective for human 5-HT2C over other human 5-HT receptors (5-HT1A, 5-HT3, 5-HT4C, 5-HT55A, 5-HT6, and 5-HT7), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT2A agonist (±)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT2A agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT2C-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT2A antagonist (R)-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT2C receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT2C receptor, with potential for the treatment of obesity.

Behavioral Effects of Low-Level Exposure to Hg" Among Dentists

Abstract Exposure thresholds for health effects associated with elemental mercury (Hg∘) exposure were examined by comparing behavioral test scores of 19 exposed (mean urinary Hg = 36 μg/l) with those of 20 unexposed dentists. Thirty-six μg Hg/l is 7 times greater than the 5 μg Hg/l mean level measured in a national sample of dentists. To improve the distinction between recent and cumulative effects, the study also evaluated porphyrin concentrations in urine, which are correlated with renal Hg content (a measure of cumulative body burden). Subjects provided an on-site spot urine sample, were administered a 1-h assessment consisting of a consent form, the Profile of Mood Scales, a symptom and medical questionnaire, and 6 behavioral tests: digit-span, symbol-digit substitution, simple reaction time, the ability to switch between tasks, vocabulary, and the One Hole Test. Multivariate regression techniques were used to evaluate dose-effects controlling for the effects of age, race, gender and alcohol consumption. A dose-effect was considered statistically significant below a p value of 0.05. Significant urinary Hg dose-effects were found for poor mental concentration, emotional lability, somatosensory irritation, and mood scores. Individual tests evaluating cognitive and motor function changed in the expected directions but were not significantly associated with urinary Hg. However, the pooled sum of rank scores for combinations of tests within domains were significantly associated with urinary Hg, providing evidence of subtle preclinical changes in behavior associated with Hg exposure. Coproporphyrin, one of three urinary porphyrins altered by mercury exposure, was significantly associated with deficits in digit span and simple reaction time. The prophyrin pooled sums of rank scores were as sensitive as the urinary Hg analyses within the cognitive and motor domains but were less sensitive for the overall battery of tests. The reported effects were detected among dentists with a mean urinary Hg level of 36 μg/l, which lies between the proposed biologic thresholds of 25 and 50 μg Hg/creatinine, suggesting the need for a more comprehensive study to determine the threshold of adverse biologic effects.

Behavioral Effects of Low-Level Exposure to Hg0 Among Dental Professionals: A Cross-Study Evaluation of Psychomotor Effects

A Across-study design was used to evaluate the sensitivities of five psychomotor tasks previously used to assess preclinical effects of low-level Hg0 (urinary < or =55 microg/l). Pooling dental professional subject populations from six studies conducted over the last 6 years, a larger study population was obtained with a high degree of uniformity (N = 230). The five psychomotor tests were: Intentional Hand Steadiness Test (IHST); Finger Tapping: The One-Hole Test: NES Simple Reaction Time (SRT); and Hand Tremor. Multivariate analyses were conducted following the hierarchical analysis of multiple responses (HAMR) approach. First, multiple scores of each test were combined into a single-factor (or related summary) variable and its reliability was estimated. Second. multiple regression analyses were conducted including log-transformed [Hg0]U levels, age, gender, and alcohol consumption in each model. Computed were both B and bu, the magnitudes of the log-Hg0 standardized coefficient. respectively uncorrected and corrected for dependent variable attenuation due to unreliability. Results indicated remarkable differences in the effects of relative level of Hg0 on psychomotor performance. Significant associations were found for the IHST factor (B = 0.415, p < 10(-6)), followed by finger tapping, which was relatively meager and insignificant (B 0.141, p = 0.17). The IHST results hold the greatest occupational relevance for dental professionals who rely on manual dexterity in restorative dentistry. Further, this statistical approach is recommended in future studies for condensation of multiple scores into summary scores with enhanced reliabilities useful in correcting for attenuation relationships (B(u)s) with exposure levels.

Issues in design and analysis of a randomized clinical trial to assess the safety of dental amalgam restorations in children

The Casa Pia Study of the Health Effects of Dental Amalgams in Children is a randomized clinical trial designed to assess the safety of low-level mercury exposure from dental amalgam restorations in children. It is being carried out in 507 students (8 to 12 years of age at enrollment) of the Casa Pia school system in Lisbon, Portugal, by an interdisciplinary collaborative research team from the University of Washington (Seattle) and the University of Lisbon, with funding from the National Institute of Dental and Craniofacial Research. Since the goal of the trial is to assess the safety of a treatment currently in use, rather than the efficacy of an experimental treatment, unique design issues come into play. The requirements to identify as participants children who have extensive unmet dental treatment needs and who can be followed for 7 years after initial treatment are somewhat in conflict, since those with the most treatment needs are usually in lower socioeconomic categories and more difficult to track. The identification of a primary study outcome measure around which to design the trial is problematic, since there is little evidence to indicate how health effects from such low-level exposure would be manifested. The solution involves the use of multiple outcomes. Since there are concerns about safety, multiple interim comparisons over time between treatment groups are called for which, in conjunction with the use of multiple outcomes, require an extension of statistical methodology to meet this requirement. Ethical questions that have to be addressed include whether assent of the children participating is required or appropriate, and whether the director of the school system, who is the legal guardian for approximately 20% of the students who are wards of the state and live in school residences, should provide consent for such a large number of children. Approaches taken to address these and other design issues are described.

The Contribution of Dental Amalgam to Urinary Mercury Excretion in Children

Background Urinary mercury concentrations are widely used as a measure of mercury exposure from dental amalgam fillings. No studies have evaluated the relationship of these measures in a longitudinal context in children. Objective We evaluated urinary mercury in children 8–18 years of age in relation to number of amalgam surfaces and time since placement over a 7-year course of amalgam treatment. Methods Five hundred seven children, 8–10 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of dental amalgam in children. Subjects were randomized to either dental amalgam or resin composite treatments. Urinary mercury and creatinine concentrations were measured at baseline and annually on all participants. Results Treatment groups were comparable in baseline urinary mercury concentration (~ 1.5 μg/L). Mean urinary mercury concentrations in the amalgam group increased to a peak of ~ 3.2 μg/L at year 2 and then declined to baseline levels by year 7 of follow-up. There was a strong, positive association between urinary mercury and both number of amalgam surfaces and time since placement. Girls had significantly higher mean urinary mercury concentrations than boys throughout the course of amalgam treatment. There were no differences by race in urinary mercury concentration associated with amalgam exposure. Conclusions Urinary mercury concentrations are highly correlated with both number of amalgam fillings and time since placement in children. Girls excrete significantly higher concentrations of mercury in the urine than boys with comparable treatment, suggesting possible sex-related differences in mercury handling and susceptibility to mercury toxicity.

Oral lichen planus and dental materials: a case‐control study

The purpose of this study was to examine the association of dental materials with oral lichen planus (OLP) and, particularly, the effects of amalgam, amalgam corrosion status, gold and dissimilar metals in continuous contact. A case‐control study was performed with 43 OLP cases from the Oral Medicine Clinic at the School of Dentistry, University of Washington and 78 controls from a general dental care‐screening clinic, also at the School of Dentistry, University of Washington. Health histories and oral examinations were obtained. Current metal or metal‐based restorations were charted, along with corrosion status of amalgams and the presence of dissimilar metals in continuous contact. Adjusted odds ratios (ORs) and 95% confidence intervals for the following potential risk factors were found to be: (1) number of teeth with amalgam, OR = 1·02 (0·92, 1·13); (2) total surfaces of amalgam, OR = 0·96 (0·92, 1·0); (3) number of teeth with gold, OR = 1·12 (0·95, 1·31); (4) mean amalgam corrosion score, OR = 5·74 (2·34, 14·1); (5) presence of dissimilar metals in contact (per quadrant), OR = 1·25 (0·81, 1·92). These findings suggest that although the presence of amalgam or gold themselves is not associated with increased risk of OLP, corrosion of amalgams and the presence of a ‘galvanic effect’ from dissimilar dental materials in continuous contact (bimetallism) are associated with an increased risk of OLP.

MODIFICATION OF NEUROBEHAVIORAL EFFECTS OF MERCURY BY A GENETIC POLYMORPHISM OF COPROPORPHYRINOGEN OXIDASE IN CHILDREN

Mercury (Hg) is neurotoxic, and children may be particularly susceptible to this effect. A current major challenge is the identification of children who may be uniquely susceptible to Hg toxicity because of genetic disposition. We examined the hypothesis that CPOX4, a genetic variant of the heme pathway enzyme coproporphyrinogen oxidase (CPOX) that affects susceptibility to mercury toxicity in adults, also modifies the neurotoxic effects of Hg in children. Five hundred seven children, 8-12 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of Hg from dental amalgam tooth fillings in children. Subjects were evaluated at baseline and at 7 subsequent annual intervals for neurobehavioral performance and urinary mercury levels. Following the completion of the clinical trial, genotyping assays for CPOX4 allelic status were performed on biological samples provided by 330 of the trial participants. Regression modeling strategies were employed to evaluate associations between CPOX4 status, Hg exposure, and neurobehavioral test outcomes. Among girls, few significant CPOX4-Hg interactions or independent main effects for Hg or CPOX4 were observed. In contrast, among boys, numerous significant interaction effects between CPOX4 and Hg were observed spanning all 5 domains of neurobehavioral performance. All underlying dose-response associations between Hg exposure and test performance were restricted to boys with the CPOX4 variant, and all of these associations were in the expected direction where increased exposure to Hg decreased performance. These findings are the first to demonstrate genetic susceptibility to the adverse neurobehavioral effects of Hg exposure in children. The paucity of responses among same-age girls with comparable Hg exposure provides evidence of sexual dimorphism in genetic susceptibility to the adverse neurobehavioral effects of Hg in children and adolescents.

Catechol O-methyltransferase (COMT) VAL158MET functional polymorphism, dental mercury exposure, and self-reported symptoms and mood.

Associations were evaluated between a functional single nucleotide polymorphism (Val158Met) in the gene encoding the catecholamine catabolic enzyme catechol O-methyltransferase (COMT), dental mercury exposure, and self-reported symptoms and mood among 183 male dentists and 213 female dental assistants. Self-reported symptoms, mood, and detailed work histories were obtained by computerized questionnaire. Spot urine samples were collected and analyzed for mercury concentrations to evaluate recent exposures, whereas a chronic mercury exposure index for all subjects was created from the work histories. COMT polymorphism status was determined using a polymerase chain reaction (PCR)-based assay. Scores for current, recent, and chronic self-reported symptom groups and six self-reported mood factors were evaluated with respect to recent and chronic mercury exposure and COMT polymorphism status. Multiple regression analysis controlled for age, socioeconomic status, tobacco and alcohol use, self-reported health problems, and medications. Separate evaluations were conducted for dentists and dental assistants. No consistent patterns of association between either urinary mercury concentration or the chronic index of mercury exposure and any category of symptoms were observed. However, consistent and significant associations were found between increased symptoms and the COMT polymorphism involving the double allelic substitution (full mutation) compared to subjects with no substitutions. Associations with mood were limited to polymorphism status among female dental assistants, and were observed for four of six mood factors and overall mood score. These findings extend evidence of genetic factors potentially affecting human susceptibility to the toxic effects of mercury and other environmental chemicals.

Examination of dietary methylmercury exposure in the Casa Pia Study of the health effects of dental amalgams in children.

This study examined methylmercury concentrations in blood of children participating in the Casa Pia Study of the Health Effects of Dental Amalgams in Children over a 1-yr period and related them to their diets in terms of fish and other seafood consumption. One hundred and fifty children between the ages of 8 and 10 yr who were residents of the Casa Pia School System of Lisbon, Portugal, participated. Parents or caregivers completed a food frequency questionnaire designed specifically for this study at baseline. Children provided urinary and blood samples for mercury determinations at baseline and at 1 yr following placement of dental tooth fillings. Mercury levels in fish samples from childrens diets were also obtained. Mercury determinations in urine, blood, and fish were performed using cold vapor atomic fluorescence spectroscopy. The mean value of baseline methylmercury concentrations in blood increased as the report of seafood consumption increased, although not statistically significantly. However, blood methylmercury and total mercury concentrations were significantly lower at 1-yr follow-up than at baseline. Sixty-one percent of parents/caregivers reported that their children consumed fish on a weekly basis. The fish offered at a sample of the schools contained low levels of methylmercury (range 13.9-23.6 ng/g). Thus, children participating in the Casa Pia dental amalgam study are exposed to low dietary levels of methylmercury by way of fish consumption, and this finding was reflected in the low mean blood methylmercury concentrations observed. The present findings indicate that dietary methylmercury is not a significant source of mercury exposure and is not likely to confound the association of dental amalgam mercury with potential health effects in the present study.

Epidemiologic assessment of measures used to indicate low-level exposure to mercury vapor (Hg°)

Mercury (Hg) concentrations in individual spot urine samples collected over consecutive 1-d periods were compared with Hg concentrations measured in combined 24-h urine samples from 69 practicing dental professionals with low exposure to Hg vapor (Hg) in order to validate the use of spot urine samples as an indicator of Hg exposure. The level of Hg in air as an exposure measure was also evaluated by comparing air concentrations of Hg in dental offices with both spot and 24-h urine Hg levels. The results showed: (1) There was little diurnal variation (approximately 9%) in urinary Hg values; (2) a strong correlation (R2 = .85) exists between the Hg concentration in the first morning void and that in a complete 24-h urine sample; (3) adjustment of urinary Hg levels for creatinine concentrations did not improve this correlation; (4) there was no added value in the speciation of total urinary Hg into the inorganic Hg fraction; and (5) concentrations of Hg in air did not significantly correlate with measures of Hg in urine at this low Hg exposure level. We conclude from this study that first morning void urine samples may be used to derive reasonably valid estimates of Hg concentrations found in the total amount of urine collected over a 24-h period. Thus, due to its comparability, ease of collection, and lower cost, the first morning urine void may be used in place of a sample collected over a full 24 h to facilitate Hg exposure assessments in epidemiologic studies that use urinary Hg levels as a measure of low-level Hg exposure.