Evidence for environmental manganese (Mn) as a neurodevelopmental toxicant is mounting; yet knowledge about Mn susceptibility remains limited and findings are inconsistent across studies. Our study aims to help clarify childhood environmental Mn neurotoxicity by understanding which domains are most sensitive to Mn exposure across childhood, and by identifying subgroups who are more susceptible to Mn-toxicity by investigating exposure timing, sex differences and interactive and joint effects of multiple metals. The proposed epidemiologic study will use previously measured data from the Public Health Impact of Mixed element Exposure (PHIME) study, comprised of 720 children (ages 10-14 years) with varied airborne exposure to ferro- manganese industry in Italy. Under the mentorship of a multidisciplinary team of experts in environmental epidemiology, neurobehavioral toxicology, exposure biology and mixtures, Julia Bauer (PI) proposes the following aims: 1) identify patterns of Mn-associated neurobehavioral decrements by estimating associations of Mn exposures in early life (measured in deciduous teeth) and adolescence (measured in hair and blood) and multiple Mn-sensitive domain-specific and global neurobehavioral tasks; 2) describe susceptibility factors of Mn neurotoxicity by estimating a) sex-specific associations of Mn in early life and adolescence, with performance on complex visuospatial abilities (measured by the Virtual Radial Arm Maze) as well as on full-scale IQ measured by the Wechsler Intelligence Scale for Children; and b) interactive and joint effects of multiple metals (Mn, Pb, Fe, Cu, Cr) on full-scale IQ. This research will inform the current understanding of Mn effects on children's neurobehavior. It also targets several NIEHS strategic goals: use of technologically advanced exposure data, understanding individual susceptibility across childhood, and accounting for mixture scenarios.

The primary objectives of this study are to characterize the effects of environmental manganese (Mn) exposure on adolescent neurodevelopment. Although long known to be a critical neurodevelopmental period in pediatrics/psychology, little is known about the impact of toxic chemical exposures on neurophenotypes in adolescence. Mn is a compelling neurotoxicant to study in adolescence because: i) Mn is an essential trace metal, but it is also neurotoxic at elevated exposures; ii) the catecholaminergic system is a well-known target of Mn, and adolescence neurodevelopment involves substantial changes to the dopaminergic system, iii) Mn exposure is more ubiquitous than previously recognized, but its role in altering brain development is not well known, and iv) there are significant knowledge gaps regarding the impacts of Mn in adolescence. Preliminary results from our initial grant period show that elevated Mn exposure during pre/early adolescence is associated with deficits in attention, neuromotor, cognitive, and olfactory function in children. We will build upon these findings in a cohort of 240 adolescents to determine: 1) the role of Mn exposure timing on adolescent executive function, behavior, and self-regulation by applying novel exposure biomarkers and statistical methods to identify and define critical developmental windows of susceptibility, and 2) functional and anatomical neurophenotypes that are impacted by Mn, focusing on brain areas that subserve executive function, behavior, and self-regulation in adolescence (e.g., PFC, ventral striatum, amygdala) using functional and anatomical MRI. Our established PHIME (Public Health Impact of Manganese Exposure) cohort presents a unique opportunity to achieve these important goals. PHIME was designed to investigate long-term Mn exposure timing; it onsists of subjects (now adolescents) living in three well-characterized communities in Northern Italy that differ in the timing and intensity of environmental Mn exposure from current or historic ferromanganese alloy plant operations. Moreover, recent innovations in exposure science now allow us to retrospectively reconstruct Mn exposure across fetal life/early childhood using a novel tooth biomarker of exposure These studies will be among the first to longitudinally assess the role of environmental Mn on adolescent neurodevelopment and behavior and to objectively determine critical exposure windows that can inform the timing of future public health interventions.

Mixtures are a common exposure scenario but are rarely studied. Metals comprise a class of chemicals to which co-exposure is common and whose neurotoxic effects, particularly, can be severe, making them a potentially paradigm class of chemicals for mixtures research. Research on individual metals, such as lead, has been robust; but research on metal mixtures is lacking. Major challenges impeding progress in mixtures research include the lack of sufficient exposure data for multiple metals. Even when multiple metal exposure biomarkers are collected, there remains a lack of analytical tools to fully evaluate interactions. Under the mentorship of Drs. Coull and Lucchini, Dr. Claus Henn (principal investigator) will overcome barriers in mixtures research by using cutting-edge statistical learning methods, combined with robust data on host exposure to mixed metals. By doing so, she will evaluate the joint effects of multiple metals on neurodevelopment. This K99/R00 application builds upon Dr. Claus Henn's experience in environmental epidemiology and exposure science to cross train her in child development and biostatistics and prepare her to become an independent investigator specializing in chemical mixtures and pediatric environmental health. In the K99 phase of the award, Dr. Claus Henn will use tutorials, didactic instruction, and seminars/conferences to receive training in: 1) advanced quantitative methods for use in analyzing combined exposures, and 2) childhood neurodevelopment from the prenatal period through adolescence. These areas of training represent a new cross-disciplinary goal for Dr. Claus Henn. Her training will subsequently be applied in an existing cohort of 750 Italian adolescents in the Public Health Impact of Mixed Element exposure (PHIME) study. Dr. Claus Henn will apply structural equation models, random forests, and kernel regression machines to examine cognitive effects of concurrent exposure to combinations of metals. In the R00 phase, Dr. Claus Henn will evaluate whether cognitive effects of exposure to combinations of metals depend on exposure timing. Using data from the Early Life Exposure in Mexico to Environmental NeuroToxicants (ELEMENT) longitudinal birth cohort, she will apply the aforementioned methods at multiple exposure time points, across the prenatal period and childhood. This cross-disciplinary proposal represents a unique opportunity to efficiently use existing data in an innovative yet cost-effective manner. The proposed training and research will provide Dr. Claus Henn with the skills to study the joint effects of multiple chemicals and multiple time points on neurodevelopment in future research. This research will 1) address the large data gap on health effects of chemical mixtures, and 2) help characterize critical windows of susceptibility to neurotoxicants; both are NIEHS strategic goals and public health priorities. Public Health Relevance: Research on chemical mixtures is a critical public health need, because the neurotoxicity of an individual chemical is likely modified in the presence of other chemicals; yet research on the effects of metal mixtures is lacking. Further complicating this issue is that timing of exposure to mixtures may also result in differential toxicity. This application will use an integrated training plan in neurodevelopment and advanced biostatistics to train Dr. Birgit Claus Henn, an environmental epidemiologist and exposure assessment expert, to conduct high-level research in metal mixtures and children's neurodevelopment at several critical time points of development, from the prenatal period through adolescence.

Emerging evidence demonstrates that elevated environmental manganese (Mn) exposure is associated with cognitive and behavioral deficits in children. However, up to now, studies in children have only been focused on exposure via ingestion pathways, although occupational research in adults has established that Mn exposure via inhalation poses a more significant risk for cognitive and motor effects than exposure via ingestion. We will assess the neurodevelopmental effects of airborne Mn exposure in 11-13 year old children living in areas impacted by current or past ferroalloy industries. We will examine 750 children across three well-characterized study sites in the province of Brescia, Italy, that differ in history of ferroalloy plant activity: 1) Bagnolo Mella, a community with an active ferroalloy plant that produces very elevated air Mn levels; 2) Valcamonica, a region with slightly elevated air Mn levels due to a history of ferroalloy plant activity that ended in 2001, and; 3) Garda Lake, a reference region with background air Mn levels and no history of ferroalloy plant activity. The project will build upon the existing EU-funded PHIME (Public Health Impact of Mixed element Exposure in susceptible populations) study that has already examined a total of 300 children from Valcamonica and Garda Lake; we propose to enroll 450 more children to achieve our aims. A strength of our proposal is the emphasis on environmental exposure assessment, utilizing measures of airborne Mn with 24 hr personal air monitors, Mn levels in home deposited dust, tap water, soil, and a detailed food frequency questionnaire, coupled with comprehensive cognitive, behavioral, motor, and sensory assessments, along with exposure biomarker measures (blood, urine, hair, nails) and Mn in shed deciduous teeth to assess early life and cumulative lifetime exposures. Preliminary results show changes in motor-coordination, odor identification and IQ among the residents in the historically exposed area of Valcamonica are associated with air Mn levels. This study will offer the unique opportunity to compare effects of Mn at distinct developmental time periods by evaluating children with three exposure histories 1) continuous high airborne Mn exposure since birth (Bagnolo Mella), 2) exposure to high air Mn levels over the first 3 - 5 postnatal years, but reduced current exposure (Valcamonica), and 3) no history of elevated airborne Mn exposure (Garda Lake). We will also address the role of genetic susceptibility to Mn via genetic variation in iron metabolism pathways genes. This will be the first study on Mn neurotoxicity in children supported by a profound characterization of environmental Mn exposure levels.