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More details regarding the adaptation of the parent curriculum are described elsewhere

Adults that were interested in the study were screened and enrolled in the study if all inclusion criteria were met. Baseline data collection was then scheduled for adults and children. After baseline data collection, study parents were informed which treatment group they were in. This intervention component sought to increase vegetable intake among children in the ECE programs. We collaborated with the Leadership Committee and Osage Nation ECE program site managers, teachers, cooks, and staff at the tribal farm to determine which produce was of interest and available for the study. Since the climate in Oklahoma is unpredictable during the intervention months, ranging from cold, icy winters to a warm, wet spring, we utilized the farm and supplemented any produce they were unable to grow from a local supermarket. More details regarding the tribal farm are described elsewhere. The farm-to-school nutrition and gardening curriculum was adapted for NA children from two curricula: Early Sprouts; and Watch Me Grow. More details regarding how we adapted the curriculum are described elsewhere. The FRESH farm to-school nutrition and gardening curriculum included knowledge, reading, gardening, and indoor and outdoor sensory activities, comprised of three themes that were taught for five weeks each: Harvest ; Explore ; and Sprout . The focus of the curriculum was on six target vegetables: tomatoes, bell peppers, spinach, squash, butter beans, and carrots. The weekly curriculum for each theme included a reading activity , indoor and outdoor sensory activity, and cooking activity, which included a take-home recipe kit. The intended duration for each activity varied: 5–30 min for reading activities,cannabis curing up to 60 min for indoor and outdoor sensory activities, and up to 75 min for cooking activities. All weekly lessons were assembled in a handbook and distributed to intervention teachers for implementation.

Garden beds for the outdoor sensory activities and cooking activities were built and maintained by the Harvest Land farm staff at Osage Nation. All intervention children also took home a family recipe kit, including ingredients and a recipe to repeat the cooking activity with their family to increase exposure to the vegetables. Although the FRESH study did not directly intervene upon dietary intake of parents, we did include a passive online and in-person hybrid parent curriculum, adapted from the Choose Health LA’s Healthy Parenting Workshops , with components from the First Nations Development Institute’s Food Sovereignty Assessment Tool and the Grassroots International’s Food for Thought and Action curriculum. The online curriculum comprised of 12 short video modules focused on providing parents with strategies to support their children in eating healthier foods and included healthy lifestyle education and healthy parenting practices. The in-person component included three in-person family night workshops that focused on food sovereignty in the community and community capacity building for health. The last component included menu modifications at the ECE programs. Further description of the menu modifications from the FRESH study can be found elsewhere. In short, fresh vegetables from Osage Nation’s Harvest Land farm were harvested and delivered to the ECEs to be incorporated into the ECE menus. The menus were modified to achieve best practices established by the Child and Adult Care Food Program , which included more vegetables and fruits as snacks, replacing whole grains for refined grains, reducing fried foods, and removing sugar-sweetened beverages. The menus included the six target vegetables from the farm-to-school curriculum provided from the Osage Nation farm two times weekly and provided to the children in meals or offered as snacks within each menu cycle.

Dietary intake for children was assessed by measuring the consumption of the six target vegetables in the FRESH farm-to-school curriculum using the weighed plate waste method to assess objective levels of vegetable consumption. During the plate waste administration, we also assessed preference, or willingness to try, target vegetables. Trained researchers rated each child’s interaction with each target vegetable using a five-point checklist to measure observed willingness to try. The rating options were: Did not remove vegetable from box, removed food, but did not bring to nose/mouth, removed food and brought to nose/mouth, but did not put food in mouth, put food in mouth, but did not swallow food , put food in mouth and swallowed. More information regarding the child food consumption methods is provided elsewhere . Dietary intake for adults was evaluated using the National Cancer Institute’s Automated Self-Administered 24 h Recall. Recalls were obtained either in-person or via phone by trained university staff. Recall data were used to estimate mean intake of total energy , total sugar , total fats , total fruits , and total vegetables between intervention and control groups. We also used the 7-item Fruit and Vegetables Behavior Checklist to assess combined fruit and vegetable intake in cups per day.The aims of the FRESH study were to improve dietary intake , BMI, systolic blood pressure , health status, and food insecurity among NA families. FRESH is one of the first comprehensive multi-component, multi-level CBPR studies to use a farm-to-school and parent curricula to build community capacity and reduce obesity risk among NA families attending ECE programs. Although the FRESH study did not improve BMI or other secondary outcomes among children, there were significant increases in vegetable intake. Previous studies looking at vegetable intake and BMI improvement among children showed varied results. Some randomized controlled trials found that nutrition interventions that resulted in significant increase of vegetable intake also found a decrease in BMI, whereas others found no change among BMI; the latter finding is consistent with our study.

One nutrition and gardening intervention that implemented a randomized controlled trial at school found that BMI significantly improved in the intervention group compared to the controls; however, this study involved older children and a longer intervention period. At follow-up, regarding the willingness to try scale, we found significant increases in scores for tomatoes for both treatment groups and increases in scores for beans in the intervention group. Our findings are similar to the Nutrition Matters! curriculum, which found a significant increase in willingness to try scores in three fruits and vegetables among the nutrition and gardening group,curing cannabis and is consistent with a previous study that found that repeated exposures to vegetables led to an increase in children’s willingness to try target vegetables. Among the adults, the FRESH study did not improve vegetable intake, BMI, blood pressure, or food security. However, in the intervention group, there was a trend toward increased fruit and vegetable intake from baseline to post-intervention. At follow-up, total sugar intake and total energy significantly improved among the intervention group compared to the controls. Our results differ from a previous online nutritional intervention among NA participants that found an increase in vegetable intake. However, as the FRESH intervention focused primarily on children with a secondary component including the parents, significant results were not expected. Our study had several strengths. This study used a randomized controlled design, able to compare intervention and control groups. Although the study was under powered, we still found a trend towards increased vegetable intake in intervention adults. In addition, we used objective measures of vegetable consumption in children rather than a dietary recall, providing a more comprehensive dietary intake. This was noted as a suggestion among authors in a systematic review on garden-based interventions among preschoolers. Another strength of our study is providing children with repeat taste exposure of vegetables, which past research has shown to be effective in increasing intake. Furthermore, our study focused on providing the ECE menus with local fresh vegetables, addressing the need for studies that intervene in the social determinants of health. The Osage Nation is a reservation that has limited access to healthy and fresh foods, and this study built upon and strengthened local resources by facilitating the process for supplying the ECEs with the local produce. The limitations of this study include the challenge of implementing some of the dietary measures among children.

For example, there were negative values on plate waste vegetables, which were determined to likely be the result of water condensation. In addition, we did not directly intervene with parents, which contributed to low participation rates in the study’s online component of the parent curriculum. Only 56% of parents attended the first week of the online curriculum and 12% attended the final week. However, in contrast, participation in the in-person component of the parent/family intervention was nearly twice as high as the online participation, although it also decreased as the intervention continued. Since the original implementation of the FRESH study, the Osage Nation Harvest Land farm built upon the lessons from the study process and findings to expand its produce to a greater number of tribal programs and services. The Harvest Land farm now features commercial-grade aquaponics systems and eight new state-of-the-art greenhouses, which are able to grow various vegetables year-round. The farm is now in the process of developing a tribally specific community-supported agriculture program, which aims to increase access and intake of fresh produce. The principal goal of environmental epidemiology is to characterize how environmental factors affect human health. Specifically, environmental epidemiologists seek to quantify a dose-response relationship between the level of a hazardous agent in the environment and the severity of its health impact on a population. To this end, epidemiologists have generally classified potential exposures to environmental agents on the basis of self-reported information. However, self-reported exposure surrogates are generally qualitative and they may not accurately reflect true environmental exposures. When the discrepancy between estimated and true exposure levels is substantial, the true relationship between exposure and disease will be obscured. As such, the development of quantitative and objective measures of exposure is a critical aspect of environmental epidemiology. Recently, investigators have considered estimating exposures to indoor contaminants using toxicant levels in residential-dust samples, because dust measurements are quantitative and objective. Although, in practice, few epidemiological studies have employed estimates of exposure using dust samples, this dissertation will show that concentrations of chemical contaminants in residential dust can be useful surrogates for indoor chemical exposures This dissertation will focus on three chemical classes which have been associated with either childhood leukemia in the Northern California Childhood Leukemia Study  or with developmental effects in other studies , namely, polybrominated diphenyl ethers , polychlorinated biphenyls , and polycyclic aromatic hydrocarbons , as well as nicotine . All of these chemicals are ubiquitous contaminants in residential dust due to their many indoor sources. Nicotine is a specific marker of cigarette smoke; PBDEs have been used as chemical flame retardants in consumer goods ; PCBs have been used in a host of consumer products, including fluorescent lights, televisions, and refrigerators ; and PAHs are produced by indoor combustion sources, including cigarette smoke, wood-burning fireplaces, and gas appliances . PBDE, PCB, PAH, and nicotine molecules on dust can enter the body via inhalation, via inadvertent ingestion after hand-to-mouth contact, or via direct absorption through the skin . For some individuals, notably children, dust likely contributes a substantial portion of the overall intake of PBDEs , PCBs , and PAHs and nicotine levels in dust offer a useful quantitative measure of cigarette smoking in the home .There have been several reviews of the use of dust as a medium for measuring chemical contamination in the home . Investigators have generally sampled residential dust by obtaining dust from subjects’ household vacuum cleaners or by collecting dust from floors, carpets or other surfaces using a standardized vacuum cleaner, such as a high volume surface sampler . Use of household vacuum cleaner bags eliminates the need for an in-home visit, which reduces study costs and minimizes the invasiveness of home sampling. In contrast, collecting dust with a standardized protocol allows investigators to know the location and time of dust collection. Some investigators have collected dust from household surfaces using a brush or broom . For example, Tan et al. collected dust from the upper surface of a fan blade to measure room-wide contamination. Researchers have made direct comparisons between chemicals measured in dust taken from household vacuum cleaners and dust collected using a standardized protocol in the same residence .

Risk assessment of OP pesticides requires knowledge of the magnitude of the exposure

It has been shown that a semivolatile pesticide such as chlorpyrifos can volatilize days after its indoor application and can be adsorbed to various surfaces . Children’s felt toys, in particular, and, to a lesser extent, plastic toys accumulated significant levels of chlorpyrifos. For a young child exhibiting typical mouthing and hand-to-mouth behavior, dermal and nondietary oral exposure to such conditions were estimated to constitute a dose of 64 µg/ kg/d under the most conservative absorption assumptions and to contribute between 40 and 60% of the total dose. This greatly exceeds the allowable daily intake of 10 µg/kg/d proposed by the US EPA.Therefore, either environmental or biological monitoring is used. In recent years, environmental monitoring has yielded information on concentrations of OP pesticides in outdoor, indoor, and personal air; indoor dust; soil; and foods and beverages . All of the measured values vary considerably, but it is difficult to determine whether they reflect mostly methodological differences or represent true differences in pesticide concentrations. Note that many of the available studies have focused on chlorpyrifos and diazinon. The US EPA eliminated essentially all indoor residential uses of these pesticides by 2002, but they continue to be used in agriculture. Several important findings have emerged from these exposure assessment studies. OP pesticides are detectable in essentially all media analyzed, including food, indoor air, dust, and soil near the home. Interestingly, OP pesticides were not detected in duplicate beverage samples in two studies , whereas others reported their detection in 4 of 21 beverage samples; 4 of 9 of the samples that included apple juice contained azinphosmethyl . Comparisons of pesticide concentrations in dust, soil,cannabis curing and surface and hand wipes have clearly indicated that exposure of agricultural families is considerably greater than that of non-agricultural reference families . This higher exposure appears to result from both take-home pathways and proximity of the residence to farmland , although the association with proximity is not a consistent finding .

Using food consumption data from the Nurses Health Study and the Health Professionals’ Follow-Up Study combined with the data from the Food and Drug Administration Total Diet Study, researchers estimated that mean daily dietary intakes of chlorpyrifos, diazinon, and malathion were 0.8, 0.5, and 5.5 µg/d for women and 0.9, 0.5, and 6.1 µg/d for men, respectively . From duplicate diet samples, adult dietary chlorpyrifos and malathion exposure has been estimated to be 0.5 and 1.3 µg/d, respectively , and dietary chlorpyrifos intake in children was estimated to be 0.263 µg/d . Mean aggregate chlorpyrifos exposure from a total of six pathways was calculated to be 1.39 µg/d ; inhalation made the greatest contribution , whereas only between 7 and 13% was attributable to pesticide residues in solid food, and the dermal route was negligible . In two studies of children’s pesticide exposure, however, solid food made the greatest contribution to the cumulative intake of chlorpyrifos, malathion, and diazinon . Interestingly, despite the high contribution that food appeared to make to aggregate chlorpyrifos exposure in the Minnesota Children’s Pesticide Exposure Study, there was a much stronger correlation between urinary metabolites of this pesticide and concentrations in personal air than with levels in the ingested solid food . Additionally, note that the estimates of dermal absorption neglected to account for the volatilized portion of chlorpyrifos. The finding of a high correlation between chlorpyrifos in indoor air and in the corresponding dermal wipes suggests that this route of exposure may be important . The reported dietary pesticide intakes were generally well within the US EPA or similar reference values . However, it has been noted that dietary intake estimates greatly depend on the assumed value of nondetect samples, with assumption of a zero value underestimating exposure by a factor of 10 to 60 . Bio-monitoring of OP pesticide exposure most commonly involves measurement of their urinary metabolites or, much more rarely,quantification of the pesticides themselves and/or some of their metabolites in plasma .

Whereas urinary dialkylphosphate metabolites are nonspecific because they can be derived from a wide variety of OP compounds, certain other urinary metabolites are specific for one or two pesticides . Recall that urinary metabolites of OP pesticides can provide only rough estimates of exposure because the amount of absorption and the fractional excretion of specific metabolites are not really known, nor have all the metabolites been identified. Additionally, it cannot be determined whether and to what extent urinary metabolites represent exposure to one or more parent compounds or direct exposure to their metabolites. Furthermore, urinary metabolite concentrations should be corrected for dilution, but the appropriate method is still under debate , particularly because marked seasonal fluctuations in creatinine levels were observed in small children . Bio-monitoring of prenatal exposure involves the measurement of pesticides and their metabolites in umbilical cord blood, amniotic fluid, or meconium. A total of eight pesticides were detectable in 45 to 77% of maternal plasma samples obtained at delivery and in a similar percentage of cord plasma samples from 230 mother–infant pairs from New York City . Their concentrations in maternal and cord plasma were similar and highly correlated, indicating the occurrence of transplacental transfer and substantial in utero exposure . A further indication for transplacental transfer comes from the finding that the DAP metabolites DEP, dimethyl phosphate, and dimethylthiophosphate were detected in 10, 10, and 5% of amniotic fluid samples, respectively . Meconium consists of fetal bile secretions along with the content of the amniotic fluid that the fetus swallowed, representing exposure from the second trimester through delivery, and is usually not excreted by the fetus until after birth. DEP and diethylthiophosphate were present in 95 and 100% of 20 meconium samples from New York newborns, respectively, whereas other OP metabolites were detected in only one or none of the samples . Similarly, the detection of diazinon , malathion , parathion , and chlorpyrifos , along with various organochlorine compounds, has been reported in meconium samples from infants in the Philippines . Up to six or seven pesticides were detected in 4 and 5% of the samples, respectively. Some investigators detected an association between reported indoor residential pesticide use and urinary concentrations of specific pesticide metabolites, but this association was not detected in several other studies of children and adults . Reported pesticide use in the garden is also not consistently associated with urinary DAP levels .

A significant correlation was reported between levels of chlorpyrifos, diazinon, and the carbamate propoxur in personal air and the concentrations of these insecticides or their metabolites in plasma obtained within a month of the personal monitoring, but there was no correlation in plasma obtained at later time-points . Because of the relatively short half-lives of these pesticides, the relevance of these correlations is difficult to evaluate without further information about the regularity or chronicity with which the women were exposed to these pesticides. Several studies in which urinary pesticide metabolite levels were measured have confirmed the findings of environmental monitoring studies that farm children are exposed to higher levels of OP pesticides compared with children from non-agricultural reference families ,drying weed particularly during periods of pesticide application . In one of these studies, azinphosmethyl was the pesticide detected with the highest frequency and at the highest concentrations in house dust and was significantly correlated with dimethyl DAP metabolites in urine . Only the study that detectedan association between house dust levels of azinphosmethyl and phosmet and proximity to farmland also found higher dimethyl DAP levels in children living near treated orchards compared to those living at a greater distance . In the same group of subjects, however, urinary levels of the major chlorpyrifos metabolite, 3,5,6-trichloro-2-pyridinol were not significantly different between children from agricultural and non-agricultural families and did not reflect distance from orchards, although chlorpyrifos was present at higher concentrations in house dust of farming families and was increased with increasing distance from pesticide-treated areas . Although studies of exposure to individual pesticides, even those considering aggregate exposure, have generally found the estimated exposure levels to be well below the RfD , there is increasing evidence from biological monitoring studies that exposure to OP pesticides overall may exceed reference doses in a substantial number of subjects from both agricultural and non-agricultural areas. Calculations of exposure using urinary DAP metabolites are difficult because these metabolites can originate from a large variety of OP pesticides with highly different chronic toxicity and RfD values. In 2- to 5-yr-old children from urban and suburban areas of Seattle, the percentage of exposure estimates exceeding US EPA guidelines ranged between 0 and 100%, depending on which pesticide was assumed to be responsible for the exposure . When pesticides commonly applied in an agricultural community in Washington were used to calculate the absorbed daily dose in children age 6 yr or younger, 9 to 56% of children from agricultural families and 0 to 44% of reference children exceeded the EPA RfD for azinphosmethyl and phosmet during the spray season . Similar calculations for the same age groups of children from Yuma County, Arizona, indicated that the highest daily dose values were 61 to 385 times higher than the EPA RfD . In a study of pregnant women in the Salinas Valley in California, the estimated exposure to OP pesticides exceeded the oral benchmark dose10 of the US EPA in 0 to 36% of the women, depending on the index chemical on which the estimate was based and exceeded the benchmark dose for 10% response in approx 15% of women regardless of the parent compound .

The benchmark doses for 10% response are doses expected to result in a 10% reduction in brain cholinesterase activity in rats. Notably, there is evidence from urinary DAP assessments that suggests that consumption of a predominantly organic diet can greatly reduce dietary exposure to OP pesticides as well as the associated risk . However, daily consumption of a single meal prepared with organically grown produce was not sufficient to significantly influence urinary levels of DAP metabolites .OP pesticides and carbamates inhibit acetylcholinesterase . Because AChE inactivates acetylcholine at neuronal junctions, its inhibition results in ACh accumulation and continued neurotransmission. Because the autonomic, the somatic, and the central nervous systems all use ACh, the symptoms of OP-mediated AChE inhibition are manifold and include dizziness, headache, confusion, convulsions, blurred vision, respiratory distress, bradycardia and hypotension, fatigue, weakness, ataxia, muscle cramps, and increased lacrimation and salivation. Although the effects of environmental OP exposure are milder, they can resemble those of acute poisoning and, incidentally, include some well-known SBS symptoms, such as tearing eyes, chest pressure/tightness, and feeling dazed . Numerous animal studies have documented the developmental neurotoxicity of gestational or early postnatal exposure to OP pesticides at relatively low levels that did not result in overt systemic toxicity and inhibited cholinesterase to a minor extent in the dam. Such exposure resulted in impairments in maze performance, locomotion, coordination and balance, righting reflexes, and cliff avoidance. The molecular and cellular changes in the fetal or newborn brain that could account for these effects include inhibition of brain AChE and choline acetyltransferase activity , alteration of muscarinic receptor function via inhibition of ligand binding and permanent reduction in the density of muscarinic cholinergic receptors , altered synaptic development and function that can persist into adulthood , decreased expression and activity of multiple components of the adenylyl cyclase cascade , impaired DNA and RNA synthesis , and reduced cellularity and brain weight in offspring. Most of these studies were performed using chlorpyrifos, but similar effects and mechanisms were observed with other OP pesticides as well as two different pyrethroids . Few studies have addressed possible neurodevelopmental effects of prenatal OP exposure in humans. Recently, the association between prenatal OP pesticide exposure and neonatal neurodevelopment as assessed by the Brazelton Neonatal Behavioral Assessment Scale was investigated in 381 full-term infants in the CHAMACOS project. Table 11 includes maternal DAP metabolite levels during pregnancy in this cohort of women, which contained a substantial portion of agricultural workers from the Salinas Valley and other women with rather high environmental exposure to pesticides because of their heavy use in this agricultural center.