Metagenomics reveals novel microbial signatures of farm exposures in house dust.

Indoor home dust microbial communities, important contributors to human health, are shaped by environmental factors, including farm-related exposures. Advanced metagenomic whole genome shotgun sequencing (WGS) improves detection and characterization of microbiota in the indoor built-environment dust microbiome, compared to conventional 16S rRNA amplicon sequencing (16S). We hypothesized that the improved characterization of indoor dust microbial communities by WGS will enhance detection of exposure-outcome associations. The objective of this study was to identify novel associations of environmental exposures with the dust microbiome from the homes of 781 farmers and farm spouses enrolled in the Agricultural Lung Health Study. We examined various farm-related exposures, including living on a farm, crop versus animal production, and type of animal production, as well as non-farm exposures, including home cleanliness and indoor pets. We assessed the association of the exposures on within-sample alpha diversity and between-sample beta diversity, and the differential abundance of specific microbes by exposure. Results were compared to previous findings using 16S. We found most farm exposures were significantly positively associated with both alpha and beta diversity. Many microbes exhibited differential abundance related to farm exposures, mainly in the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The identification of novel differential taxa associated with farming at the genera level, including Rhodococcus, Bifidobacterium, Corynebacterium, and Pseudomonas, was a benefit of WGS compared to 16S. Our findings indicate that characterization of dust microbiota, an important component of the indoor environment relevant to human health, is heavily influenced by sequencing techniques. WGS is a powerful tool to survey the microbial community that provides novel insights on the impact of environmental exposures on indoor dust microbiota. These findings can inform the design of future studies in environmental health.

Dietary omega-6/omega-3 fatty acids and risk of prostate cancer; Is there any potential interaction by organophosphate insecticides among the agricultural health study population.

BACKGROUND: In the United States (US), the average annual increase in the incidence of prostate cancer (PCa) has been 0.5% between 2013 and 2017. Although some modifiable factors have been identified as the risk factors for PCa, the effect of lower ratio of omega-6 to omega-3 fatty acids intake (N-6/N-3) remains unknown. Previous studies of the Agricultural Health Study (AHS) reported a significant positive association between PCa and selected organophosphate pesticides (OPs) including terbufos and fonofos. OBJECTIVE: The aim of this study was to evaluate the association between N-6/N-3 and PCa and any interaction between N-6/N-3 and 2 selected OPs (i.e., terbufos and fonofos) exposure. DESIGN AND PARTICIPANTS: This case-control study, nested within a prospective cohort study, was conducted on a subgroup of the AHS population (1193 PCa cases and 14,872 controls) who returned their dietary questionnaire between 1999 and 2003 MAIN OUTCOME MEASURES: PCa was coded based on the International Classification of Diseases of Oncology (ICD-O-3) definitions and obtained from the statewide cancer registries of Iowa (2003-2017) and North Carolina (2003-2014). STATISTICAL ANALYSIS: Multivariate logistic regression analysis was applied to obtain the odds ratios adjusted (aORs) for age at dietary assessment (years), race/ethnicity (white, African American, other), physical activity (hours/week), smoking (yes/no), terbufos (yes/no), fonofos (yes/no), diabetes, lycopene intake (milligrams/day), family history of PCa, and the interaction of N-6/N-3 with age, terbufos and fonofos. Pesticide exposure was assessed by self-administrated questionnaires collecting data on ever/never use of mentioned pesticides during lifetime as a yes/no variable. Assessing the P value for the interaction between pesticides and N-6/N-3, we used the continuous variable of "intensity adjusted cumulative exposure" to terbufos and fonofos. This exposure score was based on duration, intensity and frequency of exposure. We also conducted a stratified regression analysis by quartiles of age. RESULTS: Relative to the highest N-6/N-3 quartile, the lowest quartile was significantly associated with a decreased risk of PCa (aOR=0.61, 95% CI: 0.41-0.90), and quartile-specific aORs decreased toward the lowest quartile (Ptrend=<0.01). Based on the age-stratified analysis, the protective effect was only significant for the lowest quartile of N-6/N-3 among those aged between 48 and 55 years old (aORs=0.97, 95% CI, 0.45-0.55). Among those who were exposed to terbufos (ever exposure reported as yes in the self-report questionnaires), lower quartiles of N-6/N-3 were protective albeit nonsignificant (aORs: 0.86, 0.92, 0.91 in quartiles 1,2, and 3, respectively). No meaningful findings were observed for fonofos and N-6/N-3 interaction. CONCLUSION: Findings showed that lower N-6/N-3 may decrease risk of PCa among farmers. However, no significant interaction was found between selected organophosphate pesticides and N-6/N-3.

Pesticide use and incident Parkinson's disease in a cohort of farmers and their spouses.

BACKGROUND: Extensive literature suggests an association between general pesticide use and Parkinson's disease (PD). However, with few exceptions, little is known about associations between specific pesticides and PD. OBJECTIVE: We evaluated use of pesticides and incident PD in 38,274 pesticide applicators and 27,836 of their spouses in the Agricultural Health Study cohort followed over 20 years. METHODS: We used self-reported information on ever-use of 50 specific pesticides as of enrollment for both applicators and spouses, and considered intensity-weighted lifetime days (IWLD) reported at enrollment and through the first 5-year follow-up among applicators. We estimated covariate-adjusted hazard ratios (HR) and 95% confidence intervals (CI) using Cox regression. We also examined heterogeneity in associations by history of head injury and chemical resistant glove use. RESULTS: A total of 373 applicators and 118 spouses self-reported incident doctor-diagnosed PD. Ever-use of the insecticide terbufos (HR:1.31, 95%CI:1.02-1.68) and the herbicides trifluralin (HR:1.29, 95%CI: 0.99-1.70) and 2,4,5-T (HR:1.57, 95%CI:1.21-2.04) was associated with elevated PD risk. On the other hand, diazinon (HR:0.73, 95%CI: 0.58-0.94) and 2,4,5-TP (HR:0.39, 95%CI:0.25-0.62) were associated with reduced risk. We observed heterogeneity in ever-use associations by head injury and chemical-resistant glove use for some pesticides, with higher risk among those who reported a history of head injury, or who did not use gloves. PD risk was also elevated for applicators in the highest category of IWLD for dichlorvos, permethrin (animal use), and benomyl. CONCLUSIONS: We found evidence of increased PD risk for some pesticides. Our results also suggest higher susceptibility for pesticide-associated PD among individuals with head injury as well as protection with use of chemical resistant gloves, although further research is needed to understand the impact of head injury. Research on current and newer pesticides, including mechanisms relevant to PD, is important given widespread pesticide use.

Herbicide, fumigant, and fungicide use and breast cancer risk among farmers' wives.

Evidence from epidemiologic and laboratory studies relating pesticides to breast cancer risk is inconsistent. Women engaging in agricultural work or living in agricultural areas may experience appreciable exposures to a wide range of pesticides, including herbicides, fumigants, and fungicides. METHODS: We examined exposure to herbicides, fumigants, and fungicides in relation to breast cancer risk among farmers' wives with no prior history of breast cancer in the Agricultural Health Study. Women provided information on pesticide use, demographics, and reproductive history at enrollment (1993-1997) and at a 5-year follow-up interview. We used Cox proportional hazards regression to estimate associations (hazard ratios [HRs] and 95% confidence intervals [CIs]) between the women's and their husbands' self-reported use of individual pesticides and incident breast cancer risk. RESULTS: Out of 30,594 women, 38% reported using herbicides, fumigants, or fungicides and 1,081 were diagnosed with breast cancer during a median 15.3 years of follow-up. We found elevated risk in relation to women's ever use of the fungicide benomyl (HR = 1.6; 95% CI = 0.9, 2.7) and the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (HR = 1.6; 95% CI = 0.8, 3.1) and to their husbands' use of the herbicide 2-(2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP) (HR = 1.5; 95% CI = 0.9, 2.7). We observed few other chemical associations and little evidence of differential risk by tumor estrogen receptor status or linear exposure-response relationships. CONCLUSION: We did not observe clear excesses between use of specific pesticides and breast cancer risk across exposure metrics, although we did observe elevated risk associated with women's use of benomyl and 2,4,5-T and husbands' use of 2,4,5-TP.

Dicamba use and cancer incidence in the agricultural health study: an updated analysis.

BACKGROUND: The herbicide dicamba has been commonly used agriculturally and residentially. Recent approval of genetically engineered dicamba-resistant crops is expected to lead to increased dicamba use, and there has been growing interest in potential human health effects. A prior analysis in the Agricultural Health Study (AHS) suggested associations between dicamba and colon and lung cancer. We re-evaluated dicamba use in the AHS, including an additional 12 years and 2702 exposed cancers. METHODS: The AHS is a prospective cohort of pesticide applicators in Iowa and North Carolina. At enrollment (1993-1997) and follow-up (1999-2005), participants reported dicamba use. Exposure was characterized by cumulative intensity-weighted lifetime days, including exposure lags of up to 20 years. We estimated relative risks (RR) and 95% confidence intervals (CI) using multivariable Poisson regression for incident cancers diagnosed from enrollment through 2014/2015. RESULTS: Among 49 922 applicators, 26 412 (52.9%) used dicamba. Compared with applicators reporting no dicamba use, those in the highest quartile of exposure had elevated risk of liver and intrahepatic bile duct cancer (nexposed = 28, RRQ4 = 1.80, CI: 1.26-2.56, Ptrend < 0.001) and chronic lymphocytic leukaemia (CLL, nexposed = 93, RRQ4 = 1.20, CI: 0.96-1.50, Ptrend = 0.01) and decreased risk of myeloid leukaemia (nexposed = 55, RRQ4 = 0.73, CI: 0.51-1.03, Ptrend = 0.01). The associations for liver cancer and myeloid leukaemia remained after lagging exposure of up to 20 years. CONCLUSIONS: With additional follow-up and exposure information, associations with lung and colon cancer were no longer apparent. In this first evaluation of liver and intrahepatic bile duct cancer, there was an association with increasing use of dicamba that persisted across lags of up to 20 years.

Overall and cause-specific mortality in a cohort of farmers and their spouses.

OBJECTIVES: Lower mortality rates compared with the general population have been reported for Agricultural Health Study (AHS) participants (enrolled 1993-1997) followed through 2007. We extended analysis of mortality among AHS participants (51 502 private pesticide applicators, their 31 867 spouses and 4677 commercial pesticide applicators from North Carolina and Iowa) through 2015 and compared results using several analytical approaches. METHODS: We calculated standardised mortality ratios (SMRs), causal mortality ratios (CMR) and relative SMRs (rSMR) using state-specific mortality rates of the general populations as the referent. RESULTS: Over the average 16 years of follow-up (1999-2015), 9305 private applicators, 3384 spouses and 415 commercial applicators died. SMRs and CMRs, with expected deaths calculated using the person-time among the cohort and the general population, respectively, indicated lower overall mortality in all study subgroups (SMRs from 0.61 to 0.69 and CMRs from 0.74 to 0.89), although CMRs indicated elevated mortality in private applicators from North Carolina and in ever-smokers. In SMR analyses, there were fewer than expected deaths from many causes, but deaths from some external causes including transportation-related injuries and mechanical forces were elevated in private applicators. CMRs indicated higher than expected deaths from prostate cancer, lymphohaematopoietic cancers, Parkinson's and Alzheimer's disease, and chronic glomerulonephritis in private applicators, and non-Hodgkin's lymphoma in spouses (from 1.19 to 1.53). rSMR results were generally elevated, similar to CMR findings. CONCLUSIONS: AHS participants experienced lower overall mortality than the general population.Mortality from a few specific causes was increased in private applicators, specifically when CMR and rSMR approaches were used.

Pesticide use and incident hyperthyroidism in farmers in the Agricultural Health Study.

BACKGROUND: Few studies have evaluated associations between pesticides and hyperthyroidism. OBJECTIVE: We evaluated associations between specific pesticides and incident hyperthyroidism in private pesticide applicators in the Agricultural Health Study. METHODS: We used Cox proportional hazards models to estimate HRs and 95% CIs for associations between pesticide use at enrolment and hyperthyroidism (n=271) in 35 150 applicators (mostly men), adjusting for potential confounders. RESULTS: Ever use of several pesticides (organophosphate insecticide malathion, fungicide maneb/mancozeb, herbicides dicamba, metolachlor, and atrazine in overall sample and chlorimuron ethyl among those ≤62 years) was associated with reduced hyperthyroidism risk, with HRs ranging from 0.50 (95% CI 0.30 to 0.83) for maneb/mancozeb to 0.77 (95% CI 0.59 to 1.00) for atrazine. Hyperthyroidism risk was lowest among those with higher intensity-weighted lifetime days of using carbofuran and chlorpyrifos (ptrend ≤0.05). CONCLUSIONS: Observed associations between pesticides and decreased risk of hyperthyroidism warrant further investigation.

Modeling groundwater nitrate exposure in private wells of North Carolina for the Agricultural Health Study.

Unregulated private wells in the United States are susceptible to many groundwater contaminants. Ingestion of nitrate, the most common anthropogenic private well contaminant in the United States, can lead to the endogenous formation of N-nitroso-compounds, which are known human carcinogens. In this study, we expand upon previous efforts to model private well groundwater nitrate concentration in North Carolina by developing multiple machine learning models and testing against out-of-sample prediction. Our purpose was to develop exposure estimates in unmonitored areas for use in the Agricultural Health Study (AHS) cohort. Using approximately 22,000 private well nitrate measurements in North Carolina, we trained and tested continuous models including a censored maximum likelihood-based linear model, random forest, gradient boosted machine, support vector machine, neural networks, and kriging. Continuous nitrate models had low predictive performance (R2 < 0.33), so multiple random forest classification models were also trained and tested. The final classification approach predicted <1 mg/L, 1-5 mg/L, and ≥5 mg/L using a random forest model with 58 variables and maximizing the Cohen's kappa statistic. The final model had an overall accuracy of 0.75 and high specificity for the higher two categories and high sensitivity for the lowest category. The results will be used for the categorical prediction of private well nitrate for AHS cohort participants that reside in North Carolina.

Incident thyroid disease in female spouses of private pesticide applicators.

BACKGROUND: Little is known about modifiable risk factors for thyroid disease. Several pesticides have been implicated in thyroid disruption, but clinical implications are not clear. OBJECTIVE: We assessed associations between pesticide use and other farm exposures and incident hypothyroidism and hyperthyroidism in female spouses of farmers in the Agricultural Health Study (AHS). METHODS: We used Cox proportional hazards models to estimate hazard ratios (HR) and 95% confidence intervals for risk of thyroid disease in 24,092 spouses who completed at least one follow-up questionnaire. RESULTS: We identified 1627 hypothyroid and 531 hyperthyroid cases over 20 years of follow-up. The fungicides benomyl, maneb/mancozeb, and metalaxyl, the herbicide pendimethalin, and among those over 60 years of age the insecticides parathion and permethrin (applied to crops) were associated with elevated hypothyroidism risk, with HR ranging from 1.56-2.44. Conversely, the insecticide phorate, and the herbicides imazethapyr and metolachlor were associated with decreased risk (HR ranging 0.63-0.73), as were long-term farm residence and other farm-related activities (HR ranging 0.69-0.84). For hyperthyroidism, the insecticide diazinon, the fungicides maneb/mancozeb, and the herbicide metolachlor were associated with increased risk (HR ranging 1.35-2.01) and the herbicide trifluralin with decreased risk (HR: 0.57). CONCLUSIONS: Several individual pesticides were associated with increased risk of hypothyroidism and hyperthyroidism, although some pesticides were associated with decreased risk. Some of the findings, specifically associations with fungicides, are consistent with results from an earlier analysis of prevalent diseases in AHS spouses.

A prospective study of cancer risk among Agricultural Health Study farm spouses associated with personal use of organochlorine insecticides.

BACKGROUND: Organochlorine insecticides (OCs) have historically been used worldwide to control insects, although most have now been banned in developed countries. Evidence for an association between OC exposures and cancer predominantly comes from occupational and population based-studies among men. We evaluated the association between the use of specific OCs and cancer among the female spouses of pesticide applicators in the Agricultural Health Study. METHODS: At enrollment (1993-1997), spouses of private applicators in the cohort provided information about their own use of pesticides, including seven OCs (aldrin, chlordane, dieldrin, DDT, heptachlor, lindane, and toxaphene), and information on potential confounders. We used Poisson regression to estimate relative risks (RRs) and 95% confidence intervals (CIs) for cancers (n ≥ 3 exposed cases) reported to state cancer registries from enrollment through 2012 (North Carolina) and 2013 (Iowa), and use of the individual OCs, as well as use of any of the specific OCs. RESULTS: Among 28,909 female spouses, 2191 (7.58%) reported ever use of at least one OC, of whom 287 were diagnosed with cancer. Most cancers were not associated with OC use. Risk of glioma was increased among users of at least one OC (Nexposed = 11, RR = 3.52, 95% CI 1.72-7.21) and specifically among lindane users (Nexposed = 3, RR = 4.45, 95% CI 1.36-14.55). Multiple myeloma was associated with chlordane (Nexposed = 6, RR = 2.71, 95% CI 1.12-6.55). Based on 3 exposed cases each, there were also positive associations between pancreatic cancer and lindane, and ER-PR- breast cancer and dieldrin. No other associations with breast cancer were found. CONCLUSIONS: Overall, there were some associations with OC use and cancer incidence, however we were limited by the small number of exposed cancer cases. Future research should attempt to expand on these findings by assessing environmental sources of OC exposures, to fully evaluate the role of OC exposures on cancer risk in women.

High pesticide exposure events and DNA methylation among pesticide applicators in the agricultural health study.

Pesticide exposure has been associated with acute and chronic adverse health effects. DNA methylation (DNAm) may mediate these effects. We evaluated the association between experiencing unusually high pesticide exposure events (HPEEs) and DNAm among pesticide applicators in the Agricultural Health Study (AHS), a prospective study of applicators from Iowa and North Carolina. DNA was extracted from whole blood from male AHS pesticide applicators (n = 695). Questionnaire data were used to ascertain the occurrence of HPEEs over the participant's lifetime. Pyrosequencing was used to quantify DNAm in CDH1, GSTp1, and MGMT promoters, and in the repetitive element, LINE-1. Linear and robust regression analyses evaluated adjusted associations between HPEE and DNAm. Ever having an HPEE (n = 142; 24%) was associated with elevated DNAm in the GSTp1 promoter at CpG7 (chr11:67,351,134; P < 0.01) and for the mean across the CpGs measured in the GSTp1 promoter (P < 0.01). In stratified analyses, elevated GSTP1 promoter DNAm associated with HPEE was more pronounced among applicators >59 years and those with plasma folate levels ≤16.56 ng/mL (p-interaction <0.01); HPEE was associated with reduced MGMT promoter DNAm at CpG2 (chr10:131,265,803; P = 0.03), CpG3 (chr10:131,265,810; P = 0.05), and the mean across CpGs measured in the MGMT promoter (P = 0.03) among applicators >59 years and reduced LINE-1 DNAm (P = 0.05) among applicators with ≤16.56 ng/mL plasma folate. Non-specific HPEEs may contribute to increased DNAm in GSTp1, and in some groups, reduced DNAm in MGMT and LINE-1. The impacts of these alterations on disease development are unclear, but elevated GSTp1 promoter DNAm and subsequent gene inactivation has been consistently associated with prostate cancer. Environ. Mol. Mutagen. 58:19-29, 2017. © 2016 Wiley Periodicals, Inc.

Pesticide use and LINE-1 methylation among male private pesticide applicators in the Agricultural Health Study.

Cancer risk may be associated with DNA methylation (DNAm) levels in Long Interspersed Nucleotide Element 1 (LINE-1), a surrogate for global DNAm. Exposure to certain pesticides may increase risk of particular cancers, perhaps mediated in part through global DNAm alterations. To date, human data on pesticide exposure and global DNAm alterations are limited. The goal of this study was to evaluate alterations of LINE-1 DNAm by pesticides in a variety of classes. Data from 596 cancer-free male participants enrolled in the Agricultural Health Study (AHS) were used to examine associations between use of 57 pesticides and LINE-1 DNAm measured via Pyrosequencing in peripheral blood leucocytes. Participants provided information on pesticide use at three contacts between 1993 and 2010. Associations of ever/never pesticide use and lifetime days of application (years of use × days per year) and LINE-1 DNAm level were assessed using linear regression, adjusting for potential confounders (race, age at blood draw, and frequency of drinking alcohol) and other moderately correlated pesticides. After adjustment, ever application of 10 pesticides was positively associated and ever application of eight pesticides was negatively associated with LINE-1 DNAm. In dose-response analyses, increases in five pesticides (imazethapyr, fenthion, EPTC, butylate, and heptachlor) were associated with increasing LINE-1 DNAm (ptrend < 0.05) and increases in three pesticides (carbaryl, chlordane, and paraquat) were associated with decreasing LINE-1 DNAm (ptrend < 0.05). This study provides some mechanistic insight into the pesticide-cancer relationship, which may be mediated in part by epigenetics.

Use of acetochlor and cancer incidence in the Agricultural Health Study.

Since its registration in 1994 acetochlor has become a commonly used herbicide in the US, yet no epidemiologic study has evaluated its carcinogenicity in humans. We evaluated the use of acetochlor and cancer incidence among licensed pesticide applicators in the Agricultural Health Study. In telephone interviews administered during 1999-2005, participants provided information on acetochlor use, use of other pesticides and additional potential confounders. We used Poisson regression to estimate relative risks (RR) and 95% confidence intervals (95% CI) for cancers that occurred from the time of interview through 2011 in Iowa and 2010 in North Carolina. Among 33,484 men, there were 4,026 applicators who used acetochlor and 3,234 incident cancers, with 304 acetochlor-exposed cases. Increased risk of lung cancer was observed among acetochlor users (RR = 1.74; 95% CI: 1.07-2.84) compared to nonusers, and among individuals who reported using acetochlor/atrazine product mixtures (RR = 2.33; 95% CI: 1.30-4.17), compared to nonusers of acetochlor. Colorectal cancer risk was significantly elevated among the highest category of acetochlor users (RR = 1.75; 95% CI: 1.08-2.83) compared to never users. Additionally, borderline significantly increased risk of melanoma (RR = 1.61; 95% CI: 0.98-2.66) and pancreatic cancer (RR = 2.36; 95% CI: 0.98-5.65) were observed among acetochlor users. The associations between acetochlor use and lung cancer, colorectal cancer, melanoma and pancreatic cancer are suggestive, however the lack of exposure-response trends, small number of exposed cases and relatively short time between acetochlor use and cancer development prohibit definitive conclusions.

Pesticide use and incident diabetes among wives of farmers in the Agricultural Health Study.

OBJECTIVE: To estimate associations between use of specific agricultural pesticides and incident diabetes in women. METHODS: We used data from the Agricultural Health Study, a large prospective cohort of pesticide applicators and their spouses in Iowa and North Carolina. For comparability with previous studies of farmers, we limited analysis to 13 637 farmers' wives who reported ever personally mixing or applying pesticides at enrolment (1993-1997), who provided complete data on required covariates and diabetes diagnosis and who reported no previous diagnosis of diabetes at enrolment. Participants reported ever-use of 50 specific pesticides at enrolment and incident diabetes at one of two follow-up interviews within an average of 12 years of enrolment. We fit Cox proportional hazards models with age as the time scale and adjusting for state and body mass index to estimate HRs and 95% CIs for each of the 45 pesticides with sufficient users. RESULTS: Five pesticides were positively associated with incident diabetes (n=688; 5%): three organophosphates, fonofos (HR=1.56, 95% CI 1.11 to 2.19), phorate (HR=1.57, 95% CI 1.14 to 2.16) and parathion (HR=1.61, 95% CI 1.05 to 2.46); the organochlorine dieldrin (HR=1.99, 95% CI 1.12 to 3.54); and the herbicide 2,4,5-T/2,4,5-TP (HR=1.59, 95% CI 1.00 to 2.51). With phorate and fonofos together in one model to account for their correlation, risks for both remained elevated, though attenuated compared with separate models. CONCLUSIONS: Results are consistent with previous studies reporting an association between specific organochlorines and diabetes and add to growing evidence that certain organophosphates also may increase risk.