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.
- Agricultural Health Study
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What this paper adds
Previous studies have suggested that exposure to certain pesticides is associated with incident diabetes; most of these studies have been conducted among men.
Women who apply agricultural pesticides may differ from men in their response to pesticide exposure.
Among 13 637 farmers’ wives who personally mixed or applied pesticides, five specific pesticides (fonofos, phorate, parathion, dieldrin and 2,4,5-T/2,4,5-TP) were associated with incident diabetes during a 10-year follow-up period.
The findings provide additional evidence of an association between certain organochlorines and adult onset diabetes, and also support hypothesised associations between the use of specific organophosphates and incident diabetes among women.
Diabetes is believed to have both environmental and genetic causes, but the environmental factors that contribute to its pathogenesis are largely unknown. Recent reports implicate certain chemicals as potentially contributing to the development of diabetes. These chemicals include persistent organic pollutants such as dioxins,1 polychlorinated biphenyls2 and organochlorine pesticides,3 ,4 as well as agents with shorter biological half-lives such as organophosphate insecticides4 ,5 and chlorophenoxy herbicides.6 The widespread use of many pesticides increases concern about the potential influence of pesticide exposures on diabetes.
Most epidemiological studies of persistent organic pollutants and diabetes have been cross-sectional,3 ,7–10 although a few have been prospective.4 ,11 ,12 In cross-sectional studies based on the National Health and Nutrition Examination Survey, serum levels of a polychlorinated biphenyl, an organochlorine insecticide (p,p′-DDT) and a dioxin compound were all positively associated with self-reported diagnosed diabetes among adults,8 and the sum of all persistent organic pollutants measured in serum showed a positive dose–response relationship with diabetes.9 Persistent organic pollutants may increase the risk of diabetes through binding to the aryl hydrocarbon receptor, which, in turn, causes antagonistic effects on the peroxisome proliferator-activated receptor.13
By contrast, most evidence for the role of relatively short-lived organophosphate insecticides in diabetes comes from laboratory studies. Organophosphate exposure may contribute to diabetes via disruption of adipokine signalling and metabolic regulation.14 Recent experimental studies have demonstrated excess weight gain, hyperlipidaemia and hyperinsulinaemia persisting into adulthood in rats exposed neonatally to the organophosphate chlorpyrifos; the magnitude of these effects differs between males and females.15 ,16 The implications of such sex-specific metabolic programming in humans remain unknown.
The Agricultural Health Study (AHS), a large prospective cohort of pesticide applicators and their spouses in Iowa and North Carolina, presents a unique opportunity to conduct longitudinal studies of diabetes incidence among individuals with a known history of pesticide use. A previous prospective analysis4 among predominantly male licensed pesticide applicators in the AHS found elevated risk of diabetes associated with ever-use of eight pesticides (two organochlorines: chlordane and heptachlor; four organophosphates: coumaphos, phorate, terbufos and trichlorfon; and two herbicides: alachlor and cyanazine). Diabetes risk also increased with cumulative lifetime days of use of seven pesticides: aldrin, chlordane, heptachlor, dichlorvos, trichlorfon, alachlor and cyanazine.
Among women in the AHS cohort with at least one pregnancy in the 25 years prior to enrolment, gestational diabetes was twice as likely in those who reported mixing or applying any pesticides during the first trimester of pregnancy.17 Gestational diabetes also increased with lifetime ever-use of seven specific pesticides (two organophosphates, diazinon and phorate; the carbamate insecticide carbofuran; and four herbicides, atrazine and butylate, as well as the historically dioxin-contaminated herbicides 2,4,5-T and 2,4,5-TP).17
This study tests the hypothesis that use of specific pesticides, particularly organochlorine and organophosphate insecticides, by female spouses of farmers is associated with higher incidence of diabetes during the approximately 10-year follow-up period.
Spouses of licensed pesticide applicators in Iowa and North Carolina enrolled in the AHS in 1993–1997 via self-administered questionnaire (81%) or telephone interview (19%). Seventy-six per cent of eligible spouses enrolled in the study.18 At enrolment, participants provided information about demographic characteristics, lifestyle and dietary habits, personal and family medical history, and lifetime use of specific pesticides. Participants were subsequently re-contacted twice, at approximately 5-year intervals, for follow-up telephone interviews. Of the 32 126 female spouses who enrolled in the study, 23 682 (74%) participated in the first follow-up interview, and 19 876 (62%) participated in the second follow-up interview. In the follow-up interviews, participants provided information regarding changes in lifestyle or medical history. Details of the study design are reported elsewhere,19 and questionnaires are available on the study website (http://aghealth.nci.nih.gov/questionnaires.html).
To make our analysis comparable to previous studies of occupational pesticide use,4 ,20 we limited it to female spouses who reported ever mixing or applying any pesticides before enrolment (55% of all female spouses, n=17 628). Women who personally mixed or applied pesticides differed in many ways from women who did not. Specifically, women who never mixed or applied pesticides before enrolment had more risk factors for diabetes, including a significantly higher proportion with a high school education or less, and a significantly higher proportion who reported no recreational exercise per week as compared with women who personally mixed or applied pesticides (data not shown).
Our analysis of female spouses with a history of pesticide application at enrolment was further restricted to those who completed at least one of the follow-up interviews (n=15 034). As we were interested in incident disease, we also excluded those with prevalent diabetes (n=518). Prevalent diabetes was defined as either of the following: (1) participant responded ‘yes’ to the following question on the enrolment questionnaire, “Has a doctor ever told you that you had (been diagnosed with) diabetes (sugar) (other than while pregnant)?”, or (2) participant reported no diabetes at enrolment, but at one of the follow-up interviews reported an age at diabetes diagnosis that was less than the age at enrolment. Individuals who were missing information on diabetes incidence (n=57) or baseline body mass index (BMI) (n=822) were also excluded from analysis. These exclusions resulted in an eligible study population of 13 637 women.
Description of variables
At enrolment, participants provided information about their lifetime personal use of pesticides. Women were asked to report the number of years and average number of days per year that they personally mixed or applied any pesticides. We multiplied the category midpoints to create a variable for cumulative lifetime days spent mixing or applying any pesticides, and we grouped this variable into five quintile-based categories (1–9 days, 10–25 days, 26–64 days, 65–225 days, 226–7000 days) for analysis. Approximately 25% of women did not provide information on their frequency and duration of pesticide use. Women also provided information on personal ever-use of each of 50 specific pesticides; however, the questionnaire for spouses of farmers (unlike the questionnaire for the farmers themselves) did not request information about the frequency or duration of use of specific pesticides. We combined 2,4,5-T and 2,4,5-TP into one variable because these two herbicides have similar chemical structures, similar use patterns in our cohort and both contained dioxin at some points in time.21 We restricted attention to pesticide exposures with five or more exposed cases, thereby eliminating trichlorfon, ziram, aluminium phosphide and ethylene dibromide. Consequently, we evaluated 45 pesticides for association with diabetes.
Information on covariates of interest was also collected via questionnaire at enrolment. To construct our adjusted models, we produced a directed acyclic graph representing associations previously reported in the literature, which indicated that adjustment for age and state of residence (Iowa, North Carolina) would reduce bias due to confounding. We additionally adjusted for BMI at enrolment (<25, 25–29.99, 30–34.99, ≥35 kg/m2) for comparability with a previous study among applicators.4
During each of the two follow-up interviews, we asked each participant whether a doctor had ever diagnosed her with diabetes (other than while pregnant) and, if so, her age at diagnosis (given in years). Participants were not asked to report the type of diabetes diagnosed; however, in adults, it is estimated that 90–95% of all diagnosed cases of diabetes are type 2.22 If reported age at diagnosis equalled or exceeded age at enrolment, then she was considered an incident case. Age at diagnosis, reported in years, was interval-censored. Using dates of birth, enrolment and interviews for each incident case, we established a shortest age interval containing the unknown month and day of diagnosis. Typically that interval was 365 days but might be shorter if the 365-day interval happened to contain the case's enrolment or interview dates. We defined the midpoint of the interval as the age at diagnosis in days. Non-cases were censored at their age in days at last completed interview.
As described above, age at diagnosis was interval-censored and left-truncated. We are aware of no software for Cox regression that properly accommodates both these contingencies simultaneously. We opted to accommodate left-truncation and handled interval-censoring using the midpoint of the interval. Consequently, to estimate the associations between pesticide use and diabetes risk, we fit Cox proportional hazards regression models, with age in days as the time scale, allowing for left-truncation at age at enrolment. All models were adjusted for state of residence and BMI at enrolment to produce the common base model. We checked the proportional hazards assumption for each exposure variable by including a time-varying interaction term, log(age)×exposure variable, as a covariate in the models.
To address potential confounding of individual pesticide results by correlated pesticides, we employed the following two-step procedure. First, we assessed the Spearman correlations among the pesticides that had significant associations with incident diabetes. Second, for any pair of pesticide exposure variables that were correlated at a level of ρ>0.3, both correlated pesticides were included as covariates in the same model.
All statistical analyses were performed with SAS, V.9.2 (SAS Institute, Inc., Cary, North Carolina, USA) using the AHS data sets P1REL0906.00, P2REL0907.00 and P3REL0901.00.
Among the 13 637 female spouses eligible for this study, 688 (5%) reported incident diabetes during the follow-up period. Age at enrolment ranged from 17 to 88 years (mean± SD 47.0±11.0 years). Age at diagnosis among the cases ranged from 25 to 88 years (mean±SD 58.3±10.5 years). Women who developed diabetes were more likely to be older, to have a higher BMI and to be from North Carolina than those who did not (table 1). Women who developed diabetes were also more likely to have a high school education or less, to be postmenopausal at enrolment and to have a family history of diabetes. In addition, women who reported three or more hours of recreational physical activity per week during the summer were less likely to develop incident diabetes than those who reported no weekly recreational physical activity. The mean duration of follow-up was 10.0 years for all women; 6.7 years among women with diabetes and 10.2 years among women without diabetes.
We saw little evidence of an exposure–response relationship with measures of overall lifetime pesticide use (table 2). There was no exposure–response relationship for frequency of pesticide use (days/year) or lifetime days of pesticide use. However, diabetes was associated with total years of use in the highest category of lifetime years of use; women who applied pesticides for more than 30 years were 60% more likely (95% CI 1.08 to 2.38) to be diagnosed with diabetes than women who had applied pesticides for only 1 year.
HRs for diabetes were significantly elevated for use of 5 of the 45 specific pesticides evaluated in models adjusted for age, state and BMI (table 3); no pesticide had a significantly reduced HR. Four of the five pesticides associated with diabetes risk were insecticides: the organochlorine dieldrin (HR 1.99, 95% CI 1.12 to 3.54) and 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). None of the carbamate or pyrethroid insecticides were associated with diabetes. The other pesticide significantly associated with diabetes was the herbicide 2,4,5-T/2,4,5-TP (HR 1.59, 95% CI 1.00 to 2.51). Another chlorophenoxy herbicide, 2,4-D, was not associated with diabetes (HR 1.07, 95% CI 0.90 to 1.27). No fumigants or fungicides were significantly associated with incident diabetes.
Two of the five pesticides significantly associated with diabetes, fonofos and phorate, were moderately correlated with each other (ρ ≥ 0.3). After including both correlated pesticides in the same model, HRs were attenuated but remained elevated; the HR for fonofos was reduced from 1.56 (95% CI 1.11 to 2.19) to 1.35 (95% CI 0.91 to 2.00), and the HR for phorate was reduced from 1.57 (95% CI 1.14 to 2.16) to 1.42 (95% CI 0.98 to 2.05).
Individual pesticides may influence diabetes risk among farm women who apply them. Ever-use of five pesticides was positively associated with incident diabetes. Organochlorines and dioxins have been associated with diabetes previously,10 ,11 ,13 ,23 and we saw associations with the organochlorine insecticide dieldrin and the potentially dioxin-contaminated 2,4,5-T/2,4,5-TP. Organophosphate insecticides have been connected in animal studies to hyperlipidaemia and hyperinsulinaemia, conditions related to diabetes.5 We saw an increased risk of diabetes with the organophosphates fonofos, phorate and parathion. Our results on exposure–response for total lifetime pesticide use suggested that women with 30 or more years of pesticide use had higher risk of diabetes, but the number of days of use did not increase risk.
The incidence of diabetes in this study was 5% over approximately 10 years of follow-up, which was comparable to the national incidence of diabetes among women during the study period. The annual age-adjusted incidence of diabetes among women in the USA rose from 4.9 per 1000 in 1993 to 7.0 per 1000 in 2003.22
Several persistent organic pollutants, including organochlorine pesticides and dioxins, have been previously associated with adult-onset diabetes. In non-occupationally exposed adults, serum levels of the organochlorine pesticides oxychlordane and trans-nonachlor were associated with insulin resistance,24 and the sum of three organochlorine pesticides (p,p′-DDE, trans-nonachlor and hexachlorobenzene) was associated with incident diabetes.11 While we had data on seven organochlorines, only dieldrin was significantly associated with an increased diabetes risk. Heptachlor, but not dieldrin, was associated with increased diabetes risk among the AHS licensed pesticide applicators.4 We provide the previously published associations from studies of diabetes in applicators and gestational diabetes in spouses in table 4 in order to facilitate comparison of those previous findings with our results. Heptachlor and dieldrin are both cyclodienes, a category of structurally similar organochlorine insecticides. Chlordane, another cyclodiene, was associated with increased diabetes risk in the previous study of AHS applicators (table 4). The remaining cyclodiene insecticide examined here, aldrin, was associated with somewhat elevated risk of diabetes in both the present study and the previous study of applicators, although neither estimate reached statistical significance (table 4).
The herbicides 2,4,5-T and 2,4,5-TP, which were associated with diabetes here, were historically contaminated during production with dioxins.25 Dioxins have been previously linked with incident diabetes in highly exposed populations.21 ,26 2,4,5-T was associated with gestational diabetes among women in the AHS;17 however, there was no association with (non-gestational) diabetes among applicators.4 A previous study of high dioxin exposure in Seveso, Italy, showed positive associations with diabetes in women only,23 raising the possibility that dioxin-contaminated pesticides may have different effects in men and women. Another study of persistent organochlorine pesticides 2,2′4,4′5,5′-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE) observed that the strength of association between each chemical and diabetes differed between men and women.10 The possible biological mechanisms responsible for sex-specific associations between persistent organic pollutants and diabetes have not been established.
Notably, the organochlorine and dioxin-contaminated pesticides associated with diabetes in this study have been off the market in the USA for 30 years or more. Consequently, use was more common among older cohort members. Because age was the time scale in our analyses, however, confounding by age is unlikely to explain the observed associations of specific organochlorines with incident diabetes.
Organophosphate pesticides have also been linked with diabetes and associated conditions. In animal studies, exposure to the organophosphate pesticides parathion, diazinon and chlorpyrifos during the neonatal period produced insulin resistance and altered lipid metabolism later in life.5 In the AHS, certain organophosphate pesticides have been associated with diabetes previously (table 4). Ever-use of the organophosphate phorate was associated with gestational diabetes among women who used any pesticides during their first trimester of pregnancy17 and also with incident diabetes among predominantly male licensed pesticide applicators.4 While the gestational diabetes analysis was also conducted among female spouses in the AHS, it is unlikely that the current results are driven by those findings. Only 40% of the spouses in this analysis were included in the previous analysis, and only 3.6% of the gestational diabetes cases from that analysis were among the 688 incident diabetes cases included here. Two other organophosphates, coumaphos and terbufos, were also associated with increased diabetes risk in this analysis; these associations did not reach statistical significance in the present study, but the findings are consistent with the previous study of licensed pesticide applicators4 (table 4).
We considered BMI as a confounder due to its strong association with the outcome, and for the purpose of comparability with the previous study of applicators. However, some researchers have hypothesised that certain environmental pollutants may act as ‘obesogens’ and promote weight gain in exposed individuals.27 If pesticides had obesogenic effects, then BMI could actually be a causal intermediate in a hypothesised causal relationship between pesticide use and diabetes. In this scenario, adjustment for BMI could bias the effect estimate, typically (though not always) toward the null.28 Even after adjusting for BMI, however, we observed positive associations between individual pesticides and incident diabetes.
To our knowledge, this study is the largest to date of diabetes among women who applied pesticides, particularly agricultural pesticides. Its prospective design allowed us to assess incident diabetes reported 5–10 years after enrolment. Women provided detailed information on their pesticide use, so we were able to evaluate individual pesticides rather than chemical classes. The large number of women who used pesticides in this cohort allows an informative comparison with previous studies of diabetes among predominantly male pesticide users. The pesticides most frequently used by spouses in the AHS are glyphosate, carbaryl, malathion, 2,4-D and diazinon.18 None of these commonly used pesticides were associated with diabetes in the present study.
We relied on self-reported pesticide use information, which may result in some exposure misclassification. However, the reliability of self-reported ever use of specific pesticides by applicators in the AHS was high, ranging from 79% to 88% exact agreement based on two questionnaires completed 1 year apart in a sample of the cohort.29 We expect that the reliability of reporting among spouses involved in pesticide application would be similar. We see no reason to suspect that exposure misclassification would be differential with respect to subsequent diabetes diagnosis. There is little potential for recall bias in exposure reporting because pesticide exposure was recorded at enrolment before the onset of incident diabetes.
The outcome of this study was self-reported diabetes diagnosis. The validity of self-reported diabetes among postmenopausal women was assessed in another cohort.30 In that study, 74% of women who reported a diagnosis of diabetes at baseline were also found on examination to have elevated fasting glucose that met the diagnostic criterion for diabetes (at least 126 mg/dL). This result suggests that self-reported diabetes in the AHS cohort may have a similar positive predictive value for clinical diabetes.
Our model building strategy relied on the use of a common base model to evaluate each individual pesticide exposure in order to facilitate comparison of the results across pesticides. The base model used age as the time scale and included state and BMI, all three of which are known to be strongly associated with diabetes risk. If 45 independent statistical tests were run with a type 1 error rate of 0.05, between two and three of these tests would be expected to appear statistically significant by chance alone, of which approximately half would be in the inverse direction. We observed five statistically significant adjusted associations between ever-use of pesticides and incident diabetes. None of these associations were in the inverse direction. Of course, our tests were not fully independent. However, correlation among pesticides did not explain our findings.
Overall, our findings suggest that in women, as well as men, increased risk of diabetes is associated with the use of specific pesticides. Our results are consistent with previous studies reporting an association between organochlorines and diabetes and add to growing evidence that the use of certain organophosphates also may be associated with diabetes risk. The consistent associations between ever-use of specific pesticides (such as phorate) and diabetes across multiple studies in this cohort should provoke more focused investigation of these chemicals. Further research should also examine whether a dose–response relationship may exist between the use of specific pesticides and incident diabetes in women.
A poster describing these findings was presented at the 45th Annual Meeting of the Society for Epidemiologic Research in Minneapolis, MN on 27–30 June 2012, and an abstract was published in a supplement to the American Journal of Epidemiology.
Contributors JAH and APS conceived of the analysis. JAH, APS, FK, DPS and DMU contributed to the study design. APS, DMU and SL contributed to data analysis. JAH, APS, FK, DMU, DPS and SL contributed to writing and editing the manuscript. JAH is the guarantor of this work.
Funding This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01 ES049030) and National Cancer Institute (Z01 CP044008). APS was supported by an extramural award (1-F30-ES022126-01) from the National Institute of Environmental Health Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Competing interests None.
Ethics approval NIH IRB.
Provenance and peer review Not commissioned; externally peer reviewed.