Grantee Research Project Results
2013 Progress Report: New Environmental Public Health Indicator Linking Organochlorine Compounds and Type 2 Diabetes
EPA Grant Number: R834795Title: New Environmental Public Health Indicator Linking Organochlorine Compounds and Type 2 Diabetes
Investigators: Chambers, Janice E. , Crow, John Allen , Ross, Matthew K. , Wills, Robert W.
Institution: Mississippi State University
EPA Project Officer: Hahn, Intaek
Project Period: April 1, 2011 through March 31, 2014 (Extended to March 31, 2016)
Project Period Covered by this Report: April 1, 2013 through March 31,2014
Project Amount: $500,000
RFA: Exploring Linkages Between Health Outcomes and Environmental Hazards, Exposures, and Interventions for Public Health Tracking and Risk Management (2009) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Human Health
Objective:
The overall objective of this project is to develop an environmental public health indicator (EPHI) by investigating the linkage between soil residues of a class of persistent organic pollutants (i.e., organochlorine insecticides and their stable metabolites/degradates), levels of these compounds in people and a disease with which they are implicated, type 2 diabetes. The proposed EPHI would be serum levels of these organochlorine (OC) compounds.
There are two linked hypotheses: 1) Environmental (soil) levels of OC compounds and serum levels of these compounds in people residing in a region of intense agricultural crop production are greater than levels in soil and people from a less intensely farmed region. 2) A quantitative relationship exists between serum levels of OC compounds and the prevalence of type 2 diabetes.
Progress Summary:
Aspects of Aim 1, the environmental soil sampling and analysis, were conducted during the first year of the project. Soil samples from two regions in the state of Mississippi, the Delta region and a non-Delta region, were collected and analyzed for select OC compound levels. The objective of this aim was to provide the environmental data that would constitute part of the Environmental Public Health Indicator (EPHI). The hypothesis underlying this portion of Aim 1 was: environmental (soil) levels of select organochlorine (OC) compounds would be higher in the soils from the highly agricultural Delta region than in soils from the less agricultural non-Delta region. The proposed work was to collect 40 soil samples in each of the two locations. We decided to increase that number to 60 to represent more locations in the two regions.
The second year of the grant activities concentrated upon initiating the human subjects research by obtaining the IRB approvals from both Mississippi State University and the Veterans Administration (VA), obtaining approval of these approvals by EPA, obtaining a randomized patient list from which the subjects were recruited, and conducting the organochlorine compound analysis of the serum samples. The procedures at the Sonny Montgomery Veterans Administration Hospital in Jackson, MS, involved an IRB application which was approved, followed by a review by the hospital’s Research and Development Committee which approved the protocol, followed by the construction of a randomized patient list of individuals who met the criteria for the research project (males who lived in the 18-county Delta region or who lived in a select part of Mississippi that was the non-Delta region, and who were not service-connected to possible TCDD (“dioxin”) exposure during the Vietnam conflict. (Note: TCDD has been implicated in the literature for association with type 2 diabetes, so this potential confounder was eliminated). As patients who were on the randomized list were noted to be scheduled for an appointment, they were telephoned, apprised of the research project, and asked if they were interested in participating. Those who provided a positive response were contacted when they came into the hospital for their scheduled appointment, provided the information about the project including the Informed Consent Form, and had their questions answered by the Investigator at the VA (Dr. Dana Jones) or her Research Associate (both of whom were trained and approved for human subjects research). Those subjects still interested in participating signed the Informed Consent Form, were provided a copy of the form, and allowed the collection of extra tubes of blood for the purposes of this project. The blood was centrifuged and the serum collected and frozen. A code was established to provide to the subject samples and information for the purpose of deidentification of subjects. An information sheet was filled out on each subject with clinical and demographic information. The code identifying the subjects was retained at the VA under lock. The deidentified information sheet and serum samples were supplied to Mississippi State University. At the time of the submission of this report, 198 of the 300 approved subjects have been recruited and their samples analyzed.
The information from the patient information sheet was entered into a spreadsheet. The information sheets are maintained securely at Mississippi State University under lock.
Analysis of OC compounds in serum was performed by gas chromatography/mass spectrometry (GC/MS) following organic solvent extraction. The methodology, developed initially by DPX labs (Columbia, SC) for analysis of pesticides in fruits and vegetables, was modified in our laboratories for OC extraction from human plasma or serum. An internal standard containing C13 p,p’-DDT and C13 trans-nonachlor in hexane was added to 1 mL of serum. Acetonitrile was added to the sample to precipitate proteins. Following centrifugation, the resultant supernatant was mixed with deionized water was added to the supernatant and the mixture was aspirated into a DPX disposable pipette solid phase extraction column (DPX). OC compounds were eluted from the sorbent matrix, concentrated, and resuspended. Concentrations of the target OC compounds were determined by isotope dilution GC/MS. Extracts were analyzed using an Agilent Technologies 6890N gas chromatograph connected to a 5975C triple-axis mass spectrometer. A targeted mass analysis was performed in electron ionization (EI+) mode using single ion monitoring (SIM) for the analytes described above. Quantification and confirmation ions were monitored for each analyte and its respective isotopically labeled internal standard. Limits of quantitation were 100 pg/mL serum (14.8 ng/g serum lipid) for trans-nonachlor, oxychlordane, and p,p’-DDE. Areas under the curve were converted to pg/mL utilizing a standard curve. Values were adjusted for serum lipid content from the clinical data provided by the VA.
It was anticipated that the Delta soils would contain higher levels of these legacy OC insecticides (or their degradation products) than the non-Delta soils, because of the much higher historical levels of agricultural activity and pesticide use in the Delta compared to the non-Delta. The results, as reported last year, have been consistent with this anticipation, with more Delta than non-Delta soils having OC compound residues and, for the samples that did have quantifiable residues, about 10-fold higher levels of DDE in the Delta soils than in the non-Delta soils.
There were 98 diabetics and 100 non-diabetics in the subjects recruited thus far. No oxychlordane was quantifiable in any of these samples, and very few (only about 16%) of the samples had quantifiable trans-nonachlor, but the majority of the samples (about 70%) had DDE (Table 1).
Table 1: Numbers of Subjects |
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|
|
|
Above Quantitation Limits |
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|
Total Number |
Average Age |
Oxychlordane |
Trans-nonachlor |
p,p'-DDE |
Non-diabetic |
100 |
66.2 |
1 |
14 |
68 |
Diabetic |
98 |
66.1 |
0 |
18 |
70 |
A wide range of OC compound levels were observed (Table 2). There was a higher average concentration of DDE and of trans-nonachlor in diabetics than non-diabetics on both a volume of serum basis and as adjusted for lipid content; this is the relationship anticipated. However, it must be borne in mind that data collection is still incomplete and the trans-nonachlor data result from a very small number of subjects; therefore no statistical analysis was conducted at this point.
Table 2. Serum concentrations (averages and ranges) of trans-nonachlor and p,p'-DDE, expressed on both a volume basis and adjusted for serum lipid |
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|
ng/mL Serum |
ng/mL Serum |
ng/g Lipid |
ng/g Lipid |
|
Trans-nonachlor |
p,p'-DDE |
Trans-nonachlor |
p,p'-DDE |
Non-diabetic |
1.65 |
6.44 |
239.1 |
1,128 |
Diabetic |
1.70 |
7.02 |
289.9 |
1,271 |
Range |
0.19-5.12 |
0.09-59.61 |
19.4-825.3 |
8-14,391 |
Because data collection is still on-going, no statistical analysis comparing levels in diabetics to non-diabetics was conducted, nor was any analysis of the Delta compared to non-Delta subjects, or comparing African Americans to Caucasians. However the data to date are compiled into Table 3 below. As mentioned above, the overall mean of DDE is somewhat higher in the diabetics compared to the non-diabetics; while the lower end of the range of values from diabetics and non-diabetics were similar, the upper end of the range was considerably higher in diabetics than in non-diabetics. Those subjects from the agricultural Delta region showed much higher average levels of DDE than those from the non-Delta region, as expected, although the higher end of the range of values was in the non-Delta group. We have no information on residence history or occupation of the subjects in the study. Lastly the African American subjects displayed considerably higher DDE levels and displayed a much wider range of values than the Caucasian subjects.
Table 3. Serum levels of DDE from subjects residing in Mississippi |
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Group |
N |
Detects |
mean |
range |
|
N |
% |
||||
Normal |
100 |
68 |
68 |
1,128 |
8-9,365 |
T2D |
98 |
70 |
71 |
1,271 |
10-14,391 |
|
|||||
Delta |
77 |
58 |
75 |
1,500 |
8-10,572 |
Non-Delta |
121 |
77 |
64 |
976 |
10-14,391 |
|
|||||
African-American |
81 |
56 |
70 |
1,869 |
8-14,391 |
Caucasian |
117 |
79 |
67 |
727 |
10- 3,166 |
The results of both years indicate that DDE is rather generally distributed throughout soils in the Delta region, and only infrequently observable in the non-Delta region. In soils that had quantifiable levels of DDE, average levels were an order of magnitude higher in the agricultural Delta region than in the largely non-agricultural non-Delta region. Therefore the likelihood of exposure to people from dust and consequently contamination of food and water, as well as contact surfaces, will be substantially higher for residents of the Delta than the non-Delta. This environmental parameter is consistent with the hypothesis underlying this project’s proposed Environmental Public Health Indicator. Early results from the human samples indicate higher levels of DDE in the serum of diabetics compared to non-diabetics, which is consistent with the hypothesis underlying this project. If this difference between diabetics and non-diabetics is maintained through the rest of the samples, then DDE concentrations in serum may well be a possible biomarker for risk of type-2 diabetes.
Future Activities:
Subject recruitment through the VA clinic has improved, so subject recruitment will continue into the fourth year. The additional research activity will include continuing collection of the human blood samples and processing them for quantification of OC compounds. When data collection is complete, statistical analysis of the data will be conducted, including construction of statistical models that will determine whether the proposed Environmental Public Health Indictor (i.e., serum levels of OC compounds) is verified.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this projectProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.