Grantee Research Project Results
2008 Progress Report: Current-Use Pesticides: Assessing Exposure and Spermatoxicity
EPA Grant Number: R832515 aka R832098Title: Current-Use Pesticides: Assessing Exposure and Spermatoxicity
Investigators: Swan, Shanna Helen
Current Investigators: Swan, Shanna Helen , Thomas, Peter , Sparks, Amy
Institution: University of Rochester
Current Institution: The University of Texas at Austin , University of Iowa , University of Rochester
EPA Project Officer: Klieforth, Barbara I
Project Period: September 1, 2005 through August 31, 2010
Project Period Covered by this Report: January 1, 2008 through December 31,2008
Project Amount: $672,821
RFA: Application of Biomarkers to Environmental Health and Risk Assessment (2004) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Human Health
Objective:
Since our Annual Report of December 31, 2007, we received permission to obtain BPA under this grant on 333 men in addition to 256 pesticide measurements. These BPA results were received in 2008. However, only a subset of the pesticide measurements were received in 2008, so those analyses could not be carried out. Once all pesticide measurements are received (CDC has promised all results by April 2009), we will be able to carry out the analyses to meet our objectives.
The objectives in the research plan (submitted and approved in April 2005) are as follows:
Primary objectives: (additional aims in italics)
- Increase the precision of our prior estimates of exposure to current-use pesticides and compare these in agricultural (IA and MO) and urban (MN) areas.
- Estimate the strength of the associations between biomarkers of current-use pesticides and semen parameters across our three study centers, as well as within each center, and examine nine atrazine metabolites in relation to semen quality and compare to measures of association based on the mercapturate alone.
- Examine the effects of simultaneous exposure to multiple pesticides on semen quality and compare the fit of additive, sub-additive and super-additive models to these data.
- Measure associations between multiple urinary biomarkers of atrazine and serum biomarkers of atrazine.
- Measure levels of human sperm membrane progesterone receptor (hmPRα) in relation to both semen parameters and levels of current-use pesticides.
- Compare male and female levels of urine biomarkers of current use pesticides (including multiple metabolites of atrazine).
The change, approved in 2008 (described below and in our last annual report), will allow us to address the following objectives:
- Examine the effects of simultaneous exposure to multiple pesticides as well as to BPA and phthalates on semen quality and compare the fit of additive, sub-additive and super-additive models to these data, as well as the use of Structural Equation Modeling to examine the effects of these complex mixtures on semen quality. (This expands original Primary Objective 3 to a mixture including BPA).
- Examine factors that affect distribution of, and correlations between men’s exposure to these three classes of common environmental chemicals. (This expands original Primary Objective 4 to include associations among a wider class of chemicals, including BPA).
- Measure levels of human sperm membrane progesterone receptor (hmPRα) in relation to both semen parameters and levels of BPA. (This expands original Primary Objective 5 to include associations between hmPRα and BPA).
- Estimate the strength of the associations between biomarkers of BPA and semen parameters across our three study centers, as well as within each center. (This expands original Primary Objective 2 to BPA as well as pesticides. By obtaining BPA on the 77 CA men, we will be able to examine this in all four centers).
Because of the addition of BPA (and the corresponding decision not to obtain women’s pesticide measurements) we will not be able to examine prior Objective 6.
Progress Summary:
Reasons for delays in pesticide analysis
The methods for pesticide analyses at the Pesticide Laboratory at CDC, under the direction of Dana Barr, have undergone considerable development since pesticide analytes were originally obtained in this population in 2002. The analytic techniques have been improved and sensitivity and reliability are increased.
CDC has developed methods to measure nine atrazine metabolites, rather than the single metabolite (mercapturate) measured previously. This work has demonstrated that using the single (mercapturate) metabolite previously measured underestimates human exposure. Because one of our prior findings was a statistically significant association between the metabolite and semen quality, it was very important for this current analysis to include all nine metabolites. We are assured that this is now possible, and there will be no additional charge for obtaining all nine metabolites.
In addition, the large backload of samples that the laboratory faces due to the demands of analyzing NHANES samples has delayed analyses for research, including ours.
Reasons for adding BPA analysis of our male samples
It has only recently been established that the environmental chemical BPA is ubiquitous in the U.S. population. Further, although there are a large number of rodent studies suggesting its toxicity, there are almost no published human studies that have examined this potentially toxic and ubiquitous compound. There is a large body of animal data suggesting that BPA can alter sperm production, possibly through oxidative stress, but whether this occurs in humans exposed to environmental levels has not been investigated. Because we have previously obtained sperm parameters on our subjects, we will be able to address this important question. We will also be able to examine human sperm membrane progesterone receptor (hmPRα) in relation to BPA concentration.
Ability to examine a mixture of three important environmental chemical classes
We have also obtained concentrations of 15 phthalate metabolites on these men, under separate funding. Thus, under the proposed research plan, we will be able to examine the joint effects of pesticides, phthalates and BPA in men from MO, MN and IA, and for 77 men from CA, we will be able to examine phthalates and BPA. The problem of complex mixtures of environmental chemicals and their joint effects on health outcomes is perhaps the biggest challenge facing environmental epidemiology today. Recent work (e.g., Gray et al. 2006) has shown that a wide range of chemicals can act in a dose-additive manner. We are working with Sally Thurston, biostatistician, to develop Bayesian methods to address this problem. This combined data set would provide a unique opportunity to test these methods.
QUALITY ASSURANCE
Sources of data for this study are: questionnaires completed by study subjects (pregnant women, their partners and their partners mothers), men’s physical exams, men’s hormone assays (urinary FSH), and pesticide measurements (urinary metabolites). Provisions to minimize errors and maximize data accuracy and completeness were described in the application and are being followed. Dana Barr at the CDC oversees the quality control (QC) program for pesticide assays, and Antonia Calafat oversees the quality control of BPA and phthalate assays. These laboratories at the CDC are certified by auditors under the Clinical Laboratory Improvement Act (CLIA #11D0668290). These laboratories have extensive experience in analyzing toxicants in biological specimens. The QC limits and means are established by at least 200 QC sample runs and represent expected normal laboratory performance. Each batch of samples will include one blank, one QC material and 10 sample urines. Any sample associated with an out-of-control QC value will be reanalyzed or not reported. QA material values will be considered out- of control if they fall into any of these three groups: any value outside the 99% confidence limit, any two sequential values outside the 95% limit, and 12 sequential values above or below the mean. The failure rate for the laboratory is less than 3%.
YEAR 3 PROGRESS AND ACCOMPLISHMENTS:
- In the third study year, CDC provided pesticide results on 257 men (MO = 73, MN = 102, IA = 82) in August 2008 (See Table 1). Samples from an additional 83 men from IA have not yet been analyzed. In addition, the eight atrazine metabolites have not yet been received.
- Samples from the 73 MO men included in the August 2008 analyses had previously been analyzed for pesticides (2002-2003). We conducted an analysis to compare these repeat measurements and found considerable variation within the men (See Table 2). This has been discussed extensively with Dana Barr who is confident in the reliability of the 2008 values. to confirm the reliability of these, CDC has agreed to rerun, at no charge, samples from 20 men who were analyzed in August 2008. These samples have been sent to CDC and results are expected shortly.
- Because we could not conduct pesticide analyses (aside from those to examine the quality of the 2008 data), we conducted analyses on several factors potentially related to semen quality, as well as on phthalates and semen quality. We examined the comparability of the first and second semen sample (Stokes-Riner et al. 2007). We have identified several factors that affect semen quality, including the adverse effects of psychosocial stressors (Gollenberg et al. 2009), caffeine drinks (Jensen TK et al 2009), and the protective effect of antioxidants (Lewis et al. 2009). This information will allow us to increase the precision of our effect estimates for pesticides by controlling for these sources of variability.
REPORT ON REPEAT ANALYSIS OF URINE SAMPLES FROM 73 MISSOURI MEN
HISTORY:
In 2002-2003, the CDC ran pesticide analyses on a limited number of samples from MO and MN men. (That part of our study was unfunded at that time). We subsequently obtained a STAR grant that funded additional pesticide analyses. Those samples were run in August 2008. Included in the samples run in August 2008 were urine samples from 73 MO men whose urine had been previously analyzed. This gave us the opportunity to compare two estimates of pesticide concentration in the same urine sample (or possibly in a second sample from the same man). All urine samples were obtained at the first study visit and stored at UC Davis under identical conditions.
METHODS
We looked at summary statistics in the first and second analyses, and did this separately for Group I, which included 34 samples that were run as part of our initial pesticide analyses (data published in Swan et al 2003) and Group II, 39 MO men whose samples were rerun at CDC later (in part in response to queries from Monsanto). Because these first analyses were done at different times, in this report we present results separately for these groups. In this report, we show means, medians and correlations for three pesticides (atrazine, alachlor and acetochlor). It is likely that results would be similar for other pesticides. A second data analyst reran all results in this report.
RESULTS
Changes in means/medians
Table 1 shows the mean and median values for the first vs. the repeat analysis. Values in the 2008 analyses were uniformly higher than those reported initially. For example, in Group I median alachlor was 2.2X higher in the repeat analysis than measured initially, 3.2X higher for acetochlor and atrazine.
Table 1: Mean/Median for urinary metabolite concentration of three pesticides in repeated analyses of samples from 73 Missouri men.
Changes in percent less than LOD
The much higher values reported for the 2008 analyses are also reflected in the much lower percentages of samples falling below the LOD. Although some of the LODs changed between 2002 and 2008, these changes are unlikely to account for these discrepancies.
Table 2: Percent less than LOD for urinary metabolite concentration of three pesticides in repeated analyses of samples from 73 Missouri men.
Correlation between sample values
We considered the possibility that the entire distribution of pesticide values had shifted, but that the rank of an individual man (or sample) would remain relatively unchanged between the first and the repeat analysis. Therefore, we calculated (Pearson and Spearman) correlation coefficients between the ranks of the sample results on the first and second analysis. These are shown in Table 3.
Table 3: Pearson and Spearman correlation coefficients between ranks on first and second analyses of urinary metabolite concentration of three pesticides in 73 Missouri men.
Conclusion
There are large unexplained discrepancies between repeat values in samples from the same man. However, Dana Barr assures us that the methods currently used are reliable. We will send 20 samples to CDC for reanalysis. If these are in close agreement to measurements received in 2008, we will proceed and will only use 2008-09 data in future analyses.
Future Activities:
We have a commitment from Dana Barr at the Pesticides Laboratory in CDC to complete all pesticide analyses by April 2009. Therefore, in the subsequent reporting period, we expect to complete the following:
- Obtain remaining urinary metabolites of 15 pesticide metabolites in men from MO, MN and IA for whom we have measured phthalates, semen parameters and hormone levels.
- Analyze the relationships between these pesticide concentrations and sperm concentration, motility and morphology, and prepare a manuscript on this topic.
- Obtain urinary metabolites of bisphenol-A (BPA) in the same 300 men from MO, MN and IA for whom we have measured phthalates, semen parameters and hormone levels.
- Analyze the relationships between BPA concentration and sperm concentration, motility and morphology, and prepare a manuscript on this topic.
- Develop Bayesian methods using structural equation modeling to examine the effects on semen quality of exposure to mixtures of these chemicals.
- Examine associations between pesticide concentration as well as BPA concentration on hmPRα in 77 IA men and prepare a manuscript on this topic.
However, to complete this work, we request an extension in time until December 31, 2009. We are also requesting that the PI be given permission to decrease her time to 5%. We would like to also request support for a second statistical analyst@ 50% for 9 months (total cost $20,000), so that we will have the staff necessary to complete these analyses.
We will continue to use the equipment that was purchased in Year 1. Materials used for assays at CDC
will be those described in the application.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
Other project views: | All 13 publications | 12 publications in selected types | All 9 journal articles |
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Type | Citation | ||
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Cooper TG, Brazil C, Swan SH, Overstreet JW. Ejaculate volume is seriously underestimated when semen is pipetted or decanted into cylinders from the collection vessel. Journal of Andrology 2007;28(1):1-4. |
R832515 aka R832098 (2007) R832515 aka R832098 (2008) |
Exit Exit Exit |
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Gollenberg AL, Liu F, Brazil C, Drobnis EZ, Guzick D, Overstreet JW, Redmon JB, Sparks A, Wang C, Swan SH. Semen quality in fertile men in relation to psychosocial stress. Fertility and Sterility 2010;93(4):1104-1111. |
R832515 aka R832098 (2007) R832515 aka R832098 (2008) |
Exit Exit Exit |
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Jorgensen N, Liu F, Andersson A-M, Vierula M, Irvine DS, Auger J, Brazil CK, Drobnis EZ, Jensen TK, Jouannet P, Overstreet JW, Redmon JB, Sparks A, Toppari J, Wang C, Skakkebaek NE, Swan SH. Serum inhibin-b in fertile men is strongly correlated with low but not high sperm counts:a coordinated study of 1,797 European and US men. Fertility and Sterility 2010;94(6):2128-2134. |
R832515 aka R832098 (2008) |
Exit Exit Exit |
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Laumbach R, Tong J, Zhang L, Ohman-Strickland P, Stern A, Fiedler N, Kipen H, Kelly-McNeil K, Lioy P, Zhang J. Quantification of 1-aminopyrene in human urine after a controlled exposure to diesel exhaust. Journal of Environmental Monitoring 2009;11(1):153-159. |
R832515 aka R832098 (2008) R832097 (Final) R832144 (Final) |
Exit |
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Stokes-Riner A, Thurston SW, Brazil C, Guzick D, Liu F, Overstreet JW, Wang C, Sparks A, Redmon JB, Swan SH. One semen sample or two? Insights from a study of fertile men. Journal of Andrology 2007;28(5):638-643. |
R832515 aka R832098 (2006) R832515 aka R832098 (2007) R832515 aka R832098 (2008) R832515 aka R832098 (Final) |
Exit Exit Exit |
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Swan SH, Liu F, Overstreet JW, Brazil C, Skakkebaek NE. Semen quality of fertile US males in relation to their mothers' beef consumption during pregnancy. Human Reproduction 2007;22(6):1497-1502. |
R832515 aka R832098 (2007) R832515 aka R832098 (2008) |
Exit Exit Exit |
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Swan SH, Liu F, Overstreet JW, Brazil C, Skakkebaek NE. Reply: testis development, beef consumption and study methods. Human Reproduction 2007;22(9):2574-2575 (letter to the editor). |
R832515 aka R832098 (2007) R832515 aka R832098 (2008) |
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Supplemental Keywords:
RFA, Health, Scientific Discipline, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Biochemistry, Endocrine Disruptors - Human Health, pesticide exposure, altered sexual development, EDCs, endocrine disrupting chemicals, exposure studies, developmental biology, human growth and development, atrazine, agrochemicalsProgress 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.