Project Number: VOO8006 Project Title: Investigations of Pesticides as Endocrine Disruptors Division: DART Project Officer (PO): William Moorman PO Degree: M.S. PO Area of Expertise (scientific discipline): Toxicology Project Start Date: 10/97 Project End Date: 09/98 Total Budget over the Project Period: $38,159 Goal/Sub-Goal: Chronic Effects of Pesticide Exposure/Health Outcomes Research Challenge/Issue: Adolescence is a growth period where secondary sex development is under critical endocrine control. Exposures during the summer months to adolescents on the farm present a sensitive population to agrichemicals known as a group to contain numerous endocrine disruptors. Recent reports suggest that exposure to agricultural products and other environmental pollutants contribute to increasing incidences of impaired reproduction, developmental abnormalities, and cancers (breast, testis and prostate) (Colburn et al 1993; Safe, 1995). Studies evaluating health effects resulting from ED in humans often focus on evaluating endocrine levels and struggle or fail to detect the significant impact of these exposures. In 1995, 2.6 million teenagers were employed on the farm and 70 die each year (Executive Summary, NIOSH Child Labor Working Team Document) This Document states that “Etiological Research to identify Risk Factors Leading to Work Injuries and illnesses of Children and Adolescents, With Particular Attention to Risk Factors Which May be Specific to Youths” is needed. The importance of direct occupational exposure to millions of agricultural workers applying chemicals with a known potential for endocrine disruption (pesticides, herbicides, fungicides) presents a major concern to occupational reproductive health scientists. The EPA found that except for cases involving workplace exposure to chemicals, there has been no clear causal relationship shown between these chemicals and hormonal disruptions leading to adverse health effects. The importance of mixtures is emphasized by recent reports showing that combinations of two weak environmental estrogens such as dieldrin and endosulfan were 1000 times more potent than each chemical alone (Arnold et al Science Vol 272, June 7, 1996). Adolescence is therefore a time of dramatic neuroendocrine changes required for sexual maturation. Hormonal mimicking or inhibiting chemicals can present significant impairment during this critical period. Vinclozolin was shown to be an anti-androgen affecting male offspring. A majority of teenagers working on farms are male, therefore, Vinclozolin, a potent antiandrogenic fungicide identified in toxicology studies was selected for evaluation of effects on adolescence. Details of the toxicology studies were reviewed and agreed upon by a review board of the National Toxicology Program. The peripubertal phase for the rabbit was determined to be between the third and fourth months. The rabbit model was chosen because it can readily provide semen for analysis as in humans. A 2 month dosing period was therefore initiated at 3 months of
age and carried through the fourth month. The rabbits were then held until fully mature (6 months of age). Semen was collected and evaluated from sexually mature males on a weekly, schedule for 5 weeks to maximize sperm output. This project was part of an integrated system focusing on reproductive health of workers by providing very specific information (hazard identification) necessary to designing sensitive surveillance studies Activities: The DART/RHAT Program for Reproductive Health included studies of pesticides known as endocrine disrupting chemicals. An animal model was developed to evaluate male reproductive effects resulting from antiandrogenic endocrine disrupting chemicals. Mixtures are generally formulated with insecticides and fungicides that are known to be synergistic (estrogenic and antiandrogenic). Male rabbits were exposed during their adolescence to demonstrate effects and provide guidance for the human study. Resources included 38,000 in discretionary funds and 1.5 MY. Outputs: An animal model was developed to assess reproductive effects of pesticides (endocrine disruptors) during the peripubertal period. The rabbit was selected as the test species, since it has proven to be an effective species for modeling reproductive toxicant effects in the male. The peripubertal phase for the rabbit was determined to be between the 3rd and 4th months of age. The rabbit model was used to assess vinclozolin. Under the conditions of the study, vinclozolin reduced pubertal weight gain, weight of the accessory sex glands at maturity, but paradoxically was associated with increased sperm count. Additionally, a model was developed to prioritize toxic chemicals for research. This model is ideal for use to prioritize chemicals found in agriculture, especially pesticides. Journal Publications: Andrologia 32,285-293 2000, Male Adolescent Exposure to Endocrine Disrupting Pesticides: Vinclozolin Exposure in Peripubertal Rabbits. Moorman WJ, Cheever KL, Skaggs SR, Clark JC, Turner TW, Marlow KL, Schrader SM. Copy of abstracts. Male Adolescent Exposure to Endocrine Disrupting Pesticides: Vinclozolin Exposure in Peripubertal Rabbits. W. J. Moorman, K.L. Cheever, S.R. Skaggs, J.C. Clark, T.W. Turner, K.L. Marlow and S.M.Schrader List of presentations. Male Adolescent Exposure to Endocrine Disrupting Pesticides: Vinclozolin Exposure in Peripubertal Rabbits. W. J. Moorman, K.L. Cheever, S.R. Skaggs, J.C. Clark, T.W. Turner, K.L. Marlow and S.M.Schrader Presented at International meeting: Molecular Aspects of Male Reproductive Toxicology, Giessen Germany 11/13/99 Method for Quantitation of Tissue Vinclozolin and Metabolites Using a Benchmate Automated SPE with GC/MS. K.L. Cheever, K.L.Marlow, S. R. Skaggs, J.C. Clark, T.W. Turner, W. J. Moorman, D. G. Debord Presented at the Society of Toxicology Annual Meeting New Orleans LA. March 14-18 1999 Intermediate Outcomes:
Guides were provided to the identification of degradation products for biomonitoring of exposure to vinclozolin. Sentinal reproductive endpoints were identified and used in other studies. Reproductive disorders associated with antiandrogen exposure were identified. The main published article from this work has been referenced in the following journal articles: JOURNAL OF CHROMATOGRAPHY, 1115 (1-2): 8-18 MAY 19 2006 REPRODUCTION: 31 (4): 805-816 APR 2006 ANNALS NEW YORK ACADADEMY SCIENCE 1061: 18-32 2005; JOURNAL OF VET MEDICAL SCIENCE 66 (7): 847-853 JUL 2004; ENVIRON HEALTH PERSPECTIVES 109 (10): 1063-1070 OCT 2001. External Factors: Follow up phases of this research were terminated as a result of all animal and toxicological research being shifted from the Cincinnati Division (DBBS) to Morgantown (HELD). Future Directions: The study results will allow us to design and conduct human studies by providing more specific information on the specific vinclozolin target sites within the male reproductive system and information about the urinary metabolites of vinclozolin which can be used for biomoniting of worker exposure.