Male Reproductive Tract Lesions at 6,12,and 18 Months of Age Following in Utero Exposure to Di(n-butyl) Phthalate

Abstract

In utero exposure of male rats to the antiandrogen di(n-butyl) phthalate (DBP) leads to decreased anogenital distance (AGD) on postnatal day (PND) 1, increased areolae retention on PND 13, malformations in the male reproductive tract, and histologic testicular lesions including marked seminiferous epithelial degeneration and a low incidence of Leydig cell (LC) adenomas on PND 90. One objective of this study was to determine the incidence and persistence of decreased AGD, increased areolae retention, and LC adenomas in adult rats following in utero DBP exposure. A second objective was to determine whether AGD and areolae retention during the early postnatal period are associated with lesions in the male reproductive tract. Pregnant Crl:CD(SD)BR rats were gavaged with corn oil or DBP at 100 or 500 mg/kg/day, 10 dams per group. Three replicates of rats (n = 30 rats per replicate) were exposed from gestation day 12 to 21 and the male offspring allowed to mature to 6, 12, or 18 months of age. Gross malformations in the male reproductive tract and histologic lesions in the testes were similar to those previously described. However, testicular dysgenesis, a lesion of proliferating LCs and aberrant tubules that has not been previously described in DBP-exposed testes, was diagnosed. The incidence of this lesion was approximately 20% unilateral and 7—18% bilateral in the high-dose group and was similar among all ages examined, implicating a developmental alteration rather than an age-related change. AGD and areolae retention were found to be permanent changes following in utero exposure to 500 mg/kg/day of DBP. Decreased AGD was a sensitive predictor of lesions in the male reproductive tract, relatively small changes in AGD were associated with a significant incidence of male reproductive malformations. In utero DBP exposure induced proliferative developmental lesions, some of which would have been diagnosed as LC adenomas by the morphological criteria set forth by the Society of Toxicologic Pathology. However, these lesions were dissimilar to traditional LC adenomas as the LCs were poorly differentiated and the lesions contained aberrant seminiferous tubules. While the morphology and incidence of this DBP-induced testicular developmental lesion has been fully characterized by this study, the detailed pathogenesis warrants further investigation.

Keywords

Di(n-butyl)phthalate in utero exposure male reproductive development testes Leydig cell adenoma testicular dysgenesis.

References

Barlow, N.J. and P.M.D. Foster (2003). “Pathogenesis of Male Reproductive Tract Lesions from Gestation Through Adulthood Following in utero Exposure to di(n—butyl) Phthalate.” Toxicol Pathol 31 (in press).

Boorman, G.A. , R.E. Chapin , and K. Mitsumori (1990). Testis and epididymis, Pathology of the Fischer Rat. G. A. Boorman , S. L. Eustis , M. R. Elwell , C. A. Montgomery , and W. F. MacKenzie (eds.). Academic Press, Inc., San Diego.

Gray, L.E., Jr. , C. Wolf , C. Lambright , P. Mann , M. Price , R.L. Cooper , and J. Ostby (1999). “ Administration of Potentially Antiandrogenic Pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and Toxic Substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) During Sexual Differentiation Produces Diverse Profiles of Reproductive Malformations in the Male Rat.” Toxicol Ind Health 15: 94—118.

Hanley, J.A. and B.J. McNeil (1982). “The Meaning and Use of the Area Under a Receiver Operating Characteristic (ROC) Curve. ” Radiology 143: 29—36.

Herbst, A.L. , R.J. Kurman , R.E. Scully , and D.C. Poskanzer (1972). “Clear-Cell Adenocarcinoma of the Genital Tract in Young Females. Registry Report.” N Engl J Med 287: 1259—64.

Imperato-McGinley, J. , Z. Binienda , J. Gedney , and E.D. Vaughan, Jr. (1986). “Nipple Differentiation in Fetal Male Rats Treated with an Inhibitor of the Enzyme 5 α-reductase: Definition of a Selective Role for Dihydrotestosterone .” Endocrinology 118: 132—7.

International Programme on Chemical Safety ( 1997). Environmental Health Criteria 189. Di-n-butyl Phthalate . World Health Organization: Geneva .

Lambright, C.S. , V.S. Wilson , J.R. Furr , C.J. Wolf , N. Noriega , and L.E. Gray, Jr. (2003). “Effects of Endocrine Disrupting Chemicals on Fetal Testes Hormone Production.” The Toxicologist 72(S-1): 272.

Lang, P.L. (1992). “Spontaneous Neoplastic Lesions and Selected Non-Neoplastic Lesions in the Crl:CDBr Rat.” Charles River Laboratory, Raleigh, NC.

10.

Lanning, L.L. , D.M. Creasy , R.E. Chapin , P.C. Mann , N.J. Barlow , K.S. Regan , and D.G. Goodman (2002). “Recommended Approaches for the Evaluation of Testicular and Epididymal Toxicity.” Toxicol Pathol 30: 507— 20.

11.

Larsson P.A. , M. Cano , L. Grenabo , J.E. Brorson , H. Hedelin , S. Pettersson , and S.L. Johansson (1989). “Morphological Lesions of the Rat Urinary Tract Induced by Inoculation of Mycoplasmas and Other Urinary Tract Pathogens .” Urol Int 44: 210—7.

12.

Latendresse, J.R. , A.R. Warbrittion , H. Jonassen , and D.M. Creasy (2002). “Fixation of Testes and Eyes Using a Modified Davidson's Fluid: Comparison with Bouin's Fluid and Conventional Davidson's Fluid.” Toxicol Pathol 30: 524—33.

13.

McConnell, R.F. , H.H. Westen , B.M. Ulland , M.C. Bosland , and J.M. Ward (1992). Proliferative Lesions of the Testes in Rats with Selected Examples from Mice, in Standardized System of Nomenclature and Diagnostic Criteria, Society of Toxicologic Pathology. STP/ARP/ AFIP, Washington, DC.

14.

McIntyre, B.S. , N.J. Barlow , and P.M.D. Foster (2001). “Androgen-Mediated Development in Male Rat Offspring Exposed to Flutamide in Utero: Permanence and Correlation of Early Postnatal Changes in Anogenital Distance and Nipple Retention with Malformations in Androgen-Dependent Tissues.” Toxicol Sci 62: 236—49.

15.

McIntyre, B.S. , N.J. Barlow , and P.M.D. Foster (2002). “Male Rats Exposed to Linuron in utero Exhibit Permanent Changes in Anogenital Distance, Nipple Retention, and Epididymal Malformations That Result in Subsequent Testicular Atrophy. ” Toxicol Sci 65: 62—70.

16.

McMartin, D.N. , P.S. Sahota , D.E. Gunson , H.H. Hsu , and R.H. Spaet (1992). “Neoplasms and Related Proliferative Lesions in Control Sprague-Dawley Rats from Carcinogenicity Studies. Historical Data and Diagnostic Considerations.” Toxicol Pathol 20: 212—25.

17.

Mitsumori, K. and M.R. Elwell (1988). “Proliferative Lesions in the Male Reproductive System of F344 Rats and B6C3F1 Mice: Incidence and Classification.” Environ Health Perspect 77: 11—21.

18.

Mylchreest, E. , M. Sar , R.C. Cattley , and P.M.D. Foster (1999). “Disruption of Androgen—Regulated Male Reproductive Development by di(n—butyl) phthalate During Late Gestation in Rats is Different from Flutamide.” Toxicol Appl Pharmacol 156: 81—95.

19.

Mylchreest, E. , M. Sar , D.G. Wallace , and P. M. D Foster (2002). “Fetal Testosterone Insufficiency and Abnormal Proliferation of Leydig Cells and Gonocytes in Rats Exposed to di(n-butyl) phthalate.” Reprod Toxicol 16: 19— 28.

20.

Mylchreest, E. , D.G. Wallace , R.C. Cattley , and P.M.D. Foster (2000). “Dose-Dependent Alterations in Androgen-Regulated Male Reproductive Development in Rats Exposed to di(n-butyl) phthalate During Late Gestation.” Toxicol Sci 55: 143—51.

21.

National Research Council (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press: Washington, DC.

22.

Neumann, F. , R.V. Berswordt-Wallrabe , W. Elger , H. Steinbeck , J.D. Hahn , and M. Kramer (1970). “Aspects of Androgen—Dependent Events as Studied by Antiandrogens. Recent Prog Horm Res 26: 337—410.

23.

Newbold, R. (1995). “Cellular and Molecular Effects of Developmental Exposure to Diethylstilbestrol: Implications for Other Environmental Estrogens .” Environ Health Perspect 103 (Suppl 7): 83—7.

24.

Newbold, R.R. , E.P. Banks , B. Bullock , and W.N. Jefferson (2001). “Uterine Adenocarcinoma in Mice Treated Neonatally with Genistein.” Cancer Res 61: 4325—8.

25.

Newbold, R.R. , R.B. Hanson , W.N. Jefferson , B.C. Bullock , J. Haseman , and J.A. McLachlan (2000). “Proliferative Lesions and Reproductive Tract Tumors in Male Descendants of Mice Exposed Developmentally to Diethylstilbestrol .” Carcinogenesis 21: 1355—63.

26.

Newbold, R.R. , A.B. Moore , and D. Dixon (2002). “Characterization of Uterine Leiomyomas in CD-1 Mice Following Developmental Exposure to Diethylstilbestrol (DES) .” Toxicol Pathol 30: 611—16.

27.

Qureshi, S.R. , E. Perentes , R.A. Ettlin , M. Kolopp , D.E. Prentice , and A. Frankfurter (1991). “Morphologic and Immunohistochemical Characterization of Leydig Cell Tumor Variants in Wistar Rats.” Toxicol Pathol 19: 280—6.

28.

Shultz, V.D. , S. Phillips , M. Sar , P.M.D. Foster , and K.W. Gaido (2001). “Altered Gene Profiles in Fetal Rat Testes After in Utero Exposure to di(n-butyl) Phthalate.” Toxicol Sci 64: 233—42.

29.

Takeuchi, H. , M. Ueda , M. Satoh , and O. Yoshida (1991). “Effects of Dietary Calcium, Magnesium and Phosphorus on the Formation of Struvite Stones in the Urinary Tract of Rats .” Urol Res 19: 305—8.

30.

Turner, K.J. , N.J. Barlow , M.F. Struve , D.G. Wallace , K.W. Gaido , D.C. Dorman , and P.M. Foster (2002). “Effects of in utero Exposure to the Organophosphate Insecticide Fenitrothion on Androgen-Dependent Reproductive Development in the Crl:CD(SD)BR rat.” Toxicol Sci 68: 174— 83.