Title: In Vitro Analysis of the Effects of Acute and Chronic Phthalate Exposures on Leydig Cell Testosterone Production, and the Molecular Mechanisms Involved

Project Summary
By interfering with normal hormonal function and action, endocrine-disrupting chemicals (EDCs) such as the
plasticizer di-(2-ethylhexyl) phthalate (DEHP) can have negative effects on the male reproductive system. In
previous studies, Dr. Traore found that in utero exposure to DEHP can result in reduced serum testosterone
levels in the adult. In studies conducted in vitro, he found that acute (short-term) exposure to mono-2-ethylhexyl
phthalate (MEHP), a metabolite of DEHP, can inhibit testosterone production by isolated Leydig cells and
progesterone production by MA-10 mouse tumor Leydig cells. Additionally, he found that MEHP can negatively
affect mitochondrial function and cholesterol translocation in MA-10 cells. Until recently, it has not been feasible
to conduct long-term studies using Leydig cells isolated from the testis because although these cells are able to
produce high levels of testosterone, they do not do so for longer than 1-2 days. Therefore, it has not been feasible
to use isolated Leydig cells to examine the direct, long-term effects of environmentally relevant concentrations
of EDCs, or to critically evaluate mechanisms involved in phthalate actions. Dr. Zirkin’s lab at the Johns Hopkins
Bloomberg School of Public Health (JHSPH) recently developed a method by which Leydig cells can be derived
from the in vitro differentiation of stem cells associated with the seminiferous tubules, and that the derived Leydig
cells are able to produce and sustain high levels of testosterone production for several months. The availability
of these cells will be of great value for our proposed studies of the effects on testosterone production
of acute, chronic, and repeat exposures of Leydig cells to environmentally relevant phthalate
concentrations, and particularly so for addressing the molecular mechanisms involved in phthalate-
induced reductions in testosterone. The studies that we propose in this R15 renewal application will:
(1) increase our understanding of the mechanism by which environmental stressors mediate the inhibition of
testosterone hormone production; and (2) provide new insights regarding multiple exposure of Leydig cells to
phthalates, chemicals to which we are exposed throughout life; and 3) address whether early exposures
predispose susceptibility to subsequent non-toxic dose exposure later in life. Campbell University has
outstanding medical students, graduate students and undergraduates who will be involved in the proposed
research. With funds from the REAP grant that the current application seeks to renew, Dr. Traore supported and
trained many students in his lab at Campbell University, an institution located in a rural, underserved area of
North Carolina. Renewal of the grant will allow Dr. Traore to continue and greatly extend ongoing research
while increasing the interest in basic science and/or clinical research of the involved students. Moreover, his
research success undoubtedly will influence attract other Campbell University faculty to seek funding to develop
research programs in their labs.