Mark A. Kukucka and Hara P. Misra. 1993. The antioxidant defense
system of isolated guinea pig Leydig cells. Molecular and Cellular
Biochemistry, Volume 126, Number 1:1-7.

Department of Biomedical Sciences, Virginia-Maryland Regional College
of Veterinary Medicine, Virginia Polytechnic Institute & State
University, 24061 Blacksburg, Virginia, USA

Received: 23 March 1993  Accepted: 6 May 1993

Abstract: Utilization of highly enriched preparations of steroidogenic
Leydig cells have proven invaluable for studying the direct effects of
various hormones and agents on Leydig cell functionin vitro. However,
recent work indicates that isolated Leydig cells are often subjected
to oxygen (O2) toxicity when cultured at ambient (19%) oxygen
concentrations. Because intracellular antioxidants play an important
role in protecting cells against oxygen toxicity, we have investigated
the intracellular antioxidant defense system of isolated Leydig cells.
The cellular levels of several antioxidants including catalase,
glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase
(SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione
reductase and total glutathione were quantitated using enriched
populations of Leydig cells isolated from adult male guinea pig
testes. Compared to whole testicular homogenates, Leydig cells
contained significantly (P<0.01) less G-6-PDH, total SOD, glutathione
reductase and total glutathione, but significantly (P<0.001) more
glutathione peroxidase. Compared to hepatic values previously reported
in the guinea pig, Leydig cells contain nearly 400 times less
catalase, about 14 times less glutathione peroxidase and almost 11
times less glutathione reductase. Since G-6-PDH and glutathione
reductase are both necessary to regenerate reduced gluthathione (GSH)
which couples with glutathione peroxidase to breakdown hydrogen
peroxide (H2O2) under normal conditions, it is plausible that the
oxygen toxicity observed in isolated Leydig cells is due to the
intracellular accumulation of H2O2. Using the dichlorofluorescin
diacetate (DCF-DA) assay, we found that Leydig cells incubated in the
presence of 19% O2 produced significantly (P<0.001) higher levels of
H2O2 with time in culture compared to Leydig cells maintained at 3%
O2. These results support the hypothesis that the increased
susceptibility of isolated Leydig cells to oxygen toxicity may be due,
in part, to decreased amounts of certain antioxidant defenses and an
increased production of the reactive oxygen species H2O2.

Key words  Leydig cells - testes - superoxide dismutase - catalase
glutathione peroxidase - glutathione - hydrogen peroxide