[Toxicology] Potential relationship between non-enzymatic antioxidants vitamin C and oxytocin

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Sep 26, 2008, 7:47:43 AM9/26/08
to tox...@magpie.bio.indiana.edu

This is the first report regarding quantitation of enzymatic (eg. superoxide
dismutase, glutathione reductase & glucose-6-phosphate dehydrogenase) and
non-enzymatic (eg. glutathione) antioxidant levels in isolated Leydig cells
that I can find in the scientific literature:

Mechanisms by which hypoxia augments Leydig cell viability and
differentiated cell function in vitro (PhD Dissertation, 1993)

by Mark A. Kukucka, MS, DVM, PhD

Department of Biomedical Sciences
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute & State University
Blacksburg, Virginia 24061-0442


The 1980s heralded the discovery and identification of extra-pituitary
sources of the neurohypophysial hormone oxytocin in non-neural tissues of
several animal species. The presence, location and biosynthesis of
significant amounts of oxytocin in the ovarian corpus luteum was followed by
the immunocytochemical demonstration of an oxytocin-like peptide in the
testicular interstitial cells. Leydig cells, which comprise up to 80% of the
testicular intertubular cell population, are known to synthesize
testosterone in situ. Indirect evidence indicated that an oxytocin-like
peptide was also present in Leydig cells. The question arose whether this
peptide was synthesized de novo by Leydig cells or was taken up and stored
by the cells following biosynthesis at some other intra- and/or
extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are
known to augment the production of oxytocin in ovarian granulose cells,
varying concentrations of these two stimulants were used to monitor the
biosynthesis of oxytocin from isolated Leydig cells in culture.

Highly enriched populations of guinea pig Leydig cells were isolated using a
method that employed enzymatic dissociation and Percoll gradient
centrifugation. Since ambient oxygen tensions are toxic to cultured Leydig
cells leading to decreased steroidogenic capacity, the antioxidant defense
system of isolated Leydig cells was discerned. Decreased levels of several
antioxidants including superoxide dismutase, glutathione reductase,
glucose-6-phosphate dehydrogenase and total glutathione were measured. Using
the dichlorofluorescin (DCF-DA) assay, it was determined that isolated
Leydig cells were capable of accumulating hydrogen peroxide (H2O2). Leydig
cells maintained in an atmosphere composed of 19% oxygen produced H2O2 at a
faster rate than similar cells incubated at 3% oxygen.

Using a polyclonal antibody (Ab)-based immunoaffinity column, oxytocin
biosynthesis was monitored in Leydig cells incubated with a mildly
stimulating dose (0.1 ng/ml) of ovine LH for 24, 48 and 72 hours in the
presence of increasing concentrations of sodium ascorbate (1- 500 mM) under
culture conditions of hypoxia and normoxia. Following solid phase extraction
and immunoaffinity purification, sample supernatants were analyzed for both
testosterone and oxytocin content as measured by radioimmunoassay (RIA) and
high performance liquid chromatography-electrochemical detection (HPLC-ECD)
respectively. Hypoxic culture conditions and low (1-10 mM) concentrations of
sodium ascorbate augmented the production of oxytocin from Leydig cells in
culture. Higher (50-500 mM) levels of ascorbate and normoxic culture
conditions suppressed both testosterone and oxytocin production in isolated
Leydig cells. Because oxytocin synthesis was found to be
cycloheximide-sensitive, we conclude that Leydig cells possess the
biosynthetic machinery necessary to manufacture oxytocin. The isolated
oxytocin peptide was purified by HPLC with fraction collection followed by
polyclonal-Ab immunoaffinity column chromatography. Comparison of the amino
acid sequence of the isolated octapeptide with authentic oxytocin provides
unequivocal evidence that Leydig cells synthesize oxytocin de novo.
Considering the widespread use of vitamin C as a dietary supplement, the
research reported yields valuable mechanistic information on the
reproductive biologic role of vitamin C in gonadal steroid and peptide
hormone metabolism.


Vitamin C (aka ascorbate) is another non-enzymatic antioxidant found in
significant levels within the testes. As an antioxidant, ascorbate's primary
role is donate electrons to neutralize reactive species of oxygen including
superoxide (to H2O2) and hydroxyl free radicals (to H2O). When ascorbate
acts as a scavenger (by donating an electron to a free radical), ascorbate
is oxidized in the process to the ascorbate free radical and
dehydro-ascorbate. The ascorbate free radical and the dehydro-ascorbate are
reduced back to ascorbate either by NADH catalyzed by semidehydroascorbate
reductase (and forming NAD) or reduced glutathione (GSH) catalyzed by
dehydroascorbate reductase (and forming oxidized glutathione (GSSG)).

n.b. Vitamin C also works along with glutathione peroxidase (a major free
radical-fighting enzyme) to revitalize vitamin E.

Interestingly, Kukucka et. al. reported finding significant levels of
oxytocin (a disulfide containing octapeptide) in isolated Leydig cells.
Kukucka theorized in the introduction of his PhD dissertation (back in 1993)
that open chain oxytoceine (the reduced form of oxytocin) may also act as a
scavenger (by donating an electron to a free radical), oxytoceine may then
be oxidized back to oxytocin. As noted above, the ascorbate free radical and
the dehydro-ascorbate are reduced back to ascorbate either by NADH catalyzed
by semidehydroascorbate reductase (and forming NAD) or reduced glutathione
(GSH) catalyzed by dehydroascorbate reductase (and forming oxidized
glutathione (GSSG)).... why couldn't the ascorbate free radical and
dehydro-ascorbate be reduced back to ascorbate by reduced oxytoceine
(forming closed-ring oxytocin)?

Thus, the redox potential of oxytocin <---> oxytoceine may drive ascorbate
<---> dehydro-ascorbate or vice versa as part of the non-enzymatic
antioxidant defense system.

Other publications by Dr. Mark A. Kukucka

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