X-Message-Number: 25017
Date: Thu, 11 Nov 2004 20:24:19 -0800 (PST)
From: Doug Skrecky <>
Subject: ascorbyl-2-O-phosphate slows cellular senescence

[but ascorbic acid does not]

J Cell Biochem. 2004 Oct 15;93(3):588-97
Slow-down of age-dependent telomere shortening is executed in human skin
keratinocytes by hormesis-like-effects of trace hydrogen peroxide or by
anti-oxidative effects of pro-vitamin C in common concurrently with
reduction of intracellular oxidative stress.
  The cellular life-span of cultivated human skin epidermis keratinocytes
NHEK-F was shown to be extended up to 150% of population doubling levels
(PDLs) by repetitive addition with two autooxidation-resistant
derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and
Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with
Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20
microM which was non-cytotoxic to the keratinocytes, or at 60 microM
being marginally cytotoxic achieved the cellular longevity, unexpectedly,
up to 160 and 120% of PDLs, respectively, being regarded as a
hormesis-like stimulatory effect. The lifespan-extended cells that were
administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from
senescence-induced symptoms such as PDL-dependent enlargement of a cell
size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called
loss of proliferation ability as estimated with a channelizer, and
retained young cell morphological aspects such as thick and compact shape
and intense attachment to the culture substratum even upon advanced PDLs,
whereas other non-extended cells looked like thin or fibrous shape and
large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA
of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was
observed in common for each additive-given cells, but was decelerated in
the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM
H(2)O(2) > Asc = no additive, being in accord with the order of cell
longevity. Intracellular reactive oxygen species (ROS) was diminished by
Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60
microM H(2)O(2) as estimated by fluorometry using the redox indicator dye
CDCFH. There was no appreciable difference among NHEK keratinocytes that
were administered with or without diverse additives in terms of
telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times
lower for the keratinocytes than for HeLa cells which were examined as
the typical tumor cells. Thus longevity of the keratinocytes was suggested
to be achieved by slowdown of age-dependent shortening of telomeric DNA
rather than by telomerase; telomeres may suffer from less DNA lesions due
to the continuous and thorough repression of intracellular ROS, which was
realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an
antioxidant ability more persistent than Asc itself or by 20 microM
H(2)O(2) which diminished intracellular ROS assumedly through a
hormesis-like effect.

In Vitro Cell Dev Biol Anim. 2004 Mar-Apr;40(3-4):71-3.
Suppressive effects of ascorbate derivatives on ultraviolet-B-induced
injury in HaCaT human keratinocytes.
  The aging of skin, including sunburning, is caused by ultraviolet (UV)
irradiation. Here, we examined the inhibitory effect of ascorbic acid
(AsA) and its derivatives AsA 2-phosphate (AA-2P) and AsA 2-glucoside
(AA-2G) on UV-B- induced cytotoxicity in HaCaT keratinocytes. Results
show that cell viability significantly decreased when exposed to UV-B at
0.1-0.4 J/cm2 in a dose-dependent manner. In this study, AsA could not
inhibit cytotoxicity, but AA-2P and AA-2G was able to cancel the harmful
effect of UV-B when treated at high levels of 0.5-5 mM. These results
indicate that the masking of the C-2 OH group may be an effective
modification for AsA to inhibit UV-B-induced cytotoxicity in human
keratinocytes.

J Cell Biochem. 2004 Sep 9;93(4):653-663 [Epub ahead of print]
Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate
through suppression of oxidative stress that is generated immediately
after post-anoxic reoxygenation or with alkylhydroperoxides.
  Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell
death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox),
but protected by pre-Anox administration with L-ascorbic acid
(Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative,
but not by Asc itself. This cytoprotection with Asc2P was achieved in a
glucose (Glc)-lacking buffer more advantageously than in a Glc-containing
buffer where less efficiency had been demonstrated for Asc entry into
BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were
detected explosively in the extracellular space at 2-5 min after Reox
following the Anox treatment of HUVE endotheliocytes, and were thereafter
retained at levels as high as approximately one-half of the maximum level
until 60 min after Reox, as shown by cytochrome c reduction assay.
Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox
administration with Asc2P, but not with Asc or dehydro-Asc, and were not
suppressed by post-Anox administration with Asc2P; the cytoprotection may
need the intracellular accumulation of the ROS-scavenging effector Asc
that is converted from Asc2P until 3 min after Reox. The ROS-generator
tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell
viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes,
which were also protected dose-dependently with Asc2P. The cytoprotection
was attributed to reduction of intracellular ROS including
hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry
with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can
be prevented by Asc2P that suppresses ROS-generation immediately after
Reox following Anox more efficiently in the intracellular sphere rather
than in the extracellular space.

Cell Biol Int. 1999;23(4):287-98
Reduced viability of vascular endothelial cells by high concentration of
ascorbic acid in vitreous humor.
  Normal mammalian vitreous humor maintains its avascularity after
regression of hyaloid vessels. Neovascularization in adults is only
detected under pathological conditions which suggests that antiangiogenic
factors are present in the vitreous humor. To elucidate the mechanism of
vitreal angiogenic inhibition, we investigated the effect of vitreous
humor on cultured vascular endothelial cells. When bovine aortic
endothelial cells were cultured in the presence of bovine vitreous humor
in medium, a decrease in cell viability was observed within 24 h.
Ascorbic acid from vitreous humor has been identified as a cell death
inducing factor with high performance liquid chromatography (HPLC) and
molecular mass analysis. Ascorbic acid reduced endothelial cell viability
at concentrations normally present in vitreous humor. This effect was
completely inhibited by antioxidants, N-acetylcysteine and  catalase.
Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate
did not induce cell death, suggesting that the production of ascorbyl
radical is required for induction of cell death. Furthermore, capillary
formation in three-dimensional collagen gel cultures characteristic of
vascular endothelial cells were disrupted in the presence of ascorbic
acid. Since ascorbic acid is highly concentrated in ocular tissues,
especially in vitreous humor, it may function as a neovascularization
inhibitor.

Life Sci. 1998;63(11):935-48
Age-dependent telomere shortening is slowed down by enrichment of
intracellular vitamin C via suppression of oxidative stress.
  Telomeres in eukaryotic somatic cells are destined to the age-dependent
shortening, which has not been demonstrated to correlate to direct lesion
of telomeric DNA by reactive oxygen intermediates (ROI); still less
explicable is the inhibitory effect of ROI-scavenging on telomere
shortening. Here, we succeeded in artificial slowdown of age-dependent
telomere shortening to 52-62% of the untreated control, in human vascular
endothelial cells, by addition of the oxidation-resistant type of
ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), which concurrently
achieved both extension of cellular life-span and prevention of cell size
enlargement indicative of cellular senescence. The results are
attributable to a 3.9-fold more marked enrichment of intracellular Asc
(Asc(in)) by addition of Asc2P, subsequently dephosphorylated before or
during transmembrane influx, than by addition of Asc itself, and also
attributed to diminution of intracellular ROI to 53% of the control level
by Asc2P; telomerase activity was at a trace level and underwent an
age-dependent decline, which was significantly decelerated by Asc2P.
Thus, age-dependent telomere-shortening can be decelerated by
suppression of intracellular oxidative stress and/or by telomerase
retention, both of which are achieved by enriched Asc(in) but not by
extracellular Asc overwhelmingly more abundant than Asc(in).

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