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* LBSO/LCR7 No. 8-Université Joseph Fourier, F-38043 Grenoble Cedex 03, France; and
`Laboratoire Lésions des Acides Nucléiques', Département de Recherche Fondamentale sur la Matière Condensée, Service de Chimie Inorganique et Biologie, CEA/Grenoble, F-38054 Grenoble Cedex 9, France
1Correspondence: LBSO, Laboratoire de Biochimie C, C. H. U. Albert Michallon, 38043 Grenoble Cedex 03, France. E-mail:
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: DNA damage • NO • metalloenzymes • SNAP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nevertheless,
NO• appears to have both toxic and protective
effect (2)
and may have a role in the pathogenesis of some
diseases such as inflammation (3)
and cancer
(4)
.
Recent studies from Baudouin and Tachon (5)
, Villiotou and
Deliconstantinos (6)
have shown that normal human
keratinocytes constitutively expressed an isoform of nitric oxide
synthase (NOS), which is Ca2+/calmodulin
dependent. More recently, Wang et al. (7)
demonstrated
that unstimulated human skin fibroblasts expressed only
Ca2+-dependent/NOS activity whereas cells
stimulated with cytokines and lipopolysaccharides expressed both
constitutive (Ca2+-dependent/NOS) and inducible
(Ca2+ independent/NOS) ones. Although
constitutive and inducible NOS isoforms can be expressed in all cell
types of human skin (4)
, the subsequent regulatory and
cytotoxic effects of NO• in skin are not well
known. In the skin, NO• could participate to
the deleterious effect of ultraviolet (UV) irradiation, in conjugation
with others reactive oxygen species (ROS) produced during the
photo-oxidation of intracellular components by ultraviolet radiation
(UVA-2, 320–340 nm and UVA-1, 340–400 nm). Indeed, the effectiveness
of NO• as a cytotoxic molecule is modulated by
ROS such as the superoxide anion radical
(O2•-)and hydrogen peroxide
(H2O2). The interaction of
NO• with
O2•- yields the peroxynitrite
anion (ONOO-) and increases the reactivity of
the two individual molecules toward targets of cellular metabolism
(3
, 8
, 9)
. In fact, most of the cytotoxic properties of
NO• have been attributed to the formation of
ONOO- (10)
although nitrite, a
major product of NO metabolism, can also readily promote protein
nitration via reactions with peroxidases (11)
. Villiotou
and Deliconstantinos (6)
showed that UVA-irradiated human
squamous cell carcinoma (SCC-13) released reactive nitrogen oxides, as
evidenced by increased nitrite/nitrate production and products of
protein nitration. Formation and/or release of these reactive nitrogen
oxides by UVA were time and concentration dependent. Their levels were
significantly decreased by addition of a specific inhibitor of NOS,
namely, N-monomethyl-L-arginine.
In the present study, we provide evidence that L-arginine (L-Arg), the substrate of NOS, increased the deleterious effects induced by UVA irradiation in the human keratinocyte cell line HaCaT. To elucidate the respective roles of ROS and NO• in UVA/L-Arg-mediated cytotoxicity, 1) the production of NO• was measured, 2) the alterations of the intracellular antioxidant metalloenzymes were assessed, 3) DNA damage was determined, and 4) the potential role of poly(ADP-ribose) polymerase (PARP) in UVA/L-Arg-treated cells was evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Keratinocytes immortalized from a spontaneously transformed
human epithelial cell line (HaCaT) were kindly supplied to us by Dr.
Fusenig (12)
. They were routinely cultured at 37°C in a
humidified atmosphere (5% CO2 in air) in RPMI
1640 containing 1 mM L-Arg, supplemented with 10% fetal calf serum,
3.6 mM L-glutamine, 4.5 U·ml-1 penicillin, and
0.045 U·ml-1 streptomycin. For each study,
cells were plated onto 9 cm2 petri dishes from
Life Technologies, Inc. (Grand Island, N.Y.) and grown for 4 days to
near confluency. The culture medium was replaced by fresh medium
48 h before the experiment.
UVA irradiation
The UVA source was a high-pressure Tecimex apparatus (Verre &
Quartz-Dixwell, Bondy, France). The emission maximum of the lamp was
centered at 372 nm. The UVA doses effectively received from the bottom
by the cells were evaluated with a radiometer (Verre & Quartz). Prior
to irradiation, the culture medium was removed and reserved. Cells were
rinsed twice with 1.5 ml of PBS and left in 1 ml of PBS. Control cells
were similarly treated but left in the dark instead to be irradiated.
Three UVA doses were applied: 25.3, 50.5, and 67.3
J·cm-2.
NO•sources
Two different NO•-generating systems
were used. SNAP (0.02 M) was dissolved in PBS just before use and
diluted in the medium to a final concentration of 750 µM. The
solution of L-Arg, the specific substrate of NOS, was also prepared
just before use and applied on near-confluent cells to final
concentrations varying from 0.5 to 10 mM. To study the interaction
between UVA radiation and L-Arg, the amino acid was added to the
culture medium immediately after the irradiation and the cells were
then reincubated for 18 h.
Other treatments
To assess the ability of metalloenzymes to prevent UVA/L-Arg
induced damage catalase (500 U/ml) and/or SOD (0.44 U/ml) were added
immediately after the irradiation.
The stock solution of 0.1 M L-Thio, an irreversible inhibitor of NOS, was prepared in ethanol, diluted first in deionized water and then added to the culture medium (100, 250 µM) just before L-Arg treatment.
Cells were treated with 3-aminobenzamide (ABA, 1 mM) just after irradiation in order to study the link between DNA damage, cell death, and activation of the nuclear PARP.
Assay of cell viability
Cell viability was evaluated using the neutral red technique.
After irradiation and/or chemical treatment, cells were incubated for
18 h in their reserved medium. A solution of neutral red (50 mg/l)
was made with the cell culture medium and 3 ml was added to the dishes.
Then cells were reincubated for 3 h at 37°C, resulting in the
uptake of the vital dye into viable cells. The dye medium was removed
and the cells were washed rapidly with 4% formaldehyde-1%
CaCl2 to remove extraneously adhering,
unincorporated dye, according to Borenfreund and Puerner
(13)
. Neutral red was extracted into 3 ml of a mixture of
1% acetic acid-50% ethanol. After 20 min, the absorbances were
measured with a spectrophotometer set at 540 nm. The results represent
the means of three experiments with two petri dishes for each condition
(Cc, control cells; TIc, treated irradiated cells). The results were
expressed as the viability (%) evaluated from the formula (TIc/Cc) *
100.
Alkaline single-cell gel electrophoresis
The procedure already described (14)
was modified
as follows. Typically, 150 µl of 0.6% normal melting agarose in PBS
was dropped onto frosted microscope slides (Touzart et Matignon, Les
Ullis, France), covered with a coverslip, and kept at room temperature
until subsequent use. Cells in petri dishes were treated (irradiation
or/and application of L-Arg). Three hours later, HaCaT cells were
trypsinized and the cell pellet was suspended in PBS. About 20,000
cells were mixed with 75 µl of 0.6% low melting point agarose (FMC
Bioproducts, Rockland, Maine) in PBS. The slides were kept in the dark
at 4°C in order to limit nonspecific DNA damage and repair process. A
final layer of agarose (75 µl of 0.6% low melting point agarose) was
applied. Each slide was processed in duplicate, as described previously
(15)
, and examined using an epifluorescence microscope,
Zeiss Axioskop 20 (Carl Zeiss, Microscope Division, Oberkochen,
Germany), equipped with a mercury lamp HBO (50 W, 516–560 nm, Zeiss)
and filters 5 and 15 (Zeiss) at x20 magnification. Fifty randomly
selected comets on each duplicated slide were scored with a pulmix TM
765 camera (Kinetic Imaging, Liverpool, U.K.), linked to a Komet 3.0
image analysis system (Kinetic Imaging, U.K.). The software allows
measurement of the fluorescence intensity of the head and tail regions
along with determination of their length. The quantification of DNA
damage was performed using the tail moment, the product of the tail
distance, and the percent of DNA in the tail (relative to the amount of
DNA in the entire comet).
Metalloenzymes activities
Keratinocytes in 75 cm2 culture flasks
(NUNC, Grand Island, N.Y.) were incubated in the presence of either 750
µM SNAP or 5 mM L-Arg for 18 h. After two washings with PBS, the
cells were harvested and frozen-defrozen five times. The homogenates
were centrifuged for 10 min at 4000 rpm at 4°C. The supernatants were
stored at -80°C until metalloenzymes analysis. The glutathione
peroxidase (GSH-Px) activity was determined as described previously
(16)
, using tert-butyl hydroperoxide as the substrate.
This was achieved by following the decrease in NADPH concentration at
340 nm. Catalase activity was determined by the method of Beers and
Sizer (17)
by following the decomposition of
H2O2 at 240 nm. Manganese
(MnSOD) and copper/zinc (CuZn SOD) superoxide dismutase activities were
determined as described previously (18)
by monitoring the
rate of the oxidation of pyrogallol by superoxide radicals. The
specific inhibition of CuZn SOD by 9 mM potassium thiocyanate allows
MnSOD determination by the same procedure. Each sample was assayed
twice and results were expressed as SOD units (1 U is the amount of SOD
inhibiting the reaction rate by 50% in the given assay conditions).
Enzymatic activities were normalized to the soluble cell protein
content determined by the Micro BCA protein assay (Pierce, Rockford,
Ill.). The results of five replicate experiments are given.
Statistical analysis
All data, expressed as mean ± standard deviation, were
processed statistically using the Student's t test.
Differences were considered to be significant when P <
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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illustrates the promoting effect of L-Arg, added at different
concentrations to the culture medium, on the UVA cytotoxicity. Cell
viability was not modified significantly by the irradiation at the
fluences used (100.7 ± 1.5%, 93.4 ± 2.1% for 25.3 and
50.5 J/cm2, respectively). Low concentrations of
L-Arg were found to have no effect on cell viability. Treatment with
L-Arg (5 mM and 7.5 mM) immediately after UVA irradiation led to a
significant decrease in cell viability (P<0.001) compared
with irradiation alone. These results demonstrate that L-Arg
potentiates the lethal effect of UVA and suggest that NOS are
stimulated.
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NOS involvement in the lethal effects of UVA/L-Arg treatment
Under basal conditions, the release of NO•
by HaCaT keratinocytes over 24 h is very low. This was illustrated
by the fact that the level of nitrites in the culture medium was below
the detection threshold of the Greiss reaction (<3
nmol/106 cells/18 h). However, the amount of
NO• released by cells exposed to UVA/L-Arg
(25.3 J·cm-2/5 mM) treatment increased more
than sixfold (20 ± 5 nmol/106 cells/18 h).
To assess the involvement of NO• or
ONOO- in the cytotoxic effect of UVA/L-Arg
treatment, we used L-Thio, a stereospecific inhibitor
(Ki=100 µM) of the constitutive brain and
endothelial NOS isoforms. L-Thio is recognized by the active site of
NOS and acts with the heme-containing enzyme, thus giving rise to an
irreversible inhibition. From the data listed in Table 1
, it is clear that the addition of either 100 or 250 µM L-Thio
protected keratinocytes (P<0.05 and P<0.01,
respectively) against cell death induced by the sequential treatment
UVA/L-Arg (25.3 J.cm-2/5 mM). These results are
in accordance with the levels of nitrite (< 3
nmol/106 cells/18 h); L-Thio 250 µM) released
in the medium by UVA/L-Arg-treated cells. The protection increased with
the concentration of L-Thio, suggesting that NOS are implicated in the
observed cytotoxicity.
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UVA/L-Arg as SNAP treatment altered antioxidants systems
To further study the effect of L-Arg (5 mM) on HaCaT
keratinocytes, the intracellular antioxidant metalloenzymes activities
were determined 18 h after treatments. Table 2
summarizes the results. GSH-Px was strongly inhibited when cells were
either exposed to UVA radiation (77% decrease relative to control) or
cultured in the presence of L-Arg (58% decrease). The inhibition was
amplified by the combined treatment UVA/L-Arg (83% decrease). The
level of catalase was also significantly affected by UVA, L-Arg, and
both sequential treatments (36, 59, and 66% decrease, respectively,
relative to control cells). In contrast, the total activity of SOD was
not significantly modified. However, an induction of MnSOD was observed
in UVA-irradiated cells (45% increase relative to control).
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NO• has been shown to alter the activities of
several enzymes in which thiol groups, heme or nonheme iron, are
essential for the catalytic function (19
, 20
, 21)
. Thus,
the activities of metalloenzymes were determined 18 h after
treating the HaCaT cells with 750 µM SNAP (Table 3
). A significant increase in MnSOD was observed (32% relative to
control). Catalase was significantly disturbed and the GSH-Px activity
was highly inhibited after the SNAP treatment (respectively 25.5 and
92% decrease relative to control).
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Modulation of UVA/L-Arg injury by antioxidant metalloenzymes
To determine the nature of reactive species implicated in the
UVA/L-Arg cytotoxicity, the assay was conducted in the presence of
metalloenzymes (SOD and/or catalase). The results reported in
Table 4
showed that SOD protected the keratinocytes (73% protection compared
to treated cells without any antioxidant) whereas catalase was
ineffective. However, SOD amplified the deleterious effects of
irradiation alone or those of the L-Arg treatment compared to treated
cells without any antioxidant. Cell viability decreased by 25 and 48%,
respectively.
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The role of NO•and PARP in the DNA damage induced by
UVA/L-Arg
To better understand UVA/L-Arg toxicity, DNA damage was
determined. Nuclear DNA in nonirradiated cells was not altered and the
tail moment was very low (6.45 ± 0.78), showing that the
experimental procedure was tolerated well by the cells (Table 5
). The tail moment was not significantly modified in cells treated with
5 mM L-Arg alone. In contrast, even under conditions where UVA did not
induce cell death, the tail moment was fourfold greater in
UVA-irradiated cells (25.43 ± 1.23, P<0.001) than in
control cells. The combined sequential treatment involving UVA
irradiation (25.3 J/cm2) and L-Arg (5 mM)
strongly increased the number of alkali-labile sites and DNA strand
breaks (41.05 ± 3.9). In cells previously treated by 250 µM
L-Thio, the mean tail moment (9.86±0.98) was similar to those
determined in control cells. These results suggest that L-Arg via the
NOS pathway and ROS generated by UVA radiation could interact in
inducing DNA fragmentation and cell death. To determine whether PARP
activation participates in the observed DNA damage, cells were treated
with a PARP inhibitor. ABA (1 mM) was found to enhance the cell
viability up to 56.63 ± 5.75% (P<0.001 vs.
L-Arg/UVA-treated cells without ABA). In addition, ABA had a
statistically significant (P<0.001) protective effect on
UVA/L-Arg-induced DNA damage (Fig. 2
). We observed that ABA also prevented DNA damage in UVA-irradiated or
L-Arg-treated cells, but this ability was low compared with those
observed in UVA/L-Arg cells. These results support the notion that
poly-(ADP-ribosyl)-ation is the primary injury in UVA/L-Arg-treated
cells whereas other mechanisms started off in either UVA or
L-Arg-treated cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L-Arg, was found to enhance the cytotoxic effect of UVA on cultured the
human keratinocyte HaCaT cell line. This essential amino acid is
nontoxic at concentrations lower than 7.5 mM, although it is involved
in the L-Arg/NO• pathway. In contrast, supply
of UVA-irradiated cells with L-Arg increased dramatically the UVA
cytotoxicity. Recent studies have shown that UVA-irradiated human
squamous cell carcinoma (SCC-13) led to the release of the reactive
nitrogen species (NOx, ONOO-). Formation and/or
release of these reactive nitrogen species was time- and
concentration-dependently stimulated by UVA whereas their level was
decreased by N-monomethyl-L-arginine (6)
. Here it was
demonstrated that the release of NO• increased
significantly in UVA/L-Arg-treated cells (20 ± 5
nmol/106 cells/18 h). It should be remembered
that HaCaT cells released very weak amounts of
NO• in the culture medium (<3
nmol/106 cells/18 h). These observations are
consistent with previous reports on human dermal fibroblasts
(7)
and human squamous cell carcinoma (6)
.
Moreover, we demonstrated that the NO• release
was abolished in cells pretreated with L-Thio, a stereospecific
inhibitor of NOS, which also largely prevented the UVA/L-Arg
cytotoxicity. These results confirm the involvement of NOS in the
latter deleterious effect.
Reactive nitrogen species may induce DNA fragmentation
(22)
. Using the comet assay, it was demonstrated that DNA
strand breaks and alkali-labile sites induced by the combined treatment
UVA/L-Arg increased twofold compared to UVA irradiation alone. When NOS
was inhibited by L-Thio, the tail moment decreased significantly both
in L-Arg and UVA/L-Arg-treated cells, whereas in UVA-irradiated cells
the inhibition of NOS led to a significant increase in tail moment.
These paradoxical results may be related to the balance between
reactive nitrogen and reactive oxygen species. The production of
NO• is not always associated with cellular
toxicity (23)
. Indeed, whereas NO•
protects against ischemia reperfusion, peroxide-induced toxicity
(24
, 25)
, lipid peroxidation (26)
, and
myocardial injury, it can combine with
O2•- to form
ONOO-, which may be protonated to yield
peroxinitrous acid. Depending on the pH of the local environment,
peroxinitrous acid degrades to either inactive metabolites or toxic
radicals. Thus, it appears that NO• may behave
either as a potentially toxic molecule or as an oxygen free radical
scavenger (20)
. In UVA/L-Arg-treated cells, the
simultaneous production of NO• and ROS might be
led to ONOO- formation and the increase in DNA
damage might be related to this highly cytotoxic species. Ischiropoulos
et al. (9)
have reported that inhibition of
NO• synthesis by N-methyl-L-arginine increases
the level of H2O2 arising
from the dismutation of superoxide radical in murine macrophages. Thus,
we may hypothesize that under conditions where NOS are inhibited by
L-Thio, UVA-irradiated HaCaT cells produced essentially ROS
(O2•-,
H2O2, and
•OH). Indeed, L-Thio significantly reduced the
tail moment measured in UVA/L-Arg-treated cells, suggesting that the
modulation of the flux ratio of ROS relative to
NO• interfere with the otherwise damaging
potency of UVA. Although ONOO- might be formed
under basal condition and L-Arg treatment, its low production rate does
not automatically contribute to the initiation of cell death.
When SOD was added into the medium, L-Arg became cytotoxic whereas
catalase had no effect. We postulated that SOD increases the rate of
superoxide dismutation into
H2O2, leading to a
modification of the ratio between ROS and NO•.
Under this condition, the low level of superoxide did not allow the
generation of ONOO-. These results are in
accordance with Filep et al. (22)
, who reported a
cooperative effect between nitric oxide and
H2O2 in inducing both cell
lysis and DNA fragmentation in murine lymphoma cells. Nevertheless,
exogenously added SOD conferred protection to UVA/L-Arg-treated cells,
whereas catalase did not. The final outcome of the biological reactions
of NO• is likely to be determined by the rate
of NO• and superoxide production.
Several studies have reported that PARP was implicated in
NO• cytotoxicity (26
, 27)
. Mebmer
et al. (28)
reported that NO•
increases the intracellular caspase-3 by elevated expression or
activation and mediates PARP cleavage. Proteolysis results in the split
of its DNA binding vs. poly(ADP-ribose) polymerase
activities. Moreover, PARP cleavage could result in endonuclease
activation. In the present work, we confirmed that DNA damage induced
by UVA/L-Arg treatment was relevant to PARP. Indeed, the addition of
ABA, a potent inhibitor of PARP, prevented completely the formation of
DNA lesions. In addition, ABA also reduced the amount of DNA damage in
L-Arg-treated cells. It is believed that DNA damage elicits a rapid
stress response in mammalian cells, which involves attachment of PARP
to the strand breaks and extensive synthesis of short-lived polymers by
the bound enzyme. Although PARP has no direct role in DNA excision
repair, it binds tightly to DNA break and may interfere with repair if
poly(ADP-ribose) synthesis is prevented (29)
. However,
with massive PARP activation after extensive DNA damage, the ADP-ribose
donor NAD+ is depleted and subsequent ATP
deprivation may represent a signal leading to cell death (30
, 31)
. In UVA/L-Arg-treated cells, the PARP inhibitor ABA provided
a protection against cell killing (percent of cell viability: 56.63 in
ABA pretreated cells vs. 15.2 in untreated cells). Our
observations suggest that PARP inhibition might maintain DNA integrity
and protects cells against cell death. It may be one of the mechanisms
by which pharmacological inhibition of PARP during oxidative stress
exerts beneficial effects as it improves survival and prevents the
degree of peroxinitrite-induced endothelial and epithelial dysfunction
(32)
.
To better understand the cytotoxic effect of the combined treatment,
UVA/L-Arg, we measured the antioxidant metalloenzymes activities. L-Arg
treatment inactivated strongly intracellular GSH-Px. It was shown that
GSH-Px was also inactivated by SNAP, which led to an exogenous NO
generation (19)
. GSH-Px are antioxidative enzymes that
metabolize various peroxides. The level of GSH-Px is higher than that
of catalase in most cells. The Km value of catalase
for H2O2 is higher than
that of GSH-Px, suggesting the primary importance of GSH-Px in cells.
Hence, the destruction of the balance between oxidants and antioxidants
by reactive nitrogen species such as NO• and
peroxinitrite through inhibition of GSH-Px could easily affect cellular
homeostasis. In our models, we observed both GSH-Px and catalase
inactivations. As UVA also inactivated the latter enzymes, the combined
treatment strongly affected the antioxidant cell defenses. The
inactivation of GSH-Px observed in the UVA/L-Arg model could be induced
by direct binding of NO• to the amino acid
residue (selenocysteine),at the catalytic center of the molecule
(19)
. Because redox regulation of cells and GSH-Px
activity are closely tied to apoptosis, inactivation of GSH-Px by
NO• may be one cause of cell death.
Immunoprecipitation and amino acid sequencing techniques identified
manganese superoxide dismutase, the major antioxidant enzyme in
mitochondria, as one of the targets of tyrosine nitration. In
vitro studies demonstrated that peroxynitrite readily nitrates
tyrosine residue of MnSOD and inhibits its enzymatic activity
(33)
. In UVA/L-Arg-treated keratinocytes, no significant
alteration of MnSOD was observed. However, we showed that MnSOD
activity increases in irradiated cells so it could compensate a
tyrosine nitration. SNAP decomposition in the culture medium also
induced a significant increase of MnSOD in HaCaT cells.
We have demonstrated that UVA-irradiated keratinocytes HaCaT are able to generate NO• in the presence of the substrate of the NOS. The findings obtained using NOS inhibitor and SOD suggested that ONOO- formation in the UVA/L-Arg-treated cells induced both DNA damage and cell death. The extent to which ONOO- caused inactivation of metalloenzymes and DNA damage suggested that it may be a causative factor of UVA-induced skin damage. The L-Arg/NO• pathway could be the foundation for the development of new approaches to the management and treatment of skin diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 20, 1998. Revised for publication April 29, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem. 272,1402-1411This article has been cited by other articles:
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  M. A. Bachelor and G. T. Bowden Ultraviolet A-induced Modulation of Bcl-XL by p38 MAPK in Human Keratinocytes: POST-TRANSCRIPTIONAL REGULATION THROUGH THE 3'-UNTRANSLATED REGION J. Biol. Chem., October 8, 2004; 279(41): 42658 - 42668. [Abstract] [Full Text] [PDF]
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A. Cheng, S. L. Chan, O. Milhavet, S. Wang, and M. P. Mattson p38 MAP Kinase Mediates Nitric Oxide-induced Apoptosis of Neural Progenitor Cells J. Biol. Chem., November 9, 2001; 276(46): 43320 - 43327. [Abstract] [Full Text] [PDF]
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 C. Adrie, C. Richter, M. Bachelet, N. Banzet, D. Francois, A. T. Dinh-Xuan, J. F. Dhainaut, B. S. Polla, and M.-J. Richard Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes Am J Physiol Cell Physiol, August 1, 2000; 279(2): C452 - C460. [Abstract] [Full Text] [PDF]
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