(Received for publication, December 13, 1995, and in revised form, December 2, 1996)
From the Faculty of Medicine, Department of Medicine IV, Experimental Division, University of Erlangen-Nürnberg, 91054 Erlangen, Federal Republic of Germany, the § Department of Toxicology, National Health Institute, 70211 Kuopio, Finland, and ¶ Case Western Reserve University, Molecular Cardiovascular Research Center, School of Medicine, Cleveland, Ohio 44106-4958
RAW 264.7 macrophages, when challenged with a
combination of lipopolysaccharide (10 µg/ml) and interferon- (100 units/ml), respond with endogenous NO· formation, which
ultimately results in apoptotic cell death. Apoptosis is detected
morphologically by chromatin condensation. Concomitantly we noticed the
accumulation of the tumor suppressor protein p53. NO·-derived
apoptosis was blocked by the NO·-synthase inhibitor
NG-monomethyl-L-arginine.
Repetitive treatment of RAW 264.7 macrophages with
lipopolysaccharide/interferon-
, followed by subculturing viable
cells, allowed us to select resistant macrophages which we called RES.
RES cells still produced comparable amounts of nitrite/nitrate in
response to agonist treatment but showed no apoptotic markers,
i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO·, released from the NO-donors
S-nitrosoglutathione or spermine-NO. Assessment of
cytochrome c reduction established that RES cells released
twice the amount of superoxide compared to RAW 264.7 macrophages under
both resting and stimulated conditions. We linked increased superoxide
production to cellular macrophage resistance by demonstrating decreased
apoptosis after simultaneous application of
S-nitrosoglutathione or spermine-NO and the redox cycler
2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage
resistance toward NO·-mediated apoptosis is, at least in part,
due to increased superoxide formation. Therefore, the balance
between reactive nitrogen and reactive oxygen species regulates
RAW 264.7 macrophage apoptosis.
Nitric oxide (NO)1 is recognized for
its participation in diverse biological processes in nearly all aspects
of life (1-3). The formation of NO· occurs under both
physiological and pathophysiological settings. The molecule is
synthesized by a family of enzymes termed NO· synthases (NOS),
which utilize arginine as their substrate in the generation of
NO· and stoichiometric amounts of citrulline (4). For
convenience, two types of NOS are recognized; constitutive isoforms,
which are active for a relatively short time in response to
intracellular Ca2+ fluctuations, and a cytokine-inducible
isoform. For the latter to be active, mRNA translation and protein
synthesis are required. The inducible NOS generates large amounts of
NO· for an extended period. However, once NO· is produced
by the action of NOS, it is extremely susceptible to both oxidation and
reduction. This results in the concomitant formation of species with
NO+-like activity (nitrosonium ion) or NO
(nitroxyl anion), respectively (5). In addition to reacting with
oxygen, superoxide, and transition metals, NO· causes biological
signaling via interactions with sulfhydryl groups. Because multiple NO
surrogates are formed, transduction pathways are classified as either
cyclic GMP-dependent or -independent. Cyclic GMP formation
is initiated by NO· binding to the heme group of soluble
guanylyl cyclase, thus causing enzyme activation. This leads to the
conversion of guanosine triphosphate to guanosine cyclic
3
:5
-monophosphate. Formation of cyclic GMP and downstream
phosphorylation events are the basis of the transducing ability of NO,
whereby a vascular message is carried from the endothelium to vascular
smooth muscle; these events also describe the action of NO· on
platelets (6). The cyclic GMP-independent mechanisms, however, are less
well understood. Both cytostatic and cytotoxic actions have been
observed. NO·-mediated toxicity affects pathogens, tumor cells,
and susceptible host cells (1, 7, 8). In the search for an underlying mechanism, investigations are considering inhibition of iron-sulfur centers, protein modification due to S-nitrosylation or
tyrosine nitration, DNA damage, and poly(ADP-ribosylation) activation
with cellular energy deprivation (5-9).
Neurons (10, 11), pancreatic beta cells (12, 13), and macrophages (14, 15) among several other cells seem to be particularly sensitive to the toxic effects of NO·. In some systems NO· causes cell death by necrosis; in others, the progressive intra- or extracellular generation of NO· may cause apoptosis.
The most characteristic features of apoptosis are nuclear and cytoplasmic condensation, DNA fragmentation to high molecular weight fragments followed by DNA laddering, and apoptotic body formation (16, 17). Recently, we established p53 accumulation as an early event during NO·-mediated apoptosis in RAW 264.7 macrophages and the RINm5F beta cell line (18).
Recognizing NO· as an apogen in several experimental systems, we
wanted to investigate signaling pathways and, more importantly, uncover
cellular defense mechanisms against NO·-mediated apoptosis. To
this end, we have used a RAW 264.7 derived cell line, termed RES. RES
macrophages showed complete protection to apoptosis after endogenous
NO·-generation in response to lipopolysaccharide (LPS) and
interferon- (IFN-
). Cellular resistance to NO·-mediated
apoptosis was apparently conferred by increased superoxide formation.
Hoechst dye 33258, protein A-Sepharose, diphenylamine, bovine serum albumin, and cytochrome c were purchased from Sigma (Deisenhofen, Germany). Spermine-NO came from Biotrend (Cologne, Germany). Kaiser's glycerol gelatin was provided by Merck (Darmstadt, Germany). RPMI 1640 was ordered from Biochrom (Berlin, Germany). Cell culture supplements and fetal calf serum were from Life Technologies, Inc. (Berlin, Germany). DMNQ was kindly provided by Prof. Nicotera, University of Konstanz (Konstanz, Germany). All other chemicals were of the highest grade of purity commercially available.
Cell CultureThe mouse monocyte/macrophage cell line RAW 264.7 and derived RES-cells were maintained in RPMI 1640 supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum (complete RPMI), as described previously (15). All experiments were performed using complete RPMI. For nitrite/nitrate determinations cells were cultured in high glucose Dulbecco's modified Eagle's medium supplemented to make it comparable with RPMI 1640.
Nitrite/Nitrate DeterminationNitrite, a stable NO
oxidation product, was determined using the Griess reaction. Cell-free
culture supernatants were collected (200 µl), adjusted to 4 °C,
mixed with 20 µl of sulfanilamide (dissolved in 1.2 M
HCl), and 20 µl of N-naphthylethylenediamine dihydrochloride. After 5 min at room temperature the absorbance was
measured at 560 nm with a reference wavelength at 690 nm. Nitrite
concentrations were calculated using a NaNO2 standard. For
details see Ref. 15. The total production of NO· was determined
by analyzing the media for the presence of nitrite and nitrate using a
described method (19). Briefly, the assay consists of two steps; the
enzymatic reduction of nitrate to nitrite using purified nitrate
reductase (EC 1.6.6.2.; Boehringer Mannheim), flavin adenine
dinucleotide, and reduced -nicotinamide dinucleotide phosphate,
followed by derivatization and spectrophotometric detection of nitrite
after interfering NADPH has been enzymatically removed.
4 × 105 cells were grown in 12-well plates. After overnight adhesion, cells were stimulated according to experimental protocols and scraped off the culture plates followed by fixation with 3% paraformaldehyde for 5 min onto glass slides. Samples were washed with phosphate-buffered saline, stained with 8 µg/ml Hoechst dye 33258 for 5 min, washed with distilled water, and mounted in Kaiser's glycerol gelatin. Nuclei were examined using an Axiovert 35 microscope (Carl Zeiss, Oberkochen, Germany) (15).
GSNO SynthesisGSNO (S-nitroso derivative of glutathione) was synthesized as described previously (20). Briefly, glutathione was dissolved in HCl, at 4 °C, prior to the addition of NaNO2. The mixture was stirred at 4 °C for 40 min followed by the addition of 2.5 volumes of acetone. Precipitates were filtered, washed once with 80% acetone, two times with 100% acetone, three times with diethyl ether, and dried under vacuum. GSNO was characterized by high pressure liquid chromatography analysis and UV spectroscopy.
p53 QuantificationDetection of p53 was performed following previously published procedures (18). Briefly, 5 × 106 cells were lysed (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, pH 8.0) for 30 min on ice and sonicated (Branson Sonifier, 10 s, duty cycle 100%, output control 1). After the addition of SDS sample buffer, samples (equalized to the same amount of protein, 100-150 of µg protein) were heated (95 °C, 5 min), separated on 10% SDS-polyacrylamide gels, and electrophoretically transferred onto nitrocellulose sheets. Nonspecific binding was blocked with TBS-T-M (140 mM NaCl, 50 mM Tris, pH 7.2, 0.06% Tween-20, 5% nonfat-dry milk), followed by the addition (1 h) of p53 antibody (1:20 in TBS-T-M, hybridoma supernatant against p53, clone Pab122, kindly provided by H. Stahl, Universität des Saarlandes, Homburg, Germany). Transblots were washed three times (TBS-T) before adding an anti-mouse peroxidase-labeled secondary polyclonal antibody (45 min, diluted 1:5000 in TBS-T-M from Promega). Blots were washed four times, before ECL development.
Cytochrome c ReductionSuperoxide-induced reduction of
ferricytochrome c to ferrocytochrome c was
monitored spectrophotometrically at 550 nm (21). 4 × 105 cells were stimulated for 16 h with 10 µg/ml LPS
and 100 units/ml IFN-. Medium was changed to Phenol Red-free medium
and cells were further incubated for 3 h in the presence of 50 µM cytochrome c. Medium with cytochrome
c served as a control.
Each experiment was performed at least three times and statistical analysis were performed using the two tailed Student's t test. Significant differences (p > 0.05) are marked by asterisks. In case of photographs (apoptotic morphology or p53 Western blot analysis) representative data are shown.
Apoptotic Cell Death in RAW 264.7 and RES Macrophages in
Response to Endogenously Formed NO·A combination of 10 µg/ml LPS and 100 units/ml IFN-
caused apoptotic cell death in RAW
264.7 macrophages (Fig. 1, A and
B). Based on characteristic apoptotic morphology, that is
chromatin condensation visualized by Hoechst dye 33258 staining, the
apoptotic response to LPS/IFN-
was completely suppressed by the
addition of the NOS inhibitor NMMA. Thus, endogenous NO formation
fully accounted for apoptotic cell death in RAW 264.7 macrophages. Fig. 1A gives statistical analysis, whereas Fig. 1B
demonstrates the response of RAW 264.7 and RES macrophages to
LPS/IFN-
or to the combination of LPS/IFN-
/NMMA.
Although we observed extensive apoptotic cell death in RAW 264.7 macrophages after endogenous NO· production, some cells survived the damaging insult. These viable cells were subcultured until confluence was reached, then these cells were treated with LPS/IFN- again. Repeated addition of LPS/IFN-
and subculturing of surviving cells was performed 10 times. Finally, when cells were challenged with
the agonist combination, macrophages were totally resistant, no longer
showing an apoptotic response (Fig. 1). Because of their altered
cellular behavior toward endogenously generated NO·, these cells
were termed "resistant macrophages," abbreviated as RES.
It is known that RAW 264.7 macrophages accumulate the tumor suppressor
gene product p53 after endogenous generation of NO· (18). The
data in Fig. 2 confirm p53 accumulation in RAW 264.7 macrophages after LPS (10 µg/ml)/IFN- (100 units/ml) treatment. Unstimulated controls and NMMA-treated samples showed a very faint p53
protein band on the Western blot, which significantly increased in
response to LPS/IFN-
addition. Agonists in the presence of the NOS
inhibitor NMMA were ineffective in causing p53 accumulation in RAW
264.7 macrophages, thereby establishing a causative relationship between an active, inducible NOS and tumor suppressor protein accumulation. A time-response analysis revealed maximal p53
accumulation 14 h after the initial challenge.
We did not observe a p53 response in RES macrophages, and 14 h
after LPS/IFN- addition to RES cells p53 was still undetectable (Fig. 2). Therefore, based upon morphological criteria and biochemical analysis, RES macrophages showed no apoptotic markers after
LPS/IFN-
stimulation. This is in contrast to the parental RAW 264.7 macrophages.
To confirm that comparable effective signaling mechanisms were present
in both RAW 264.7 and RES macrophages, we determined nitrite and
nitrate accumulation in the cell supernatant. Increased nitrite/nitrate
values in response to LPS/IFN- are indicative of inducible NOS
induction as well as functional transcriptional/translational control
mechanisms after LPS/cytokine treatment. Table I gives the response of RAW 264.7 and RES macrophages to LPS (10 µg/ml)/IFN-
(100 units/ml) with or without NMMA addition. Both RES
and RAW parent cells showed a massive 24 h accumulation of
NOx values after the LPS/IFN-
challenge. These effects are
suppressed by inducible NOS inhibition.
|
RES cells, in general, produced less nitrite, however, the difference is not significant. Furthermore, we analyzed the media for the presence of nitrate by culturing cells in Dulbecco's modified Eagle's medium, correcting for low levels of naturally occurring nitrate. Under resting conditions no nitrate was detectable, neither in RAW nor in RES cells. After the agonist challenge, RAW macrophages produced less nitrate compared with RES cells although differences never reached statistically different levels.
Superoxide Production and Apoptotic Cell Death in RES MacrophagesSuspecting that macrophages produce not only reactive
nitrogen species but also reactive oxygen species upon activation, we determined the rate of superoxide production or more correctly, the
release of O2 to the cell exterior in both cell systems. RAW
264.7 macrophages released 0.03 ± 0.01 nmol of
O
2/106 cells × min under resting conditions.
Unexpectedly, RES cells released nearly 2-3 times the amount of
cytochrome c reductive material under resting conditions
(Table II). As a control, we showed complete inhibition
of cytochrome c reduction by 500-1000 units/ml superoxide
dismutase (data not shown). To determine superoxide formation in RAW
264.7 and RES macrophages upon agonist addition, we included NMMA to
prevent the possible interference of ONOO
with our
detection system. In agreement with published data (22) we noticed an
interference of the cytochrome c reduction method, when
allowing the simultaneous generation of nitric oxide. As proposed, NO
formation and ONOO
production led to an underestimation
of rates of superoxide production (22).
|
After stimulation with LPS (10 µg/ml)/IFN- (100 units/ml) in the
presence of 1 mM NMMA, RAW 264.7 and RES macrophages
responded with increased superoxide release. Similar to the control
situation, agonist-activated RES cells produced significantly more
superoxide compared to RAW 264.7 macrophages. Superoxide generation,
under control and stimulated conditions, was roughly doubled in RES compared to RAW 264.7 cells (Table II). For comparison, we also determined the rate of nitrite formation. Determinations were carried
out in the linear phase of radical production, between 15 and 18 h
post-agonist challenge.
Considering the increased superoxide formation to be related to decreased NO·-mediated cellular toxicity, we probed for the apoptotic response of unstimulated RES macrophages in the presence of the NO donor S-nitrosoglutathione (GSNO) and the redox cycler DMNQ (Table III). RES cells, when exposed to exogenously derived NO·, showed substantial DNA fragmentation, which was quantified using the diphenylamine assay. DNA fragmentation became obvious using concentrations of 300-750 µM of the NO donating compound. In an additional, supportive observation, GSNO induced p53 accumulation in RES cells (data not shown). Redox cycling quinones like DMNQ, depending on the concentration used, are reported to result in either cell proliferation or apoptotic or necrotic cell death. Employing DMNQ at a concentration of 1 or 2 µM resulted in neither apoptotic nor necrotic RES cell death. Using the superoxide producing DMNQ in combination with GSNO lowered the apoptotic inducing ability of GSNO significantly.
|
During our studies we carefully examined for, and discriminated
between, apoptosis and necrosis by microscopy. The decreased apoptotic
response therefore refers to inhibition of apoptosis without
compensating cellular necrosis. Low levels of the redox cycling
compound suppressed DNA fragmentation in combination with all tested
GSNO concentrations. Although substantial reduction of GSNO-induced
apoptosis became apparent, we never decreased the signal to control
values. With increasing concentrations of DMNQ (5 µM)
we noticed necrotic RES cell death. Assuming that increased superoxide
formation in the presence of a NO donor is protective, this also should
apply for RAW 264.7 macrophages. Therefore, experiments were
subsequently performed with parent RAW 264.7 cells. Again coincubation
of GSNO and DMNQ resulted in decreased macrophage apoptosis (Table
III). Similar results were obtained using the NO·-releasing
compound spermine-NO. Spermine-NO induced more than 40% DNA
fragmentation in RAW cells. Values were decreased to nearly 20% in the
presence of DMNQ (Table III). Overall these experiments suggest that
macrophage apoptotic cell death in response to exogenous NO·,
derived from GSNO or spermine-NO, is partially decreased in the
presence of low concentrations of a redox cycling compound like DMNQ.
DMNQ releases approximately 260 pmol of O
2/106
cells × min under these experimental conditions.
In further experiments we assayed for RES cell apoptosis after GSNO
addition under both resting and stimulated conditions (Fig.
3). When RES macrophages were incubated with increasing concentrations of GSNO, dose-dependent DNA fragmentation
was observed.
In contrast, stimulation of RES cells for 15 h with LPS, IFN-,
and NMMA, followed by an 8-h exposure to GSNO, totally blocked DNA
cleavage. Obviously, preactivation of RES cells confers resistance to
exogenously added and otherwise apoptogenic GSNO.
Apoptosis elicited by endogenous or exogenously derived NO·
may play a role in pathologic conditions ranging from neurodegeneration to pancreatic beta cell death and activated macrophage self-destruction (12-15). Apoptosis is an active process with specific defined
morphological and biochemical features. RAW 264.7 macrophages in cell
culture respond to endogenously generated NO· with characteristic
apoptotic cell death (15, 18). Although many cells respond to LPS
and/or cytokine treatment with NOS induction and massive NO generation, not all of them are susceptible to NO toxicity. Whereas RAW 264.7 macrophages, RINm5F cells, and authentic rat beta cells are responsive to NO·-mediated apoptosis, hepatocytes are spared the toxic
effects of endogenous NO· formation (7). Similar differences in
toxicities hold for exogenously supplied peroxynitrite. Cerebrocortical
cultures show apoptotic cell death in response to low concentrations of ONOO (10 µM) and respond with necrotic
death at 100 µM ONOO
(23). PC12 cells
undergo apoptosis in response to 850 µM
ONOO
(24), whereas some endothelial cell lines are
completely insensitive to ONOO
-induced apoptosis (25).
These observations are consistent with the notion that in the brain,
NO-producing cells are selectively spared the toxic effects of
NO· (26).
Not only does cellular susceptibility to NO· differ among cells,
the ultimate damaging molecule remains ambiguous. Although exogenously
added ONOO causes, depending on the concentration used,
apoptosis or necrosis, at the same time it is also considered a
biological mediator exhibiting physiological NO·-related
reactions (20, 27, 28). By analogy, the production of NO· is not
always associated with cellular toxicity. Increased NO· formation
protects against conditions like ischemia-reperfusion, peroxide-induced
toxicity, lipid peroxidation, and myocardial injury (29-32).
Our strategy of subculturing RAW 264.7 macrophages that survived
LPS/IFN- treatment allowed us to select for macrophages with
resistance to NO·-mediated apoptosis. Resistance was complete when NO· was endogenously generated, but was barely detectable after exogenous GSNO addition (Table III). This substantiated a specific agonist-inducible protective cellular response. Decreased inducible NOS induction cannot account for cellular resistance, as both
RES and RAW 264.7 macrophages clearly responded to these agonists.
Consistent with published observations (33, 34), RAW 264.7 macrophages
produced approximately equal amounts of both nitrite and nitrate. The
increased production, which is not statistically significant, of
nitrate in RES cells over RAW 264.7 macrophages might be related to
increased ONOO
formation, as the decomposition of
ONOO
under cellular conditions is rather complex,
depending upon the production rate of both O
2 and NO·
radicals. Both nitrite and nitrate are expected decomposition products
of ONOO
(33, 34). Unexpectedly, RES cells consistently
produced more superoxide under both non-stimulated and stimulated
conditions. Formation of O
2 in RES cells, under stimulated
conditions with inducible NOS being inhibited, is in the range of
approximately 0.2 nmol of O
2/106 cells per min.
Previous reports for RAW 264.7 macrophages indicated that the formation
of both nitrite and nitrate was in the range of 0.1-0.25 nmol
NO
2 and NO
3/106 cells per min (Refs. 32
and 35, and references cited therein). These findings are in agreement
with our measurements in RAW and RES cells (Tables I and II). As the
formations of both O
2 and NO (as assayed from the sum of
NO
2/NO
3) are approximately equal, one would assume
efficient signaling by modulating the relative production rate of one
of the radicals.
To substantiate a link between increased steady-state production of
superoxide and protection against NO·-derived toxicity, we chose
to incubate a redox cycler in the presence of a NO-liberating agent
while assaying for apoptotic cell death. Although the correlation may
or may not demonstrate cause, supporting evidence for protection against GSNO-induced apoptosis came from a coculture with the redox
cycler DMNQ. As expected, this experimental setting protected both RES
and RAW 264.7 macrophages from the effects of exogenously generated
NO·. Similar results were obtained with spermine-NO/DMNQ. To
further strengthen the idea of inducible resistance toward NO· we
treated RES cells with agonists in the presence of NMMA, followed by
the addition of GSNO. This conferred complete protection of RES cells
toward exogenously added NO (GSNO). Further investigations are
necessary to relate protection from apoptosis to increased O2
under these experimental conditions.
We suggest that modulating the flux ratio of O2 relative to
NO· interferes with the otherwise damaging potency of NO·.
NO· and superoxide produce ONOO
in a nearly
diffusion-controlled reaction (36, 37). Although ONOO
might be formed under our experimental conditions, a low production rate of ONOO
does not automatically contribute to the
initiation of apoptosis. However, in agreement with published data, a
higher rate of ONOO
production results in substantial
cell death (23, 24) indicating severe cellular damage in response to
massive NO·/O
2 production. It is most likely that, under
these conditions, cellular rescue/repair mechanisms are overwhelmed,
either by superoxide formation, NO production, or a combination of
product generation.
Our results describe cellular resistance to NO·-mediated
apoptotic cell death and p53 accumulation through low level
superoxide generation. A steady-state level of superoxide generation
may channel NO· toxicity through pathways that allow cellular
defense mechanisms to cope with an otherwise lethal insult. This seems especially relevant for systems that are equipped to produce NO· and superoxide under inflammatory conditions, such as astrocytes, endothelial cells, mesangial cells, or macrophages. The situation seen
in RES cells is analogous to chondrocyte apoptosis (38). Chondrocytes
treated with sodium nitroprusside undergo apoptosis, whereas endogenous
NO· formation in response to IL-1 failed to do so, because
endogenous production of oxygen radicals seemed to cause protection.
Our results show that cells may acquire a defense mechanism in order to
protect themselves against NO toxicity. Cellular resistance of
NO·-producing cells relative to NO· target cells may
therefore be linked to compensating O2 formation.
As described for O2, the controlled production of the radical
might allow cells to proliferate, inhibit growth, or to enter the
apoptotic/necrotic pathway depending on the concentration and probably
the flux rate of its formation (39). A similar scenario might apply for
the NO signaling system. The final outcome of biological reactions of
NO· will be determined by the rate of NO· and O
2
production. The generation of superoxide relative to NO·
compensates for an otherwise NO·-mediated apoptotic insult. Cellular protection may be due to the elimination of adverse effects associated with reactive nitrogen species due to O
2-dampening effects. It may well be that RES cells have been selected on the basis
of their increased superoxide formation after repetitive stimulation.
Therefore, RES cell sensitivity to NO donors like GSNO may be due to
the lack of compensating O
2 formation. Consistent with this,
GSNO or spermine-NO in the presence of chemically generated superoxide
produces less apoptotic cell death.
Decreased apoptosis in response to endogenously generated NO is also observed after Bcl-2 gene transfer (40). However, protection afforded by Bcl-2 still allowed accumulation of p53, whereas down-regulated apoptosis in RES cells correlated with a suppressed tumor suppressor response. Further studies will examine whether this points to a redirection of the DNA-damaging species in RES cells normally accounting for the positive p53 response.
Increased superoxide production has also been linked to the development
of nitrate tolerance (41) and emphasizes the nondestructive mechanisms
of NO· formation under conditions of enlarged superoxide generation. In the macrophage system, the simultaneous formation of
NO· and O2 protect, at least in part, cells from
NO-induced apoptosis. This is clearly the case when looking at
apoptotic signals like chromatin condensation or p53 accumulation. The
controlled formation of reactive nitrogen species and reactive oxygen
species may play a fundamental role in controlling human diseases by
determining the apoptogenic activity of endogenously generated
radicals.
We thank Prof. P. Nicotera for providing DMNQ.