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Department of Molecular and Cell Biology, University of California, Berkeley/CHORI, Oakland, California 94609, USA
1Correspondence: CHORI, 5700 Martin Luther King Jr. Way, Oakland, CA, 94609-1673, USA. E-mail:bnames{at}uclink4.berkeley.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: NtBHA • senescence • proteasome • growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The role of mitochondria in the
process of the age-dependent deterioration of tissues has become the
focus of many studies (3
4
5
6)
. The age-dependent changes in
mitochondria are characterized by a high rate of generation of reactive
oxygen species (ROS), a decline in the activity of electron transport
complexes, and a decrease in cardiolipin, an essential phospholipid
for normal function of mitochondria (7
8
9
10
11)
. During ATP
production by oxidative phosphorylation, electrons from NADH or
succinate in the mitochondrial matrix are transferred through the
electron transport chain (ETC) (complexes I through IV) and reduce
molecular oxygen to water (12)
. In this process, 
2% of
the electrons leak and reduce O2 to
O.-2 radical and
H2O2 (13)
. The
leakage of oxidants from the ETC appears unavoidable (14
, 15)
and mitochondria are considered the main endogenous source
for the formation of the superoxide radical (16
, 17)
. The
combination of continuous formation of
O.-2 and a limited antioxidant
capacity of mitochondria (18)
make them vulnerable to
oxidative damage. For example, mitochondria lack catalase, the ability
to synthesize GSH, the ability to transport GSSG out of the matrix, and
possibly chelators for transition metals, all of which act as elements
to decrease ROS production (19
, 20)
.
Supplementation with 
-phenyl-N-t-butyl nitrone (PBN) and
acetyl carnitine or lipoic acid and acetyl carnitine
(21
22
23)
, which target mitochondria, ameliorated the
age-dependent decline of mitochondria in rats. PBN is a nitrone that
traps free radicals and protects against damage in different models
such as inflammation, ischemia reperfusion, and aging
(24)
. Recently we showed that the decomposition product of
PBN, N-t-butyl hydroxylamine (NtBHA), mimics PBN and is much
more effective in delaying senescence of IMR90 cells (25)
.
The ability of several N-hydroxylamine derivatives to delay cellular
senescence in human lung fibroblasts was the subject of our previous
study (25)
. In those experiments, NtBHA proved to be the
most effective compound in delaying senescence of human lung
fibroblasts. NtBHA appears to act on mitochondria to delay
age-dependent alterations in function [such as the decline of the
respiratory control ratio (RCR), 
, and some enzymatic
activities]. The significance of these studies is that they open the
door for developing and optimizing intervention therapies that can
delay or prevent age-related deterioration of tissues.
In the present study, the effect of supplementation with NtBHA in an animal model and a tissue culture model maintained under suboptimal condition of growth factors was investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
UCB Animal Care and Use committee approved the animal
experimentation performed in this study. Young (age 3 months; Simonsen,
Gilroy, CA) and old (24 months, National Institute of Aging animals
colonies) male Fisher 344 rats were divided equally into control and
NtBHA treatment groups. At commencement of the study, each treatment
group consisted of four or five rats housed together in large cages in
order to minimize stress, in conditions of controlled temperature
(25°C) and a 12 h light/dark cycle (6:00 h to 18:00 h). The rats
were allowed ad libitum access to standard Purina rodent chow. NtBHA
was administered to the rats in double distilled water at a final
concentration of 1 mM for a period of 25 days. The salinity of the
drinking water was adjusted to 1 µmol NaCl/ml and sodium hydroxide
was used to adjust the water to pH 6 for all groups. Fresh water with
or without NtBHA was supplied daily. Body weight was measured weekly
and food and water intake was measured daily. Chow or water intake was
measured at the beginning and end of every 24 h period and the
difference was divided by the number of the animals in the cage. At the
end of the experiment, the rats were anesthetized with ether and killed
by cardiac puncture. The liver was resected and placed in ice-cold
mitochondrial isolation buffer containing 210 mM mannitol, 70 mM
sucrose, 5 mM HEPES, and 1 mM EDTA, pH 7 (MSH/EDTA). The liver was
homogenized immediately and the mitochondrial fraction was isolated by
differential centrifugation as described (26)
.
Mitochondrial respiration supported by succinate 5 mM, phosphate (4
mM), and ADP (0.15 mM) was measured in 125 mM KCl and 5 mM Tris, pH 7.4
by a Clark Oxygen electrode (Yellow Springs Instruments, Yellow
Springs, OH) in the presence of 4 µM rotenone.
This experimental protocol was repeated in a group of old rats (n=10) that were either untreated or administered NtBHA for 25 days. The results of the two experiments were pooled for data analysis, except for the RCR.
Ambulatory activity
On day 21 of the study, rats were transferred to individual
cages (48 cm long x 25 cm wide x 20 cm high) for
measurements of ambulatory activity. Rats were acclimatized to their
new surroundings for at least 4 h before monitoring. Rats had ad
libitum access to food and water. The room was on a 12 h
light/dark cycle (lights on 6:00 to 18:00). At 20:00 h, a very low
intensity light illuminated the rats for video tracking. Monitoring of
ambulatory activity began at 21:00 h and continued for 4 h. One
hour later, the low light was turned off and the standard light cycle
was continued. The ambulatory activity of each rat was recorded for
four consecutive nights. A video signal from a camera suspended
directly above the individual cages was connected to a Videomex-V
(Columbus Instruments, Columbus, OH) computer system running the
Multiple Objects Multiple Zones software. The system quantified
ambulatory activity parameters and was calibrated to report distance
traveled in centimeters.
Determination of free GSH and protein-mixed disulfides in liver
A 200 µl aliquot of liver homogenate was immediately
transferred into 50 µl of 1 M MSA and 2.5 mM DTPA and stored at
-80°C until analysis. The proteins from the MSA homogenate were
precipitated by centrifugation at high speed. The supernatant was used
for quantification of free GSH. The pellet was washed three times by
resuspending in ice-cold PBS. The final pellet was resuspended in 100
µl of ice-cold 0.1 M Tris and 50 mM DTT (pH 8.3) and incubated on
ice. After 1 h incubation, 20 µl of 1 M MSA and 2.5 mM DTPA were
added to precipitate the proteins and stabilize GSH. The pellet was
used for protein quantification (Bio-Rad protein assay, Bio-Rad); the
supernatant was filtered and used for quantification of the GSH that
was liberated from the mixed disulfides in the proteins. Both
supernatants were filtered through 30,000 cutoff filters before
injection into an HPLC column. The amount of protein injected was 5–10
µg or 1–3 µg for GS-SR and free GSH, respectively. Free GSH and
GSH liberated from protein-mixed disulfides after reduction by DTT was
determined by HPLC-EC detection, as described (25)
. The
activities of glutamate dehydrogenase and glucose-6-phosphate
dehydrogenase were assayed as described previously (27
, 28)
.
Experiments on IMR90 cells
Experiments on IMR90 cells were started from low passages, the
cells being split into two groups. One group maintained in DMEM medium
supplemented with nFBS (29)
. The second group was
maintained under suboptimal conditions of growth by maintaining
cultures in medium supplemented with FBS that had been stripped of
growth factors by passing through c/dFBS (30)
. Each group
was split into subgroups for further treatment. Cells grown in nFBS
were split into groups of control, treated with thyroid hormones (80 nM
T3/1.2 µM T4), and treated with NtBHA (100 µM). Cells grown in
c/dFBS were split into groups of control, treated with thyroid hormones
(80 nM T3/1.2 µM T4), treated with NtBHA (100 µM), and a
combination of NtBHA/thyroid hormones. The treatment with T3/T4 or the
combination T3/T4+NtBHA began at the 3rd week of the experiment. Twice
a week, the used media were removed and fresh media were added to the
cells (on the day of the split and 3 days later); the media were
supplemented with fresh chemicals when necessary. The effect of
different growth conditions on population doublings (PDLs) and on
mitochondria and proteasomal activity (31)
was tested and
compared with the control.
Flow cytometric analysis of mitochondria in IMR 90 cells
After experimental treatments, the cells were harvested,
counted, and analyzed within 60 min for NAO retention by mitochondria
as described (32
33
34
35)
, with minor modifications. Aliquots
of 0.5 x 106 cells of each treatment were
transferred to separate polystyrene round-bottom tubes, centrifuged,
the supernatant removed, and the cells suspended in 980 µl of DEMEM
medium without FBS. The cells were then loaded with NAO at a final
concentration of 1.5 µM (20 µl of NAO from 75 µM stock of NAO in
N,N-dimethylformamide). The cells were incubated in the dark for 15 min
at room temperature, followed by centrifugation. Then the cells were
washed once with 2 ml of PBS and resuspended in 400 µl of PBS. All
centrifugations were for 7 min at 350 g. The fluorescence
and light scattering properties of the NAO-loaded cells were assayed
with a FACSort flow cytometer (Becton Dickinson, Rutherford, NJ), and
the data were analyzed with Cell Quest software running on a Macintosh
computer. The dye was excited with a 488 nm argon laser and the emitted
NAO fluorescence was gathered by the FL-1 photomultiplier (530±15 nm).
Before analysis, the voltages for FL-1 were set using LinearFlowTM
calibration beads (Molecular Probes) to compensate for any day-to-day
fluctuations in photomultiplier sensitivity. A density plot was used to
simultaneously assay light scattering properties and a region was drawn
around the target cells. Fluorescence properties were obtained for the
target region and histogram plots were used to determine the
fluorescence intensity for each cell population. Mean fluorescence
values were compared between treatment groups to determine the effects
of treatment on mitochondria in the cell.
Effect of hydrogen peroxide on mitochondrial hyperstaining by NAO
IMR90 cells (2x106/ml) were treated with
increasing doses (0–800 µM) of hydrogen peroxide for 20 min in
DMEM/20 mM HEPES without FBS at 37°C. Hydrogen peroxide was washed
out and the cells were incubated for 10 min with 1 µM NAO, followed
by washes as described above. The cells were resuspended into 500 µl
of ice-cold Hanks buffer. The florescence from NAO was measured by FACS
analysis as described above. Protection by NtBHA of
H2O2-induced toxicity to
mitochondria was evaluated by incubating the cells with NtBHA for 2
min, followed by hydrogen peroxide treatment.
HPLC-EC method for detection of NtBHA and its interaction with
IMR90 cells
Authentic NtBHA was prepared in 0.2 M MSA. Separation was
achieved by HPLC using a Suplecosil LC18-DB 3 µm column (150x4.6 mm;
Supelco, Bellefonte, PA). The mobile phase consisted of 20 mM
NaH2PO4, 5 mM octane
sulfonic acid, and 2% methanol, pH 2.7 (phosphoric acid) at a flow
rate of 1 ml/min. NtBHA was detected by electrochemical detection using
an ESA model 5100A Coulochem detector and model 5010 analytical cell
combination. Oxidation potentials of 0 V and 0.65 V were used for
electrodes 1 and 2, respectively. Full-scale output was 10 µA. A
linear standard curve for NtBHA was established between 50 and 2000
pmol. Experiments on permeability and interaction of NtBHA with cells
were made in control cells that were maintained in medium supplied with
normal FBS. For permeability, IMR90 cells
(
3x106/ml) were incubated at room temperature
with 1 mM NtBHA for different intervals. Then the cells were washed
once with cold PBS, resuspended in 200 µl of ice-cold 0.2 M MSA and
0.5 mM DTPA, and allowed to stand for 10 min at room temperature. The
interaction of NtBHA with IMR90 cells was studied using different
inhibitors of mitochondria. The cells were incubated separately with
each inhibitor for 5 min, then 1 mM NtBHA was added for 30 min. Complex
I was inhibited by 4 µM rotenone, cytochrome c oxidase by
5 mM KCN, and complex III by 14 µg/ml antimycin A. The uncoupler CCCP
(3 µM) and NH4Cl (20 mM) were used to oxidize
mitochondrial NADH, then protein was precipitated by 0.2 M MSA/DTPA and
pelleted by centrifugation. The supernatant was filtered with 30,000 Da
MW cutoff Ultrafree filters (Millipore, Bedford, MA), and a volume
equivalent to 40–50 µg protein was injected onto the HPLC.
Statistical analysis
Statistical analysis using the Student’s two-tailed
t test or nonparametric Mann-Whitney test was performed with
an Instat Statistical Analysis program (Instat, San Diego, CA) or
multiple regression analysis was done by STATISTICA version 5 for PC
computer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, B
, D
). The increase in daily food intake by young rats
treated with NtBHA vs. young control rats was not statistically
significant (Fig. 1A
, C
). NtBHA has no effect on the body
weight of the rats from either age group. For example, the body weight
for the control group in the first and last week of the study was
408 ± 27 and 407 ± 23 (n=5) and for the
NtBHA-treated group, 413 ± 24 and 418 ± 28
(n=5).
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An age-dependent decrease in ambulatory activity from 417 ± 56 (n=5) to 143 ± 17 cm/h/d (P<0.001, n=8) was observed when young and old rats were compared. NtBHA treatment significantly improved ambulatory activity in old rats to 296 ± 7 cm/h/d (P<0.0001, n=9) compared with the appropriate control, but remained unchanged in young rats (399±40 cm/h/d, n=5).
NtBHA modulates age-related oxidative changes in liver of old rats
Glutathione-mixed disulfides (bound GSH) were measured as a marker
for oxidative stress. The pool of glutathione-mixed-disulfides in the
liver increased significantly with age and was reversed by treatment
with NtBHA (Fig. 2
). The level of free glutathione was not affected by age and NtBHA
showed no effect on the levels of GSH in the liver.
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Old (but not young) rats fed for 25 days with NtBHA possessed more
coupled mitochondria than their controls. The RCR values for experiment
I were 3.62 ± 0.061 vs. 4.25 ± 0.100 (P<0.01,
rats for group were 3–4) and for experiment II were 4.87 ± 0.44
vs. 5.62 ± 0.51 (P<0.04; rats for group were 5) for
control and NtBHA treated, respectively. NtBHA partially reversed the
age-dependent decline in GDH (36)
but had no effect on the
age-dependent decline in G6PDH activity (37
38
39
40
41)
or on
catalase activity (data not shown).
NtBHA prevents senescence induced by charcoal/dextran-treated FBS
in IMR90 cells
IMR90 cells maintained in medium supplemented with c/dFBS senesce
faster than control cells that are maintained in medium supplemented
with nFBS (Fig. 3
). When the c/dFBS medium was supplemented with 100 µM NtBHA, the
prosenescence effect of c/dFBS was prevented and the cells gained more
PDLs, achieving as many PDLs as the controls in nFBS (Fig. 3)
.
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To counteract the prosenescence effect of c/dFBS, we supplemented the cells grown in c/dFBS medium with thyroid hormones (T3/T4) to levels seen in nFBS. Despite our expectations, resupplementation of only T3/T4 did not improve the growth conditions of the cells, and a combination of NtBHA and T3/T4 added only 1–2 PDLs on top of the gain achieved by using NtBHA alone.
A senescence-dependent increase in the ability of the cells to retain
NAO was observed (Fig. 4
). We refer to this phenomenon as hyperstaining of mitochondria. In
medium supplemented with c/dFBS vs. nFBS, there were higher levels of
NAO fluorescence, both at low PDLs and as the cells senesced (Fig. 4)
.
Consistent with the gain in PDLs, NtBHA prevented the c/dFBS-induced
hyperstaining of mitochondria (Fig. 4)
. These effects of NtBHA were
observed also in control cells maintained in normal FBS.
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We also observed hyperstaining of mitochondria in young IMR90 cells as
a result of treatment with hydrogen peroxide (Fig. 5
). However, when NtBHA was used to pretreat the cells before and during
exposure to 200 µM hydrogen peroxide, a total prevention of
hyperstaining of mitochondria was achieved (Fig. 5)
. When higher
concentrations of hydrogen peroxide were applied, NtBHA was less
effective (Fig. 5)
.
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The effect of NtBHA on the activity of proteasomes from IMR90 cells
Proteasomal activity was determined in cells at the PDLs between
50 and 75. When cells were maintained in a medium supplemented with
NtBHA, a 30–40% increase in the activity of the proteasome was
observed regardless of the type of media (Fig. 6
).
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The steady-state level of NtBHA is maintained by mitochondrial NADH
Human lung fibroblasts (IMR90) were used as a model to elucidate
interaction of NtBHA at the cellular level. NtBHA readily penetrates
and accumulates in intact cells (Fig. 7
). Assuming a fibroblast volume of 500 fl and 5 x
106 cells/mg protein, it was estimated that NtBHA
accumulates at 10- to 15-fold above the extracellular concentration (1
mM). We conclude that the intracellular steady-state concentration of
NtBHA was maintained by mitochondrial NADH, since oxidation of
mitochondrial NADH by ammonium chloride or CCCP decreased the
intracellular concentration of NtBHA by 95% and 50%, respectively
(Fig. 8
). Antimycin A, an inhibitor of ubiquinol:cytochrome c
oxidoreductase (complex III), decreased the intracellular concentration
of NtBHA by 40%, whereas other mitochondrial inhibitors such as
rotenone and KCN had no effect on the level of intracellular NtBHA
(Fig. 8)
. These reagents did not affect NtBHA in a cell-free system,
which indicates they do not react with NtBHA (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 42)
. Mitochondria are vulnerable to oxidants because
they are the major source of free radicals in the cell and are limited
in their ability to cope with oxidative stress (43
, 44)
.
Several basic functions of mitochondria have been reported to decline
with age (such as RCR, , and activity of complexes I, III, and
IV) as a result of cumulative oxidative damage. Thus, the age-dependent
decline in the normal functions of tissue can be attributed in part to
changes in the mitochondria. Some of the age-related changes to
mitochondria have been prevented or delayed by dietary supplementation
(21
22
23
, 45)
with the mitochondrial metabolites, R-lipoic
acid, and acetyl-L-carnitine, which benefited old rats after a few
weeks of daily intake. These two metabolites are known to play a role
in the basic biochemistry of mitochondria, with R-lipoic acid also
acting as an antioxidant (46)
. Thus, age-related changes
to mitochondria can be attenuated by appropriate supplementation that
is accompanied by major benefits to the entire organism (21
22
23
, 47
, 48)
.
When fed to rats, PBN (a spin trap) was shown to be an alternative to
lipoic acid as a mitochondrial antioxidant. We showed that its
breakdown product, NtBHA, was much more active than PBN in vitro
(25)
. When we tested its biological activity in vivo, we
found it reversed the age-related decline in food consumption and
ambulatory activity in rats, although it is not clear which of the two
parameters is primarily affected. Body weight of the rats was not
affected by NtBHA, which suggests that the increase in food consumption
did not cause a buildup of fat tissue. NtBHA did not affect the daily
water consumption by rats and had no observable side effects on either
age group.
In our present study, we found that NtBHA fed to old rats improves the mitochondrial RCR, prevents the loss of GDH, and decreases glutathione-mixed disulfides in proteins from liver. On the other hand, NtBHA showed no significant effect on liver mitochondria of young rats, even though the dose these rats were receiving was 40% higher than the old rats (as the young consume more water). These observations suggest that NtBHA acts partly as an antioxidant that protects mitochondria and proteins from oxidative damage.
To assess further the effect of NtBHA on mitochondria, we maintained
IMR90 cells in a medium that possesses suboptimal levels of growth
factors. These conditions were established by using medium supplemented
with 10% c/dFBS. This is a well-known approach to minimize growth
factors (thyroid hormones, insulin, and steroids) in serum
(30)
. The levels of micronutrients, vitamins, and minerals
were not different from the control medium (nFBS), because they either
were not affected by charcoal/dextran or were supplemented in DMEM.
IMR90 cells grown in medium supplemented with c/dFBS exhibited
considerable senescence-dependent hyperstaining by NAO and senesce
before their same-batch controls, suggesting a profound influence on
mitochondria (Figs. 3
, 4)
. The effect of c/dFBS on the cells may be
explained by the low level of hormones (thyroid hormones, insulin, and
steroids) vs. nFBS (30)
. These hormones can interact with
and affect mitochondria (49
50
51
52)
. However, in contrast to
our expectations, reconstitution of only T3/T4 hormones to c/dFBS did
not restore the PDLs or improve the mitochondrial status (Fig. 4)
,
which emphasizes the significance of the other missing growth factors
for normal senescence of these cells.
As fibroblasts senesce, they retain high levels of
mitochondrial-specific dyes such as Rh123 (10
, 25
, 53
54
55)
or NAO (Fig. 4)
. Mitochondrial hyperstaining was also observed in a
fraction of hepatocytes isolated from old rats (10)
and in
fibroblasts from older humans (54)
. We demonstrated that
hyperstaining of mitochondria by NAO is not limited to senescence, as
similar effects were seen when hydrogen peroxide (Fig. 5)
, valinomycin,
antimycin A, and ceramide were applied for a short period to young
cells (data not shown). Thus, it is conceivable that hyperstaining of
mitochondria is a marker for stressed mitochondria in the cell (H.
Atamna and B. N. Ames, unpublished results). The model of
acceleration of cellular senescence (c/dFBS) probably shares common
parameters with the normal mechanism of cellular senescence (nFBS), and
both negatively affect mitochondria. Consistent with our current
observation with NAO, we had previously demonstrated that NtBHA
prevented the age-related hyperstaining of mitochondria from IMR90
cells as evaluated by Rh123 (25)
.
Proteasomal activity is increased in IMR90 cells by NtBHA (Fig. 6)
. The
activity of the proteasome was consistently higher in cells maintained
in NtBHA regardless of the type of media used. Since proteasomes are
susceptible to oxidative damage induced by an oxidizing agent
(31)
, we assume that NtBHA protects the enzyme from
endogenous oxidants. We have shown that NtBHA increases the ratio
GSH/GSSG and protects IMR90 cells from
H2O2 (25)
. The
protection of proteasomal activity by N-hydroxyl amines could have a
marked effect on aging and age-related disorders.
NtBHA is a reducing agent that can undergo two steps of one-electron
oxidation (56
57
58
59)
to produce N-t-butyl
hydronitroxide (from one-electron oxidation) and
2-methyl-2-nitrosopropane (from two electron oxidation). NtBHA also
reacts with reactive aldehydes (measured by HPLC-EC), and thus could
prevent damage from lipid peroxidation to cellular macromolecules;
NtBHA reacts with hydrogen peroxide, superoxide, and oxidized
cytochrome c as measured by EPR (E. Ho. H. Atamna, and
B. N. Ames, unpublished results).
The reduced NtBHA is predominant in the cell as a result of the reduced
intracellular environment. Mitochondrial NADH maintains NtBHA in its
reduced form, as evident from CCCP- and
NH4Cl-dependent oxidation of mitochondrial NADH
(60
, 61)
. Ammonium chloride (NH4Cl),
causes more than a 95% decrement in the intracellular steady-state
concentration of NtBHA. Oxidation of mitochondrial NADH by
NH4Cl renders the mitochondrial ETC (including
cytochrome c) and other cytosolic factors almost completely
oxidized. It could be that intracellular NtBHA is oxidized by the
mitochondrial electron transport chain. Cytochrome c, which
has been shown to slowly oxidize NtBHA in a cell-free system
(25)
, could play some role in the intracellular oxidation
of NtBHA. Complex IV then oxidizes reduced cytochrome c and
forms water. On the other hand, CCCP, a mitochondrial uncoupler that
partially oxidizes mitochondrial NADH and the ETC (62)
,
causes only a 50% decrease in the steady-state level of intracellular
NtBHA (Fig. 8)
. These observations support the notion that
intracellular NtBHA shuttles between reduced and oxidized forms and
that mitochondrial NADH plays a role in maintaining and recycling of
NtBHA. The difference in the efficiency in oxidizing NtBHA between
ammonium chloride and CCCP (Fig. 8)
could be explained by
NH4Cl oxidizing NADH and minimizing the reduction
of NAD by consuming -ketoglutarate, thus inhibiting the
tricarboxylic acid cycle (TCA), leaving all the ETC components
completely oxidized. CCCP, on the other hand, oxidizes NADH by
dissipating mitochondrial and has no inhibitory effect on the
TCA cycle, which continues to reduce NAD to NADH. Thus, cellular
components involved in reduction of the oxidized form of NtBHA (i.e.,
N-t-butyl hydronitroxide and/or 2-methyl-2-nitrosopropane)
are likely to be the same systems that use NADH.
Antimycin A inhibits complex III and almost completely prevents
oxidation of NADH and succinate by complexes I and II. As a result,
cytochrome c and complex IV are oxidized, mitochondrial
pyrimidine nucleotides are reduced, and electrons leak from
ubisemiquinone to form free radicals (63
, 64)
. The 40%
decline in NtBHA induced by antimycin A could be explained as a result
of oxidation of NtBHA by ubisemiquinone and/or ROS, which are generated
from its auto-oxidation. Oxidized cytochrome c could also
contribute in part to the oxidation of NtBHA, which is induced by
treatment with antimycin A. Since mitochondrial NADH is not affected by
antimycin A, 60% of the intracellular steady-state level of NtBHA
remained reduced. This again suggests that NtBHA is recycled through
steps of oxidization and reduction that are controlled by NADH. Under
low mitochondrial NADH, a net oxidation of NtBHA dominates and appears
not be detected easily by electrochemical detection. Thus, NtBHA is
indirectly maintained in the reduced form in cells (Fig. 7)
by
mitochondrial NADH.
KCN and rotenone, inhibitors of complex IV and complex I, respectively,
have no effect on intracellular NtBHA. KCN keeps the mitochondrial ETC
fully reduced by inhibiting complex IV; rotenone inhibits only complex
I and has no effect on the rest of ETC. NtBHA is a classical
antioxidant in that it reacts with free radicals (59)
to
form N-t-butyl hydronitroxide, a stable free radical, and
reacts with reactive aldehydes (E. Ho, H. Atamna, and B. N. Ames,
unpublished results). NtBHA could be recycled by mitochondria through
N-t-butyl hydronitroxide and 2-methyl-2-nitrosopropane as
intermediates. NtBHA or the intermediates could participate in
oxidation reduction reactions at the site of electron leakage (i.e.,
ubisemiquinone), thus competing with molecular
O2. Then NtBHA could be oxidized enzymatically
(i.e., complex IV), which results in electron shuttling between
ubisemiquinone and complex IV, which could prevent electron leak to
O2. We are preparing N-t-butyl
hydronitroxide in order to establish the intracellular components
involved in its reduction to NtBHA.
Evidence suggests that the mitochondria in old (vs. young) tissue
exhibit morphological and biochemical modifications that accelerate
leakage of electrons from ETC to O2 to form
superoxide and hydrogen peroxide. The interaction between NtBHA and
H2O2 provides protection to
mitochondria as shown by its preventing
H2O2-induced hyperstaining
of mitochondria in IMR90 cells (Fig. 5)
. The influence of feeding NtBHA
in high doses at the levels of damage in rats brain and biochemical
functionality is under study.
We are grateful to Ann Fischer (Tissue Culture Facility, NIEHS Center, University of California, Berkeley) and Bluma Lesch for their assistance. We thank Dr. P. Walter for comments on the manuscript. This work was supported by NIA grant AG17140, Ellison Medical Foundation Senior Investigator grant Ss-0422–99, Department of Energy grant DE-FG03–00ER62943, Tobacco-Related Disease Research Program grant 7RT-0178, Wheeler Fund for the Biological Sciences grant at the University of California Berkeley, National Foundation for Cancer Research grant M2661, and National Institute of Environmental Health Sciences Center grant ES01896 to B.N.A.
Received for publication April 4, 2001.
Revision received June 15, 2001.
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