
Indian Journal of Biochemistry & Biophysics
Vol. 27, October 1990, pp. 269-274


H2O2 has a role in cellular regulation


T. Ramasarma
Department of Biochemistry
Indian Institute of Science
Bangalore 560 012



Abstract:

H2O2, in addition to producing highly reactive molecules through 
hydroxyl radicals or peroxidase action, can exert a number of direct 
effects on cells, organelles and enzymes.  The stimulations include 
glucose transport, glucose incorporation into glycogen, HMP shunt 
pathway, lipid synthesis, release of calcium from mitochondria and of 
arachidonate from phospholipids, poly ADP ribosylation, and insulin 
receptor tyrosine kinase and pyruvate dehydrogenase activities.  The 
inactivations include glycolysis, lipolysis, reacylation of 
lysophospholipids, ATP synthesis, superoxide dismutase and protein 
kinase C.  Damages to DNA and proteoglycan and general cytotoxicity 
possibly through oxygen radicals were also observed.  A whole new 
range of effects will be opened by the finding that H2O2 can act as a 
signal transducer in oxidative stress by oxidizing a dithiol protein 
to disulphide form which then activates transcription of the stress 
inducible genes.  Many of these direct effects seem to be obtained by 
dithiol-disulphide modification of proteins and their active sites, 
as part of adaptive responses in oxidative stress.

   +        +         +         +         +         +         +


Molecular oxygen, also termed dioxygen, has two unpaired electrons.  
These go into separate antibonding n-orbitals which parallel spins.  
The stability and paramagnetic property of oxygen are due to this.  
The reductions of O2 to superoxide, hydrogen peroxide and water are 
made possible by adding one, two and four electrons to the anti-
bonding orbitals of dioxygen (1).  These reactions are shown in Fig. 
1 along with two dismutation reactions for superoxide and hydrogen 
peroxide.

The formation of H2O2 in cellular oxidation is known to occur by 
direct 2-electron reduction by flavoprotein oxidases (2), or by 1-
electron reduction to superoxide anions, two of which dismutate 
yielding a molecule each of H2O2 and O2 by the enzyme superoxide 
dismutase (3).  By its facility for electron exchange H2O2 can act 
both as an oxidant and a reductant typically found in catalase 
reaction itself.  In presence of Fe2+ and other metal ions, H2O2 can 
also generate hydroxyl radicals which are known to cause molecular 
damage.  H2O2 is toxic to cells and is indeed responsible for killing 
internalized bacteria in phagocytosis (4).  This led to the 
misconcept that H2O2 is undesirable by-product of oxidase reactions 
that the aerobic cells tackle by providing themselves with high 
concentrations of degrading enzymes such as catalase and glutathione 
peroxidase, which ensure adequate protection.  Peroxidases are of 
ubiquitous occurrence and utilize H2O2 to oxidize a wide range of 
compounds to yield important metabolites.  Therefore generation of 
H2O2 in cellular processes seems to be purposeful, and has been found 
to be widespread in occurrence in aerobic cells and cellular 
organelles (5,6).  But reduction of oxygen to H2O2 by cytochrome 
oxidase, the major O2 user, had over-shadowed the importance of the 
qualitatively minor pathways.

Generation of H2O2 appears to be a natural process in aerobic cells 
as part of the of the reactions of a number of oxidases and 
dehydrogenases, essential in cellular activities.  Only the 
endomembranes, plasma membranes (7,8) and microsomes (9), have the 
special property of dormant NAD(P)H oxidation that can lead to very 
high rates of H2O2 generation in presence of decavanadate (10) or in 
phagocytosis (11).  Under normal conditions the rates are small and 
account for H2O2 no more than 2% of total O2 consumed.  Thus, in the 
presence of excess catalase and glutathione peroxidase in cells, the 
limited H2O2 has little chance of exhibiting its purported toxicity.

With respect to mitochondria the accumulated information indicates 
the presence of H2O2 generator distinct from the respiratory chain 
(12).  The parallel utilization of substrates has provided a false 
facade of sharing dehydrogenases.  The two activities, substrate-
dependent dye reduction and H2O2 generation, respond differently.  
Only the H2O2 generation is inhibited by phenolates (12), increased 
in cold exposure (13) and noradrenaline treatment (14) and decreased 
in heat exposure (15,16).  This regulated activity therefore must 
have a meaningful physiological role.

A specific need for H2O2 in killing the phagocytosed bacteria is 
established.  While lysosomes undertake the task of dissolving out 
the components of the injected particles, the killing of pathogenic 
bacteria requires a H2O2 dependent reaction, yet to be defined.  This 
process utilizes the latent capacity of NAD(P)H oxidation of the 
plasma membrane unmasked by a serum component picked up during 
opsonization and requires the phagosome structure (17).  The 
explanation for these peculiar features is not available (18).

Intrinsic high rates of H2O2 generation, an apparent metabolic 
necessity, seems to be a characteristic of protozoa.  Parasitism in 
the case of trypanosoma and plasmodium may indeed by characterized by 
the removal by the host cell of such metabolically generated H2O2, 
otherwise self-destructive in view of the absence of H2O2 detoxifying 
systems in these protozoa.  This is exemplified by the decreased 
survival of these disease-causing parasites in the host cells with 
defective H2O2 scavenging mechanism or on treatments that lead to 
increased H2O2 generation (19).

Since seventies it is increasingly realized that H2O2 is not a mere 
wasteful by-product but fulfills functional, metabolic needs.  Inter-
relationship of hormone H2O2 dithiol proteins-metabolic control is 
suggested in the case of insulin-mimicking action of H2O2 (20).  The 
hormonal response of NADH dehydrogenase of plasma membrane (21) that 
is known to generate H2O2 (22) is documented.  An ubiquitous, 
regulated phenomenon must have a role in cellular activities.  The 
small rates, in fact, are best designed for that purpose in view of 
its toxicity and high reactivity.  A number of direct effects of H2O2 
on metabolism and enzyme activities are described (Table 1) and this 
review projects the importance of H2O2 in this regard.


Carbohydrate Metabolism

As early as 1958 Warburg and coworkers (23) and Holzer and Frank (24) 
recognized that the presence of H2O2 depressed gycolytic flux.  This 
direct effect on tumour cells, confirmed by others (25,26), can be 
partially reversed by addition of endogenous NAD (24,25).  
Interestingly this effect was traced to decrease in activity 
specifically of glyceraldehyde-3-phosphate dehydrogenase (GAPD) 
raising the possibility of an oxidative inactivation by H2O2 of this 
known sulphydryl enzyme (27,28).

In a comprehensive study with P388D1 cells, Hyslop et al. (29) showed 
that a large, rapid inhibition of GAPD was obtained with IC50 of 100 
uM concentration of H2O2.  Purified rabbit muscle enzyme was 
inhibited completely at this concentration.  Similar inibition on 
exposure of cells or tissue to H2O2 of this enzyme was reported for 
human lung carcinomal cells (30) which can be partially reversed by 
DTT, and for rat heart which cannot be reversed by DTT (31).  In 
these studies on treatment with H2O2, Hyslop (29) and Radda (32) and 
coworkers found that only GAPD showed rapid decreases (Fig.2) but 
some glycolytic enzymes, among the following tested, remained 
unaffected: hexokinase, phosphoglucose isomerase, 
phosphofructokinase, aldolase, triose-P-isomerase, kinases of 
pyruvate and phosphoglycerate, enolase and dehydrogenases of G-6-P 
and lactate.  As expected the fructose 1, 6-diphosphate and aldolase-
products (triose phosphates) accumulated in cells under conditions of 
inhibition of GAPD by H2O2.  Some indication of decrease in hexose 
monophosphates as well as glucose-1, 6-diphosphate was obtained with 
P388D1 cells which appears to be more due to lack of ATP than by 
modifications of the enzymes involved.

H2O2 was shown to stimulate transport of glucose (33) and glucose C-1 
oxidation (34) as well as glucose incorporation into glycogen (35) in 
rat adipocytes, and insulin-responsive tissue.  These effects follow 
the known stimulation of HMP shunt activity in such as tissue by 
oxidants and H2O2 (36,37).

In P388D1 cells treated with H2O2, the net glucose uptake decreased, 
coinciding with decrease in lactate production, but not the glucose 
transporter rate (29).  It appears that G-6-P-dehydrogenase was not 
the target of action of increased overall activity of HMP shunt and 
the step affected is yet to be identified.

In intact spinach chloroplasts, H2O2 treatment caused drastic 
inhibition of CO2 fixation that can be reversed by catalase or DTT 
(38).  This resulted in increase of incorporation of 14CO2 in hexose 
and heptulose bisphosphates and pentose phosphates, and decrease in 
hexose monophosphates and ribulose 1,5-bisphosphate.  Since oxidative 
pentose phosphate cycle and G-6-P-dehydrogenase are known to be 
inactivated by dithiols (39), the H2O2 activation is conjectured to 
be a reversal of this effect by 'oxidation of light-generated SH-
groups'.


Lipid Metabolism

H2O2 was found to inhibit lipolysis stimulated by theophylline (40) 
or isoproterenol (41).  Some of these compounds used are prone to 
oxidation by H2O2 and thus in principle the effect of H2O2 may simply 
be to destroy the stimulator.  Using ritodrine (100 nM), a B-
adrenergic agonist resistant to oxidative destruction, and glucagon 
(1nM), Little and deHaen (42) were able to show that stimulated 
lipolysis in epididymal fat cells was indeed inhibited by H2O2 
similar to insulin.

On H2O2 treatment stimulation of [14C]glucose incorporation into 
lipids, particularly glyceride-fatty acids, had been reported similar 
to insulin response (43,44).  Accompanying this effect the active 
form of pyruvate dehydrogenase showed rapid increase, without 
changing the total amount of the enzyme protein (44).  This 
stimulation, like with insulin, was found to occur in the absence of 
glucose in the medium and therefore is independent of increased 
glucose due to its enhanced transport (33), also known to stimulate 
the active form of enzyme (45).  The response of pyruvate 
dehydrogenase increase was obtained as early as 5 min after treatment 
of adipocytes with H2O2 (0.31 mM) and was maximal at 15 min followed 
by decrease consequent to degradation of H2O2 (Fig. 2).  These and 
other experiments led May and deHaen (20,44) to propose that H2O2 
plays a second messenger role.  In further experiments deHaen and 
coworkers (46) found that in cells treated with 100 nM of phenyl 
(isopropyl) adenosine, a potent inhibitor of lipolysis, and exposed 
to insulin in the presence of medium glucose, glycerol production and 
cyclic AMP concentrations were unaffected, whereas free fatty acid 
release was inhibited coinciding with increase in H2O2 production.  
Therefore they considered that "H2O2 production is a metabolic 
consequence of insulin action distal to the receptor and is 
correlated with the fall of free fatty acids."

Irreversible brain injury during ischemia is thought to be due to 
released unsaturated fatty acids through their peroxidation products.  
The fatty acid hydroperoxides (LOOH) were found to inhibit 
reacylation of phospholipid in neural membranes (47), an essential 
step in repair of damaged membranes.

H2O2 treatment of alveolar macrophages inihibited 5-lipoxygenase and 
stimulated release of arachidonic acid and synthesis of thromboxane 
A2 (48).  Conditions that promote lipid peroxidation, however, 
stimulated lipoxygenase activity (49).

In the case of soybean lipoxygenase, H2O2 behaves as a potent 
activator (5).


ATP and NAD Metabolism

One of the striking effects of H2O2 treatment of cells is the rapid 
depression of intracellular ATP (51,52) and NAD+ (refs 53,54) 
concentrations.  In P388 d1 cells, the t 1/2 for decrease of levels 
of ATP and NAD+ were found to be about 15 and 4 min, respectively, on 
treatment with 50 uM concentrations of H2O2.  Calculations of data on 
ADP phosphorylation in these experiments revealed that both 
glycolytic and mitochondrial contributions were inhibited and results 
in loss of pool of ATP and eventual cellular death.  The decline in 
ADP phosphorylation appears to be related more to inactivation of the 
ATPase-synthase rather than to the decline in the rate of electron 
transport according to Hyslop et al. (29).

Both NAD+ and NADH concentration decline in H2O2 treated cells.  This 
appears to be due to the use in ADP ribosylating nuclear proteins 
during this stress (55) on activation of the nuclear enzyme, poly 
(ADP ribose) polymerase, also known to occur (53,54).


Protein Phosphorylation

Another relationship exists between H2O2 and insulin through the 
mechanism of protein phosphorylation.  Insulin receptor is a self 
phosphorylating insulin-sensitive protein kinase.  This protein 
phosphorylation was found to be dramatically potentiated by H2O2 in 
intact Fao cells (56), and was inhibited by antagonists such as 
phorbol ester and cyclic AMP.  Such effects were also obtained with 
vanadate (26) which was found in our laboratory to generate H2O2 on 
oxidation of NADH by plasma membranes (8).  Thus, the effects with 
reduced naphthoquinones (57) and vanadate (58) on stimulation of 
protein tyrosine phosphorylation in plasma membrane appear to depend 
on generation of H2O2.  Further studies by Heffetz et al. (59) 
indicated that H2O2 (3mM) and vanadate (0.1 mM) in combination far 
exceeded insulin in stimulating phosphorylation of four proteins in 
Fao cells and part of this effect was obtained by marked inhibition 
of protein-tyrosine phosphate hydrolysis.

Purified protein kinase C was found to be inactivated by H2O2 and the 
susceptibility increased in the presence of calcium ions and phorbol 
ester (6).  This phenomenon seems to be complex because mild 
oxidation showed a small increase but further oxidation damaged both 
regulatory and catalytic domains.  Also, the membrane-bound enzyme, 
which increased on activation of x-adrenergic receptor by adrenergic 
agonists (61) and also by decavanadate (62), was more susceptible to 
inactivation by H2O2 produced in situ as a result of such treatment 
(14,63).  Intracellular free calcium (64) itself registered fast rise 
on H2O2 treatment and also in synaptosomes on addition of menadione 
bisulphite which released endogenous H2O2.  Thus, all the effects of 
H2O2 seem to favour inactivation of protein kinase C to keep the 
dependent signal transduction inoperative.


Damage to Biopolymers and Cytotoxicity

Damage to DNA on H2O2 treatment of cells had been noted in several 
systems (51-53,66).  This effect may occur through calcium, as 
indicated by its prevention by its intracellular chelator, Quin 2 
(ref. 67).

Hyaluronic acid in proteoglycan aggregates was found to be fragmented 
on H2O2 treatment of neonatal human articular-cartilage.  This effect 
was apparently obtained through hydroxyl radicals and also involved 
cleavage of link protein to remove a trideca-peptide as well as 
modification of His (16) and Ala and Asn (21) to Asp (68).

Inactivation of superoxide dismutase of the Cu-Zn and Fe-types, but 
not Mn-type, occurred on treatment with H2O2 (69,70) and in the case 
of the bovine liver enzyme release of copper was responsible for 
this.

The above effects contribute to the cytotoxicity of H2O2.  The 
reactive oxygen radicals generated from H2O2 in presence of iron or 
trace metal ions are likely to cause strand breaks in DNA (71) or 
leaky membranes (72) or cytoskeletal plasma membrane perturbations 
(73).  H2O2 insult to mammalian (74) and bacteria (75) cells leading 
to killing include a variety of processes such as DNA strand breaks, 
poly ADP ribosylation, protein modifications, membrane perturbations 
and energy transducing systems.  Cell survival seems to depend on its 
ability to restore the cellular reductive process and thiol status 
(76).


Thiol-disulphide Status of Proteins

Glutathione redox cycle was affected in presence of H2O2 and 
intracellular thiols were oxidized (29,77,78).  The effects of such 
oxidations of proteins sulphydryls will be seen in their respective 
activities and in metabolism involving them.  This was established in 
cases of GAPD and pyruvate dehydrogenase described above.  It is 
apparent that H2O2 in small quantities generated in cells can exert 
powerful regulatory actions by modifying enzymes capable of redox 
changes of thioldisulphide type.  Ziegler (79) presented a case for 
such regulation of enzyme activity.  The enzymes thus affected are: 
phosphorylase a, fructose bisphosphatase, G-6-Pase, G-6-P 
dehydrogenase, acetyl CoA hydrolase, and pyruvate dehydrogenase are 
increased, while glycogen synthetase, phosphofructokinase, 
hexokinase, phosphoenol-pyruvate carboxykinase, GAPD, HMGCoA 
reductase, N-acetyl tranferase, protein kinase, guanylate cyclase and 
mevalonate kinase are decreased.

The cellular response to oxidative stress in the first place is 
adaptive and is likely to use redox reaction for counteracting the 
stress.  An excellent example of this is provided by the studies of 
Ames and coworkers (80) on direct activation by oxidation of a 
protein responsible for transcription of oxidative stress-inducible 
genes.  They found that the gene product of oxy R, a 34 kDa protein 
oxy R, which binds with promoter region of the oxy R, was oxidized 
rapidly and reversibly to disulphide form when the bacterial cells 
were exposed to H2O2 and was then able to activate transcription for 
at least 9 proteins, including catalase.  The purified oxy R, protein 
was found to bind to DNA in both reduced-inactive form and oxidized-
active form, albeit differently as characterized by the foot-
printing.  While both oxidized and reduced forms of the protein oxy R 
repress own expression in vitro, only the oxidized form was capable 
of stimulating expression of katG gene in a concentration dependent 
fashion that was sensitive to DTT.  It may be expected that other 
such proteins will be discovered where oxygen species are involved in 
metabolic regulation.

Long exposures and high concentrations of H2O2 do destroy the 
biological structures and lead to irreversible damage.  It appears 
that such lethal actions are initiated by oxygen radical species.  
This happens only in certain conditions such as phagocytosis.  Under 
normal physiological conditions, H2O2 is generated in small 
quantities and is rapidly used or degraded.  It is now clear that 
this regulated generation of H2O2 is not only used as a substrate for 
peroxidases, where present, but also for protein-thiol oxidation.  
The use of H2O2 for this additional role in cellular regulation has 
only revealed a vignette of its vast potential in modification of 
proteins and their activities.  H2O2 can perform a role similar to 
protein phosphorylations in cellular regulations.


Acknowledgment

The financial support from the Department of Science and Technolgy, 
Government of India, New Delhi is acknowledged.


Figure 1.

Reduction of oxygen [The reductions of dioxygen by 1.2 and 4 
electrons to superoxide, hydrogen peroxide and water, respectively 
are shown.  It may be noted the O-O distance progressively increases 
on reduction.  The two dismutations of superoxide and hydrogen 
peroxide by enzymes are indicated.  The formation of radical species 
of hydroxyl and lipid hydroperoxide are also shown]


Figure 2.

The changes in activities of glyceraldehyde-3-phosphate dehydrogenase 
and pyruvate dehydrogenase on incubation with H2O2 [The data are 
adapted from Hyslop et al. (2) for P388 D1 cells, Chatham et al. (32) 
for heart tissue and May and deHaen (44) for adipocytes]


Table 1.  Metabolic effects of H2O2 treatment

(Some of the effects described in the text for direct effects of H2O2 
treatment of tissues/cells/enzyme systems are summarized.  The time 
periods and mode of treatment are different in each case.


Tissue/cells      H2O2        Test System          % Control     Ref.
                  conc.                                           No.
                  mM
---------------------------------------------------------------------
Adipocytes, rat   0.20     [U-14C]Glucose -->         170          44
                            TG-fatty acids
                  0.31     Pyruvate dehydrogenase     185          44
                  0.06     Lipolysis, glycerol        Decreased    42
                            release (B-adrenergic
                            stimulated)

P3888 D1, cells   0.10     Net glucose uptake         40           29
                  0.10     Glucose-->lactate          50           29
                  0.10     HMP shunt pathway          510          29
                  0.10     Glyceraldehyde-3-P         50           29
                            dehydrogenase

Carcinoma cells   1.0      Glyceraldehyde-3-P         Decreased    30
                            dehydrogenase
                  (ROOH)   Glyceraldehyde-3-P         Decreased    31
                            dehydrogenase

Heart, rat        0.15     Glyceraldehyde-3-P         25           32

Fao cells         3.0      Insulin-receptor           Potentiated  56
                            tyrosine phosphorylation
                  3.0      Protein-tyrosine-P-        50           59
                            phosphatase

Protein kinase    5.0      Ca-dependent protein       20           60
 C                          phosphorylation

ADP-ribose        --       Poly ADP ribosylation      Increased    54
 polymerase                 of proteins

Pseudomonas       0.42     Dismutation of             50           60
 superoxide                 superoxide
 dismutase

Chloroplasts,     0.6      CO2 fixation               10           38
 spinach                    into sugar
                            phosphates

Soybean           0.5      5-Lipoxygenase             Increased    50

Synaptosomes,     0.025    Reacylation of             50           47
 rat brain

E. coli           0.06     Transcription of           Increased    80
                            oxy R gene controlled
                            oxidative stress
                            inducible genes



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Received 24 May 1990

