H₂O₂ has a role in cellular regulation
 

Indian Journal of Biochemistry & Biophysics

Vol. 27, October 1990, pp. 269-274

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 Omitted

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 Omitted

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]

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