Halliwell B,et, al

(FEBS Letters, 486(1), 2000) (Federation of European Biochemical Societies)

Abstract

Because hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent, levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed. Levels of H2O2 in the body may be controlled not only by catabolism, but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Urinary H2O2 levels are influenced by diet, but under certain conditions might be a valuable biomarker of `oxidative stress'.

1. Introduction

Hydrogen peroxide is freely miscible with water and is apparently able to cross cell membranes readily, high (usually 50+ micro-M) levels being cytotoxic. It is therefore widely thought that H2O2 is very toxic in vivo and must be rapidly eliminated, employing enzymes such as catalases, peroxidases (especially glutathione peroxidases) and thioredoxin-linked systems. Paradoxically, however, in chemical terms, H2O2 is poorly reactive: it can act as a mild oxidizing or as a mild reducing agent, but it does not oxidize most biological molecules readily, including lipids, DNA and proteins (unless the latter have hyper-reactive thiol groups or methionine residues). The danger of H2O2 largely comes from its ready conversion to the indiscriminately reactive hydroxyl radical, either by exposure to ultraviolet light or by interaction with a range of transition metal ions, of which the most important is probably iron.

Living organisms have evolved mechanisms to sequester transition metal ions into protein-bound forms that cannot catalyze hydroxyl radical formation and other free radical reactions in vivo. These mechanisms are especially important in such extracellular fluids as the blood plasma. Nevertheless, H2O2 can contribute to Fenton chemistry not only by being one of the substrates but also by providing the other, e.g. by liberating iron from heme proteins. Addition of H2O2 to cells in culture can lead to transition metal ion-dependent hydroxyl radical-mediated oxidative DNA damage, although this damage appears to be rapidly repaired provided that the cells are not rendered non-viable by an excess of H2O2. (Spencer J, et al, Biochem. Biophys. Res. Commun, 224, 1996)

However, levels of H2O2 at or below about 20-50 micro-M seem to have limited cytotoxicity to many cell types. Indeed, there is a growing literature showing that H2O2 can be used as an inter- and intra-cellular signalling molecule. At sites of inflammation, H2O2 generated by activated phagocytes appears to modulate the inflammatory process, e.g. by up-regulating expression of adhesion molecules, controlling cell proliferation or apoptosis and modulating platelet aggregation.

2. Exposure of human tissues to H2O2

Hydrogen peroxide is generated in vivo by the dismutation of superoxide radical both non-enzymatically and catalyzed by superoxide dismutase enzymes. Hydrogen peroxide is also directly produced by a range of oxidase enzymes including glycollate and monoamine oxidases as well as by the peroxisomal pathway for beta-oxidation of fatty acids. With the apparent exception of cardiac muscle, mitochondria in most tissues appear to have limited capacity to remove H2O2, in that they readily generate substantial amounts of H2O2 in vitro and probably in vivo. Although mitochondria contain glutathione peroxidase and thioredoxin-linked peroxidase activities, the efficiency of these enzymes in removing H2O2 is uncertain given the ease with which mitochondria release H2O2. It thus seems likely that most or all human cells are exposed to some level of H2O2, with the mitochondria being an important source. However, certain tissues may be exposed to higher H2O2 concentrations.

2.1. The oral cavity, oesophagus and stomach

Several beverages commonly drunk by humans can contain H2O2 at concentrations above 100 micro-M, including green and black tea and especially instant coffee. When such beverages are ingested, the H2O2 they contain presumably rapidly diffuses into the cells of the oral cavity and upper part of the gastrointestinal tract. Oral bacteria also produce H2O2, although the resulting levels of exposure of the oral tissues are uncertain. It is often suggested that H2O2 released into saliva is used by salivary peroxidase to oxidize thiocyanate into products toxic to certain bacterial strains.

2.2. The respiratory system

The cells lining the respiratory system, in common with the oral and oesophageal epithelium, are exposed to high O2 concentrations (21%) as compared with most other body tissues. Hydrogen peroxide is present in exhaled air of humans from phagocytes (e.g. alveolar macrophages, neutrophils in the oral cavity, or neutrophils recruited to the lungs in inflammatory lung diseases) or other lung cells. Amounts of exhaled H2O2 appear greater in subjects with inflammatory lung diseases.

2.3. The kidney, urinary tract and bladder

Substantial quantities of H2O2, at concentrations sometimes exceeding 100 micro-M can be detected in freshly voided human urine, even in babies. The H2O2 detected in human urine appears to arise, at least in part, by superoxide radical-dependent auto-oxidation of urinary molecules, some of which originate from diet. Traces of superoxide dismutase are present in urine: this enzyme, as well as the acidic pH of urine, should facilitate both enzymic and non-enzymic dismutation of superoxide radicals to H2O2. The high levels of H2O2 that can be detected in some urine samples, strongly suggest that at least some H2O2 generation occurs within the bladder. Hydrogen peroxide has an antibacterial effect and it may be that its presence at high levels in urine could be advantageous in diminishing infections of the bladder and urinary tract. Indeed, there are suggestions that H2O2 is involved in modulation of renal function. Excretion of H2O2 may also represent a metabolic mechanism for controlling its levels in the human body, a valuable tool for assessment of `oxidative stress'.

2.4. Vascular endothelial and circulating blood cells

Some studies have claimed substantial levels of H2O2 (up to ~35 micro-M) in human blood plasma, but others have claimed levels to be very low. In part, it is degraded by the traces of catalase present, but H2O2 can also react with heme proteins, ascorbate, and protein-SH groups. In vivo, H2O2 generated in plasma could also diffuse into erythrocytes, white cells, endothelial cells and platelets for metabolism. Human plasma may be continuously generating H2O2, at least under pathological conditions.

2.5. Ocular tissues

The presence of H2O2, at widely varying levels (in some cases, 100 micro-M or more), has been reported in human and other animal aqueous and vitreous humors. Any impairment in the capacity of the lens epithelium, retina or other ocular tissues to dispose of H2O2 would then result in its accumulation. The origin of this H2O2 is uncertain, but oxidation of glutathione or ascorbate is one possibility.

3. Conclusion

Hydrogen peroxide appears to be a ubiquitous molecule. We exhale it, excrete it and take it in from diet. It can be detected in drinking water, rain-water and sea water (Willey J et al, Eco. Prog Ser. 178, 1999; Fujiwara K, Nippon Bunseki Kagakkai 9, 1999.) These data emphasize the importance of metal ion sequestration in preventing the toxicity of H2O2 in vivo by decreasing the occurrence of Fenton chemistry.

CAUTION: Hydrogen peroxide is an irritant of the eyes, mucous membranes, and skin. Inhalation of high concentrations of the vapor or mist may cause extreme irritation of the nose and throat. Severe systemic poisoning may cause headache, dizziness, vomiting, diarrhea, tremors, numbness, convulsions, pulmonary edema, unconsciousness, and shock. Exposure for a short period of time to the mist or spray may cause stinging and tearing of the eyes. Skin contact with liquid hydrogen peroxide causes a temporary whitening or bleaching of the skin; if the skin is not washed promptly, redness and blisters may develop. Ingestion of hydrogen peroxide may cause irritation of the upper gastrointestinal tract and severe damage to the esophagus and stomach. (Hathaway G et al. Proctor and Hughes' chemical hazards of the workplace. Van Nostrand Reinhold. 1991) Splashes of high concentrations of hydrogen peroxide in the eyes may cause severe corneal damage. At very low concentrations (1 to 3 percent), instillation of hydrogen peroxide into the eye causes severe pain that later subsides. (Grant W, Toxicology of the eye. Charles C Thomas, 1986)

For more information on some the possible uses of hydrogen peroxide please click here.