Fundamental and Molecular Mechanisms of Mutagenesis ELSEVIER Mutation Research 350 (1996) 103-108 Antioxidant defences against reactive oxygen species causing genetic and other damage Diana Anderson Accepted 3 June 1995 K~~rwrds: Free radical damage: Antioxidant; DNA peroxidation; Cancer 1. Introduction Oxygen. present as 20% of the atmosphere, is a terminal oxidant essential for respiration and other oxidative reactions in all aerobic organisms. During the reduction of molecular oxygen, reactive oxygen species are formed. These free radicals are impli- cated in many diseases including atherosclerosis (Witzum. 19941, respiratory tract disorders (Cross et al., 1994), neurodegenerative disease (Jenner, 1994), inflammatory bowel disease (Grisham, 1994), cancer (Ames. 1989: Cerutti. 1994) and also in ageing (Ames, 1989. 1988a, b). A free radical is any atom or molecule that con- tains one or more unpaired electrons (Halliwell, 1994; Halliwell and Gutteridge. 1989). The unpaired elec- trons alter the chemical reactivity of an atom or molecule, usually making it more reactive than the corresponding non-radical. The chemical reactivity of radicals varies enormously. The hydrogen radical (H ‘) containing one proton and one electron is the simplest free radical. By the removal of (H ‘) from Corresponding author. Fax: + 44 I8 1 66 17039. other molecules, chain reactions are often initiated e.g. during lipid peroxidation. In the human body. solar radiation or low wavelength electromagnetic radiation. such as gamma-rays. from the environment can split water to generate the hydroxyl radical, OH ‘, which is very reactive at the site of formation. The body also makes another oxygen radical where the unpaired electron is located on oxygen. superox- ide (02 ). but this is poorly reactive. Active phago- cytes including neutrophils, monocytes. macrophages and eosinophils. generate large amounts of superox- ide in the killing of foreign organisms. However, with chronic inflammation this normal protective mechanism may itself be damaging (Grisham, 1994). Another physiological free radical is nitric oxide (NO ‘) which is made by the vascular endothelium as a relaxing factor, but excessive nitric oxide can be toxic (Moncada and Higgs. 1993). Superoxide can react with iron and copper ions to make hydroxyl radicals (Fig. I) or can combine with nitric oxide Oi- + NO + ONOO- (peroxynitrite). This decom- poses into toxic products including nitrogen dioxide gas (NO,), hydroxyl radical and nitronium ion (NO; ). Since most molecules in the body are not radicals. any reactive free radical generated is likely to react 0027-5 107/96/$I5.00 0 1996 Elsevier Science B.V. All rights reserved SSIII 0017-5 107(95)00096-8
Transcript
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER Mutation Research 350 (1996) 103-108
Antioxidant defences against reactive oxygen species causing genetic and other damage
Diana Anderson
Accepted 3 June 1995
K~~rwrds: Free radical damage: Antioxidant; DNA peroxidation; Cancer
1. Introduction
Oxygen. present as 20% of the atmosphere, is a terminal oxidant essential for respiration and other oxidative reactions in all aerobic organisms. During the reduction of molecular oxygen, reactive oxygen species are formed. These free radicals are impli- cated in many diseases including atherosclerosis (Witzum. 19941, respiratory tract disorders (Cross et al., 1994), neurodegenerative disease (Jenner, 1994), inflammatory bowel disease (Grisham, 1994), cancer (Ames. 1989: Cerutti. 1994) and also in ageing (Ames, 1989. 1988a, b).
A free radical is any atom or molecule that con- tains one or more unpaired electrons (Halliwell, 1994; Halliwell and Gutteridge. 1989). The unpaired elec- trons alter the chemical reactivity of an atom or molecule, usually making it more reactive than the corresponding non-radical. The chemical reactivity of radicals varies enormously. The hydrogen radical (H ‘) containing one proton and one electron is the simplest free radical. By the removal of (H ‘) from
Corresponding author. Fax: + 44 I8 1 66 17039.
other molecules, chain reactions are often initiated e.g. during lipid peroxidation. In the human body. solar radiation or low wavelength electromagnetic radiation. such as gamma-rays. from the environment can split water to generate the hydroxyl radical, OH ‘, which is very reactive at the site of formation. The body also makes another oxygen radical where the unpaired electron is located on oxygen. superox- ide (02 ). but this is poorly reactive. Active phago- cytes including neutrophils, monocytes. macrophages and eosinophils. generate large amounts of superox- ide in the killing of foreign organisms. However, with chronic inflammation this normal protective mechanism may itself be damaging (Grisham, 1994). Another physiological free radical is nitric oxide (NO ‘) which is made by the vascular endothelium as a relaxing factor, but excessive nitric oxide can be toxic (Moncada and Higgs. 1993). Superoxide can react with iron and copper ions to make hydroxyl radicals (Fig. I) or can combine with nitric oxide Oi- + NO + ONOO- (peroxynitrite). This decom- poses into toxic products including nitrogen dioxide gas (NO,), hydroxyl radical and nitronium ion (NO; ).
Since most molecules in the body are not radicals. any reactive free radical generated is likely to react
0027-5 107/96/$I5.00 0 1996 Elsevier Science B.V. All rights reserved
SSIII 0017-5 107(95)00096-8
6-l : Woo*
,’ + M’+ Mn
SPONTANEOUS SOD I /
CAT
HP0 + %O>
Fig. I. The interaction of active oxygen species. M, metal ion:
with a non-radical. Attack of reactive radicals on membranes or lipoproteins starts lipid peroxidation which is implicated in atherosclerosis development (Witzum, 1994).
2. Antioxidant defences against reactive oxygen species
Defence mechanisms have evolved within the body to limit the levels of reactive 0, species and the damage they induce. Superoxide dismutase (SOD) can convert superoxide to hydrogen peroxide (H 202) (30,-+ 2HL+ H,O, + 0,). These enzymes are found in mitochondria and cytosol. Catalases remove hydrogen peroxide. are found in peroxisomes in most tissues and probably remove peroxide generated by peroxisomal oxidase enzymes (Chance et al.. 1979). Glutathione transferases and glutathione peroxidases are usually associated with detoxication by conjuga- tion of xenobiotic electrophiles (Ketterer and Meyer. 1989). They remove hydrogen peroxide generated by SOD in the mitochondrial and cytosol (Chance et al.. 1979). They transform the tripeptide glutathione (GSH) into its oxidised form (GSSG) 2GSH + H ?O, + GSSG + 2H,O. Two types of GSH peroxidase occur in the cell, both of which detoxify fatty acid hydroperoxides. thymine hydroperoxide and DNA
hydroperoxides. The glutathione peroxidase that re- moves hydrogen peroxide contains selenium which is essential for catalytic function at its active site. The non-selenium dependent GSH peroxidase does not detoxify H,O, but has a high activity towards DNA hydroperoxide (Ketterer and Meyer, 1989). GSH also has important roles in leukotriene synthe- sis and xenobiotic metabolism and is found at minute concentrations in all human cells (Chance et al.. 1979). Reactive radicals, such as NO,, OH’ or
CCI,O;. abstract an atom of hydrogen from poly- unsaturated fatty acid side chains in membranes or lipoproteins. This leaves an unpaired electron on carbon. The carbon radical reacts with oxygen and the resulting peroxyl radical attacks adjacent fatty acid side chains to generate new carbon radicals and so the chain reaction continues. The attack of one reactive free radical can oxidise multiple fatty acid side chains to lipid peroxides, damaging membrane proteins. This makes the membrane leaky and even- tually causes complete membrane breakdown (Hal- liwell, 1994).
Since iron and copper ions are powerful promot- ers of free-radical damage causing formation of hy- droxyl radicals and accelerating lipid peroxidation, there is a complex system of storage and transport proteins to ensnare these essential metals. Iron within ferritin which is the usual storage form of iron will not stimulate free-radical reactions (Halliwell and
Gutteridge. 1989). Repair enzymes also exist that destroy free-radical
damaged proteins (Stadtman and Oliver. 1991). re- pair free-radical damage to DNA (Breimer, 1991) with excretion of oxidised bases in urine (Ames. 1989: Stillwell et al.. 1989) and remove oxidised
fatty acids from membranes (Maiorino et al., 1991). Such reactions are intracellular.
Some antioxidant defences are extracellular in- cluding the plasma iron-transport-protein transferrin and the iron-binding protein lactoferrin. When iron is bound in this way it cannot catalyse free-radical damage (Halliwell and Gutteridge. 1989). These pro- teins are found in body secretions, such as tears. Albumin can scavenge several radicals and binds copper ions (Halliwell and Cutteridge. 1989) and caeruloplasmin is a safe transport form of copper and helps load iron onto transferrin (Gutteridge and Stocks. 1981). Carnosine which is present in high
D. Atldersm/ Mutntion Research 350 flYY61 103-108 105
concentrations in human muscle and brain chelates copper and iron ions so as to inhibit oxidative reac-
tions (Kohen et al., 1988). Other antioxidant defences are found both intra-
and extracellularly. a-Tocopherol occurs in mem- branes and lipoproteins. It prevents the chain reac- tion of lipid peroxidation by scavenging intermediate peroxyl radicals. Vitamin C can convert the toco- pherol radical. which is much less reactive in attack- ing adjacent fatty acid side-chains, back to (Y- tocopherol. a-Tocopherol is important in protection against cardiovascular disease and neurodegeneration
(Muller and Goss-Sampson. 1990). Reduced glu- tathione is found in trace amounts in the body except
in the respiratory tract (Slade et al.. 1993) where it scavenges oxidative toxins such as NO;. ozone and free radicals in cigarette smoke. Vitamin C also helps in this instance as does p-carotene. Urate is the end product of purine metabolism and it scav- enges free radicals (Kaur and Halliwell. 1990). Ascorbate, a-tocopherol, GSH and water remove free radicals by reacting directly with them non-cata- lylically.
Plants contain many phenolic compounds, such as
flavonoids. that inhibit lipid peroxidation and lipooxygenases (Laughton et al.. 1991). A diet rich in vegetables. fruits. nuts and grains with a reduction in fat intake seems to protect against several human diseases including cardiovascular disease and cancer (Ames. 1983; Coghlan. 1991; Caragay. 1992). An increased dietary intake of vitamin E seems to de- crease death from myocardial infarction (Byers. 1993). Epidemiological studies on serum anti- oxidants and diet suggest that an increased level of vitamin E and p-carotene reduces mortality from cancer in the lung and colon (Menkes et al.. 1986; Blot et al., 1993).
3. Radical interactions resulting in genetic damage
Because of the time between generation of oxi- dants and their destruction by a defence mechanism, low levels can exist for sufficient time to produce damage to cellular macromolecules (Chance et al.. 1979). For nuclear DNA, the mammalian cell has
three levels of defence (Ames. 1989). First, nuclear DNA is compartmentalised away from mitochondria and peroxisomes where most oxidants are probably generated. Secondly, most non-replicating nuclear DNA is surrounded by histones and polyamines which may protect against oxidants. Thirdly, most of
the types of DNA damage produced can be repaired by efficient enzyme systems (Breimer, 1991; Still- well et al.. 19891. The overall result of this multi-level defence is that nuclear DNA is very well, but not completely. protected from oxidants.
When hydroxyl radicals are generated close to
DNA, purine and pyrimidine bases are attacked so causing mutations where, for example, guanine is converted to 8-hydroxyguanine and other products
(Dizdaroglu. 1993). One view of the somatic damage theory of cancer and ageing is that the amount of maintenance and repair of somatic tissues is always less than that required for indefinite survival. Thus some DNA damage in somatic cells induced by endogenous mutagens will accumulate with time (Ames. 1989).
There are 4 important endogenous processes lead- ing to significant DNA damage. These are oxidation (Totter. 1980; Ames. 1983: Cutler, 1984), methyla- tion. deamination and depurination (Saul and Ames. 1986). Supporting this are the existence of specific DNA-repair glycosylases for oxidative, methylated and deaminated adducts. and a repair system for apurinic sites produced by spontaneous depurination
(Lindhal, 1982). Ames (1989) has shown that oxida- tion is a major type of DNA damage and has calcu- lated that the total number of oxidative hits of DNA per cell per day in man may be more than 10’. He has promoted the use of a simple urinary assay for measuring the oxidised base 8-hydroxydeoxyguano-
sine (oh8dG) (Cundy et al., 1988; Shigenaga et al., 1989). This base is present at a level of I / 130 000 bases in nuclear DNA and l/8000 bases in mito- chondrial DNA. This very high level in mitochon- drial DNA may be caused by the extensive oxygen metabolism, relatively inefficient DNA repair and the absence of histones in mitochondria. Energy supplies to the cell could be compromised since mitochondria might be accumulating mutations with age (Ames, 1989).
During DNA peroxidation. DNA peroxides so formed reside in the pyrimidine fraction and involve
thymine. These are either products of the reaction of O2 with radicals resulting from the addition of OH. across the thymine 5, 6-double bond to give ring- saturated thymine hydroxy-hydroperoxides or with the radical produced by hydrogen abstraction from the thymine 5-methyl group to give %hydroper- oxymethyl acid (Schulte-Frohlinde and von Sonntag. 1985). These hydroperoxides are relatively unstable
and in the presence of metal ions, notably copper ions, are mutagenic (Thomas et al.. 1976).
If mild oxidative stress occurs. tissues can re- spond by increasing antioxidant defences, but severe oxidative stress can cause cell injury and death. Such cell death can progress as necrosis or apotosis. Cer- tain cells appear to encode free radical scavengers in anti-apoptosis genes (Sarafian and Bredesen. 1994).
Several possibilities relate oxidative endogenous DNA damage to cancer (Ames. 1989; Cerutti, 1994)
and also ageing (Ames. 1989). One is that mutagens react with nuclear DNA to produce somatic muta- tions, including point mutations and somatic events such as deletions. Mutagenic oxidants could include long-lived ones generated outside the nucleus in the mitochondria and cytoplasm which are capable of crossing the nuclear membrane, lipid soluble oxi- dants generated in the nuclear membrane itself and oxidants generated within the nucleus. Spontaneous methylation of DNA could cause both point muta- tions and apurinic sites which lead to breaks and clastogenic events. Somatic mutation could disrupt
cells by altering their regulation or gene production. Another possibility is that the high rate of oxidative damage to mitochondria causes an accumulation of mutations with age in mitochondrial DNA that re- sults in energy deficiencies in old cells. In addition to mutation, DNA damage can prevent DNA replica- tion leading to cell death. This can cause neighbour- ing cells to proliferate which can be a promotional stimulus for carcinogenesis in cells that do not un- dergo DNA replication.
Mutation can occur in cancer-related genes. The most important cancer-related genes are ras-family proto-oncogenes and the pS3 tumour suppressor gene. Skin cancer is related to solar radiation and near-ul- traviolet light (290-380 nm) damages DNA partly by oxidative mechanisms. G + T transversions are frequently observed in the middle position of hot spot codon 12 of Ki-ras and H-t-as in non-melanoma
skin tumours (Daya-Grosjean et al., 1993). G + T transversions have also been observed in several ~53
codons in smoking related lung carcinoma (Taka- hashi et al.. 1989) and could be produced by the release of oxy-radicals into the tissue by inflamma- tory leucocytes (Cerutti. 1994).
Oxidation or methylation could also cause a loss of S-methylcytosine, an epigenetic change. S-Meth- ylcytosine is involved in turning off genes in differ-
entiation so that its loss by DNA damage could cause de-differentiation and contribute to cancer and ageing (Wilson et al.. 1987; Holliday. 1987). Simi- larly, %hydroxyguanosine and 7-methylcytosine are likely to interfere with maintenance methylation at neighbouring or base-paired cytosines and cause loss of 5-methylcytosine.
4. Importance of oxidative damage to cancer and other disease states
Halliwell and Gutteridge (1984) emphasised that oxidative damage could be just as much a conse- quence of tissue injury as a cause of it. Tissue injury almost certainly leads to oxidative stress. This could then contribute significantly to worsening the tissue
injury or it might be irrelevant. However, the disease states listed earlier have accumulating evidence that free-radical damage is important. It is also important to understand how antioxidant defences are useful in protecting against such diseases, e.g.. carcinogenesis is a multiple stage event and antioxidants could act at any stage but they may not be anticarcinogenic in every instance. High antioxidant capacity shields DNA from oxidative damage and mutagenesis. but at the same time it may protect initiated cells from excessive oxidant toxicity and apoptosis and favour their clonal expression in tumour promotion (Cerutti and Trump, 1991). No clear answer can be given about the role of individual antioxidants on human cancer. The outcome may depend on the multiple, interacting oxidant components of the target tissue and the particular carcinogen to which it is exposed (Cerutti, 1994). However, antioxidant defences per- haps can be enhanced by appropriate diets (Ames. 1983; Coghlan. 1991; Caragay. 1992) and vitamin supplementation; see earlier.
D. Anderson/Mutation Research 350 (1996) 103-108 107
5. Conclusion
Thus, it would seem that reactive oxygen species play an important role in cancer and various other disease states in man, and means are now available for beginning to understand their contribution to such diseases. In addition, the role of antioxidant defences in preventing such diseases is becoming better understood. Although in vitro methods have been used to examine oxygen-mediated genetic dam- age for many years (e.g., Phillips et al., 19841, new and simple methods such as the COMET assay are available for measuring genetic damage caused by oxidative events in human cells and the modifying responses produced by antioxidants (Anderson et al., 1994).
Chance. B., H. Siers and A. Boveris (1979) Hydroperoxide
metabolism in mammalian organs. Phys. Rev.. 59. 527-605.
Coghlan. A. (1991) Europe’s search for the winning diet. New
Scientist. 29-33. Cross, C.E.. A. van der Vliet, C.A. O’Neill and J.P. Eisenrich
(1994) Reactive oxygen species and the lung. Lancet. 344.
930-932.
Cundy. K.. R. Kohen and B.N. Ames (1988) Determination of
8-hydroxydeoxy-guanosine in human urine: A possible assay
for in viva oxidative DNA damage, in: M.G. Simic. K. Taylor.
J.F. Ward and C. van Sonntag (Eds.). Oxygen Radicals and
Biology and Medicine. Plenum Press. New York. pp. 479-482.
Cutler. R.G. (1984) Antioxidants, aging and longevity, in: W.A.
Pryor (Ed.), Free Radicals in Biology, Vol. 6. Academic Press.
New York.
References
Ames, B.N. (1983) Dietary carcinogens and anticarcinogens. Oxy-
gen radical and degenerative diseases. Science. 221, 1256-
1246.
Ames, B.N. (1988a) Measuring oxidative damage in humans:
Relation to cancer and aging, in: H. Bartsch, K. Hemminki
and I.K. O’Neill (1988) Methods for detecting DNA damaging
agents in humans: Applications in cancer epidemiology and
prevention (IARC Sci. Publ. No. 89) International Agency for
Research on Cancer, Lyon. pp. 407-416.
Ames. B.N. (198%) The measurement of oxidative damage in
humans: Relation to cancer and aging. in: T. Yoshikawa (Ed.),
Medical, Biochemical and Chemical Aspects of Free Radicals.
Elsevier. Amsterdam.
Daya-Grosjean, L.. C. Robert, H. Drougard. A. Suarez and H.
Sarasin (1993) High mutation frequency in ras genes of skin
tumours isolated from DNA repair deficient Xeroderma pig-
mentosum patients. Cancer Res.. 53, 1625-1629.
Dizdaroglu, M. (1993) Chemistry of free radical damage to DNA
and nucleoproteins, in: B. Halliwell and 0.1. Aruoma (Eds.),
DNA and Free Radicals. Chicester, Ellis Harwood, pp. 19-39.
Grisham, M.B. t 1994) Oxidants and free radicals in inflammatory
bowel disease. Lancet, 344. 859-86 I Gutteridge, J.M.C. and J. Stocks (198 I) Caeruloplasmin: physio-
logical and pathological perspectives. Crit. Rev. Clin. Lab.
Sci.. 14. 257-329.
Halliwell, B. (1994) Free radicals, antioxidanta and human dis-
ease: curiosity. cause or consequence? Lancet. 344. 73 I-723.
Halliwell. B. and J.M.C. Gutteridge (19841 Lipid peroxidation,
oxygen radicals. cell damage and antioxidant therapy. Lancet.
i. 1396- 1398.
Halliwell, B. and J.M.C. Gutteridge (1989) Free Radicals in
Biology and Medicine. 2nd Edn. Oxford, Clarendon Press.
Holliday. R. (1987) The inheritance of epigenetic defects. Science.
238. 163-169. Ames, B.N. (1989) Endogenous DNA damage as related to cancer
and aging. Mutation Res., 214. 41-46.
Anderson. D.. T.W. Yu. B.J. Phillips and P. Schmezer (1994) The
effects of various antioxidants and other modifying agents on
oxygen-radical generated DNA damage in human lymphocytes
in the COMET assay. Mutation Res., 307, 261-271.
Blot, W.. J.-Y. Li. P. Taylor et al. (1993) Nutrition intervention
trials in Linxian. China. Supplementation with specific/vita-
min mineral combination cancer incidence and disease-specific
mortality in the general population. J. Natl. Cancer Inst.. 85, 1383-1492.
Jenner. P. (1994) Oxidative damage in neurodegenerative disease.
Lancet. 344. 796-798.
Breimer, L. (1991) Repair of DNA damage induced by reactive
