Indian Journal of Experimenta l Biology Vol. 40, June 2002, pp. 656-667

Oxidative stress and experimental carcinogenesis

Mohammad Athar

Department of Medical Elemento logy and Toxicology, Jamia Hamdard (Hamdard Uni versity), Hamdard Nagar, New Delhi I 10 062, Indi a

Fax : 00-9 1-608 8874; E-Ma il : ma493 @columbia.edu

The focus of this rev iew is to provide state-o f-the-art know ledge on the invo lve ment o f oxygen free rad icals (OFR) in carcinogenesis with a particul ar reference to skin model sys te m as the process of cancer deve lopment is best understood in this organ. Howe ver, a bri ef descripti on of the role o f OFR in other organs is also provided. The term OFR refers to fo rms of oxygen exhi biting high reacti vity and having at least one unpaired e lec tron. The ro le of OFR in di fferent stages o f carcino­genesis such as in iti at ion, promotion and progression is described. Out o f many mechani sms descri bed for the chemi ca l ini ­tiation o f tumori genesis, a number of the m may involve free radica ls in the cascade of reac tions. Evidences that support the in vo lvement of free radicals in tumor promotion include (i) a number of free rad ical-generating compounds are found to be tumor promoters in vari ous animal model systems, (i i) ROS generating systems can mimic the biochemical action o f tumor promoters, (iii ) some tumor promoters stimul ate the producti on of ROS, (iv) tumor promoters modulate the cell ular anti oxi ­dant defense systems, and (v) free rad ical scavengers, detoxiliers and antiox idan ts inhi bit the process of tumc·r promotion. The role of ROS in the progression stage of carcinogenesis is ev ident from the fact that a number of different free radical genera ting compounds enhance the malig nant conversion of beni gn papillo mas into carcinoma and their effectiveness may be related to the type o f rad icals produced into the biological sys te m.

During the last decade, considerable attention has been focussed on the involvement of oxygen free radi cals (OFR) in various diseases t-4. OPR are continuously generated in cells exposed to an aerobic environment during the course of normal metabolism. Despite the presence of strong anti ox idan t defence mechanism to counteract the OFR and minimise the plausible oxidati ve damage5

.6

, OFR-dependent damage of proteins?, DNA 8 and other biomolecules accumulate during lifetime of organi sms. It has been postulated that age-dependent di seases as athero­scelorosis, arthriti s, neuro-degenerati ve disorders and cancer involve OPR at least at some stage of their developmen{2. The focus of thi s rev iew is to provide state-of-the-art knowledge on the involvement of OFR in carcinogenesis with a particular reference to skin model system as the process of cancer development is best understood in thi s organ. However, a brief description of the role of OFR in other organs is also provided in thi s review.

Skin is the largest body organ, which is in direct and continuous contact of numerous potentially haz­ardous environmental pollutant including radi at ion. Many of these agents directl y or indi rectl y generate OFR, while they are absorbed th rough ancl/or are me­tabolised by the skin and contribute to the develop­ment of the most malignant neoplasm. Cancer deve-

lopment is now commonly recognised as a micro­evolutionary process that requires the cumulati ve ac­tion of multiple events. These events may occur in a single cell clone and can be explained by a simpli fied th ree-stage model. These stages include (a) the induc­ti on of DNA mutation in a somatic cell known as ini­ti ation, (b) stimulation of the initi ar.ed cell and its clonal ex pansion referred as promotion, and (c) ma­li gnant conversion of the benign tu mor into cancer termed as progression. OFR have been shown to stimulate cancer development by playing a ro le at all the three stages namely , initi ation, promotion and progress ion9

•LO

•

Aetiology of oxygen-free radicals The term OPR refers to forms of oxygen exhibiting

high reactivity and having at least one unpaired elec­tron. However, other reacti ve fo rms of oxygen are also known which are non-free radical. Both of these forms are collecti vely referred as reactive oxygen species (ROS) and include singlet oxygen, superoxide anion, hydrogen peroxides, hydroxyl radical, etc. singlet oxygen is formed by the transfer of radi ant energy to the oxygen molecule whic is triplet and paramagnetic at ambient temperatures. The stepwise uni valent reducti on of oxygen leads to the fo rmation of superoxide anion, hydrogen peroxide and hydroxyl

ATHAR: OXIDATIVE STRESS & EXPERIMENTAL CARCINOGENESIS 657

radical. The oxidation potential and reactivity of vari­ous ROS may be given in the following order ll

:

0; < HP2 <' O2 <'OH

OFR are short-lived species that are generated ill situ in normal cells under pathological conditions. In addition, the metabolism of xenobiotics and/or expo­sure to ionising radiation also generate these species. An important feature of free radical reactions with non-radical species is the formation of new radical species. Free radical driven reactions are usually chain reactions l2

• Electron acceptors such as molecu­lar oxygen react easily with free radicals become radical themselves, the OFR. This explains why, in aerobic life, where molecular oxygen is ubiquitous, OFR become the primary mediators of cellular free radical injury.

Oxidative stress in carcinogenesis In aerobic biota, the OFR are formed in normal cell

metabolism from molecular oxygen . Despite tight antioxidant defences, these OFR cause constant dam­age to oxidizable molecules, which are repaired or replaced in a dynamic equilibrium. The condition of cellular oxidative stress arises either from the over production of OFR or from the deficiency of antioxi­dant defences or repair mechanism(s) and results in reversible or irreversible tissue injury. Examples of short-term oxidative stress are the ischemia­reperfusion injury syndrome, acute inflammation and hyperoxia (due to the exposure to hyperbaric oxygen). In addition, important endogenous manifestation of chronic oxidative stress is inflammatory disorders3.

Activated leukocytes generate 0 ; and HOCI, repre­

senting an important source of OFR in situ which are intended to kill or destroy target site/organisms 13

.

However, OFR not only mediate the killing/ destruction of target sites but also induce oxidative stress in adjacent cells. Activated neutrophils have been shown to stimulate mutagenesis in vitro l4

, and oxidative stress from chronic inflammation favours

cancer development in many organs I4-16

. A few important examples of common exogenous causes of oxidative stress and their consequences are shown in Table 1. Smoking, the oxidative stress from tobacco smoke arises through (a) a potent mixture of reactive oxidants, in particular nitrogen oxides and the hy­droxyl radical, (b) the depletion of the intracellular antioxidant, glutathione (GSH) by reactive aldehydes, and (c) the induction of chronic inflammation 18-20.

Both ultraviolet light and ionising radiation of higher energy (X-rays, gamma radiation) stimulate DNA damage by generating OFR in situ which include hy­droxyl radical and the free radicals of biomolecules21

.

The pattern of oxygen independent free radical reac­tions-mediated DNA lesions is qualitatively different from that observed during the course of oxidative stress. Evidence is there to suggest that OFR from lipid peroxidation reactions are responsible for the association between the fat intake and colorectal can­cer22. Iron (Fe2+) is an important factor that enhances the production of OFR23. Copper (Cu+) is almost as effective as iron as a catalyst in the Fenton's reaction but is a more potent mutagen than iron as observed under the in vitro experimental conditions24 . This may be due to the direct interaction of copper with bases of DNA 4. Ethanol is another major cancer risk factor that may, in part, act through free radical mechanism. Free radicals generated as a result of the metabolism of alcohol are shown to be responsible for augmentation of hepatic lipid peroxidation. It has been speculated that OFR are involved in ethanol-mediated liver car­cinogenesis25 .

The role of OFR in different stages of carcinogene­sis is described as follows:

Role of OFR in initiation Evidences have accumulated to suggest that ROS

play an important role in tumor initiation by enhancing or facilitating, the metabolic activation and/or initiating effects of carcinogens. Out of many mechani sms described for the chemical initiation of

Table I-Major exogenous cause of oxidative stress involved in carcinogenesis

Cause of oxidative stress

Tobacco smoke

Ultraviolet light

Fatty acids in food

Iron and copper ions

Ethanol

Oxygen free radicals

NO';OH

'OH, organic radicals

Lipid peroxides

'OH

Lipid peroxides

Cancer associated with exposure

Bronchogenic carcinoma

Melanoma and other skin cancers

Colorectal cancer, breast cancer

Colorectal cancer

Hepatocellular carcinoma, breast cancer

658 INDIAN J EXP BIOL, JUNE 2002

tumorigenesis, a number of them may involve free radicals in the cascade of reactions 10. Initiators are in general metabolized to an ultimate carcinogen, which usually forms adduct(s) with DNA. A number of initiators have been shown to produce free radicals by themselves26-28. DNA is the potential target for initiators of carcinogenesis. Free radicals cause DNA base damage, DNA single strand and double strand breaks, cross-linking between DNA and proteins or DNA and chromosomal aberration. One of the major products of base damage in DNA is thymine g lycol, which is a consequence of chemical oxidation and ionizing radiation exposure. It has been shown that chemical carcinogens that can generate free radical often induce the formation of thymi ne glycol29 . For example, N-hydroxy-2-naphthylamine and benzo(a) pyrene have been shown to produce thymine glycol in a dose-dependent manner following their treatment in cultured fibroblasts or DNA in solution3o. Superoxide anion generated from hypoxanthine-xanthine oxidase system induces chromosomal aberration in cultured Chinese hamster cells31 and V79 cells32. In addition, it has also been shown to act as a weak complete carcinogen in other model systems33. Hydrazine and its derivatives, dimethyl hydrazine and isoniazid, which produce ROS27 have been shown to induce sister chromatid exchange in CHO cells34. Chromosomal breakage, rearrangement and sister chromatid exchange are also formed as result of photochemical or enzymatic generation of superoxide radicals35. It has been implicated that H20 2 and hydroxyl radical, which are fo rmed within the cells of cultured human IMR-90 fibroblasts by the exposure to fluorescent light were responsible for the induction of DNA damage36

• H20 2 has also been implicated as a causative agent in the induction of chromosomal damage37 and found associated with the induction of cancer in animal model systems38.39. H20 2 induces molecular damage leading to induction of transfor­mation in the normal cells in vitro 40.

It is also known that free radicals produce single strand and double strand DNA breaks in biological system. Potassium superoxide and H20 2 induce single strand and double strand DNA breaks in calf thymus DNA, xanthine-xanthine oxidase in E.coli DNA, hy­droxyl radicals in pBR 322 plasmid DNA (ref. 41 and references therein), ferric nitrilotriacetate and H20 2 in DNA fragments from human c-H-ras-l, protoonco­gene42, and N-OH-naphthylamine in human fibro­blasts43. The mutagenicity of bleomycin is caused by

the production of superoxide radicat44 . Free radicals in cigarette smoke have been also shown to cause tu­morigenesis. The peroxyl radicals, which are formed by spontaneous or enzyme catalyzed auto-oxidation of unsaturated fatty acids have been shown to be in­volved in the activation process of many carcinogens such as benzo(a) pyrene, aromatic ami nes (e.g. naph­thylamine, acety laminoflourene, etc.) amino azo compounds, 4-nitroquinoline-l-oxide and n-n itro compounds lO

•45. ROS induce changes in intracellular

pH, which is known to be one of the early cellular responses to mutagenic signals, suggesting that under certain predi sposed conditions, ROS are mutagenic and mediate growth-associated gene expression46.

Strong evidence for the involvement of free radi­cals in free radicals-induced DNA damage and conse­quent carcinogenesis comes from the fact that these processes can be abolished by free radical scaven­gers/antioxidants47.48. Potent inhibitors of skin tumor initiation in mice include antioxidants (butylated hy­droxytoluene, butylated hydroxyanisole and sele­nium), flavones (7,8-benzoflavone and quercetin) and vi tamins (A, C and E) etc. Some of the flavones and antioxidants appear to inhibit skin carcinogenesis by inhibiting the metabolism of a procarcinogen to its ultimate carcinogenic form49. The antioxidant, buty­lated hydroxyanisol (BHA) is widely used as food preservative and has been shown to inhibit chemically induced lung, liver, mammary, forestomach, colon and liver cancer in experimental animals. Similarly, inhibitory effects of selenium and vitamins E and C have been demonstrated5o. Katiyar et al. 51 have shown that the possible inhibition of N-niirosodiethylamine and benzo(a) pyrene induced forestomach and lung tumorigenesis by some polyphenolic fraction isolated from green tea, is due the induction of antioxidant enzymes by these fraction. Based on these findings, involvement of ROS in the process of tumor initia­tion52 can be deemed due to one or many of the fol­lowings:

1. Free radicals may be involved in metabolism re­quired for the activation of procarcinogen.

2. Reactions involving metabolic activation and/or degradation of carcinogen may release free radi­cals that can attack DNA.

3: Metabolites of carcinogen may themselves become free radicals and may interact directly with DNA.

4 . Following binding of ultimate carcinogen to DNA, DNA-carcinogen adducts may produce free radicals in vicinity of DNA.

ATHAR: OXIDATIVE STRESS & EXPERIMENTAL CARCINOGENESIS 659

Role of OFR in tumor promotion The role of free radicals in tumor promotion has

been reviewed53 . Following ev idences support the invol vement of free radicals in tumor promotion:

A number of free radical-generating compounds are found to be tumor promoters in various animal model systems.

2 ROS generating systems can mimic the biochemi­cal action of tumor promoters.

3 Some tumor promoters stimulate the production of ROS.

4 Tumor promoters modulate the cellular antioxi­dant defense systems.

5 Free radical scavengers, detoxifiers and antioxi­dants inhibit the process of tumor promotion .

Each of these observations is described in a greater detail in the following section.

Free radical-generating compounds as tumor promoters

There is a long list of oxidants with tumor promot­ing properties. H20 2, peroxyacetic acid, chlorobenzoic acid, benzoyl peroxide, decanoyl peroxide, cumene hydroperoxide, lauryl peroxide, dicumyl peroxide, p-nitroperbenzoic acid and periodic acid are demon­strated to be tumor promoters in cultured mouse epi­dermal cell and in mouse skin models54. Benzoyl per­oxide, !auryl peroxide, decanoyl peroxide and cumene hydroperoxide have been shown to be potent tumor promoters in the murine skin initiation-promotion model , while tert-butyl hydroperoxide and methyl ethyl ketone peroxide are weak promoters55.58. H20 2 is only marginally active. In the multi-stage promotion protocol, H20 2 was found to be effective stage-I tu­mor promoter59. H20 2 has also been reported to be a complete promoter in rat duodenum6o. Hyperbaric oxygen and traces of active oxygen present in air may exert promoting effect in lung carcinogenesis6

'. Pro­moting potentials of these peroxides is attributed to their ability to generate ROS within the biological system. Although these peroxides are quite stable to unimolecular homolysis at body temperature, many of them have been demonstrated to undergo immediate decomposition in the presence of hematin to form radical species that can be detected by employ ing spin trapping technique on an electron spin resonance spectrometer62. Keratinocytes and epidermal cells ex­posed to hydroperoxide tumor promoters have been shown to generate free radicals63.65 . Taffe et ai. have demonstrated the generation of alkyl radical and

alkoxy l radical by incubating different organic hy­droperoxide with isolated mouse keratinocytes64

.

These alkyl radicals have also been shown to partici­pate in hydrogen abstraction or addition66. Alkylation especially methylation of cellular components has been implicated in eukaryotic gene expression

67.

The generation of free radicals from a variety of oxidant tumor promoters has also been studied in other biological systems such as liver microsomes and cytosolic fraction68.7' . The antipsoriatic agent, an­throne derivatives such as anthralin and chrysarobin that can generate directly free radical by the simple oxidation in presence of air and light, have been shown to be mouse skin tumor promoters. The ul ti­mate promotional ability of these compounds has been suggested to be due to the production of super­oxide radical72

.74 . The metabolism of benzo(a) pyrene

has been shown to be accompanied initially by the

generation O ~ and subsequently by H20 2 and ' OH ,

and the involvement of these radicals in tumor promo­tion has also been suggested75 . Iron overload enhances dimethylbenz(a) anthracin (DMBA)-induced skin tumorigenesis76. In addition that iron overload aug­ments BPO and TPA-mediated tumor promotion in DMBA-initiated mouse skin77

•78

•

In kidney, nickel when administered as nickel sub­sulphide produces carcinoma. Athar et al. 79 have for the first time shown that nickel gererates hydroxyl radical79 and leads to the tissue accumulation of redox active iron80, which plays an important role in con­junction with glutathione in causing oxidative in­jur/ '. Iron-nitrilotriacetate (Fe-NTA), a water pollu t­ant is a complete renal carcinogen. Further, it may act as a potent renal82 and hepatic83 tumor promoter and acts by generating oxidative stress84. The other similar compound, Cu-NTA promotes renal tumorigenesis by a distinct free radical mechanism85 . Additionally, KBr03, a food additive and a free radical generating compound has been shown to be a renal tumor pro­moter and acts by generating oxidative stress in kid­ne/6

.

Free radical generating systems that mimic the action of tumor promoters

Free radical generating systems have been shown to mimic the action of tumor promoters. Zimmerman and Cerutti 33 have shown that ROS produced extracel­lularly by xanthine-xanthine oxidase system act as a promoter of transformation in mouse C3H\lOTl\2\CI8 fibroblasts cells in culture33. Many known tumor pro-

660 INDIAN J EXP SIOL, JUNE 2002

moters like TPA, mezerin and BPO have been ob­served to induce irreversible transformation of the murine JB6 epidermal cell lines to an anchorage inde­pendent growth phenotype87

. Similar transformation was also observed to occur by xanthi ne-xanthine oxi­dase systems. Additional similarity between known tumor promoter and free radical generati ng systems is their ability to induce ornithine decarboxylase (ODC) activity. ODC, which is a rate limiting enzyme in polyamine biosynthesi s, is induced during the process of tumor promotion88

. There is an excellent correla­tion between the tumor promoting ability of various tumor promoting compounds and their potential as inducers of ODC89

. Xanthine-xanthine oxidase was found to induce the activity of ODC in primary mur­ine keratinocytes in a manner analogous to that ob­served with TPA54

• Fatty acid hydroperoxides, the autooxidation products of unsaturated fatty acids have been shown to stimulate DNA synthesis and ODC induction9o

• We have shown that Fe-NT A induces ODC act ivity both in liver and kidnel2

•83

. Addition­ally, oxidants have also been shown to stimulate PKC activity similar to that can be observed following TPA application9 1

.92

. Analogous to TPA, ROS generated by xanthine-xanthine oxidase stimul ate expression of early response genes such as c-fos and c-myc in addi­tion to inducing DNA sy nthesis in JB6 cells93

•94

• It has also been reported that treatment of quiescent Balb 3T3 cells with xanthine-xanthine oxidase has a com­petence fac tor like effect95

. H20 2 has also been shown to activate these genes in mouse osteoblastic cells96

.

Porphyrin administration followed by exposure to visible light is utilized for the photodynamic therapy of cancer. However, the major drawback for this mo­dality of cancer treatment is the extended skin photo­sensitivity, which may also involve oxidative stress. Superoxide anion radical and other ROS are involved in this process97

.99

. Sustained generation of ROS has been demonstrated in mouse skin using porphyrin and light, which acts as stage-I, stage-II and complete tu-

. . k' 100 10 1 I ' d mor promoter III munne Sill ' . n situ generate ROS augments TPA-mediated skin tumor promo­tion lO2

. In a separate study, dihematoporphyrin ether (DHE)-mediated photosensitization was shown to . d 1 103 III uce ear y respo.nse genes .

Activation of reactive oxygen by tumor promoters It is now known that tumor promoters act at least in

part by inducing a cellular prooxidant state. Many tumor promoters accentuate the elaboration of ROS by the endogenous sources to create a cellular prooxi-

dant state. TPA stimu late leukocytes and macro­phages for the increased consumption of oxygen and in turn they generate ROS. It has been suggested that ROS derived from these pro-inflammatory cells are critical component of the tumor promotion process 104.

Another potential source of ROS in some target tis­sues is xanthine oxidase. Reiners et al.105 have ob­served a 2-3 fold increase in xanthine oxidase activity in keratinocytes following TPA treatment 105 . Kerati­nocytes exposed to hydroperoxide tumor promoters generate free radicals 106.108 . The levels of hydroperox­ides are increased gradually by successive applica­tions of TPA until a plateau is reached 107. In vivo, TPA stimulates the infiltration of neutrophils and as a result myeloperoxidase activity in dermis is enhanced. It is known that it also enhances the formation of H 0 d 'd' d DNA b . 'd . 109 11 0 2 2 an OXI Ise ases III epl ermls . . Tumor promotion process is media ted through the interaction of TPA with the PKC receptor. The tumor promoting activity of different analogs of TPA was found to be in concordance with their ability to act as stimulators of PKC90

.92

. The other classes of epider­mal tumor promoters such as mezerein and indole alkaloids, which are chemically different from TPA,

also activate PKC and stimulate O ~ production I ".

The weak endogenous skin tumor promoter, diacyl­glycerol also st imulates superoxide generation from neutrophils ll2

. BPO does not bind to PKC, but acti­vates it indirectly' 13. Some tumor promoters such as anthralin, indolacetic acid and tween 60, which do not activate PKC, do not evoke these response. Further, other tumor promoters such as poly toxi n and thapsi­gargain also evoke oxidative burst in neutrophils but apparently not through the PKC signal transduction casade l14

. In addition to PKC, other protein kinases have been shown to get activated under the predis­posed conditions of oxidative stress. For example, oxidants have been found to activate the rat insulin receptor kjnase l15

. 11 6 and stimulate phosphorylation of the ribosomal protein through Ca2+-dependent event l17

.

The promoters of tumors in di fferent organs also induce prooxidant state. For example, the known he­patic tumor promoter, phenoborbi tal induces cyto­chrome P-450 enzymes. It is presumed that as a result of induction of cytochrome PA50, microsomal and mitochondrial electron transport are perturbed, and prooxidant state is generated 1 18. 119. The tumor promot­ing potential of peroxisomes proliferators is also known 120. 121. Peroxisome proliferators such as clofibrate,

ATHAR: OXIDATIVE STRESS & EXPERIMENTAL CARCINOGENESIS 661

nafedopin and phthalate derivative induce overpro­duction of H20 2 (ref. 121). In carbon tetrachloride­mediated tumor promotion of liver, trichloromethyl radical initiates lipid peroxidationl22. Cytochromc P-450 was found to metabolize ethanol, a hepatic tumor promoter by generating free radicals 123

• Paraquat is known for its tumor promoting potential in urethane­induced tumorigenesis in mouse lung 124. The bipyridylium radical generated as a result of para­guat's reduction participates in the univalent reduc­

tion of O2 to 0 ; and causes lipid peroxidation. In situ

delivery of glutathione protects against the paraquat­mediated lung inj ury l25. The renal tumor promoter, KBr03 has been reported to increase renal lipid per­oxidation significantly 126.127. Subsequently, Sai et al. 128 have suggested that ROS generated by the interaction of Kbr03 and kidney cells may be in­volved in this process. These results are parallel to the studies of Giri et al.86 who observed a reduction in renal antioxidant levels following KBr03 treatment. Estradiol, bile acids and androgens are known to pro­mote a variety of human cancers including breast, colon and prostatic cancer. These promoters through redox cycli ng, induce prooxidant state and thereby participate in the enhancement of tumor develop­ment l29 . Steroids such as estradiol are metabolically oxid ized to catechol estrogens, which are capable of redox cycling 130. Although free radical generation by redox cycling between 2- or 4-hydroxyestradiol and corresponding qui nones has not yet been demon­strated in situ , an increae in 8-0H-dG in kidney of hamsters treated with l7-estradiol has been proposed as an evidence of such free radical generation 131.

Modulation of cellular antioxidant defense systems by tumor promoters

In addition to the production of free radicals, many tumor promoters have been shown to modulate the cellular antioxidant mechanism. Solanki et al.132 have shown that in mouse epidermis TPA, anthralin, non­phorbol-ester tumor promoter, etc cause a rapid and sustained decrease in the activities of superoxide dis­mutase, and catalase. Similarly, the TPA-mediated changes in glutathione peroxidase activity were found to be time-dependent and are transiently increased within 30 min of TPA treatment which is followed by a depression between 1 to 12 hr. Glutathione reduc­tase activity was also depressed throughout the multi­ple exposure of TPA 133

• Consistent with these enzy­matic changes, a four-fold increase in the levels of

oxidized glutathione was observed 134. In mouse epi­dermal JB6 cells also showed that TPA treatment re­duces SOD, CAT, and GPX activities 135

• A known liver tumor promoter, clofibrate modulates antioxi­dant enzyme activities. Catalase activity per unit vol­ume of peroxisome has been shown to decrcase con­tinuosuly during the treatment of clofibrate in the male rat liver l36. Ciriolo et al. 136 have reported that clofibrate and other peroxisome proliferator tumor promoter, fenofibrate lowered the activities of GPX, and SODI36. In addition, clofibrate and liprofi­brate137.138 also decrease GST activities. Similarly, another hepatic tumor promoter, phenobarbital in­crease liver catalase activity after 6 weeks of its treatment in rat. In contrast to CAT, GPX is reduced by the treatment of phenobarbital 139. Estrogen has been reported to change the activities of free radical detoxifying enzymes in kidney of male syrian ham­sters. It decreases renal CAT activity but increases GPX activity however, SOD remains unchanged 140. Iqbal et al.84 observed that Fe-NT A administration reduces antioxidant enzy mes in both liver and kidney .

Antioxidant as antipromoters Kensler et al. 141 have postulated that if tumor pro­

motion is related to the increase in the intracellular free radical level, the application of free radical scav­enger or antiox idant could conceivable modulate tu­mor growth . Indeed, exogenous addition to mouse skin of a lipophilic biomimetic SOD, Cu(II)(3,5 -diisopropyl salicylic acid), Cu DIPS , inhibits TPA­induced ODC activity and tumor formationI 42. 143. In experiments by Friedman and Cerutti 144, scavengers of ROS(SOD, CAT and mannitol) displayed simil ar effects by antagonizing PM A-induced ODC activity in mouse mammary tumor cells.

Antioxidants such as GSH, cysteine, and (X­

tocopherol were shown to prevent the TPA-mediated decrease in the ratios of reduced to oxidized g lu­tathione in mouse epidermal cells. In the same sys­tem, these antioxidants inhibit TPA-induced ODC activity as well as tumor growth l45 . OberJey and Ober­ley l46 have demonstrated the role of antioxidant en­zymes in cell immortalization, transformation and carcinogenesis. Furthermore, the free radical scaven­gers such as BHA and BHT, which are known inhibi­tors of lipid peroxidation, were shown to inhibit TPA­and BPO-induced ODC activity and tumor promotion in mouse skin l47 . Additionally, these compounds and their analogs were found positive for their inhibito ry

662 INDIAN J EXP BIOL, JUNE 2002

effects of TPA-stimulated chemiluminescence in PMNs. Various antioxidants were fou nd to prevent Fe-NTA-mediated induction of ODC and eH] thymidine incorporation both in liver and kidney cell s82

.83

. There is also evidence that contrary to their antioxidant properties, these antioxidants under cer­tain circumstances may act tumor promoter. For ex­ample, BHA has been shown to promote forestomach and urinary bladder tumorigenesis, whi le it was found to inhibit liver and mammary gland carcinogenesis. BHT on the other hand, promoted carcinogenesis of urinary bladder, thyroid and esophagus, while it inhib­ited ear duct and mammary gland carcinogenesis. It has been reported that BHT and vitamin E inhibited the promoting action of polyunsaturated fatty acids in mammary gland carcinogenesis in rats initiated with DMBA. The effect has been proposed to be caused by their ability to block prostaglandin synthesis or by inhibition generation of oxidative products of fatty acids, which may playa role in tumor promotion by blocking oxidative metabolism of polyunsaturated fatty acids. The naturally occurring plant products such as nordihydroguaiaretic acid and diallyl sulfide having antioxidant prodperties, abrogate the tumor promoting effects of BPO in murine skin 148. Ascorbic acid l49 and a-tocopherol have been shown to have inhibitory effects on the mouse skin tumor promotion by TPA. Sarcophytol, a marine product has been re­ported to inhibit the two-stage carcinogenesis in DMBA initiated telocidinerrPA promoted mouse skinl 5o. The anti-tumor promotional activity is pro­posed to be due to their ability to suppress the PMN infiltration, ROS generation and oxidative DNA dam­age. Several free radical scavengers have been shown to inhibit chyrosarobin- and anthralin-induced tumor promotion. The known renal tumor promoter, potas­sium bromate-mediated DNA damage is inhibited by glutathione, cysteine and vitamin C151 . The same anti­oxidants have been shown to have protective effect against the KBrOJ· mediated induction of micronuclei in rat peripheral blood reticulocytes 152

• The dietary antioxidant, tannic acid suppresses DMBA-induced skin and bcnzo(a) pyrene-induced lung and forestom­ach tumorigenesis153

.

Role of ROS in tumor progression Rotstein et al.154 have shown that low frequency of

conversion of papillomas to carcinomas can be in­creased by treating papilloma-bearing mice with free radical generating compound, benzoyl peroxide.

These results prompted many other researchers to fur­ther investigate the role of ROS in the progression stage of carcinogenesis. It has also been shown that BPO enhances the invas iveness of mouse epidermal

. II I· 155 156 M . . . I h carCInoma ce Ines . . any 111ltlators a so ave potential to enhance the conversion of papilloma to

. 157 I .. d I carCInoma . n a two-stage carclllogenesls mo e , exposure to ionizing radiation as source of free radi­cals augmented the malignant conversion although, it did not alter the incidence of papillomas 155

. Athar / 155 h et a . ave demonstrated the efficacy of a number

of different free radical generating compounds to en­hance the malignant conversion of benign papillomas into carcinoma. They have also suggested that the effectiveness of these compounds may be related to the type of radicals produced into the biological sys­tem. Further, it has been shown that organic hydrop­eroxides are metabolized into free radicals by normal mouse skin keratinocytes and by the human carci-

k · 158 Th b . noma eratlllocytes . ese 0 servatIOns suggest that prooxidant compounds havi ng ability to be me­tabolized into free radicals may enhance the rate of progression of benign tumors to malignant neoplasms involving free radicals. These findings suggest that tumor progression rate may be sens iti ve to the ROS­mediated genetic alterations. It was also postulated that progression of cancer may involve free radical­induced clastogenic changes leading to the activation and/or inactivation of various cellular genes in the experimental carcinogenesis. Ruggeri et al. 159 showed inactivation of p53 tumor suppressor gene during the progression stage. This inactivation may lead to onset genes coding metalloproteases, which are involved in the invasion and/or metastasisI 60.16 1. Athar et al. 162

have reported the protective effect of all trans-retinoic acid (RA) in the free radicals-mediated conversion of chemically-induced and UV-B radiation-induced skin papillomas to carcinomas 162. It is believed that RA may act in many ways, to afford protection by inter­fering with the free radical-mediated enhancement of

. 162 F h h ·d d· tumor progressIOn . urt er, t e superoxI e Ismu-tase mimic, Cu(II)-(3,5-diisopropylsalicylate)2 has becn shown to inhibit BPO-i nduced skin tumor pro-

. 163 Th I f ·d· . gressIOn . e ro e 0 OX) atlve stress In tumor pro-gression is also supported by the findings that di­ethyl maleate increases, whereas GSH and disu lfiram decrease the rate of ski n tumor progression 164. The free radical scavenger, N-aeyl dehydroalanin inhibits the carcinoma format ion, though it does not alter the TPA-mediated ODC induction or papilloma forma­tion l65 . Similarly, tea polyphenols also showed a de-

ATHAR: OXIDATIVE STRESS & EXPERIMENTAL CARCINOGENESIS 663

crease in the rate of progression of benign papillomas to carcinomas l66

. All these evidences are suggestive of the fact that ROS or redox state of the cell may af­fect the cascade of events related to the progression stage of carcinogenesis.

In summary, free radicals and/or oxidative stress seems to alter experimental carcinogenesis in animal model systems. Free radical scavengers and antioxi­dants have protective effect against chemically­physically- or biologically-induced cancers in these model systems. However, ex trapolation of these data in human settings did not always show such a direct correlation. Many dietary and synthetic agents, proved successful in animal experimentation, are underway for clinical trials in human intervention studies.

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