A direct assay for clastogenic activity, which is not complicated by cellular uptake consid- erations, involves the induction of single-strand breaks in puc18 DNA by hydrogen perox- ide in the presence of ferrous sulfate in deaerated sodium phosphate buffer at pH 7.4 (the Fenton reaction).14 In the absence of oxidative damage, one observes a high concentration
of highly twisted circular DNA (HT or RF I DNA). The presence of oxidative damage results in open DNA with single nicking (RF II DNA). Further damage results in double- stranded nicking, producing linear (L) DNA. Antioxidants protect DNA against this form of damage in a concentration-dependent manner. The DNA forms are separated by agarose gel electrophoresis and the DNA strands are detected by staining with ethidium bromide. Quantitation is achieved by scanning using the MACROS gel reading program. Only water-soluble compounds are conveniently assayable in this system because cosolvents such as dimethyl sulfoxide, ethanol, and methanol interfere, giving artifactual protection figures.Figure 10.8 represents a typical developed gel andFigure 10.9illustrates the scans which translate the zones into comparable quantitative numbers.Table 10.1records the values obtained at 50 µM concentrations each.
FIGURE 10.7
Conversion of green tea catechins to black tea theaflavins, etc., by the action of Camellia sinensis polyphenoloxidase.
©2000 by CRC Press LLC
FIGURE 10.8
A developed gel electrophoresis of DNA following oxidative damage by the Fenton reagent.
FIGURE 10.9
Densitometer scans of developed gel electrophoresis of DNA following oxidative damage by the Fenton reagent. ©2000 by CRC Press LLC
The individual and collective green tea catechins are significantly more antioxidant at this concentration level than any of the other agents, and EGCG leads the list. D-Tocopherol was insufficiently soluble to give useful assay results in this test, and ascorbic acid was actually clastogenic — possibly artifactually as it is capable of recycling the catalytic iron in this assay. Reduced glutathione and sulforaphane (from cruciferous vegetables) were roughly competitive with some of the green tea catechins, but other antioxidants such as ellagic acid and N-acetyl-L-cysteine were clearly less powerful.
In Table 10.2, a comparison is made of the antioxidant power of various teas brewed identically and then fractionated to remove caffeine and pigments and to concentrate the catechins. The purified catechin fractions were all very similar in their antioxidant power, but green tea produced more than twice as much catechins. Comparatively, green tea drinkers receive approximately twice the antioxidants that drinkers of oolong or black tea drinkers receive.
Superoxide, the product of the reaction of phenazine methosulfate-NADH, is a reactive oxygen species which can cause DNA damage of the type involved in Tables 10.1 and
TABLE 10.1
Summary of Densitometer Scanning Results of DNA Damage When Subjected to the Fenton Reaction in the Presence of Various Antioxidants (at a concentration of 50 µM each)
Compound % DNA Damaged % DNA Protection
No oxidant 0 100
Hydrogen peroxide control 100 0
EGCG 11 89
GCG 28 72
ECG 30 70
Reduced glutathione 39 61
Sulforaphane 41 59
Mixed green tea catechinsa 43 57
EC 58 42
Ellagic acid 69 31
EGC 89 11
N-Acetyl-L-cysteine 96 4
Ascorbic acid 0 0b
a A commercially available decaffeinated mixture of green tea catechins sold as TeGreen 97 (Pharmanex Corporation, Simi Valley, CA). The mixture was arbitrarily assigned the molecular weight of epigallocat- echin gallate as an approximation.
b DNA damage greater than that from the control was observed. This is speculated to be due to ascorbate recycling Fe(III) back to Fe(II).
TABLE 10.2
A Comparison of the Antioxidant Power of Various Teas Brewed under Identical Conditions, Decaffeinated, and the Catechins Isolated
Tea Catechin Fraction, mg Antioxidant Power Normalization Ratio
Green tea 7.8 0.75 5.9 1.00
Oolong tea 3.3 0.74 2.4 0.41
Black tea 3.7 0.66 2.4 0.41
Note: Antioxidant power was determined as in Table 10.1 but at 0.5 µg/reaction. Normal- ization was achieved by multiplying the antioxidant power by the quantity of catechins isolated and normalizing with the largest value (green tea, in this case) arbitrarily assigned a value of 1.00.
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10.2.15 It was of interest, therefore, to measure the comparative interceptive ability of vari-
ous antioxidants toward this species. The data are presented in Table 10.3. Once again, the green tea catechins lead the list with EGCG being the most potent. Resveratrol, one of the antioxidants present in red wine and associated with the protective effects of this dietary constituent (the “French paradox”),7 is about two thirds as protective compared to EGCG,
and ascorbic acid, BHT, D-tocopherol, and BHA cluster at roughly half of the potency of EGCG in this test.
Another in vitro test system which generates superoxide anion as a by-product of the enzymatic oxidation of xanthine to uric acid involves the action of xanthine oxidase on this end product of purine metabolism.15A potential complication is that enzyme inhibition
might give the appearance of consumption of superoxide, whereas it could actually mean simply lessened production. To measure this effect, the potential inhibition of uric acid for- mation by the enzyme acting on xanthine was measured. Active inhibitors in this system were quercetin, sulforaphane, N-acetyl-L-cysteine, selenium powder, reduced glutathione, and curcumin (Table 10.4). Thus, the data for these agents inTable 10.5are partially artifac- tual in that they not only intercept superoxide anion but also inhibit the enzyme which gen- erates it. Thus, the figures for these particular agents may overstate their antioxidant power
TABLE 10.3
Scavenging Activity of Various Antioxidants at 80 µM Each Against Superoxide Anions Generated by the Phenazine Methosulfate–NADH System
Substance % Protection Substance % Protection Control 0 Ellagic acid dihydrate 0
EGCG 94 Reduced glutathione 0
GCG 90 N-Acetyl-L-cysteine 0
EGC 87.5 Sulforaphane 0
ECG 74.5 Quercetin dihydrate 0
EC 68 Curcumin 0
Resveratrol 62 Troloxa 0
Ascorbic acid 53 Selenium powder 0
BHT 52
D-Tocopherol 50
BHA 47
Note: Superoxide reacts with nitroblue tetrazolium to produce a deep purple color measured at 560 nm.
a 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
TABLE 10.4
Inhibition of Enzymatic Uric Acid Production in the Presence of Various Antioxidants at 80 µM Each in the Xanthine-Xanthine Oxidase System
Substance % Enzyme Inhibition
Quercetin 80 Sulforaphane 20 N-Acetyl-L-cysteine 15 Selenium powder 8 Reduced glutathione 4 Curcumin 4
Note: This is a control for inhibition of the enzyme. Uric acid production is measured at 295 nm.
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in this system. The figures for the other antioxidants are, however, not artifactual. A graph of some of the data obtained for enzyme inhibition is shown in Figure 10.10.
Once again, the green tea catechins, particularly EGC, GCG, and EGCG, are the most powerful of this group of widely reported antioxidants. The data leading to this conclusion are not inflated by enzyme inhibition at these concentrations. The nature of the data is illus- trated inFigure 10.11.
TABLE 10.5
Scavenging Potential of Various Antioxidants at 80 µM Each Against Superoxide Anions Generated by the Xanthine-Xanthine Oxidase System
Substance % Interception of Superoxide Anion
EGC 70 GCG 62 EGCG 55 ECG 31 Quercetin 30a BHA 28 BHT 24 EC 20 Sulforaphane 20a N-Acetyl-L-cysteine 15a Trolox 6 Resveratrol 2 D-Tocopherol 1
Note: Detection is based on reduction in the purple color measured at 560 nm and produced by the reaction be- tween superoxide and nitroblue tetrazolium.
a The starred figures overstate the actual antioxidant power of the agents involved. SeeTable 10.4.
FIGURE 10.10
Inhibition by a variety of antioxidants of the conversion of xanthine to uric acid catalyzed by xanthine oxidase.
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Having demonstrated that many of these agents are antioxidants in the sense that they consume superoxide anion and that they also can protect DNA against cleavage by reactive oxygen species, we turned to a whole-cell system. The Ames test is widely employed as a rapid and quantitative measure of the ability of various chemicals to cause genetic dam- age.16 The basic system uses one of a variety of strains of Salmonella typhimurium which
have been mutated in the histidine biosynthesis pathway so that they can no longer biosyn- thesize this essential amino acid (his–) and, therefore, are unable to produce colonies in the
absence of histidine in the medium. When subjected to the action of mutagens, a percent- age of the cells undergo genetic damage which results in the reversion to his+ cells, which
are capable of producing their own histidine. These revertants constitute only a small num- ber of cells, but they are able to reproduce and when cultured suitably will produce colo- nies which can be counted. The number of these revertants produced in the presence of an added test substance, minus the number of spontaneous revertants, provides an index of mutagenicity. We employ a modification of this test in which a second agent that is capable of intercepting the mutagen (desmutagenic effect) or inducing cellular repair processes which diminish the damage brought about by the mutagen (true antimutagenic effect), or a combination of these features is added to the cells. This decrease indicates the antimu- tagenic ability of the cytoprotective agent. Since dead cells neither mutate nor produce revertant colonies, controls must be run that demonstrate which concentrations of antimu- tagen may be safely employed without producing artifactual results.
An example of the data produced in the viability control phase and the revertants are shown in Figure 10.12. Hydrogen peroxide was used as the insulting agent, and tester strain TA-102, which is specially sensitive to hydrogen peroxide, was used as the test organism. The results of these assays at 10, 20, and 40 µM concentrations are given in Table 10.6. The green tea catechins individually (except for epicatechin) and collectively lead in antimutagenic potential over all of the other agents throughout this concentration range. At 10 µM concentration, a concentration where all of the agents are sufficiently non- toxic to allow for a side-by-side comparison, resveratrol, quercetin, and selenium powder FIGURE 10.11
Interception by various antioxidants of superoxide anions generated by the catalytic action of xanthine oxidase in converting xanthine to uric acid.
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are about one third as potent as EGCG, followed by trolox and curcumin. In particular, the well-known natural antioxidants D-tocopherol and ascorbic acid trail badly in potency at this concentration. When the concentrations are increased, BHA and BHT are too toxic to be used. As would be expected, all of the agents increase in their antimutagenic properties at higher concentrations. Some of the compounds in the midrange change their relative FIGURE 10.12
(Top) Toxicity of various resveratrol concentrations against Salmonella typhimurium tester strain TA-102 in a histidine-containing medium. (Bottom) Antimutagenic effect of resveratrol concentrations in a modified Ames test employing strain TA-102 and hydrogen peroxide.
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positions (D-tocopherol and Trolox), but none approaches the activity of the green tea cat- echins, nor do the five agents at the bottom of the list (including reduced glutathione and
L-ascorbic acid) supplant any of the midrange agents. At the highest reported concentra-
tion, a further increase in antimutagenic/antioxidative power is observed for virtually all of the agents (among other things, a further indication of lack of toxicity) and almost with- out exception they preserve their relative rankings.
Since whole cells are involved in this test, cellular penetration factors must be added to the underlying phenomena dictating the results. Further, it is unclear what comparative roles are played by prevention of the formation of reactive oxygen species generation, interception of the radical species produced, and the induction and operation of cellular intercepting and repair mechanisms. It is highly suggestive, however, to note from the data in the various tables that those agents best able to protect naked DNA from superoxide rad- icals, and those which are best at interfering with production of superoxide in in vitro sys- tems, are the same agents which excel at protecting this procaryotic test system from hydrogen peroxide. It seems reasonable to conclude that the primary property at work in all of these systems is an antioxidant action. This, further, is consistent with the general belief that the protective effect exerted by green tea catechins, and to a lesser extent by the other antioxidants, in the many other in vitro and animal studies, and inferred from the clin- ical data, are likewise manifestations of the same phenomenology. The chemistry respon- sible for these effects would seem to be ready donation of electrons to reactive oxygen species (ROS) by green tea catechins which then quenches the ROS and produces more sta- ble and thus less-damaging radical species. One notes that, in the green tea catechins, the presence of gallate moieties is characteristic of the best antioxidants, as is the cis-relation- ship of the phenolic appendages, allowing these functions to interact intermolecularly in much the same manner as they are seen to do when being enzymically converted to black tea catechins if the polyphenolases are not promptly heat-inactivated following picking.
TABLE 10.6
Percent Inhibition of His+ Revertants Induced
by 1.4 mM Hydrogen Peroxide by Various Concentrations of Selected Antioxidants
Compound 10 µM 20 µM 40 µM
EGCG 58 65 70
EGC 42 47 61
Green tea catechins 37 49 59
ECG 29 45 53 GCG 29 43 52 Resveratrol 20 31 44 Selenium powder 20 29 33 Curcumin 15 20 32 Quercetin 19 25 28 D-Tocopherol 5 16 28 Trolox 16 25 28 Sulforaphane 6 10 22
BHA 8 Toxic Toxic
BHT 8 Toxic Toxic EC 3 5 12 Reduced glutathione 0 0 4 Ellagic acid 3 2 6 L-Ascorbic acid 1 1 2 N-Acetyl-L-cysteine 0 0 1
Note: Spontaneous revertants were subtracted.
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Other chemical mechanisms must be involved with the other antioxidants in this study because of their differing chemical constitution. The degree to which these various agents induce or activate preexisting oxidative protection mechanisms in cells remains specula- tive at present.
At best, then, consumption of green tea beverage or capsules containing the concentrated catechins as a cytoprotective and antioxidant measure is logical and supported by much data. At worst, this is a pleasant custom which appears to do no harm in the vast majority of cases.