Fd Chem. Toxic. Vol. 29, No. I, pp. l~i, 1991 Printed in Great Britain.All rights reserved
0278-6915/91 $3.00+0.00 Copyright © 1991PergamonPress pie
Research Section ANTIOXIDANT BEHAVIOUR OF CAFFEINE: EFFICIENT SCAVENGING OF HYDROXYL RADICALS X. SHI and N. S. DALAL* Department of Chemistry, West Virginia University, Morgantown, WV 26506 and A. C. JAIN Section of Cardiology, Department of Medicine, West Virginia University School of Medicine, Morgantown, WV 26506, USA (Received 15 June 1990; revisions received 21 September 1990)
Abstract---Considerable controversy exists in the literature regarding the toxicity of coffee, including its possible carcinogenic and anticarcinogenic properties. This study reports on the reaction of 1,3,7trimethylxanthine (caffeine) with the hydroxyl radical (.OH), as investigated by electron spin resonance (ESR) spin trapping. The .OH was generated by the Fenton reaction (Fe2+ + H202) as well as by the reaction of chromium(V) with H202. The results show that caffeine effectively scavenges .OH with a reaction rate constant of ~ 5.9 x 109M 1SeC 1that is comparable with those of other efficient •OH radical scavengers. ESR measurements provide evidencethat a caffeine-derivedoxygen-centred radical is formed in the reaction of caffeine with •OH and suggest a biochemicalbasis for the understanding of the reported anticarcinogenic properties of caffeine and related methylxanthine compounds. -
-
INTRODUCTION
(Booth and Boyland, 1953; Leiter and Shear, 1943; Weil-Malherbe, 1946). For example, the effectiveness of benzo[a]pyrene as a carcinogen was reduced when it was administered by subcutaneous injection together with caffeine (Leiter and Shear, 1943). In vivo tumour production has been shown to be partially suppressed by theophylline, an analogue of caffeine (Reddi and Constantinides, 1972). It has also been demonstrated that caffeine inhibits the carcinogenic action of cigarette smoke condensate fractions when added to the fractions before application (Rothwell, 1974). The above experimental evidence seems to indicate that caffeine may have an anticarcinogenic property, although the underlying mechanism is not clear. In this context we noted that uric acid, which is structurally similar to caffeine, is an important scavenger for the hydroxyl radical (.OH) (Ames et al., 1981; Davies et al., 1986; Maples and Mason, 1988). Since •OH is implicated in many carcinogenic and cytotoxic processes, the study presented here investigated the reaction of caffeine with .OH. It was found that caffeine scavenges •OH as efficiently as other commonly used .OH scavengers, which thus suggests a basis for the reported anticarcinogenic properties of caffeine and its analogous drugs.
During the late 1970s and early 1980s, it was reported that there were associations between coffee-drinking and cancers of the bladder (Cole, 1971), pancreas (MacMahon et al., 1981) and ovaries (Trichopoulos et al., 1981). On the other hand, recent animal and human epidemiological studies have shown that coffee is not carcinogenic (Grice, 1987; Mohr et al., 1984; Rosenkranz and Ennever, 1987; Wiirzner et al., 1977; Yamaguchi and Iki, 1986). Some experimental evidence indicates, in fact, that coffee might be anticarcinogenic. For example, Sprague-Dawley rats that received coffee for 2 yr showed significantly lower tumour incidence that depended on the level of coffee intake (Mohr et aL, 1984). A dose-dependent reduction in tumour incidence for a range of neoplasms has also been described in a 2-yr study in rats (Wiirzner et al., 1977). Coffee extracts have been reported to show inhibitory effects on the mutagenicity of 3-amino-l-methyl-5H-pyrido(4,3-b)indoleand benzo[a]pyrene (Yamaguchi and Iki, 1986). The six chemicals (H202, 1,3,7-trimethylxanthine (caffeine), chlorogenic acid, methylglyoxal, glyoxal and caffeic acid) that have been identified in coffee solution are all mutagenic, except for caffeine (Ariza et al., 1988). Moreover, there is some evidence that caffeine protects against the effects of chemical carcinogens
MATERIALS AND METHODS
*To whom all correspondence and reprint requests should be addressed. Abbreviations: DMPO = 5,5-dimethyl-1-pyrroline-N-oxide; DMSO = dimethylsulphoxide; ESR = electron spin resonance; . OH = hydroxyl radical. F~ 29/t--A
Caffeine, hydrogen peroxide (H202), ethanol, dimethylsulphoxide (DMSO), mannitol and sodium formate were purchased from Aldrich (Milwaukee, WI, USA). Potassium dichromate (K2Cr207) and 1
2
X. Sm et al.
sodium sulphite (Na2SO3) were purchased from Fisher (Pittsburgh, PA, USA). N A D P H was purchased from Sigma Chemical Co. (St Louis, MO, USA). The spin trap 5,5-dimethyl-l-pyrroline-Noxide (DMPO) was also purchased from Aldrich and was purified by charcoal decolorization before use (Buettner and Oberley, 1978). A standard solution of 0.3 M-DMPO was prepared in phosphate buffer, pH 7.2. All electron spin resonance (ESR) spectra were obtained at X-band ( ~ 9 . 7 G H z ) using a Bruker ER200 ESR spectrometer. For accurate measurements of the g values and hyperfine splittings, the magnetic field was calibrated with a self-tracking nuclear magnetic resonance gaussmeter (Bruker, model ER035 M) and the microwave frequency was measured with a frequency counter (HewlettPackard, model 5340A). An ASPECT 2000 microcomputer was used for data acquisition and analysis, and for ESR spectral simulations to deduce the splitting constants. The spectrometer settings are provided in the Figure legends. The concentrations given in the Figure legends are the final concentrations. All experiments were carried out in the phosphate buffer solution (pH 7.2) at room temperature. RESULTS AND DISCUSSION
In order to study the reaction of. OH with caffeine, we first calibrated our ESR detection system using the well known Fenton reaction (Fe 2+ + H 2 0 2 - ~
a ,v,..,-,.J~ , ~ J I S
I S
I b -- - _. c~
(
~ . - DMPO+F'eZ÷-i-Hz02
w
---.-'----- -------"--- (a] +0.16MCaffeine a
°-4--
)
+
0.08MCaffeine
.---.••.[a]
+ 0.04MCaffeine
~ ( a )
1-0.02M Caffeine
(a] +0.16M Caffeine "-"-- r-'---V~added after 20eee 15G
Fig. I. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2) of 0.075 M-DMPO with 2.5mM-Fe 2+ and 2.5 mM-H202. (b) As for (a), hut with
0.16 M-caffeine. (c) As for (a), but with 0.08 M-caffeine. (d) As for (a), but with 0.04 M-caffeine. (e) As for (a), but with 0•02 M-caffeine. (f) As for (a), but with caffeine (0.16M) added after 20 sec of reaction. Spectrometer settings were: receiver gain, 2.5 x 105; modulation amplitude, 1.0 G; scan time, 200 sec; field, 3480 + 50 G; time constant, 0.5 sec.
/ b
~
~
/t
~
~ ' - - ' - " I al + 0.14M Caffeine
C~ d
DMPO+NADPH
(0)I'0.07 MCaffeine
~ ( a ) + 0 . 0 3 5
e~
(
o
)
MCaffeine +0.02 MCaffeine
15G
Fig. 2. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2), of 0.065 M-DMPO with 2.0 mM-NADPH, 2.0 n~-K2Cr:O7, and 2.0 mM-H202. (b) As for (a), but with 0.14 M-caffeine.(c) As for (a), but with 0.07 M-caffeine.(d) As for (a), but with 0.035 M-caffeine.(e) As for (a), but with 0.02 M-caffeine. Spectrometer settings were: receiver gain, 5.0x 105; modulation amplitude, 1.25 G; scan time, 2 min; field, 3480 __ 50 G; time constant, 0.5 sec. Fe 3+ + .OH + O H - ) as a source of .OH. As shown in Fig. la, the reaction of F C + with H202 in the presence of the spin trap DMPO generates a 1 : 2: 2: 1 quartet with hyperfine splittings of a N = a l l = 14.9(G). This spectrum was assigned to the D M P O - O H adduct and constitutes the evidence for •OH generation (Kadiiska et al., 1989). As may be noted from Fig. lb, 0.16u-caffeine in the reaction system of Fig. la effectively scavenges the D M P O OH spin adduct. The scavenging behaviour of caffeine exhibited a dose dependence (Fig. l b - l e ) . In order to check whether the decreased intensity was related to the reaction of caffeine with the D M P O OH spin adduct, measurements were made on solutions in which Fe ~+ was reacted with H202 in the presence of DMPO first, and then 0.16 u-caffeine was added 20 sec later• As shown in Fig. lf, the intensity of the D M P O - O H spin adduct was esentially the same as that in Fig. la without caffeine• This result shows that the intensity of reduction observed in the reaction of F C + with H2 02 in the presence of caffeine (Fig. l b - l e ) was not related to the reaction of caffeine with D M P O - O H adduct, but to the scavenging of .OH. Additional evidence for the scavenging of" OH by caffeine was obtained by using a different source of •OH: the reaction of chromium(VI) with a solution of N A D P H containing H202 (Shi and Dalal, 1989 and 1990a). Figure 2a shows the spin trapping evidence of •OH generation in this reaction, the 1 : 2: 2: 1 quartet. It can be noted from Fig. 2b-2e, that caffeine effectively scavenges •OH in a dose-dependent manner. As discussed in the previous paragraph, caffeine did not have any effect on the D M P O - O H adduct itself. As reported earlier (Shi and Dalal, 1990a), the reaction of Cr(VI) with N A D P H generates chromium(V) intermediates that subsequently react with H20~ to generate .OH (equation 1): Cr(V) + H 202 - , Cr(VI) + O H - + . OH
(1)
Antioxidant behaviour of caffeine of 0.6 M-caffeine. The spectrum in Fig. 3b is identical to that in Fig. 3a, which shows that in the presence of NADPH, caffeine does not react with Cr(V). As a third check, we evaluated the possibility of the ~ ~~ Cr[VI)+ NADPH direct reaction of caffeine with H202, because any depletion of H202 will suppress .OH generation. Figure 4 shows the dependence of. OH generation on H202 concentration in the Cr(V)-NADPH system. In Fig. 4a, Cr(V) was not observable in the presence of ~ ,,,,. (o)+O.6MCaffeine a high (2.5 mM) concentration of H202. However, the intensity of D M P O - O H spin adduct spectrum decreases and that of Cr(V) increases on reducing the H202 concentration (from 2mM to 0.2raM) (Fig. 4b,c). In the absence of H202, the D M P O - O H signal becomes very weak while the Cr(V) signal becomes strong (Fig. 4d), but with 0.32 M-Caffeine, 56 neither D M P O - O H spin adduct nor Cr(V) was obFig. 3. (a) ESR spectrum of 25 mM-K2Cr207 and 25 mM- servable (Fig. 4e). If the depletion of D M P O - O H was NADPH in a phosphate buffer solution (pH 7.2). (b) As for related to the depletion of H202 by caffeine prior to (a), but with 0.60 M-caffeine. the formation o f . OH, a strong Cr(V) peak should have been observed; its absence shows that the depletion of DMPO--OH by caffeine was not related to We checked the possibility of whether the complexing any reaction of caffeine with H202. of caffeine with Cr(V) itself might be the cause of the We also examined whether caffeine has any effect reduction in •OH concentration. As shown in Fig. 3a, on spin trap itself, so as to reduce its ability to trap the reaction between Cr(VI) and N A D P H generated a free radical. To evaluate this, we generated the a spectrum centered at g = 1.9792 with five principal DMPO-SO~- adduct and examined the effect of components having a 0.84 G spacing. This g value is caffeine in the formation of this adduct (Shi and typical of that of a Cr(V) complex, and the splittings Dalai, 1990b). As shown in Fig. 5a, the reaction of are characteristic of the superhyperfine interaction of Cr(VI) with SO~3- in the presence of D M P O generates nearby hydrogens (Shi and Dalai, 1990a). Thus, this a strong spin adduct signal with hyperfine splittings spectrum was assigned to a Cr(V)-NADPH complex. of ar~ = 14.7 G and aH = 16.0 G, which was assigned Figure 3b shows the spectrum that was obtained by to DMPO-SO;- spin adduct according to earlier the reaction of Cr(VI) with N A D P H in the presence analyses (Shi and Dalai, 1990b). When this reaction was carried out in the presence of caffeine, the same spin adduct signal with essentially the same intensity was observed (Fig. 5b), which shows that the presence DMPO+Cr (VI)+ of caffeine did not have any effect on the ability of NADPH+2.SmM Hz02 DPMO to trap a free radical. On the basis of the control experiments discussed above, we conclude that caffeine must have an ability DMPO+Cr (VI) + to scavenge .OH. Currently, formate, ethanol, NADPH+ 0.6mM HzOz DMSO and mannitol are frequently used as .OH DMPO+Cr (VI) +
NADPH+ 0.2 mMH202 .PO+SO
-+c.v,l
o ------J DMPO+Cr(VI} +
NAOPH d
~
(a) +0.32 M Coffelne
Fig. 4. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution (pH 7.2) of 0.065 M-DMPO with 10 mM-NADPH, 10 mM-K2Cr207 and 2.5 mM-H202. (b) As for (a), but with 0.625 mM-H202. (c) As for (a), but with 0.2 mM-H202. (d) As for (a), but without H20:. (e) As for (a), but with 0.25 M-caffeine. Spectrometer settings were: receiver gain, 1.6 × 105; modulation amplitude, 0,8 G; scan time, 200 see; field, 3480 4- 75 G; time constant, 0.5 see.
b
~
) +0.32 MCaffeine
15G Fig. 5. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution of 0.065 M-DMPO with 0.1 MNa2SO 3 and 2 mu-K2Cr2OT. (b) As for (a), but with 0.25 Mcaffeine. Spectrometer settings were: receiver gain, 2.5 x 105; modulation amplitude, 1 G; scan time, 200see; field, 3480 4- 50 G; time constant, 0.5 see.
X. Sm et al. 4 o
T 2 >1> 1
~
(0)+0.1MFormate 0 .0
c /
d~ e
0.25 M EtOH
+
0
.
2
5
~
MMonnltot (o)+02.5MDMSO (0) + 0.07 M
I I 0.6 0.9 [Coffelne]/[DMPO]
I 1.2
Fig. 7. Inhibition of hydroxyl radical by caffeine. The hydroxyl radical was produced by the reaction of 2.5 mMFe2+ with 2.5mM-H202 containing 0•075 M-DMPO. The data were plotted according to V / 0 - 1 =k2[caffeine]/ kl IDMPO], as explained in the text• represent the rate of spin trapping in the absence and presence of caffeine, respectively, one obtains equation 7:
Caffeine
f
I 0.3,
V/v = 1 + k2[caffeine]/ki [DMPO]
(7)
or V -
Fig. 6. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2) of 0.075 M-DMPO with 2.5 mM-Fe2+ and 2.5 mM-H202. (b) As for (a), but with 0.1 M-formate. (c) As for (a), but with 0.25 M-ethanol,(d) As for (a), but with 0.25 M-mannitol. (e) As for (a), but with 0.25 M-DMSO. (f) As for (a), but with 0.07 M-caffeine. The spectrometer settings were the same as those in Fig. 1. scavengers (Finkelstein et al., 1980; Morehouse and Mason, 1988; Shi and Dalai, 1990a). We compared these with caffeine in their ability to scavenge •OH. As shown in Fig. 6, the caffeine scavenges the .OH to a level comparable with these scavengers. To further characterize the. OH scavenging property, we carried out kinetic studies following the methods described earlier for the reaction of ethanol with .OH (Finkelstein et al., 1980; Morehouse and Mason, 1988). The reaction steps may be written as: kl •OH + DMPO , DMPO-OH (2) k2
•OH + caffeine d[.OH] - dt
= k~
, product
(3)
[DMPO] [. OH] + k2 [' OH] [caffeine]
d[DMPO-OH] dt = k~[DMPO] [. OH]
(4)
(5)
Dividing equation 4 by equation 5, one obtains equation 6: - d[. OH]/dt d[DMPO-OH]/dt
=
1 +
k2 [caffeine] k~[DMPO]
(6)
At a saturating level of DMPO and in the absence of caffeine, the rate of spin trapping is equal to the rate o f . OH generation, d[. OH]/dt. Thus, if V and o
o
k2 [caffeine] -
1 =
kl [DMPO]
(8)
Figure 7 shows the inhibition o f . OH by caffeine. The data were plotted according to equation 8, and a straight line with a slope of 2.8 was obtained. The ratio k2/k I is the slope of the straight line in Fig. 7. Using the value of kt =2.1 x 10aM-~SCC-l for the trapping of .OH by DMPO (Buettner, 1982), the value of k 2 is obtained as follows: k 2 = 2.8 x kl = 2.8 x 2.1 x 109M-l sec -l = 5.9
x
1 0 9 M -1 s e c -1
This result is comparable to that (3.0 x 109 M - 1 SeC -1 ) for the well known .OH scavenger, formate (Buettner, 1982; Farhataziz and Ross, 1977)• We note that the rate constant k2 as calculated by this method may not be very accurate• For example, the above model neglects the decay of the spin adduct after its generation (Marriott et al., 1980). However, it does provide a convenient method for obtaining relative values. In the above equations, we have shown that caffeine does have an ability to scavenge .OH. If caffeine reacts with .OH as we have demonstrated, then a likely product of such a reaction could be a caffeine-derived radical• This radical may be trapped by DMPO. As shown in Fig. 8a, a six-line spin adduct spectrum was obtained from the reaction mixture of DMPO, H:O2, Fe 2+ and caffeine. Analysis of the spectrum in Fig. 8a yields the hyperfine coupling constants, aN= 15.7G and a , = 18.7G. It may be noted that the hyperfine splitting constants for the DMPO adduct of a carbon centred radical (R.) are in the range of ar~ = 14.0 to 16.5 G and a H= 19.0 to 23.5 G (Buettner, 1987), and those of an oxygen centred radical (RO .) are in the range of aN = 14.5 to 16.0G and a . = 18.0 to 19.0G (Buettner, 1987)• Thus, the spin adduct spectrum observed in Fig. 8a
Antioxidant behaviour of caffeine
°|r-~
A^III f) AA °,,o+,;, VI/--" IC"---H,O,*
V
)V
V
o6.c...,..
N b
A.
51"
W//-
]~.
I /'J l,/'~
DMPO+ Fe + V~''-HzOz-t"
15G
Fig. 8. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution (pH 7.2) of 0.1 M-DMPO with 4mM-Fe 2+, 5 mM-H202, and 0.6 M-caffeine. (b) As for (a) but with 0.14M-caffeine. A flat cell was used for these measurements. Spectrometer settings were: receiver gain, 5 x 105; modulation amplitude, 1.0 G; scan time, 600 sec; field, 3480 ___50; time constant, 0.5 sec.
is most likely to be the result of an oxygen-centred radical. Based on literature studies of similar radicals (Simic et al., 1989), we suggest the scheme in Fig. 9 for the species responsible for the spectrum shown in Fig. 8a. When the caffeine concentration was decreased to 0.14 M, both the spin adduct of the caffeine-derived radical and that of .OH could be detected simultaneously (Fig. 8b). The signals from .OH are highlighted by asterisks. We note, however, that with spin trapping the relative concentrations of spin-trapped radicals need not reflect the relative concentrations of the initial radicals (Flitter and Mason, 1989). Thus, while we cannot yet state that this caffeine-derived radical is the major reactive intermediate of the reaction between. OH and caffeine, it seems to be one of the significant products. The above results clearly show that caffeine efficiently scavenges •OH. This is an important observation since toxicity by oxygenated radicals has been suggested as a likely cause of cancer, heart disease and ageing (Ames, 1989; Fridovich, 1978; Jamieson, 1989; Oberley et al., 1980; Simic, 1988; Sevanian et al., 1985; Totter, 1980; Zweier et aL, 1988). The .OH scavenging ability of caffeine may be one of the mechanisms responsible for the earlier reported effect of caffeine against some chemical carcinogens. Other components of coffee such as HzO2 (a solution of 1 mg coffee contains 12 nmol H20:) (Ariza et al., 1988; Peinado et aL, 1986) might be mutagenic and carcinogenic. For example, it has been suggested that H20~ produced from coffee or tea may react with metal ions either in the coffee or in cellular systems to generate. OH (Alejandre-Duran et al., 1987; Ariza
O O" CH3. U /CHa CH3~N..~ HO.+"NL~l~==_.=.~ ===-=~ CHa
N/CH3
CHa
Fig. 9. Schematic diagram illustrating the reaction between caffeine and 'OH to produce an oxygen-centred radical.
et al., 1988). The. OH was hypothesized to be at least partially responsible for the genotoxicity of coffee (Ariza et al., 1988). Caffeine may scavenge this radical and reduce the genotoxic effect of coffee. However, the current study did not rule out other mechanisms through which caffeine may give rise to the toxicity of coffee. For example, caffeine may potentiate the mutagenic and lethal effects of genotoxic agents by the inhibition of D N A repair (Selby and Sancar, 1990). Thus, caffeine could have both a toxic and beneficial effect depending on the environment. In a cellular system, these two aspects would ;e competitive, and this might explain some of the controversial literature on the adverse effects of caffeine (Ariza et al., 1988; Booth and Boyland, 1953; Cole, 1971; Grice, 1987; Leiter and Shear, 1943; MacMahon et al., 1981; Mohr et al., 1984; Reddi and Constantinides, 1972; Rosenkranz and Ennever, 1987; Rothwell, 1974; Selby and Sancar, 1990; Trichopoulos et al., 1981; Weil-Malherbe, 1946; Wiirzner et al., 1977; Yamaguchi and Iki, 1986).
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0278-6915/91 $3.00+0.00 Copyright © 1991PergamonPress pie
Research Section ANTIOXIDANT BEHAVIOUR OF CAFFEINE: EFFICIENT SCAVENGING OF HYDROXYL RADICALS X. SHI and N. S. DALAL* Department of Chemistry, West Virginia University, Morgantown, WV 26506 and A. C. JAIN Section of Cardiology, Department of Medicine, West Virginia University School of Medicine, Morgantown, WV 26506, USA (Received 15 June 1990; revisions received 21 September 1990)
Abstract---Considerable controversy exists in the literature regarding the toxicity of coffee, including its possible carcinogenic and anticarcinogenic properties. This study reports on the reaction of 1,3,7trimethylxanthine (caffeine) with the hydroxyl radical (.OH), as investigated by electron spin resonance (ESR) spin trapping. The .OH was generated by the Fenton reaction (Fe2+ + H202) as well as by the reaction of chromium(V) with H202. The results show that caffeine effectively scavenges .OH with a reaction rate constant of ~ 5.9 x 109M 1SeC 1that is comparable with those of other efficient •OH radical scavengers. ESR measurements provide evidencethat a caffeine-derivedoxygen-centred radical is formed in the reaction of caffeine with •OH and suggest a biochemicalbasis for the understanding of the reported anticarcinogenic properties of caffeine and related methylxanthine compounds. -
-
INTRODUCTION
(Booth and Boyland, 1953; Leiter and Shear, 1943; Weil-Malherbe, 1946). For example, the effectiveness of benzo[a]pyrene as a carcinogen was reduced when it was administered by subcutaneous injection together with caffeine (Leiter and Shear, 1943). In vivo tumour production has been shown to be partially suppressed by theophylline, an analogue of caffeine (Reddi and Constantinides, 1972). It has also been demonstrated that caffeine inhibits the carcinogenic action of cigarette smoke condensate fractions when added to the fractions before application (Rothwell, 1974). The above experimental evidence seems to indicate that caffeine may have an anticarcinogenic property, although the underlying mechanism is not clear. In this context we noted that uric acid, which is structurally similar to caffeine, is an important scavenger for the hydroxyl radical (.OH) (Ames et al., 1981; Davies et al., 1986; Maples and Mason, 1988). Since •OH is implicated in many carcinogenic and cytotoxic processes, the study presented here investigated the reaction of caffeine with .OH. It was found that caffeine scavenges •OH as efficiently as other commonly used .OH scavengers, which thus suggests a basis for the reported anticarcinogenic properties of caffeine and its analogous drugs.
During the late 1970s and early 1980s, it was reported that there were associations between coffee-drinking and cancers of the bladder (Cole, 1971), pancreas (MacMahon et al., 1981) and ovaries (Trichopoulos et al., 1981). On the other hand, recent animal and human epidemiological studies have shown that coffee is not carcinogenic (Grice, 1987; Mohr et al., 1984; Rosenkranz and Ennever, 1987; Wiirzner et al., 1977; Yamaguchi and Iki, 1986). Some experimental evidence indicates, in fact, that coffee might be anticarcinogenic. For example, Sprague-Dawley rats that received coffee for 2 yr showed significantly lower tumour incidence that depended on the level of coffee intake (Mohr et aL, 1984). A dose-dependent reduction in tumour incidence for a range of neoplasms has also been described in a 2-yr study in rats (Wiirzner et al., 1977). Coffee extracts have been reported to show inhibitory effects on the mutagenicity of 3-amino-l-methyl-5H-pyrido(4,3-b)indoleand benzo[a]pyrene (Yamaguchi and Iki, 1986). The six chemicals (H202, 1,3,7-trimethylxanthine (caffeine), chlorogenic acid, methylglyoxal, glyoxal and caffeic acid) that have been identified in coffee solution are all mutagenic, except for caffeine (Ariza et al., 1988). Moreover, there is some evidence that caffeine protects against the effects of chemical carcinogens
MATERIALS AND METHODS
*To whom all correspondence and reprint requests should be addressed. Abbreviations: DMPO = 5,5-dimethyl-1-pyrroline-N-oxide; DMSO = dimethylsulphoxide; ESR = electron spin resonance; . OH = hydroxyl radical. F~ 29/t--A
Caffeine, hydrogen peroxide (H202), ethanol, dimethylsulphoxide (DMSO), mannitol and sodium formate were purchased from Aldrich (Milwaukee, WI, USA). Potassium dichromate (K2Cr207) and 1
2
X. Sm et al.
sodium sulphite (Na2SO3) were purchased from Fisher (Pittsburgh, PA, USA). N A D P H was purchased from Sigma Chemical Co. (St Louis, MO, USA). The spin trap 5,5-dimethyl-l-pyrroline-Noxide (DMPO) was also purchased from Aldrich and was purified by charcoal decolorization before use (Buettner and Oberley, 1978). A standard solution of 0.3 M-DMPO was prepared in phosphate buffer, pH 7.2. All electron spin resonance (ESR) spectra were obtained at X-band ( ~ 9 . 7 G H z ) using a Bruker ER200 ESR spectrometer. For accurate measurements of the g values and hyperfine splittings, the magnetic field was calibrated with a self-tracking nuclear magnetic resonance gaussmeter (Bruker, model ER035 M) and the microwave frequency was measured with a frequency counter (HewlettPackard, model 5340A). An ASPECT 2000 microcomputer was used for data acquisition and analysis, and for ESR spectral simulations to deduce the splitting constants. The spectrometer settings are provided in the Figure legends. The concentrations given in the Figure legends are the final concentrations. All experiments were carried out in the phosphate buffer solution (pH 7.2) at room temperature. RESULTS AND DISCUSSION
In order to study the reaction of. OH with caffeine, we first calibrated our ESR detection system using the well known Fenton reaction (Fe 2+ + H 2 0 2 - ~
a ,v,..,-,.J~ , ~ J I S
I S
I b -- - _. c~
(
~ . - DMPO+F'eZ÷-i-Hz02
w
---.-'----- -------"--- (a] +0.16MCaffeine a
°-4--
)
+
0.08MCaffeine
.---.••.[a]
+ 0.04MCaffeine
~ ( a )
1-0.02M Caffeine
(a] +0.16M Caffeine "-"-- r-'---V~added after 20eee 15G
Fig. I. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2) of 0.075 M-DMPO with 2.5mM-Fe 2+ and 2.5 mM-H202. (b) As for (a), hut with
0.16 M-caffeine. (c) As for (a), but with 0.08 M-caffeine. (d) As for (a), but with 0.04 M-caffeine. (e) As for (a), but with 0•02 M-caffeine. (f) As for (a), but with caffeine (0.16M) added after 20 sec of reaction. Spectrometer settings were: receiver gain, 2.5 x 105; modulation amplitude, 1.0 G; scan time, 200 sec; field, 3480 + 50 G; time constant, 0.5 sec.
/ b
~
~
/t
~
~ ' - - ' - " I al + 0.14M Caffeine
C~ d
DMPO+NADPH
(0)I'0.07 MCaffeine
~ ( a ) + 0 . 0 3 5
e~
(
o
)
MCaffeine +0.02 MCaffeine
15G
Fig. 2. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2), of 0.065 M-DMPO with 2.0 mM-NADPH, 2.0 n~-K2Cr:O7, and 2.0 mM-H202. (b) As for (a), but with 0.14 M-caffeine.(c) As for (a), but with 0.07 M-caffeine.(d) As for (a), but with 0.035 M-caffeine.(e) As for (a), but with 0.02 M-caffeine. Spectrometer settings were: receiver gain, 5.0x 105; modulation amplitude, 1.25 G; scan time, 2 min; field, 3480 __ 50 G; time constant, 0.5 sec. Fe 3+ + .OH + O H - ) as a source of .OH. As shown in Fig. la, the reaction of F C + with H202 in the presence of the spin trap DMPO generates a 1 : 2: 2: 1 quartet with hyperfine splittings of a N = a l l = 14.9(G). This spectrum was assigned to the D M P O - O H adduct and constitutes the evidence for •OH generation (Kadiiska et al., 1989). As may be noted from Fig. lb, 0.16u-caffeine in the reaction system of Fig. la effectively scavenges the D M P O OH spin adduct. The scavenging behaviour of caffeine exhibited a dose dependence (Fig. l b - l e ) . In order to check whether the decreased intensity was related to the reaction of caffeine with the D M P O OH spin adduct, measurements were made on solutions in which Fe ~+ was reacted with H202 in the presence of DMPO first, and then 0.16 u-caffeine was added 20 sec later• As shown in Fig. lf, the intensity of the D M P O - O H spin adduct was esentially the same as that in Fig. la without caffeine• This result shows that the intensity of reduction observed in the reaction of F C + with H2 02 in the presence of caffeine (Fig. l b - l e ) was not related to the reaction of caffeine with D M P O - O H adduct, but to the scavenging of .OH. Additional evidence for the scavenging of" OH by caffeine was obtained by using a different source of •OH: the reaction of chromium(VI) with a solution of N A D P H containing H202 (Shi and Dalal, 1989 and 1990a). Figure 2a shows the spin trapping evidence of •OH generation in this reaction, the 1 : 2: 2: 1 quartet. It can be noted from Fig. 2b-2e, that caffeine effectively scavenges •OH in a dose-dependent manner. As discussed in the previous paragraph, caffeine did not have any effect on the D M P O - O H adduct itself. As reported earlier (Shi and Dalal, 1990a), the reaction of Cr(VI) with N A D P H generates chromium(V) intermediates that subsequently react with H20~ to generate .OH (equation 1): Cr(V) + H 202 - , Cr(VI) + O H - + . OH
(1)
Antioxidant behaviour of caffeine of 0.6 M-caffeine. The spectrum in Fig. 3b is identical to that in Fig. 3a, which shows that in the presence of NADPH, caffeine does not react with Cr(V). As a third check, we evaluated the possibility of the ~ ~~ Cr[VI)+ NADPH direct reaction of caffeine with H202, because any depletion of H202 will suppress .OH generation. Figure 4 shows the dependence of. OH generation on H202 concentration in the Cr(V)-NADPH system. In Fig. 4a, Cr(V) was not observable in the presence of ~ ,,,,. (o)+O.6MCaffeine a high (2.5 mM) concentration of H202. However, the intensity of D M P O - O H spin adduct spectrum decreases and that of Cr(V) increases on reducing the H202 concentration (from 2mM to 0.2raM) (Fig. 4b,c). In the absence of H202, the D M P O - O H signal becomes very weak while the Cr(V) signal becomes strong (Fig. 4d), but with 0.32 M-Caffeine, 56 neither D M P O - O H spin adduct nor Cr(V) was obFig. 3. (a) ESR spectrum of 25 mM-K2Cr207 and 25 mM- servable (Fig. 4e). If the depletion of D M P O - O H was NADPH in a phosphate buffer solution (pH 7.2). (b) As for related to the depletion of H202 by caffeine prior to (a), but with 0.60 M-caffeine. the formation o f . OH, a strong Cr(V) peak should have been observed; its absence shows that the depletion of DMPO--OH by caffeine was not related to We checked the possibility of whether the complexing any reaction of caffeine with H202. of caffeine with Cr(V) itself might be the cause of the We also examined whether caffeine has any effect reduction in •OH concentration. As shown in Fig. 3a, on spin trap itself, so as to reduce its ability to trap the reaction between Cr(VI) and N A D P H generated a free radical. To evaluate this, we generated the a spectrum centered at g = 1.9792 with five principal DMPO-SO~- adduct and examined the effect of components having a 0.84 G spacing. This g value is caffeine in the formation of this adduct (Shi and typical of that of a Cr(V) complex, and the splittings Dalai, 1990b). As shown in Fig. 5a, the reaction of are characteristic of the superhyperfine interaction of Cr(VI) with SO~3- in the presence of D M P O generates nearby hydrogens (Shi and Dalai, 1990a). Thus, this a strong spin adduct signal with hyperfine splittings spectrum was assigned to a Cr(V)-NADPH complex. of ar~ = 14.7 G and aH = 16.0 G, which was assigned Figure 3b shows the spectrum that was obtained by to DMPO-SO;- spin adduct according to earlier the reaction of Cr(VI) with N A D P H in the presence analyses (Shi and Dalai, 1990b). When this reaction was carried out in the presence of caffeine, the same spin adduct signal with essentially the same intensity was observed (Fig. 5b), which shows that the presence DMPO+Cr (VI)+ of caffeine did not have any effect on the ability of NADPH+2.SmM Hz02 DPMO to trap a free radical. On the basis of the control experiments discussed above, we conclude that caffeine must have an ability DMPO+Cr (VI) + to scavenge .OH. Currently, formate, ethanol, NADPH+ 0.6mM HzOz DMSO and mannitol are frequently used as .OH DMPO+Cr (VI) +
NADPH+ 0.2 mMH202 .PO+SO
-+c.v,l
o ------J DMPO+Cr(VI} +
NAOPH d
~
(a) +0.32 M Coffelne
Fig. 4. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution (pH 7.2) of 0.065 M-DMPO with 10 mM-NADPH, 10 mM-K2Cr207 and 2.5 mM-H202. (b) As for (a), but with 0.625 mM-H202. (c) As for (a), but with 0.2 mM-H202. (d) As for (a), but without H20:. (e) As for (a), but with 0.25 M-caffeine. Spectrometer settings were: receiver gain, 1.6 × 105; modulation amplitude, 0,8 G; scan time, 200 see; field, 3480 4- 75 G; time constant, 0.5 see.
b
~
) +0.32 MCaffeine
15G Fig. 5. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution of 0.065 M-DMPO with 0.1 MNa2SO 3 and 2 mu-K2Cr2OT. (b) As for (a), but with 0.25 Mcaffeine. Spectrometer settings were: receiver gain, 2.5 x 105; modulation amplitude, 1 G; scan time, 200see; field, 3480 4- 50 G; time constant, 0.5 see.
X. Sm et al. 4 o
T 2 >1> 1
~
(0)+0.1MFormate 0 .0
c /
d~ e
0.25 M EtOH
+
0
.
2
5
~
MMonnltot (o)+02.5MDMSO (0) + 0.07 M
I I 0.6 0.9 [Coffelne]/[DMPO]
I 1.2
Fig. 7. Inhibition of hydroxyl radical by caffeine. The hydroxyl radical was produced by the reaction of 2.5 mMFe2+ with 2.5mM-H202 containing 0•075 M-DMPO. The data were plotted according to V / 0 - 1 =k2[caffeine]/ kl IDMPO], as explained in the text• represent the rate of spin trapping in the absence and presence of caffeine, respectively, one obtains equation 7:
Caffeine
f
I 0.3,
V/v = 1 + k2[caffeine]/ki [DMPO]
(7)
or V -
Fig. 6. (a) ESR spectrum recorded 2 min after mixing in a phosphate buffer solution (pH 7.2) of 0.075 M-DMPO with 2.5 mM-Fe2+ and 2.5 mM-H202. (b) As for (a), but with 0.1 M-formate. (c) As for (a), but with 0.25 M-ethanol,(d) As for (a), but with 0.25 M-mannitol. (e) As for (a), but with 0.25 M-DMSO. (f) As for (a), but with 0.07 M-caffeine. The spectrometer settings were the same as those in Fig. 1. scavengers (Finkelstein et al., 1980; Morehouse and Mason, 1988; Shi and Dalai, 1990a). We compared these with caffeine in their ability to scavenge •OH. As shown in Fig. 6, the caffeine scavenges the .OH to a level comparable with these scavengers. To further characterize the. OH scavenging property, we carried out kinetic studies following the methods described earlier for the reaction of ethanol with .OH (Finkelstein et al., 1980; Morehouse and Mason, 1988). The reaction steps may be written as: kl •OH + DMPO , DMPO-OH (2) k2
•OH + caffeine d[.OH] - dt
= k~
, product
(3)
[DMPO] [. OH] + k2 [' OH] [caffeine]
d[DMPO-OH] dt = k~[DMPO] [. OH]
(4)
(5)
Dividing equation 4 by equation 5, one obtains equation 6: - d[. OH]/dt d[DMPO-OH]/dt
=
1 +
k2 [caffeine] k~[DMPO]
(6)
At a saturating level of DMPO and in the absence of caffeine, the rate of spin trapping is equal to the rate o f . OH generation, d[. OH]/dt. Thus, if V and o
o
k2 [caffeine] -
1 =
kl [DMPO]
(8)
Figure 7 shows the inhibition o f . OH by caffeine. The data were plotted according to equation 8, and a straight line with a slope of 2.8 was obtained. The ratio k2/k I is the slope of the straight line in Fig. 7. Using the value of kt =2.1 x 10aM-~SCC-l for the trapping of .OH by DMPO (Buettner, 1982), the value of k 2 is obtained as follows: k 2 = 2.8 x kl = 2.8 x 2.1 x 109M-l sec -l = 5.9
x
1 0 9 M -1 s e c -1
This result is comparable to that (3.0 x 109 M - 1 SeC -1 ) for the well known .OH scavenger, formate (Buettner, 1982; Farhataziz and Ross, 1977)• We note that the rate constant k2 as calculated by this method may not be very accurate• For example, the above model neglects the decay of the spin adduct after its generation (Marriott et al., 1980). However, it does provide a convenient method for obtaining relative values. In the above equations, we have shown that caffeine does have an ability to scavenge .OH. If caffeine reacts with .OH as we have demonstrated, then a likely product of such a reaction could be a caffeine-derived radical• This radical may be trapped by DMPO. As shown in Fig. 8a, a six-line spin adduct spectrum was obtained from the reaction mixture of DMPO, H:O2, Fe 2+ and caffeine. Analysis of the spectrum in Fig. 8a yields the hyperfine coupling constants, aN= 15.7G and a , = 18.7G. It may be noted that the hyperfine splitting constants for the DMPO adduct of a carbon centred radical (R.) are in the range of ar~ = 14.0 to 16.5 G and a H= 19.0 to 23.5 G (Buettner, 1987), and those of an oxygen centred radical (RO .) are in the range of aN = 14.5 to 16.0G and a . = 18.0 to 19.0G (Buettner, 1987)• Thus, the spin adduct spectrum observed in Fig. 8a
Antioxidant behaviour of caffeine
°|r-~
A^III f) AA °,,o+,;, VI/--" IC"---H,O,*
V
)V
V
o6.c...,..
N b
A.
51"
W//-
]~.
I /'J l,/'~
DMPO+ Fe + V~''-HzOz-t"
15G
Fig. 8. (a) ESR spectrum recorded 2 rain after mixing in a phosphate buffer solution (pH 7.2) of 0.1 M-DMPO with 4mM-Fe 2+, 5 mM-H202, and 0.6 M-caffeine. (b) As for (a) but with 0.14M-caffeine. A flat cell was used for these measurements. Spectrometer settings were: receiver gain, 5 x 105; modulation amplitude, 1.0 G; scan time, 600 sec; field, 3480 ___50; time constant, 0.5 sec.
is most likely to be the result of an oxygen-centred radical. Based on literature studies of similar radicals (Simic et al., 1989), we suggest the scheme in Fig. 9 for the species responsible for the spectrum shown in Fig. 8a. When the caffeine concentration was decreased to 0.14 M, both the spin adduct of the caffeine-derived radical and that of .OH could be detected simultaneously (Fig. 8b). The signals from .OH are highlighted by asterisks. We note, however, that with spin trapping the relative concentrations of spin-trapped radicals need not reflect the relative concentrations of the initial radicals (Flitter and Mason, 1989). Thus, while we cannot yet state that this caffeine-derived radical is the major reactive intermediate of the reaction between. OH and caffeine, it seems to be one of the significant products. The above results clearly show that caffeine efficiently scavenges •OH. This is an important observation since toxicity by oxygenated radicals has been suggested as a likely cause of cancer, heart disease and ageing (Ames, 1989; Fridovich, 1978; Jamieson, 1989; Oberley et al., 1980; Simic, 1988; Sevanian et al., 1985; Totter, 1980; Zweier et aL, 1988). The .OH scavenging ability of caffeine may be one of the mechanisms responsible for the earlier reported effect of caffeine against some chemical carcinogens. Other components of coffee such as HzO2 (a solution of 1 mg coffee contains 12 nmol H20:) (Ariza et al., 1988; Peinado et aL, 1986) might be mutagenic and carcinogenic. For example, it has been suggested that H20~ produced from coffee or tea may react with metal ions either in the coffee or in cellular systems to generate. OH (Alejandre-Duran et al., 1987; Ariza
O O" CH3. U /CHa CH3~N..~ HO.+"NL~l~==_.=.~ ===-=~ CHa
N/CH3
CHa
Fig. 9. Schematic diagram illustrating the reaction between caffeine and 'OH to produce an oxygen-centred radical.
et al., 1988). The. OH was hypothesized to be at least partially responsible for the genotoxicity of coffee (Ariza et al., 1988). Caffeine may scavenge this radical and reduce the genotoxic effect of coffee. However, the current study did not rule out other mechanisms through which caffeine may give rise to the toxicity of coffee. For example, caffeine may potentiate the mutagenic and lethal effects of genotoxic agents by the inhibition of D N A repair (Selby and Sancar, 1990). Thus, caffeine could have both a toxic and beneficial effect depending on the environment. In a cellular system, these two aspects would ;e competitive, and this might explain some of the controversial literature on the adverse effects of caffeine (Ariza et al., 1988; Booth and Boyland, 1953; Cole, 1971; Grice, 1987; Leiter and Shear, 1943; MacMahon et al., 1981; Mohr et al., 1984; Reddi and Constantinides, 1972; Rosenkranz and Ennever, 1987; Rothwell, 1974; Selby and Sancar, 1990; Trichopoulos et al., 1981; Weil-Malherbe, 1946; Wiirzner et al., 1977; Yamaguchi and Iki, 1986).
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