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Dietary carotenoids protect human cells from damage
Journal
ELSEVIER
Dietary
carotenoids
Jane H. Tinkler”,
of Photochemistry and Photobiology B: Biology 26 (1994) 283-285
protect
human cells from damage
Fritz B6hmb, W. Schalch”,
T. George
Truscotta
“Department of Chemistry, Keele University, Staffordrhire ST5 5BG, UK bDepartment of Dermatology (Char&!), Humboldt University, 10117 Berlin, Germany ‘Department of Human Nutrition, F. Hoffmann-La Roche, CH-4002 Basle, Switzerland Received 28 April 1994; accepted 22 July 1994
Abstract A physical chemistry technique based on singlet oxygen luminescence at about 1270 nm and a biological cell membrane technique were used to study the quenching of singlet oxygen by four carotenoids bound to the surface of lymphoid cells. All the carotenoids studied showed a beneficial effect in cell protection, but there were subtle differences between them. Keywordx
Carotenoids;
Diet; Oxidative damage; Singlet oxygen
1. Introduction
Many common foods contain carotenoids; for example, there are large amounts of p-carotene in carrots and many other vegetables, lycopene in tomatoes and astaxanthin is used to colour farm salmon. In addition, carotenoids are often used as natural, non-toxic food colourants in foods, drinks, animal feedstuffs and even cosmetics. It is accepted that carotenoids are efficient quenchers of a highly damaging form of oxygen, known as singlet oxygen, and there has been debate over the relative efficiency of singlet oxygen quenching by different carotenoids [1,2]. Also, other reactive oxygen species, such as the superoxide radical (Oz.-), react with different carotenoids in markedly different ways
[31In a preliminary communication [4], we reported that singlet oxygen in vitro is quenched by all-trans-p-carotene when the carotenoid is bound to the surface of lymphoid cells obtained from human blood. We now report a more detailed study of this quenching by pcarotene and an extension to other carotenoids, namely astaxanthin, lycopene and canthaxanthin. The data reported in this work are based on the monitoring of the time-resolved near IR luminescence of singlet oxygen and are compared with biological studies based on cell destruction. 2. Experimental
details
Lymphoid cells were taken from healthy human blood and prepared as described previously [5]. Briefly, we
lOll-1344/94/$07.00 0 1994 Elsevier Science S.A. Ail rights reserved SSDI 1011-1344(94)07049-O
diluted the anticoagulated blood using heparin 1:l with Dulbecco buffer, passed it over a FicollR density gradient of 1.076 g ml-’ (Ficoll-PaqueR, Pharmacia, Uppsala, Sweden) and collected the huffy coat. After centrifugation (200 g, 30 min, 22 “C), the cells were washed three times with D,O-Dulbecco buffer, centrifuged again (2oOg, 10 min, 22 “C) and divided into two. One was treated with the carotenoid and the other was used as a control. For the cells treated with carotenoid, incubation was performed for 60 min at 22 “C with saturated solutions of water-soluble p-carotene (22 PM), astaxanthin (27 PM), lycopene (50 PM) or canthaxanthin (3 PM). These water-soluble carotenoids were obtained as beadlets from Hoffmann-La Roche, Basle, Switzerland, and experiments with blank beadlets showed no effect. Subsequently, the cells were washed three times with D,O-Dulbecco buffer to remove any unbound carotenoid. The carotenoid bound to the cells was estimated by extraction with dimethylsulphoxide and conventional UV-visible spectroscopy. Typically, we used 1O-8-1O-9 mol of carotenoid in 200 ~1 of cell suspension which contained 2 X lo6 cells. Cell staining was performed by the trypan blue exclusion test to estimate the lethally destroyed cell fraction. Control experiments, without carotenoid, were performed on cells, prepared and washed as above, without incubating with the carotenoid. Rose bengal (RB) or meso-tetra(4-sulphonatophenyl)porphine (TPPS) was added to both the control and carotenoidcontaining cells as a sensitizer of singlet oxygen.
284
J.H. Tidier
et al. I J. Photochem.
Some experiments were undertaken with in vivo coated lymphoid cells extracted from the blood of healthy individuals who had been treated orally, three times daily, with 25 mg p-carotene as CarotabeneR (Hetmal, Germany) over 4 weeks. The cells were washed three times with D,O-Dulbecco buffer prior to use. The carotene concentration was not readily obtained, but was estimated to be lo-* mol or less in 200 ~1 of cell suspension. We also obtained blood plasma from healthy humans who had or had not been treated with CarotabeneR by simple centrifugation of the blood (4OOg, 10 min, 22 “C). The plasma, without CarotabeneR, was incubated with a saturated carotenoid solution for 60 min at 22 “C and then used in the experiment. Two methods were used to study the effect of the carotenoid bound to the cells. For both, we mixed 80 ~1 of the appropriate cell suspension (or D,O-Dulbecco buffer) with 20 ~1 of sensitizer and the sample was then irradiated with 10 pulses of a 200 mJ, 532 nm laser line. One method was based on the singlet oxygen lifetime (T) using the average of 10 pulses, while the second was based on a cell counting technique and on the additive effect of the 10 pulses on a given sample. Singlet oxygen was detected via its luminescence at about 1270 nm using a time-resolved technique. Briefly, the apparatus consisted of a germanium diode linked to a Judson amplifier with excitation from a frequency doubled Nd:YAG laser (15 ns pulse at 532 nm).
3. Results and discussion Simple calculations of the amount of carotenoid bound to the cells, based on the number and size of the cell particles and the carotenoids, showed that, in all cases, about 1% of the cell surface was covered by the carotenoid. Nevertheless, all four carotenoids, when bound to the cell surface, showed a quenching of singlet oxygen. The quenching by cells with no bound carotenoid was much smaller. Figs. l(a)-(d) show the singlet oxygen decay as a function of time due to the different carotenoid quenchers. As can be seen, p-carotene, astaxanthin and lycopene quench singlet oxygen. This figure also shows that the cells without carotenoid show little or no quenching. Canthaxanthin was less soluble than the other carotenoids studied, but the amount bound to the cells was similar. We observed less singlet oxygen quenching by this carotenoid. Fig. 2 compares the quenching of singlet oxygen by cells coated in vivo with p-carotene with the quenching by cells only. From previous work (not shown), the cells without carotenoid give virtually the same result as the buffer alone. Clearly, the quenching ability in this in vivo experiment depends on the presence of the carotenoid on the cell. The significance of this
Photobiol. B: Biol. 26 (1994) 283-285 Fig la
Fig lb
1.4 ASTAXANTHIN
0.9 ‘5 ; 0.4
-0.1 0
0.5
0
1.5
1
1.5
1
0.5
2
Seconds x IO”
Seconds x 1N4
Fig Id
Fig Ic 1.4
CANTHAXANTHIN
LYCOPENE
0 .9
b x
$ 0.4
-0.l
+ 0
1
0.5 Seconds
a -no
1.5
0
2
x 1O-4
0.2
0.4
Seconds
0.6
0.8
1
x 1O-4
cells
b - -. . .
cells without
c-
cells with carotenoid
-
carotenoid
Fig. 1. Singlet oxygen quenching by carotenoids given as the luminescence intensity as a function of time: (a) no cells; (b) cells without carotenoid; (c) cells with carotenoid.
1.4
1 II
>
E 0.4
-1 -0.1
I
,
,
,
,
0
0.5
1
1.5
2
Seconds x 10e4 a b-
cells without carotenoid cells with carotenoid
Fig. 2. Singlet oxygen quenching by all-rranF-p-carotene bound to cells in vivo: (a) cells without carotenoid; (b) cells with carotenoid.
J.H. lkkler
et al. I J. Photochem.
result is that a concentration of p-carotene which may easily be obtained in humans is sufficient to quench singlet oxygen. When the plasma corresponding to these in vivo samples was studied, we found no quenching of singlet oxygen. Also, using samples of plasma incubated in vitro with the other carotenoids studies, no singlet oxygen quenching was observed. Thus the carotenoid must be bound to the cell to be an effective quencher. In our biological experiments based on the detection of cell membrane destruction using the trypan blue staining method, we have compared the number of cells destroyed in the absence and presence of carotenoid. The corresponding ratio is called the protection factor (PF). For TPPS as sensitizer, we obtained PF values of 2.05, 2.32, 2.73 and 1.15 and, for RB, we obtained PF values of 1.86, 2.72, 3.09 and 1.68 for p-carotene, astaxanthin, lycopene and canthaxanthin respectively. As can be seen, all carotenoids protected the cells against the photodynamic reaction sensitized by either RB or TPPS, and the highest protection was given by lycopene followed by astaxanthin, then p-carotene, with only a small PF for canthaxanthin. The two techniques show the two different sides of the quenching of the photodynamic reaction. For the measurement of the singlet oxygen lifetimes, we observe the quenching in the bulk medium. The cell staining technique shows the protection effect of the carotenoid against cell membrane destruction due to the generated
Photobiol. B: Biol. 26 (1994) 283-285
?.a5
singlet oxygen and/or radicals at the membrane surface. These two quite different techniques show the same overall effect of carotenoids against cell damage, although the cell staining results refer to all forms of reactive oxygen species as well as singlet oxygen. Our results show that all the carotenoids studied have a beneficial effect in protecting human life, but there may be subtle differences between the different dietary carotenoids. Acknowledgement
We thank Hoffman-La
Roche for financial support.
References
Ill
P.F. Corm, W. Schalch and T.G. Truscott, The singlet oxygen and carotenoid interactions, J. Photochem. Photobiol. B: Biol., I1 (1991) 41-47. 121 P. DiMascio, S. Kaiser and H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys., 274 (1989) 532-538. [31 P.F. Corm, C. Lambert, E.J. Land, W. Schalch and T.G. Truscott, Carotene+xygen radical interactions, Free Rad Res. Commun., 16 (6) (1992) 401-408. [41 F. Bohm, J. Haley, T.G. Truscott and W. Schalch, Cellular bound &carotene quenches singlet oxygen in man,1 Photochem. Photobiol. B: Biol., 21 (1993) 219-221. 151 F. Bdhm, H. Meffert and E. Bauer, PUVA therapy damages psoriatic and norma lymphoid cehs within milliseconds, Anh. Dermatol. Rex, 279 (1986) 16-19.
ELSEVIER
Dietary
carotenoids
Jane H. Tinkler”,
of Photochemistry and Photobiology B: Biology 26 (1994) 283-285
protect
human cells from damage
Fritz B6hmb, W. Schalch”,
T. George
Truscotta
“Department of Chemistry, Keele University, Staffordrhire ST5 5BG, UK bDepartment of Dermatology (Char&!), Humboldt University, 10117 Berlin, Germany ‘Department of Human Nutrition, F. Hoffmann-La Roche, CH-4002 Basle, Switzerland Received 28 April 1994; accepted 22 July 1994
Abstract A physical chemistry technique based on singlet oxygen luminescence at about 1270 nm and a biological cell membrane technique were used to study the quenching of singlet oxygen by four carotenoids bound to the surface of lymphoid cells. All the carotenoids studied showed a beneficial effect in cell protection, but there were subtle differences between them. Keywordx
Carotenoids;
Diet; Oxidative damage; Singlet oxygen
1. Introduction
Many common foods contain carotenoids; for example, there are large amounts of p-carotene in carrots and many other vegetables, lycopene in tomatoes and astaxanthin is used to colour farm salmon. In addition, carotenoids are often used as natural, non-toxic food colourants in foods, drinks, animal feedstuffs and even cosmetics. It is accepted that carotenoids are efficient quenchers of a highly damaging form of oxygen, known as singlet oxygen, and there has been debate over the relative efficiency of singlet oxygen quenching by different carotenoids [1,2]. Also, other reactive oxygen species, such as the superoxide radical (Oz.-), react with different carotenoids in markedly different ways
[31In a preliminary communication [4], we reported that singlet oxygen in vitro is quenched by all-trans-p-carotene when the carotenoid is bound to the surface of lymphoid cells obtained from human blood. We now report a more detailed study of this quenching by pcarotene and an extension to other carotenoids, namely astaxanthin, lycopene and canthaxanthin. The data reported in this work are based on the monitoring of the time-resolved near IR luminescence of singlet oxygen and are compared with biological studies based on cell destruction. 2. Experimental
details
Lymphoid cells were taken from healthy human blood and prepared as described previously [5]. Briefly, we
lOll-1344/94/$07.00 0 1994 Elsevier Science S.A. Ail rights reserved SSDI 1011-1344(94)07049-O
diluted the anticoagulated blood using heparin 1:l with Dulbecco buffer, passed it over a FicollR density gradient of 1.076 g ml-’ (Ficoll-PaqueR, Pharmacia, Uppsala, Sweden) and collected the huffy coat. After centrifugation (200 g, 30 min, 22 “C), the cells were washed three times with D,O-Dulbecco buffer, centrifuged again (2oOg, 10 min, 22 “C) and divided into two. One was treated with the carotenoid and the other was used as a control. For the cells treated with carotenoid, incubation was performed for 60 min at 22 “C with saturated solutions of water-soluble p-carotene (22 PM), astaxanthin (27 PM), lycopene (50 PM) or canthaxanthin (3 PM). These water-soluble carotenoids were obtained as beadlets from Hoffmann-La Roche, Basle, Switzerland, and experiments with blank beadlets showed no effect. Subsequently, the cells were washed three times with D,O-Dulbecco buffer to remove any unbound carotenoid. The carotenoid bound to the cells was estimated by extraction with dimethylsulphoxide and conventional UV-visible spectroscopy. Typically, we used 1O-8-1O-9 mol of carotenoid in 200 ~1 of cell suspension which contained 2 X lo6 cells. Cell staining was performed by the trypan blue exclusion test to estimate the lethally destroyed cell fraction. Control experiments, without carotenoid, were performed on cells, prepared and washed as above, without incubating with the carotenoid. Rose bengal (RB) or meso-tetra(4-sulphonatophenyl)porphine (TPPS) was added to both the control and carotenoidcontaining cells as a sensitizer of singlet oxygen.
284
J.H. Tidier
et al. I J. Photochem.
Some experiments were undertaken with in vivo coated lymphoid cells extracted from the blood of healthy individuals who had been treated orally, three times daily, with 25 mg p-carotene as CarotabeneR (Hetmal, Germany) over 4 weeks. The cells were washed three times with D,O-Dulbecco buffer prior to use. The carotene concentration was not readily obtained, but was estimated to be lo-* mol or less in 200 ~1 of cell suspension. We also obtained blood plasma from healthy humans who had or had not been treated with CarotabeneR by simple centrifugation of the blood (4OOg, 10 min, 22 “C). The plasma, without CarotabeneR, was incubated with a saturated carotenoid solution for 60 min at 22 “C and then used in the experiment. Two methods were used to study the effect of the carotenoid bound to the cells. For both, we mixed 80 ~1 of the appropriate cell suspension (or D,O-Dulbecco buffer) with 20 ~1 of sensitizer and the sample was then irradiated with 10 pulses of a 200 mJ, 532 nm laser line. One method was based on the singlet oxygen lifetime (T) using the average of 10 pulses, while the second was based on a cell counting technique and on the additive effect of the 10 pulses on a given sample. Singlet oxygen was detected via its luminescence at about 1270 nm using a time-resolved technique. Briefly, the apparatus consisted of a germanium diode linked to a Judson amplifier with excitation from a frequency doubled Nd:YAG laser (15 ns pulse at 532 nm).
3. Results and discussion Simple calculations of the amount of carotenoid bound to the cells, based on the number and size of the cell particles and the carotenoids, showed that, in all cases, about 1% of the cell surface was covered by the carotenoid. Nevertheless, all four carotenoids, when bound to the cell surface, showed a quenching of singlet oxygen. The quenching by cells with no bound carotenoid was much smaller. Figs. l(a)-(d) show the singlet oxygen decay as a function of time due to the different carotenoid quenchers. As can be seen, p-carotene, astaxanthin and lycopene quench singlet oxygen. This figure also shows that the cells without carotenoid show little or no quenching. Canthaxanthin was less soluble than the other carotenoids studied, but the amount bound to the cells was similar. We observed less singlet oxygen quenching by this carotenoid. Fig. 2 compares the quenching of singlet oxygen by cells coated in vivo with p-carotene with the quenching by cells only. From previous work (not shown), the cells without carotenoid give virtually the same result as the buffer alone. Clearly, the quenching ability in this in vivo experiment depends on the presence of the carotenoid on the cell. The significance of this
Photobiol. B: Biol. 26 (1994) 283-285 Fig la
Fig lb
1.4 ASTAXANTHIN
0.9 ‘5 ; 0.4
-0.1 0
0.5
0
1.5
1
1.5
1
0.5
2
Seconds x IO”
Seconds x 1N4
Fig Id
Fig Ic 1.4
CANTHAXANTHIN
LYCOPENE
0 .9
b x
$ 0.4
-0.l
+ 0
1
0.5 Seconds
a -no
1.5
0
2
x 1O-4
0.2
0.4
Seconds
0.6
0.8
1
x 1O-4
cells
b - -. . .
cells without
c-
cells with carotenoid
-
carotenoid
Fig. 1. Singlet oxygen quenching by carotenoids given as the luminescence intensity as a function of time: (a) no cells; (b) cells without carotenoid; (c) cells with carotenoid.
1.4
1 II
>
E 0.4
-1 -0.1
I
,
,
,
,
0
0.5
1
1.5
2
Seconds x 10e4 a b-
cells without carotenoid cells with carotenoid
Fig. 2. Singlet oxygen quenching by all-rranF-p-carotene bound to cells in vivo: (a) cells without carotenoid; (b) cells with carotenoid.
J.H. lkkler
et al. I J. Photochem.
result is that a concentration of p-carotene which may easily be obtained in humans is sufficient to quench singlet oxygen. When the plasma corresponding to these in vivo samples was studied, we found no quenching of singlet oxygen. Also, using samples of plasma incubated in vitro with the other carotenoids studies, no singlet oxygen quenching was observed. Thus the carotenoid must be bound to the cell to be an effective quencher. In our biological experiments based on the detection of cell membrane destruction using the trypan blue staining method, we have compared the number of cells destroyed in the absence and presence of carotenoid. The corresponding ratio is called the protection factor (PF). For TPPS as sensitizer, we obtained PF values of 2.05, 2.32, 2.73 and 1.15 and, for RB, we obtained PF values of 1.86, 2.72, 3.09 and 1.68 for p-carotene, astaxanthin, lycopene and canthaxanthin respectively. As can be seen, all carotenoids protected the cells against the photodynamic reaction sensitized by either RB or TPPS, and the highest protection was given by lycopene followed by astaxanthin, then p-carotene, with only a small PF for canthaxanthin. The two techniques show the two different sides of the quenching of the photodynamic reaction. For the measurement of the singlet oxygen lifetimes, we observe the quenching in the bulk medium. The cell staining technique shows the protection effect of the carotenoid against cell membrane destruction due to the generated
Photobiol. B: Biol. 26 (1994) 283-285
?.a5
singlet oxygen and/or radicals at the membrane surface. These two quite different techniques show the same overall effect of carotenoids against cell damage, although the cell staining results refer to all forms of reactive oxygen species as well as singlet oxygen. Our results show that all the carotenoids studied have a beneficial effect in protecting human life, but there may be subtle differences between the different dietary carotenoids. Acknowledgement
We thank Hoffman-La
Roche for financial support.
References
Ill
P.F. Corm, W. Schalch and T.G. Truscott, The singlet oxygen and carotenoid interactions, J. Photochem. Photobiol. B: Biol., I1 (1991) 41-47. 121 P. DiMascio, S. Kaiser and H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys., 274 (1989) 532-538. [31 P.F. Corm, C. Lambert, E.J. Land, W. Schalch and T.G. Truscott, Carotene+xygen radical interactions, Free Rad Res. Commun., 16 (6) (1992) 401-408. [41 F. Bohm, J. Haley, T.G. Truscott and W. Schalch, Cellular bound &carotene quenches singlet oxygen in man,1 Photochem. Photobiol. B: Biol., 21 (1993) 219-221. 151 F. Bdhm, H. Meffert and E. Bauer, PUVA therapy damages psoriatic and norma lymphoid cehs within milliseconds, Anh. Dermatol. Rex, 279 (1986) 16-19.