Journal of Photochemistry and Photobiology B: Biology 64 (2001) 176–178www.elsevier.com/ locate / jphotobiol

Dietary uptake of lycopene protects human cells from singlet oxygen andnitrogen dioxide – ROS components from cigarette smoke

a,b , c c c*¨Fritz Bohm , Ruth Edge , Marc Burke , T.G. TruscottaMeoclinic Berlin, Friedrichstrasse 71, 10117 Berlin, Germany

b ´Department of Dermatology (Charite), Humboldt University, 10117 Berlin, GermanycSchool of Chemistry and Physics, Keele University, Keele, Staffordshire ST5 5BG, UK

Abstract

There is current interest in the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations suchas smokers. Here we report in vivo protection of human lymphocytes, conferred by dietary supplementation of lycopene rich foods against

. 1the reactive oxygen species, NO radical (by electron transfer) and O (by energy transfer). It was found that a lycopene rich diet,2 2

maintained for 14 days, increased the serum lycopene level 10 fold compared to serum obtained after the same period, where a typical. 1western European diet had been consumed. Relative lymphocyte protection factors of 17.6 and 6.3 against NO radical and O ,2 2

respectively, were obtained, which re-enforce epidemiological data, showing protection against several chronic diseases by tomatolycopene. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Lycopene; Bio-availability; Reactive oxygen species; Protection factor

1. Introduction carotenoids protect human lymphocytes from damage by1singlet oxygen ( O ) and that lycopene was more effective2

Intervention studies in humans on the effects of than b-carotene in cell protection against the nitrogen?lycopene supplementation have not been undertaken and dioxide radical (NO ) [7]. In these experiments lympho-2

present knowledge relies on studies of dietary lycopene cytes were incubated with water-solubilised carotenoids? 1intakes or plasma levels [1]. A recent study compared fat prior to exposure to NO or O – components that can2 2

samples from 1379 men who had suffered heart attacks arise from cigarette smoke. We have recently shown [8]with those of healthy men [2]. This trial indicated that that incubation of b-carotene with cells provides similar

?those with high lycopene levels were half as likely to protection to ingested b-carotene against NO . We now2?suffer an attack compared to those with low lycopene report new results on the protection against NO as well as2

1levels, other carotenoids studied showed no effect. Several O , for lymphocytes in which lycopene was increased by2

trials have shown evidence of an inverse association dietary means.between high intake of tomato products and prostatecancer risk. The association is most marked for processedor cooked tomato products due to the low bio-availability 2. Materials and methodsof lycopene from uncooked tomatoes [3–5]. About 85% ofdietary lycopene is derived from tomatoes or tomato Human lymphoid cells were taken from the peripheralproducts and care must be taken that beneficial effects blood of volunteers who had consumed 500 ml /day tomatoclaimed for dietary lycopene are not simply a reflection juice for 2 weeks. In vivo lycopene uptake (from 0.15 tothat those in the population with a high intake of tomatoes 1.5 mM in the serum) was achieved by consumption ofhave a generally healthier lifestyle. Thus, it is important to tomato juice which had been boiled for ¯3 min with 5 mlunderpin the epidemiological data with scientific data in a olive oil to aid the lycopene bio-availability [5,9]. Essen-range of biological environments. tially the same lycopene uptake and protection results were

Previously, we have reported [6] that several dietary obtained when the tomato juice was replaced by tomatosoup (400 g per day).

As we described previously [8], nitrogen dioxide was*Corresponding author. Tel.: 149-30-2094-4053.¨E-mail address: info@meoclinic.de (F. Bohm). formed via electron transfer from sodium nitrate to 1-

1011-1344/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S1011-1344( 01 )00221-4

¨F. Bohm et al. / Journal of Photochemistry and Photobiology B: Biology 64 (2001) 176 –178 177

Table 1 increase (10%) in the rate of decay of the luminescenceCell membrane protection by lycopene was observed between the control and the lycopene-loadedReactive % Stained In vivo In vitro cells. However, the bulk of the luminescence signal arisesspecies cells protection protection from the rose bengal sensitiser in the D O rather than at2

factor factor the cell surface so the observed quenching by lycopene?NO 3.560.5; p50.002 17.6 8.2 [7]2 appears to be rather small.

(61.569.3 without; The difference in cell membrane destruction betweenp50.005) ? 1

1 NO and O in the absence of lycopene may simply2 2O 8.761.7; p50.007 6.3 3.1 [6]2 1reflect the relatively short lifetime of O in D O (about 60(55.164.4 without; 2 2

p50.014) ms). For the cells containing lycopene quite distinctmolecular protection mechanisms will arise. Singlet oxy-Cell staining [8] with eosin (2 ml of 1% eosin to 50 ml cell suspension,gen will be quenched via energy transfer producing the5 min incubation) was used to show membrane destruction leading to cell

death. All results (mean of at least 24 measurements for each experi- lycopene triplet state, which will reconvert to the groundment), were corrected for the small percentage of stained cells (,6) due state. However, it is well established [10] that under suchto the preparation technique, the laser alone and after incubation with the conditions lycopene is susceptible to loss by irreversiblereactants prior to irradiation, and show the significant difference between

oxidation, thus losing its efficacy as a cell protector.protected and unprotected cells (u-test after Mann-Whitney).Nitrogen dioxide will be quenched via electron transfergenerating the lycopene radical cation, as shown previous-

nitronaphthalene triplet (generated via 355 nm pulsed laser ly [11] and, in the ‘in vivo’ situation, reducing agents suchexcitation): as ascorbic acid can ‘repair’ the lycopene radical cation to

its original protective form without loss [12,13].3 ?2 ? 1NN 1 NaNO → NN 1 NO 1 Na2 2 A preliminary aspect of our new data is that the ‘in

vivo’ experiment was repeated on all volunteers four days1and O via rose bengal triplet sensitisation with 532 nm2 after the high lycopene intake ceased and the same resultpulsed laser excitation. Singlet oxygen was monitored by

in terms of serum concentration, cell membrane destruc-its time-resolved luminescence at 1270 nm [6].

tion and protection factors was obtained, suggesting thatCell staining was used to estimate the immediate cell

the lycopene protection is maintained for some consider-membrane destruction (see Table 1 legend for further

able period after the dietary regime ceases.details) by the reactive species generated by the laser

Understanding downstream molecular events followingtechniques. Dye exclusion tests, for example with eosin, ?reactions of an oxidising radical, such as NO , with a2are well established methods, as recommended by the NHI.

carotenoid such as lycopene, are critical in avoiding anyIt should be noted that such tests are used to detect

deleterious dietary effects [14]. The previous work [7] hasdamage, leading to cell death, or no damage on each

shown such reactions are based on electron transferindividual cell and not the damage within such a cell.

processes (initiated, for example, by ROS in cigaretteClearly, this approach leads to a linear relationship be-

smoke and other environmental hazards, such as UVtween staining and cell damage.

exposure) leading to carotenoid radical cations.

? ?1]NO 1 LYC → NO 1 LYC2 2

3. Results and discussionThe next downstream event will involve the fate of the

?Table 1 compares the membrane damage by NO and lycopene radical cation. This is an oxidising species but the21O for cells from the enhanced lycopene diet with those strength of its oxidising potential is unknown. We have2

of the control. There is a marked protection from both measured the efficiency of formation of lycopene radicaltoxic species, which is much greater than previously cation from the radical cation and neutral radical of thereported for the ‘in vitro’ study where the lycopene was amino acid tryptophan, as a function of pH. Since theincubated with the cell suspension [6,7]. The cellular oxidising potential of the tryptophan radical cation doesuptake of lycopene from the tomato juice by the intestinal not depend on pH (as there is no proton involved), but thatmucosa is clearly more effective than the ‘in vitro’ uptake of the neutral radical does (decreases by 59 mV per pHfrom water solubilised lycopene, which is likely to aggre- unit), we have been able to establish, for the first time, thegate. By contrast, on comparing ‘in vivo’ and ‘in vitro’ absolute reduction potential of the lycopene radical cationcellular protection by b-carotene [8], protection is ob- as 1.0 V610% [15]. We have confirmed this extremely

?served for NO damage in both cases but there is no strong oxidising behaviour by observing the oxidation of2

improvement in the ‘in vivo’ experiments compared with the amino acids tyrosine and cysteine at pH 7 by thethe ‘in vitro’. lycopene radical cation, thus showing a major potentially

1During cell exposure to O the time-resolved decay of deleterious downstream molecular mechanism is mem-21the O luminescence was also monitored. Only a small brane protein damage and, of course, subsequent lipid2

¨178 F. Bohm et al. / Journal of Photochemistry and Photobiology B: Biology 64 (2001) 176 –178

concentrations but rapidly lose this capacity at higher doses, Freeperoxidation, leading to a number of diseases. Of course,Rad. Res. 30 (1998) 141–151.the protective effects we report here and the epidemiologi-

[2] L. Kohlmeier, J.D. Kark, E. Gomez-Gracia, C.B. Martin, S.E. Stack,cal results on prostate cancer and heart disease [2,4] A.F.M. Kardinaal, J. Ringstad, M. Thamm, V. Masaev, R. Riemer-require other downstream molecular events to avoid this sma, J.M. Martin-Moreno, J.K. Huttunen, F.J. Kok, Lycopene andpotential damage. We have previously shown in both myocardial infarction risk in the UERAMIC study, Am. J. Epi-

demiol. 146 (1997) 618–626.methanol and micellar environments, that vitamin C quen-[3] H. Gerster, The potential role of lycopene for human health, J. Am.ches carotenoid radical cations [12] and we propose that at

Coll. Nut. 16 (1997) 109–126.normal vitamin C concentrations such processes avoid [4] E. Giovannucci, S.K. Clinton, Tomatoes, lycopene and prostratesuch amino acid oxidation. However, in the case of low cancer, Proc. Soc. Exp. Biol. Med. 218 (1998) 129–139.vitamin C (e.g. smokers) damaging effects may be due to [5] W. Stahl, H. Sies, Uptake of lycopene and its geometrical isomers is

greater from heat-processed than from unprocessed tomato juice insuch carotenoid radical cations.humans, J. Nut. 122 (1992) 2161–2166.It may be claimed that hydrocarbon carotenoids, such as

¨[6] J.H. Tinkler, F. Bohm, W. Schalch, T.G. Truscott, Dietary carot-lycopene and b-carotene, lie in hydrophobic membrane enoids protect human cells from damage, J. Photochem. Photobiol.environments and therefore will not be repaired by the B: Biol. 26 (1994) 283–285.

¨water-soluble vitamin C. However, the radical cation will [7] F. Bohm, J.H. Tinkler, T.G. Truscott, Carotenoids protect againstcell membrane damage by the nitrogen dioxide radical, Nature Med.be more polar than the parent hydrocarbon carotenoid,1 (1995) 98–99.making reorientation within the membrane possible, thus

¨[8] F. Bohm, R. Edge, D.J. McGarvey, T.G. Truscott, b-Carotene withallowing regeneration of the parent carotenoid by vitamin Vitamins E and C offer synergistic cell protection against NO ,XC; indeed for b-carotene in DPPC liposomes, we have FEBS Lett. 436 (1998) 387–389.shown efficient repair by vitamin C [16]. [9] W. Stahl, H. Sies, Perspectives in biochemistry and biophysics,

Arch. Biochem. Biophys. 336 (1996) 1–9.In conclusion, our results on the marked protection of[10] R. Edge, D.J. McGarvey, T.G. Truscott, The carotenoids as anti-lymphoid cells from two specific reactive oxygen species

oxidants – a review, J. Photochem. Photobiol. B: Biol. 41 (1997). 1(NO and O ) by dietary lycopene underpins the epi-2 2 189–200.demiological data, and possibly the recently characterised [11] J.H. Tinkler, S.M. Tavender, A.W. Parker, D.J. McGarvey, L.therapeutic benefits of tomato lycopene against prostate Mulroy, T.G. Truscott, An investigation of carotenoid radical

cations and triplet states by laser flash photolysis and time-resolvedcancer [17]. The reduction potential we have measured forresonance Raman spectroscopy: observation of competitive energythe lycopene radical cation demonstrates the importance ofand electron transfer, J. Am. Chem. Soc. 118 (1996) 1756–1761.

interactions with vitamin C in the downstream events, and ¨[12] F. Bohm, R. Edge, E.J. Land, D.J. McGarvey, T.G. Truscott,the membrane protein damage that will result from low Carotenoids enhance vitamin E anti-oxidant efficiency, J. Am.vitamin C levels as will arise in smokers. Furthermore, in Chem. Soc. 119 (1997) 621–622.

[13] A. Mortensen, L.H. Skibsted, T.G. Truscott, The interaction ofusing lycopene for the treatment of different diseases it isdietary carotenoids with radical species, Arch. Biochem. Biophys.necessary to take account of the different molecular385 (2001) 13–19.

mechanisms that may arise (energy or electron transfer) [14] G.S. Omenn, G.E. Goodman, M.D. Thornquist, J. Balmes, M.R.which, in turn, may lead to different therapeutic outcomes. Cullen, A. Glass, J.P. Keogh, F.L. Meyskens, B. Valanis, J.H.

Williams, S. Barnhart, S. Hammar, Effects of a combination ofb-carotene and vitamin A on lung cancer and cardiovascular disease,New Engl. J. Med. 334 (1996) 1150–1155.Acknowledgements

[15] M. Burke, R. Edge, E.J. Land, D.J. McGarvey, T.G. Truscott,One-electron reduction potentials of dietary carotenoids in aqueous

R. Edge and T.G. Truscott thank Hoffmann–La Roche micellar environments, FEBS Lett. 15 (2001) 132–136.for financial support and F. Boehm thanks Dr. Linus [16] M. Burke, R. Edge, E.J. Land, T.G. Truscott, Characterisation of

carotenoid radical cations in liposomal environments: interactionLanger for useful discussions. TGT also thanks the WCRFwith vitamin C, J. Photochem. Photobiol. B: Biol. 60 (2001) 1–6.and the National Lotteries Charity Board for support.

[17] O. Kucuk, F.H. Sakr, F.H. Sarkar, Z. Djuric, Y.-W. Li, F. Velazquez,M. Banaejee, J.S. Bertram, J.D. Crissman, D.P. Wood, J. Karmanos,90th Ann. Meeting Am. Assoc. Can. Res., 1999. (Abstract).

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