Antioxidant inhibition of porphyrin-induced cellular phototoxicity

Journal of Photochemistry and Photobiology B: Biology 65 (2001) 177–183www.elsevier.com/ locate / jphotobiol

Antioxidant inhibition of porphyrin-induced cellular phototoxicitya , b b ,1 a b*¨Fritz Bohm , Ruth Edge , Sarah Foley , Linus Lange , T. George Truscott

a ´Department of Dermatology (Charite), Humboldt University, Schumannstrasse 20/21, 10117 Berlin, GermanybSchool of Chemistry and Physics, Keele University, Keele, Staffordshire ST5 5BG, UK

Received 9 September 2001; accepted 27 October 2001

Abstract

Porphyrins such as protoporphyrin IX (PP IX) and uroporphyrin I (UP I) can be phototoxic to human cells. To study the protectiveability of antioxidants (b-carotene, lycopene, ascorbic acid and a-tocopherol), against such porphyrin phototoxicity, membranedestruction experiments (Jurkat cells) and human cell cultures (fibroblasts) were performed. Both b-carotene and lycopene and also thecombination of b-carotene, ascorbic acid and a-tocopherol offered cell protection against PP IX phototoxicity. Investigations of both cellmembrane protection and of cell growth showed differences in terms of the protection afforded by the anti-oxidants. Thus, for PP IX,carotenoids alone, and in combination with ascorbic acid and a-tocopherol, showed higher protection factors in general than UP I.However, for membrane protection there was significant protection against UP I by the combination of b-carotene, ascorbic acid anda-tocopherol but not by any of these anti-oxidants alone. The membrane protection against PP IX by b-carotene, and especially lycopene,is significant presumably because of the high lipophilicity of all these molecules. However, the hydrophilic UP I will cause phototoxicitymainly via H O , radical or singlet oxygen production in the aqueous phase, and these reactive species may be generated some distance2 2

from the cell membrane. This may lead to the little or no protection observed for UP I by the individual antioxidants. Nevertheless, acombination of b-carotene, ascorbic acid and a-tocopherol offers membrane protection against the phototoxicity of both porphyrins. Thisis believed to occur as a result of synergistic processes. Our results suggest that the treatment of porphyria cutanea tarda anderythropoietic protoporphyria may be improved by the use of a combination of the antioxidants studied. 2001 Elsevier Science B.V.All rights reserved.

Keywords: Protoporphyrin IX; Uroporphyrin I; Phototoxicity; Carotenoids; Antioxidants; Porphyria therapy

1. Introduction leads to tissue damage as depicted by those patientssuffering from porphyria. Erythropoietic protoporphyria

Porphyrins are natural metabolites or derivatives of (EPP) is characterised by a build-up of protoporphyrin IXnatural metabolites, which are intermediates in the bio- (PP IX) in the red blood cells, plasma and stools ofsynthesis of haem in mammalian organisms. Haem, which patients, resulting in acute skin photosensitivity [1]. Por-is the end product of the pathway, is a tetrapyrrole in phyria cutanea tarda (PCT) is another form of porphyriawhich protoporphyrin IX is chelated with ferrous iron. which also results in photosensitivity as well as actinicHeam functions in numerous metabolic pathways of living elastosis, and it is characterised by unusually high levels oforganisms due to its ability to bind and release oxygen. uroporphyrin I (UP I) in both plasma and urine [1].The porphyric group of diseases result from either inher- Patients with EPP suffer from skin burning, followed byited or acquired enzymatic abnormalities in haem syn- erythema and edema, when exposed to sunlight, whereasthesis. The interaction between excited porphyrins and the skin of PCT sufferers is fragile and is left scarred uponoxygen produces reactive oxygen species, which ultimately sun exposure [2]. The photosensitivity is caused primarily

1by the production of singlet oxygen ( O ) from the triplet2

excited states of the porphyrins, which are themselves*Corresponding author. Tel.: 149-30-2094-4053; fax: 149-30-2094- formed upon exposure to light. However, other reactive

4132. oxygen species may also be produced, such as the superox-¨E-mail address: [email protected] (F. Bohm).

1 ide radical anion, hydrogen peroxide, hydroxyl radicals´ ´ ´Present address: Laboratoire des Materiaux et Procedes Mem-and peroxyl radicals, which can also cause cell membranebranaires, UMR 5635 CNRS, 1919 Route de Mende, F-34293 Montpel-

lier Cedex 05, France. damage [1,3,4]. b-Carotene has proved effective in reliev-

1011-1344/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S1011-1344( 01 )00259-7

¨178 F. Bohm et al. / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 177 –183

ing the skin photosensitivity symptoms of EPP [5–7], mM PP IX (0.1% DMSO), or 0.1 mM UP I) and the cellswhereas it does not appear to protect to any significant were incubated for a further hour.degree against the photosensitivity in PCT [8]. However, itis possible that other carotenoids may be more effective 2.3. Irradiation procedures and cell countingand that combinations of antioxidants may enhance theprotective effects. In this study the ability of b-carotene The irradiation procedure for the human fibroblastand lycopene to protect cells from damage due to por- proliferation studies used Narva (Berlin, Germany) bluephyrin phototoxicity has been evaluated, together with the light (LS 18) for the cells incubated with PP IX (Fig. 1)protective effect of the antioxidant ascorbic acid and a- and a Philips UVA TLD lamp for the UP I experimentstocopherol in combination with b-carotene. (Fig. 2). The lamps were both kept at a constant distance

from the samples (23 mm from the bottom of the cultureplates) and were switched on for at least 30 min prior toirradiation. The outputs of the lamps were measured using

2. Experimental detailsa MSS 2040 Spectral-Analyser (MSS Electronik GmbH,Bielefeld, Germany) and these are given in Figs. 1 and 2,

2.1. Cells and cell culturetogether with the ground state absorption spectra of PP IXand UP I. The subconfluent and cell monolayer were

Jurkat cells (human T lymphocyte cell line) and Fi 301washed after incubation with the sensitiser and placed in 12cells (human embryonic lung fibroblasts; passages 11–15 )ml DPBS in open culture plates, which were covered with

were grown in culture flasks in Minimum Essentialquartz plates during the irradiation. After irradiation, the

Medium (MEM) with Earl’s salts or RPMI 1640 mediumcells were cultured again and were counted on the 1st, 3rd

(Gibco), supplemented with 7.5–10% foetal calf serumand 5th days of culture (8 counts /well) within an area of

(FCS), 2 mM glutamin, 100 mg/ml streptomycin, 100 20.5 mm , using a phase contrast microscope (OlympusI.E. /ml penicillin and 25 I.E. /ml nystatin, and were

T041).incubated at 378C in 100% humidity and 5% CO .2 To determine protection from immediate lethal mem-

brane damage, a 355 nm Nd:YAG laser line was used as2.2. Antioxidants and incubation the irradiation source (5 min at a repetition rate of 45

7pulses per min) and 1 ml of cells in DPBS (10 cells /ml)The antioxidants in medium without FCS were added to was irradiated in a quartz fluorescence cuvette. In experi-

the cells after washing twice with Dulbecco’s phosphate ments with UP I we could only achieve a sufficient cell killbuffered saline (DPBS, Sigma) and they were incubated when UP I was present in the solution at 0.1 mM.for 1 h, at room temperature for the Jurkat cells and at However, with PP IX the cells were washed after incuba-378C and 5% CO for the fibroblasts. Combinations of tion and before irradiation. The percentage cell survival2

antioxidants were added and incubated together. All-trans- was determined with a cell staining technique using 1%b-carotene and lycopene (water-soluble beadlets, Hoff- eosin; 1 ml of the dye was mixed with 50 ml of the cellmann–La Roche) were dissolved in DPBS and used at a suspension (yielding 0.02% eosin), incubated for 5 minconcentration of 5 mM, for the fibroblasts, and 20 mM for and then counted.the Jurkat cells (evaluated spectrophotometrically). DL-a- Statistics were performed with the U-test after Mann–Tocopherol (Lancaster, purified to .99% by HPLC before Whitney.use) was dissolved in dimethyl sulphoxide (DMSO, Sigma)and diluted in DPBS to yield a concentration of 35 mM(0.1% DMSO) and L(1) ascorbic acid (Sigma, recrystal- 3. Results and discussionlised from ethanol) was dissolved in DPBS and added tothe medium as 100 mM. The amounts of a-tocopherol, Table 1 shows the results obtained for the specificascorbic acid and carotenoids used were higher than membrane protection of the cells, against porphyrin photo-normal plasma levels (but are easily obtainable with a toxicity, by the various antioxidants. All the results are thesupplemented diet), since high levels of b-carotene are mean of at least four separate measurements with fourneeded to observe protection in patients with EPP. counts in each (n516) and are corrected for any cell kill

After the addition and incubation of the antioxidants, the during the preparation, incubation and laser irradiation.sensitisers were added to the fibroblasts (20 nM PP IX Controls showed no significant cell kill with the laser alone(0.03% DMSO), or 5 mM UP I) or to the Jurkat cells (5 (i.e. without porphyrins). Incubation with the antioxidants

in the dark and laser excitation of cells incubated withantioxidants stained fewer than 5% of the cells. Empty2These diploid fibroblasts were isolated in 1990 at the Robert-Koch-beadlets of the same type as those used to solubilise theInstitut, Berlin. Differentiation should be possible up until the fifthcarotenoids showed no effect.passage. After this time no differentiation of these cells has been

observed. The lifespan in vitro is limited to the 20th passage. With regard to the antioxidant protection of the cell

¨F. Bohm et al. / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 177 –183 179

Fig. 1. Spectral irradiation intensity of the Narva LS 18 blue light tube (columns) and the ground state absorption spectra of Protoporphyrin IX (line).

21 21 7 3 21 21membranes, it can be seen from Table 1 that a-tocopherol mol s [9] and 0.84–1.06310 dm mol s [10] in9and ascorbic acid do not exert a very large protective effect pyridine, respectively, compared with values of 13.5310

3 21 21 9 3 21 21for PP IX phototoxicity, but the carotenoids do. The reason dm mol s and 16.8310 dm mol s for b-for this may well be that a-tocopherol and ascorbic acid carotene and lycopene in benzene [11]. Since ascorbic acid

1are both less lipophilic than either b-carotene or lycopene, is more hydrophilic than a-tocopherol and is a poorer O2

with ascorbic acid being the most hydrophilic. Also, both quencher, its lower protective effect against PP IX photo-1

a-tocopherol and ascorbic acid quench O less efficiently toxicity is to be expected. As for UP I phototoxicity, no28 3than the carotenoids, with k values of 2.5310 dm protection was obtained for a-tocopherol, ascorbic acid,q

Fig. 2. Spectral irradiation intensity of the Philips TLD UVA-1 light tube (columns) and the ground state absorption spectra of Uroporphyrin I (line).


Table 1 However, b-carotene and lycopene alone do not offer anyCell membrane protection (Jurkat cells) by antioxidants against porphyrin protection against UP I phototoxicity with regard tophototoxicity

membrane destruction. One reason why the carotenoidsSensitiser Antioxidant(s) % Stained Protection may not protect cells from immediate membrane UP I

cells factor (PF) phototoxicity is that the phototoxicity may well be me-PP IX (5 mM) Without antioxidants 75.863.6 diated by hydrogen peroxide, whereas the phototoxicity of

1PP IX (5 mM) b-Carotene (20 mM) 22.760.8 3.3 PP IX is mediated by O . Menon et al. showed that the2PP IX (5 mM) a-Tocopherol (35 mM) 51.168.0 1.5H O production during irradiation of PP IX is only 38%2 2PP IX (5 mM) Ascorbic acid (100 mM) 67.6610.3 1.1

a of that produced from UP I irradiation [3]. Another reasonPP IX (5 mM) Triple combination 14.061.3 5.4PP IX (5 mM) Lycopene (20 mM) 16.360.7 4.7 could be that the high lipophilicity of b-carotene and

lycopene prevent them from reacting as efficiently with theUP I (0.1 mM) Without antioxidants 26.061.8 reactive oxygen species produced by UP I in the aqueousUP I (0.1 mM) b-Carotene (20 mM) 26.862.0 1.0

phase. The fact that lycopene is a more effective protectorUP I (0.1 mM) a-Tocopherol (35 mM) 25.961.5 1.0than b-carotene against PP IX phototoxicity is possiblyUP I (0.1 mM) Ascorbic acid (100 mM) 26.162.7 1.0

1aUP I (0.1 mM) Triple combination 5.060.6 5.2 surprising since it quenches O with more or less the2UP I (0.1 mM) Lycopene (20 mM) 25.862.2 1.0 same efficiency in a variety of solvents (the difference of

a 10% being unlikely to be biologically relevant). On theTriple combination5b-carotene (20 mM)1a-tocopherol (35 mM)1ascorbic acid (100 mM). Error5S.D. other hand, lycopene has been shown to be a better

1protector of lymphocytes against O damage sensitised by2

b-carotene or lycopene. However, Sinclair et al. [12] rose bengal or TPPS [15]. However, lycopene is a lessshowed that 85% of the untreated patients with PCT in efficient quencher of PP IX dimethyl ester triplet state than

9 9 3 21 21their study had low plasma ascorbate levels (,23 mM) b-carotene (1.2310 and 1.8310 dm mol s , respec-while 62% were deficient (,11 mM), and the remaining tively [16]), possibly indicating that protection proceeds

1 315% of the patients were taking multivitamin supplements. predominantly by reaction with O rather than with PP2

It has also been demonstrated that ascorbic acid inhibits IX. Although, again in the biological situation there isthe chemically induced accumulation of UP I but does not probably no significance to the difference between 1.2 and

9 3 21 21affect induced PP IX accumulation [13,14]. Both of the 1.8310 dm mol s .carotenoids studied individually protect efficiently against Figs. 3 and 4 present the data for the protection ofcell membrane destruction by PP IX with lycopene yield- proliferating human fibroblasts, counted on the 5th day ofing the higher protection factor (P,0.05, see Table 1). culture after irradiation, and Table 2 gives the corre-

Fig. 3. The proliferation of human fibroblasts with and without protection by various antioxidants on the 5th day of culture after irradiation with blue light(Narva LS 18) and incubation with PP IX (20 nM, 1 h); n524, error bars5S.D. *Triple combination5b-carotene (5 mM)1a-tocopherol (35mM)1ascorbic acid (100 mM).


Fig. 4. The proliferation of human fibroblasts with and without protection by various antioxidants on the 5th day of culture after irradiation with UVA-1light (Philips TLD) and incubation with UP I (5 mM, 1 h); n524, error bars5S.D. *Triple combination5b-carotene (5 mM)1a-tocopherol (35mM)1ascorbic acid (100 mM).

sponding protection factors obtained for each of the reactions are well established for porphyrins [17]. Theantioxidants. The results are the mean of three separate singlet oxygen quantum yield for UP I is also slightlymeasurements with eight counts in each (n524). These lower than that of PP IX (f 50.71 and 0.77, respectivelyD

proliferation experiments show the total effects occurring [18,19]). However, the differing phototoxicities of the twodue to the phototoxicity, e.g. membrane and DNA damage porphyrins may well be caused by their differing lipo-and enzyme inactivation, as well as repair effects. In philicities, since UP I is hydrophilic and does not aggre-comparison, the experiments on the Jurkat cells show the gate in aqueous solutions [20], whereas PP IX is lipophilic.immediate destruction of the cell membranes by the Hence, studies on the binding of porphyrins by plasmareactive species, without any repair processes. proteins have shown that PP IX binds to albumin and

As can be seen from the cell proliferation results UP I (5 hemopexin but UP I either does not bind at all or is onlymM) is less phototoxic than PP IX (20 nM) (compare Figs. loosely bound [20,21]. Thus, it is to be expected that PP IX3 and 4). In fact, in membrane damage experiments is more phototoxic to cells than UP I, and studies using(results not shown), with 50 mM UP I no significant cell other in vitro cellular systems agree with this findingkill was observed, and 0.1 mM UP I was found to produce [2,22]. In order to suppress the cell growth of the fi-the most stained cells, suggesting that it is also acting as an broblasts, we found the optimum concentration for PP IXantioxidant at higher concentrations. This may be related phototoxicity was 20 nM and for UP I it was 5 mM (resultsto triplet UP I quenching by UP I ground state; such not shown). This is because of the sensitivity of the

fibroblasts, as well as the influence of medium com-ponents.Table 2

b-Carotene offers some protection (P,0.05) in the cellProtection factors for the various antioxidants against PP IX and UP Iphototoxicity as measured by cell proliferation of human fibroblasts proliferation studies, although, lycopene is more efficient

against both PP IX (P,0.05) and UP I (but not significant)Antioxidant Protection factor (PF)phototoxicity (Table 2). When the antioxidants b-carotene,

20 nM PP IX 5 mM UP Ia-tocopherol and ascorbic acid were used together, the(10 min irradiation) (30 min irradiation)protection factor was similar to that for b-carotene against

b-Carotene (5 mM) 1.9 1.3 UP I but higher against PP IX (P,0.05). We have noLycopene (5 mM) 2.4 1.5

a explanation as to why the antioxidant mixture gave a lowTriple combination 2.4 1.4protection against UP I phototoxicity for the proliferation

PF, % cell proliferation with antioxidants /% cell proliferation withoutstudies, despite giving a high protection against membraneantioxidants.

a damage.Triple combination5b-carotene (5 mM)1a-tocopherol (35 mM)1ascorbic acid (100 mM). The significantly higher protection by the antioxidant


photoporphyrin and uroporphyrin on bovine corneal endothelium,mixture against PP IX phototoxicity both for membranein: K.J.A. Davies (Ed.), Oxidative Damage and Repair (5th Int. Soc.protection and proliferation, as well as that for UP I againstFree Radical Res. Bienn. Meeting), Pergamon Press, Oxford, 1991,membrane phototoxicity, can be explained by antioxidantpp. 321–325.

synergism, as already described [23]. If the antioxidants [2] H.W. Lim, I. Gigli, S.I. Wasserman, Differential effects of protopor-1simply quench O or the triplet states of PP IX or UP I by phyrin and uroporphyrin on murine mast cells, J. Invest. Dermatol.2

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Antioxidant inhibition of porphyrin-induced cellular phototoxicity