THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 68:157-160, 1977 Copyright \0 1977 by The Williams & Wilkins Co.

Vol. 68, No.3 Printed in U.S.A.

PHOTOPROTECTION IN ERYTHROPOIETIC PROTOPORPHYRIA: MECHANISM OF PHOTOPROTECTION BY BETA CAROTENE

ALAN N. MOSHELL, M.D.* , A.ND LoRING BJORNSON , PH.D.t

Departments of Dermatology (ANMJ and Medicine (LBJ, New York University School of Medicine , New York , New York , U . S. A .

Erythropoietic protoporphyria is a genetic disease caused by the accumulation of protopor­phyrin IX. This molecule absorbs 400-nm light and its presence is at times associated with severe cutaneous photosensitivity. The only effective treatment for this disease is oral administration of beta carotene.

Several possible mechanisms of photo protection by beta carotene were investigated using the photohemolysis of red blood cells as an in vitro model. Additional studies were done in an in vivo model which involves lethal hematoporphyrin photosensitization of white mice. The photoprotective effects of beta carotene were compared with those of alpha tocopherol, an agent which possesses some but not all the properties that have been implicated in explain­ing the known effectiveness of beta carotene. In the photohemolysis model, both compounds demonstrated partial protection. In hematoporphyrin-photosensitized mice, tocopherol showed some protection at high doses, while beta carotene showed greater protection at lower concentrations.

Although these results suggest that photoprotection was due to free radical scavenging or singlet oxygen quenching, properties common to both agents, they do not rule out the possible role of 400-nm light absorption, a property of beta carotene alone.

Erythropoietic protoporphyria \EPP) is a domi­nantly inherited defect in porphyrin metabolism resulting in the accumulation of protoporphyrin IX 11 ,2], a water-insoluble porphyrin demonstra­ble in the patients' erythrocytes that strongly ab­sorbs light of wavelength 400 nm. Clinically, these patients have severe photosensitivity character­ized by marked burning, pruritus, and edema fol­lowing brief exposure to intense 400-nrn light ll-3]. Avoidance is very difficult and most topical preparations are of little value in protecting pa­tients with EPP, but oral beta carotene has proved very effective.

Several mechanisms for this protection have been postulated. Beta carotene strongly absorbs 400-nm light, and simple absorption of light by beta carotene in the stratum corneum may pre­vent penetration and symptoms. Porphyrin-in­duced photosens itivity in laboratory models is an oxygen-dependent process 14- 6) involving an ex-

Manuscript received March 13, 1975: accepted for publication October 15, 1976.

This investigation was supported in part by USPHS Grants ES01041 , HE 06481, and Dermatology Training Grant. AM 05326 13.

* Present address: National Naval Medical Center, Department of Dermatology, Bethesda, Maryland 20014.

t Present address: Long Island College Hospital, Special Chemistry Laboratory, 340 Henry Street, Brooklyn, New York 11201.

Reprint requests to: Dr. A. N. Moshell , National Naval Medical Center, Department of Dermatology, Bethesda, Maryland 20014.

cited singlet oxygen intermediate l7]. Free radi­cals are generated following photoexcitation of various porphyrin derivatives in vitro 18-11]. Beta carotene is very efficient in trapping singlet ex­cited oxygen and quenching free radicals [7.12]. Either of these properties may explain beta caro­tene's efficacy. Finally, beta carotene may act in some undefined way to prevent or repair the pho­totoxic damage. Alpha tocopherol also traps free radicals and quenches singlet oxygen [13], but does not absorb 400-nm light. Alpha tocopherol and beta carotene were studied and compared us­ing in vivo and in vitro techniques previously described [5,6,9,14-20] in an attempt to evaluate the mechanisms involved in photoprotection.

MA TERlALS AND METHODS

Photohemolysis Studies

Protection against. in vitro photohemolysis of red cells from patients with EPP, and of normal red cells photosensitized with hematoporphyrin was st.udied, us­ing techniques previously described, with minor modi­fications ]15.16.21 ].

Preparation of erythrocytes. A hexane solution of al­pha tocopherol (1.5 mg) or beta carotene (0.5 mg) was evaporated to dryness with nitrogen in a 25-ml Erlen­meyer flask. Five milliliters of whole blood was added to the flask and incubated for 6 to 12 hr in a water bath at 37°C with constant shaking. After incubation, red blood cell CRBC) concentrations of alpha tocopherol and beta carotene ranged from 20 to 60 and 0.2 to 0.8 J.Lg/ml of packed RBC, respectively. The analytical procedures for these determinations are described elsewhere 122J. Control erythrocytes were incubated identically, but in

157

158 MOSHELL AND BJORNSON

the a bsence of the protective agent. All cells were then washed 3 times in normal saline, and suspended at a 1:800 dilution in either isotonic phosphate-buffered sa­line, pH 7.4 <EPP cells), or in identical buffered solution to which 0. 1 mg% hematoporphyrin had been added (normal cells).

Photoprotection assay. Three milliliters of the RBC suspensions were placed in a 3-ml quartz cuvette 2.5 em in diameter with an optical pathway of 1 em. The cu­vettes were then exposed to 400-nm radiation from a bank of 3 General Electric 20-watt bluelight fluorescent tubes (spectral range 380-520 run) at a distance of 10 em (output 696 J.LW/cm2

) for various time periods. Hemoly­sis was determined by means of direct cell counts in a hemocytometer before, during, and after irradiation. Additional control samples were kept in the dark, and hemolysis determined at the conclusion of each photo­hemolysis experiment.

Animal Studies

Hematoporphyrin phototoxicity in mice. The approxi­mate dose of hematoporphyrin intraper itoneally ad­ministered required to kill half the exposed animals 24 hr after exposure (LD;,,/1 day) was determined as de­scribed previously !21], except that 2 hr of bluelight fluorescent tube (see above) exposure replaced the blacklight fluorescent tubes.

Toxicity studies of alpha tocopherol and beta carotene. Using identical exposure conditions (2 hr bluelight at 10 em) mice were exposed 24 hr after the intraperitoneal administration of either agent in various dosages ad­justed to maintain the total intraperitoneal volume at or below 1 mi. No toxicity was observed at any of the dosages used.

Photoprotection studies using beta carotene and alpha tocopherol. Two hundred and thirteen mice were irradi­ated in groups as summarized in the Table. Fifty-nine animals received beta carotene, 0.2 mg/gm body weight in Emulfor intraperitoneally 24 hr prior to irradiation, and hematoporphyrin. 0.1 mg/gm intra peritoneally, 30 min before irradiation. Fifty animals received alpha tocopherol. 1 mg/gm in Emulfor intraperitoneally <high dose) 24 hr prior to irradiation, and hematoporphyrin. 0.1 mg/gm intraperitoneally 30 min before irradiation. Thirty animals received alpha tocopherol. 0.5 mg/gm in Emulfor intraperitoneally tlow dose) 24 hr prior to irra­diation, and hematoporphyrin. 0.1 mg/gm intraperi to­neally 30 min before irradiation. In all animals, the total volume injected intraperitoneally 24 hr before ir­radiation was approximately 0.5 ml. Seventy-four ani­mals served as controls and received the vehicle, Ernul­for alone, 0.5 ml intraperitoneally 24 hr before irradia­tion, and hematoporphyrin, 0.1 mg/gm 30 min before

TABLE. Photoprotectiue effects of intraperitoneal administration of alpha tocopherol and beta carotene in hematoporphyrin-photosensitized mice after 2 hr of light

exposure Experi- Control"

Photoprotective agent mental

# % # %

Tocopherol (1 mg/gm)1' 18/50 36 11/50 22 o/C

Tocopherol (0.5 mg/gmJ 10/30 33 9/24 38 Carotene (0.2 mg/gm)'' 32/59 54 15/54 28

a Control animal received only Emulfor vehicle and hematoporphyrin.

b Significant at 0.05 > p > 0.025 level by chi square. ' Significant at 0.005 > p level by chi square.

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irradiation. All animals were exposed to light in groups of 5 or 6 on successive days, and in each case at least one group of control animals was exposed simultaneously with the experimental groups. No anesthesia was re­quired during either the intraperitoneal administration of the various agents or during the exposure to blue­light. During the light exposure, the animals were individually restrained under half-inch wire mesh holders. Viability was assessed after 24 and 48 hr.

RESULTS

Photohemolysis

The results of various photohemolysis experi­ments, using both normal red cells photosensitized with hematoporphyrin and red cells from patients with EPP, are summarized in Figures 1-4. These results are expressed as percent hemolysis per unit time. The hemolysis was determined by direct cell counts. The time course of hemolysis of the controls was adjusted by varying the total irradia­tion time so that the hemolysis could be conven­iently observed. Control studies, using cells treated in a manner identical to the experimental groups but without irradiation, showed no signifi­cant hemolysis. Similarly , normal cells, in the absence ofhematoporphyrin but in the presence of beta carotene or alpha tocopherol and irradiation, showed negligible hemolysis. As noted in Figure 1, the protection afforded by beta carotene against photohemolysis was demonstrated by the prolon­gation of t he time required for 100% hemolysis of the red cells from 20 to 24 hr. No signif1cant hemol­ysis was observed during the first 10 hr in either the experimental or control red cells. As shown in Figure 2, essentially the same data were obtained when alpha tocopherol was used as the protective agent. In Figures 3 and 4, when the dose of radia­tion was increased by lengthening the exposure time from 20 min io 1 hr, complete hemolysis was noted in less than 8 hr. In both studies, the alpha tocopherol-treated cells were protected, as indi­cated by the prolongation of the time required for complete hemolysis.

Animal Studies

The resuJts of the photoprotection studies in mice are summarized in the Table and Figure 5. Survival in control animals, given only hemato­porphyr in and Emulfor 1 day after exposure to

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Pre. 1. Photoprot.ection by beta carotene against pho­tohemolysis of EPP red cells. Summary of 3 experi­ments each using at least 6 groups. Data obtained by direct cell counts. Cells exposed to light for 20 min.

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FIG. 2. Photoprotection by alpha tocopherol against photohemolysis of EPP red cells. Summary of 4 experi­ments each using at least 6 exeerimental groups. Data obtained by direct cell counts. Cells exposed to light for 20 min.

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FIG. 3. Photoprotection by alpha tocopherol against photo hemolysis of EPP red cells. Summary of 4 exper i­ments each using at least 6 groups. Data obtained by direct cell counts. Exposed to light for 1 hr.

bluelight, ranged from 22 to 38%- of the animals. Beta carotene showed the best protection (54t.k- vs 27o/c) at a dose !0.2 mglgm) that was significantly lower than either a lpha tocopherol dose tested. Low doses !0.5 mg/gml of alpha tocopherol showed no photoproteclion f33o/c) . High doses (1 mg/gml, however, showed some protection (36%- vs 22o/cl. There is an apparent discrepancy in the number of control animals as listed in the Table and F igure 5 and as stated in Materials and Methods. This resulted from the processing of several experimen­tal groups simuhaneou sly with a single control group. Thus, the same animals may have served as con trols for several but not all exper iments. Animals were tabulated as controls only when processed simultaneously with the corresponding exper imental animals. The solubilizer (Emulfor) a lone h ad no protective effect. In the absence of hematoporphyrin, none of the animals succumbed. Similarly, in the absence of exposure to 400-nm light, no animals succumbed.

DISCUSSION

Previous investiga tions have demonstrated the value of red blood cells from EPP patients and photosensitized normal red cells in the study of the mechanism of cell membrane damage from 400-nm radiation 14,8,14,16- 18,20.23). This photohemol­ysis is oxygen dependent 14-6], associated with free radical l8-11) and peroxide !6,8,24) formation, and the observed cell destruction is due to colloid osmotic hemolysis 114,17] initiated by the forma-

PHOTOPROTECTION IN EPP 159

tion of holes in the cell membrane under 10 A in size 117.1.

The red cell photoprotective agents discovered in the studies have infrequently been tested in animal experimen ts. A recent report by Harber et aJ 121) demonstrated that ruthiothreitol and glyc­erol act synergistically to protect hematopor­phyrin-photosensitized mice and inhibit the photo­hemolysis of red cells. In addition. beta carotene was shmvn to protect hematoprophyrin-photosen­sitized mice prior to its very successful use in pa tients with EPP 125].

In the present study, we were able to evaluate and compare beta carotene and alpha tocopherol not only in vitro in red cell photohemolysis, but also in hematoporphyr in-photosensitized mice studies.

Beta carotene contains 11 conjugated double bonds, and shows significant absorption at 400 run. It is an active free radical t rap and singlet excited oxygen quencher 112]. Here and in previously pre­sented data, beta carotene has been demonstrated to be effective in preventing t he deleterious effects of porphyrin derivatives plus 400-nm ligh t in red cell photohemolysis models, photosensitized labo­ratory animals, and EPP patients [14,19,25). Al­pha tocopherol contains only 3 conjugated double bonds, does not absorb 400-nm light (it has no significant absorption above 310 nml, less avidly quenches singlet excited oxygen, but is an even more active free radical trap t han is beta carotene (A. A. Lamola, personal communication) 113].

The similarity in the results of the photohemol­ysis experiments using beta carotene and alpha

F1a. 4. Photoprotection by alpha tocopherol against photohemolysis of hematoporphyrin-photosensitized red cells. Summary of 4 experiments each using 6 groups. Data obtained by direct cell counts. Cells ex­posed to light for 1 hr.

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.F~a. 5. _Photoprotective effects of intraperitoneal ad­m1mstrat10n of alpha tocopherol and beta carotene in hematoporphyrin-photosensitized mice after 2 hr of light exposure.

160 MOSHELL AND BJORNSON

tocopherol as protective agents would suggest that free radicals do take part in the reaction and that quenching these free radicals can inhibit mem­brane damage [21). These results also suggest that photon absorption of 400-nm light is not the only mechanism by which beta carotene may exert a protective effect.

The animal experiments demonstrate beta caro­tene to be a more effective photoprotective agent than alpha tocopherol. This implies that free radi­cal scavenging is not the only mechanism of beta carotene's photoprotection, for were this the case, one would expect alpha tocopherol, the more avid free radical scavenger, to be superior . However, there are several other possible explanations for this result. In this animal model, the tested agents were administered intraperitoneally. Active drug must reach the site of photo toxic damage in order to be protective. Major differences in absorption, distribution. or metabolism could alter the concen­tration of active drug at this site. overshadowing any differences in free radical scavenging or sin­glet oxygen quenching. Some observations as to the differences in transport and storage of these two agents in man appears in another report by our group l22].

The available data are insufficient to comment on the effect of drug localization on photoprotec­tion, although the drug must be in close proximity to any short-lived free radicals or excited singlet oxygen generated in the phototoxic reaction in order to prevent irreversible damage. Simple ab­sorption of 400-nm light by beta carotene seems an unlikely explanation for the prime mechanism of photoprotection, as the patients are ra rely grossly carotenemic at doses that prove protective, and topical application is totally ineffective as it is rapidly bleached upon exposure to intense light l25]. Thus, it seems likely that both free radical trapping and singlet excited oxygen quenching are involved in beta carotene's photoprotection in EPP, a lthough the relative importance of these two properties cannot be evaluated at present, and the simple absorption of 400-nm light. a lthough unlikely, cannot be ruled out.

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