Clinica Chimica Acta, 202 (1991) 237-242 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
237
CCA 05107
Role of iron in the photosensitization by uroporphyrin I. Aravind Menon ‘, Mary Ann C. Becker 2 and Herbert F. Haberman
2
Departments of’ Medicine and Ophthalmology, 2 Medicine, Universiry of Toronto, Toronto ON (Canada)
(Received 8 April 1991; revision received and accepted 23 April 1991) Key words: Porphyrin; Photosensitizer;
Light; Hydrogen peroxide; Singlet oxygen; Hydroxyl
Summary The role of iron in the mechanism of photosensitivity due to uroporphyrin was investigated. There is frequently increased levels of Fe in the serum from patients with porphyria cutanea tarda, where the photosensitivity is due to uroporphyrin. It has been reported that H,O, has a major role in the uroporphyrin induced photosensitivity. Hence we examined the hypothesis that Fe would catalyze the production of ‘OH from H,O, and the ‘OH thus formed may have a significant role in the uroporphyrin photosensitivity. This was examined by studying the effects of the Fe chelating compound deferoxamine in an in vitro system. Our results show that deferoxamine inhibited the uroporphyrin photosensitivity, but not the photosensitivity due to protoporphyrin. This indicates that Fe may play a role in the uroporphyrin photosensitization in the skin, by accelerating the formation of ‘OH, which may be a major reactive species responsible for the photosensitization in porphyria cutanea tarda.
Introduction Porphyrins occur widely in the mammalian body as metabolites or derivatives of natural metabolites. They are intermediates in the biosynthesis of heme and cytochromes. Free porphyrins are potent photosensitizers [l-3]. There are several types of disorders of porphyrin metabolism, termed ‘porphyrias’. Classifications of porphyrias vary according to the clinical symptoms, the organs involved, the
Correspondence and requests for reprints to: LA. Menon, Clinical Sciences Division, Medical Sciences Building, University of Toronto, ON, M5S lA8 Canada.
238
biochemical manifestation etc. and depending upon authors’ preferences [l-7]. Cutaneous photosensitivity is a feature of certain of the clinical porphyrias viz. (erythropoetic protoporphyria), porphyria cutanea tarda, congenital erythropoetic protoporphyria, erythrohepatic protoporphyria, erythropoetic coproporphyria, porphyria variegata and acute coproporphyria. The tissue accumulation as well as urinary and fecal excretion patterns of these porphyrins differ. For instance erythro-poetic proto-porphyria is characterized by the accumulation of abnormally high levels of protoporphyrin (PP) in red blood cells and often in the plasma and feces, whereas high levels of uroporphyrin (UP) and coproporphyrin in plasma and urine are characteristic of porphyria cutanea tarda [6,71. Porphyria cutanea tarda is precipitated by the administration of estrogen, and or abuse of ethanol and aggravated by iron overload. Clinically these patients demonstrate fragility of the skin, hypertrichosis, hyperpigmentation and photosensitive eruption characterized by bullae etc [8,9]. The classical therapy for porphyria cutanea tarda is phlebotomy, thought to be due to iron removal. A potent Fe chelator, deferoxamine (DM), has also been reported to be effective [9-111. There is substantial evidence to show that singlet oxygen is formed during the irradiation of the porphyrins, presumably by the transfer of the excess energy of the porphyrins in the excited states to oxygen. Several reports have indicated that singlet oxygen may be involved in the photosensitization by the porphyrins [3,4,12]. We have previously reported that the photosensitization by UP is at least partially mediated by H,O,; on the other hand no evidence was observed to indicate the role of H,O, in the photosensitization by PP [13]. Moreover, it has also been shown that irradiation of UP produces more H,O, than irradiation of PP [14]. The clinical picture of porphyria cutanea tarda differs considerably from that of erythro-poetic proto-porphyria. The above differences in the mechanism of photosensitivity due to UP and PP are probably responsible for some of the differences in the clinical photosensitivity of these two porphyrias. In this paper the role of iron in the mechanism of photosensitivity in porphyrias was investigated using an in vitro model. There is frequently increased levels of ferritin in the serum and probably in the skin of patients with porphyria cutanea tarda [9,15]. We examined the following hypothesis for the role of Fe in the photobiological activity of UP. Since H,O, has a distinct role in the UP photosensitivity, it is possible that ‘OH derived from H,O, may have a major role in this phenomenon. It is widely known that the formation of ‘OH from H,O, is catalyzed by Fe by means of the Fenton reaction. This hypothesis for the role of Fe in the UP photosensitivity was examined by studying the effect of deferoxamine on an in vitro system. Materials and methods Materials
The porphyrins, PP and UP were obtained as free acids from Porphyrin Products Inc (Logan UT, USA). DM was purchased from Sigma Chemical Company (St. Louis, MO).
239
Stock solutions of PP and UP were made in 3.0 mol/l and 1.0 mol/l HCl, respectively. These solutions were brought to pH 7 with NaOH. Appropriate dilutions were made with 0.01 mol/l PO, pH 7.4; 0.3 ml of this solution was added to the irradiation medium. All porphyrin solutions were contained in opaque glassware and stored in the dark. Ehrlich ascites carcinoma (EAC) was maintained in mice. The tumor cells were isolated, suspended in medium 199 and iabelled with ‘lCr as previously described D31. Irradiation
Various concentrations of PP and UP, in the presence or absence of DM as mentioned in each section, were irradiated for the specific periods, using a Westinghouse Mercury Vapour lamp. This source emits discontinuous radiation (320 to 700 nm), with peaks at 360, 400, 430, 550 and 580 nm 116,171. The irradiance was measured using either a narrow band filter (365 nm) or no filter. The irradiance with the filter was 23 f 2.98 Watts/sqM, and without filter 170 f 9.75 Watts/sqM. All solutions were irradiated without the filter. Determination of cell lysis
After the irradiation, the cell suspensions were centrifuged at 1,000 x g and the supernatants were separated from the cells. The radioactivities of the supernatants were determined using a gamma counter (Intertechnique CG automatic gamma spectrometer). The 5’Cr released in each sample was expressed as the percentage of the 51Cr released with respect to the total 51Cr present in a suspension containing the same number of cells. The ‘iCr release for the control sample, kept on ice during the irradiation or incubation of the experimental samples, was subtracted from the ‘lCr release for each experimental sample. This value was taken as a measure of the cell lysis induced by the porphyrin photosensitization. The control samples were either (a) incubated in the dark with one of the porphyrins or (b) irradiated in the absence of either porphyrin [13]. Analysis of results
Each experiment was performed at least four times, using triplicates for each sample. The mean cell lysis was calculated for each sample. The mean and standard error of the mean @EM) for each sample from all similar experiments were calculated. Results Effects of deferoxamine on cell lysis by uropolphyrin
The effect of DM on the cell lysis induced by UP and light is shown in Table I. There was only very low cell lysis and there was no significant difference among
240 TABLE I Effects of various concentrations of deferoxamine mesylate on cell lysis in the presence of uroporphyrin UP +.mol/l)
DM (mmol/l)
Dark: 0.0 0.0
0.0
1.0 1.0 1.0 Light: 0.0 0.0
1.0 1.0 1.0
% Lysis (Mean f SEMI
8.20 9.69 8.90 9.96
% Lysis after subtracting blank (Mean k SEM)
% Inhibition
40.25 + 3.51 34.14k6.62 28.54 + 3.67
0.0 15.2 29.1
+ 0.42 + 0.84 + 0.55 rt 0.61
1.0 0.0 0.01 I.0
10.41 + 0.57
0.0
12.32 + 4.60
1.0 0.0 0.01 1.0
9.17*0.88 52.57 + 4.65 48.00 f 7.28 40.86 + 5.22
The samples were incubated in the dark or irradiated at 37 ’ C for 90 min. The % lysis is expressed as mean+SEM. The % cell lysis for the blank in the light in the absence of UP or DM was subtracted from the % cell lysis for the samples with as well as without DM. The % inhibition was then calculated with respect to the resultant value for the control (1.0 pmol/l UP without DMI.
the values for the lysis under the following conditions: incubation in the dark without UP, incubation with UP, incubation with DM, incubation with UP as well as DM and irradiation with DM. When the samples were irradiated in the presence of UP, considerable cell lysis was observed. When the samples were irradiated in the presence of UP and DM, the cell lysis was less than when the samples were irradiated with UP alone. It was found that 0.1 and 1.0 mmol/l DM inhibited the cell lysis by 15.2% and 29.1%, respectively. Effects of deferoxamine on cell lysis by protoporphyrin
The effect of DM on the cell lysis induced by PP and light is shown in Table II. Incubation of the cells in the dark (a) in the presence of PP alone as well as (b) in the presence of PP and DM produced very low cell lysis. When the samples were irradiated in the presence of PP, considerable cell lysis was observed. It was found that 0.1 and 1.0 mmol/l DM did not inhibit the cell lysis; on the contrary DM produced a slight increase in the cell lysis. Discussion
It has been shown previously that the photosensitization due to UP is at least due to H,O,. In this paper we examined the possibility that the effects of H,O, may indeed be due to the ‘OH formed via a Fenton type reaction, which would be catalyzed by Fe. If this is the case, the photosensitization by UP would be expected to be inhibited by the removal of Fe, while Fe would not have any role in the photosensitization by PP. The role of Fe was tested by studying the effect of an Fe
241 TABLE II Effects of various concentrations phyrin
of deferoxamine
PP (~mol/l)
DM (mmol/l)
% Lysis (Mean f SEMI
Dark: 0.0 0.0
0.0
8.43 f 2.30
1.0 0.0 0.01 1.0
8.18k2.14 7.78 + 2.08 8.50 + 2.08 8.78 + 2.33
0.0
7.40 + 1.36
0.1 0.1 0.1 Light: 0.0 0.0
0.1 0.1 0.1
1.0 0.0 0.01 1.0
7.61+ 1.25 63.11+0.83 67.97 f I.57 68.00 f 2.02
mesylate on cell lysis in the presence of protopor-
% Lysis after subtracting blank (Mean k SEM)
% Inhibition
55.71*0.54 60.57 f 0.20 60.60 * 0.66
0.0 -8.7 -8.8
The samples were incubated in the dark or irradiated at 37 ’ C for 90 min. The % lysis is expressed as mean*SEM. The % cell lysis for the blank in the light in the absence of PP or DM was subtracted from the % cell lysis for the samples with as well as without DM. The % inhibition was then calculated with respect to the resultant value for the control (0.1 pmol/l PP without DM).
chelator, DM. Our results show that DM inhibits the cell lysis produced by UP but not that by PP. The lack of inhibition by DM on the PP-induced cell lysis shows that the inhibitory effect of DM on the UP-induced lysis is not due to a nonspecific effect such as absorption of the radiation. Moreover since the PP-induced cell lysis has been shown to be mediated by singlet oxygen, the lack of effect of DM in this system indicates that DM does not interfere with singlet oxygen or affect the photo-induced formation of PP triplet. These findings indicate that DM may not affect the formation of similar triplet from UP or the possible singlet oxygen mediated component of cell lysis in the UP photosensitization. Our results indirectly point to the possibility that UP photosensitized cell lysis in vitro is at least partially mediated by ‘OH. If the results from this in vitro model could be extrapolated to in vivo conditions, such as clinical porphyrias, it may be concluded that the cutaneous photosensitivity due to UP, as in the case of porphyria cutanea tarda may also be at least partially mediated by ‘OH. There have been numerous studies on the role of Fe in porphyria cutanea tarda. The disease is frequently associated with increased levels of Fe in plasma and tissues, e.g. liver. It has been shown that Fe inhibits liver uroporphyrinogen decarboxylase [181. It has also been suggested that Fe could oxidize uroporphyrinogen to uroporphyrin thereby increasing the uroporphyrin levels in the blood and skin [19]. However the complete role(s) of Fe in porphyria cutanea tarda is not clear. Our results indicate that Fe, in addition to its effects on the liver, may play a role in the UP photosensitization in the skin by accelerating the formation of ‘OH,
242
which may be the major reactive species responsible for the photosensitization porphyria cutanea tarda.
in
Acknowledgements
This work was supported by a grant from the Medical Research Council of Canada. H.F.H. is an Associate of Ontario Cancer Treatment and Research Foundation. References 1 Harber LC, Baer RL, Bickens DR. Fundamental and clinical aspects of the porphyrias. In: Pathak MA, Harber LC, Seiji M, Kukita A, eds. Sunlight and the man. Tokyo: University of Tokyo Press, 1972;631-653. 2 Slater TF. Free radical mechanisms in tissue injury. London: Pion Limited, 1972. 3 Valenzeno DP. Photomodification of biological membranes with emphasis on singlet oxygen mechanisms. Photochem Photobiol 1987;46:147-160. 4 Girotti AW. Mechanisms of photosensitization. Photochem Photobiol 1983;38:745-751. 5 Spikes JD. Photosensitization in mammalian cells. In: Parrish JA, Kripke ML, Morison WL. eds. Photoimmunology, New York: Plenum Medical Book Co., 1983;23-49. 6 Eales L. Clinical chemistry of the porphyrias. In: Dolphin D, ed. The porphyrins. Vol. VI. New York: Academic Press, 1979;663-804. 7 Hindmarsh JT. The porphyrias. Clin Biochem 1986;32:125.5-1263. 8 Haberman HF, Rosenberg F, Menon IA. Porphyria cutanea tarda: comparison of cases precipitated by alcohol and estrogens. Can Med Assoc J 1975;4:653-655. 9 Rocchi E, Gibertini P, Cassanelli M, et al. Iron removal therapy in porphyria cutanea tarda: phlebotomy versus slow subcutaneous desferrioxamine infusion. Br J Dermatol 1986;114:621-629. 10 Gibertini P, Rocchi E, Cassanelli M, Pietrangelo A, Ventura E. Advances in the treatment of porphyria cutanea tarda. Effectiveness of slow subcutaneous desferrioxamine infusion. Liver 1984;4:280-284. 11 Marchesi L, Di PC, Cainelli T, et al. A comparative trial of desferrioxamine and hydrochloroquine for treatment of prophyria cutanea tarda in alcoholic patients. Photodermatology 1984;1:286-292. 12 Spikes JD. Photosensitization, In: Smith KC, ed. The science of photobiology. New York: Plenum Press, 1989;79-110. 13 Menon IA, Persad S, Haberman HF. A comparison of the phototoxicity of protoporphyrin, coproporphyrin and uroporphyrin using a cellular system in vitro. Clin Biochem 1989;22:197-200. 14 Menon IA, Becker MAC, Persad SD, Haberman HF. Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin. Clin Chem Acta 1989;186:375-381. 15 Rocchi E, Gibertini P, Cassanelli M, Pietrangelo A, Borghi A, Ventura E. Serum ferritin in the assessment of liver iron overload and iron removal therapy in porphyria cutanea tarda. J Lab Clin Med 1986;107:36-42. 16 Menon IA, Persad S, Ranadive NS, Haberman HF. Effects of ultraviolet-visible irradiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells. Cancer Res 1983;43:3165-3169. 17 Persad S, Menon IA, Haberman HF. Comparison of the effects of UV-visible irradiation of melanins and melanin-hematoporphyrin complexes from human black and red hair. Photochem Photobiol 1983;37:63-68. 18 Mukerji S, Pimstone N, Burns M. Dual mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron: its potential role in the genesis of porphyria cutanea tarda. Gastroenterology, 1094;87:1248-1254. 19 Mukerji S, Pimstone N. Free radical mechanism of oxidation of uroporphyrinogen in the presence of ferrous iron. Arch Biochem Biophys 1990;281:177-184.
237
CCA 05107
Role of iron in the photosensitization by uroporphyrin I. Aravind Menon ‘, Mary Ann C. Becker 2 and Herbert F. Haberman
2
Departments of’ Medicine and Ophthalmology, 2 Medicine, Universiry of Toronto, Toronto ON (Canada)
(Received 8 April 1991; revision received and accepted 23 April 1991) Key words: Porphyrin; Photosensitizer;
Light; Hydrogen peroxide; Singlet oxygen; Hydroxyl
Summary The role of iron in the mechanism of photosensitivity due to uroporphyrin was investigated. There is frequently increased levels of Fe in the serum from patients with porphyria cutanea tarda, where the photosensitivity is due to uroporphyrin. It has been reported that H,O, has a major role in the uroporphyrin induced photosensitivity. Hence we examined the hypothesis that Fe would catalyze the production of ‘OH from H,O, and the ‘OH thus formed may have a significant role in the uroporphyrin photosensitivity. This was examined by studying the effects of the Fe chelating compound deferoxamine in an in vitro system. Our results show that deferoxamine inhibited the uroporphyrin photosensitivity, but not the photosensitivity due to protoporphyrin. This indicates that Fe may play a role in the uroporphyrin photosensitization in the skin, by accelerating the formation of ‘OH, which may be a major reactive species responsible for the photosensitization in porphyria cutanea tarda.
Introduction Porphyrins occur widely in the mammalian body as metabolites or derivatives of natural metabolites. They are intermediates in the biosynthesis of heme and cytochromes. Free porphyrins are potent photosensitizers [l-3]. There are several types of disorders of porphyrin metabolism, termed ‘porphyrias’. Classifications of porphyrias vary according to the clinical symptoms, the organs involved, the
Correspondence and requests for reprints to: LA. Menon, Clinical Sciences Division, Medical Sciences Building, University of Toronto, ON, M5S lA8 Canada.
238
biochemical manifestation etc. and depending upon authors’ preferences [l-7]. Cutaneous photosensitivity is a feature of certain of the clinical porphyrias viz. (erythropoetic protoporphyria), porphyria cutanea tarda, congenital erythropoetic protoporphyria, erythrohepatic protoporphyria, erythropoetic coproporphyria, porphyria variegata and acute coproporphyria. The tissue accumulation as well as urinary and fecal excretion patterns of these porphyrins differ. For instance erythro-poetic proto-porphyria is characterized by the accumulation of abnormally high levels of protoporphyrin (PP) in red blood cells and often in the plasma and feces, whereas high levels of uroporphyrin (UP) and coproporphyrin in plasma and urine are characteristic of porphyria cutanea tarda [6,71. Porphyria cutanea tarda is precipitated by the administration of estrogen, and or abuse of ethanol and aggravated by iron overload. Clinically these patients demonstrate fragility of the skin, hypertrichosis, hyperpigmentation and photosensitive eruption characterized by bullae etc [8,9]. The classical therapy for porphyria cutanea tarda is phlebotomy, thought to be due to iron removal. A potent Fe chelator, deferoxamine (DM), has also been reported to be effective [9-111. There is substantial evidence to show that singlet oxygen is formed during the irradiation of the porphyrins, presumably by the transfer of the excess energy of the porphyrins in the excited states to oxygen. Several reports have indicated that singlet oxygen may be involved in the photosensitization by the porphyrins [3,4,12]. We have previously reported that the photosensitization by UP is at least partially mediated by H,O,; on the other hand no evidence was observed to indicate the role of H,O, in the photosensitization by PP [13]. Moreover, it has also been shown that irradiation of UP produces more H,O, than irradiation of PP [14]. The clinical picture of porphyria cutanea tarda differs considerably from that of erythro-poetic proto-porphyria. The above differences in the mechanism of photosensitivity due to UP and PP are probably responsible for some of the differences in the clinical photosensitivity of these two porphyrias. In this paper the role of iron in the mechanism of photosensitivity in porphyrias was investigated using an in vitro model. There is frequently increased levels of ferritin in the serum and probably in the skin of patients with porphyria cutanea tarda [9,15]. We examined the following hypothesis for the role of Fe in the photobiological activity of UP. Since H,O, has a distinct role in the UP photosensitivity, it is possible that ‘OH derived from H,O, may have a major role in this phenomenon. It is widely known that the formation of ‘OH from H,O, is catalyzed by Fe by means of the Fenton reaction. This hypothesis for the role of Fe in the UP photosensitivity was examined by studying the effect of deferoxamine on an in vitro system. Materials and methods Materials
The porphyrins, PP and UP were obtained as free acids from Porphyrin Products Inc (Logan UT, USA). DM was purchased from Sigma Chemical Company (St. Louis, MO).
239
Stock solutions of PP and UP were made in 3.0 mol/l and 1.0 mol/l HCl, respectively. These solutions were brought to pH 7 with NaOH. Appropriate dilutions were made with 0.01 mol/l PO, pH 7.4; 0.3 ml of this solution was added to the irradiation medium. All porphyrin solutions were contained in opaque glassware and stored in the dark. Ehrlich ascites carcinoma (EAC) was maintained in mice. The tumor cells were isolated, suspended in medium 199 and iabelled with ‘lCr as previously described D31. Irradiation
Various concentrations of PP and UP, in the presence or absence of DM as mentioned in each section, were irradiated for the specific periods, using a Westinghouse Mercury Vapour lamp. This source emits discontinuous radiation (320 to 700 nm), with peaks at 360, 400, 430, 550 and 580 nm 116,171. The irradiance was measured using either a narrow band filter (365 nm) or no filter. The irradiance with the filter was 23 f 2.98 Watts/sqM, and without filter 170 f 9.75 Watts/sqM. All solutions were irradiated without the filter. Determination of cell lysis
After the irradiation, the cell suspensions were centrifuged at 1,000 x g and the supernatants were separated from the cells. The radioactivities of the supernatants were determined using a gamma counter (Intertechnique CG automatic gamma spectrometer). The 5’Cr released in each sample was expressed as the percentage of the 51Cr released with respect to the total 51Cr present in a suspension containing the same number of cells. The ‘iCr release for the control sample, kept on ice during the irradiation or incubation of the experimental samples, was subtracted from the ‘lCr release for each experimental sample. This value was taken as a measure of the cell lysis induced by the porphyrin photosensitization. The control samples were either (a) incubated in the dark with one of the porphyrins or (b) irradiated in the absence of either porphyrin [13]. Analysis of results
Each experiment was performed at least four times, using triplicates for each sample. The mean cell lysis was calculated for each sample. The mean and standard error of the mean @EM) for each sample from all similar experiments were calculated. Results Effects of deferoxamine on cell lysis by uropolphyrin
The effect of DM on the cell lysis induced by UP and light is shown in Table I. There was only very low cell lysis and there was no significant difference among
240 TABLE I Effects of various concentrations of deferoxamine mesylate on cell lysis in the presence of uroporphyrin UP +.mol/l)
DM (mmol/l)
Dark: 0.0 0.0
0.0
1.0 1.0 1.0 Light: 0.0 0.0
1.0 1.0 1.0
% Lysis (Mean f SEMI
8.20 9.69 8.90 9.96
% Lysis after subtracting blank (Mean k SEM)
% Inhibition
40.25 + 3.51 34.14k6.62 28.54 + 3.67
0.0 15.2 29.1
+ 0.42 + 0.84 + 0.55 rt 0.61
1.0 0.0 0.01 I.0
10.41 + 0.57
0.0
12.32 + 4.60
1.0 0.0 0.01 1.0
9.17*0.88 52.57 + 4.65 48.00 f 7.28 40.86 + 5.22
The samples were incubated in the dark or irradiated at 37 ’ C for 90 min. The % lysis is expressed as mean+SEM. The % cell lysis for the blank in the light in the absence of UP or DM was subtracted from the % cell lysis for the samples with as well as without DM. The % inhibition was then calculated with respect to the resultant value for the control (1.0 pmol/l UP without DMI.
the values for the lysis under the following conditions: incubation in the dark without UP, incubation with UP, incubation with DM, incubation with UP as well as DM and irradiation with DM. When the samples were irradiated in the presence of UP, considerable cell lysis was observed. When the samples were irradiated in the presence of UP and DM, the cell lysis was less than when the samples were irradiated with UP alone. It was found that 0.1 and 1.0 mmol/l DM inhibited the cell lysis by 15.2% and 29.1%, respectively. Effects of deferoxamine on cell lysis by protoporphyrin
The effect of DM on the cell lysis induced by PP and light is shown in Table II. Incubation of the cells in the dark (a) in the presence of PP alone as well as (b) in the presence of PP and DM produced very low cell lysis. When the samples were irradiated in the presence of PP, considerable cell lysis was observed. It was found that 0.1 and 1.0 mmol/l DM did not inhibit the cell lysis; on the contrary DM produced a slight increase in the cell lysis. Discussion
It has been shown previously that the photosensitization due to UP is at least due to H,O,. In this paper we examined the possibility that the effects of H,O, may indeed be due to the ‘OH formed via a Fenton type reaction, which would be catalyzed by Fe. If this is the case, the photosensitization by UP would be expected to be inhibited by the removal of Fe, while Fe would not have any role in the photosensitization by PP. The role of Fe was tested by studying the effect of an Fe
241 TABLE II Effects of various concentrations phyrin
of deferoxamine
PP (~mol/l)
DM (mmol/l)
% Lysis (Mean f SEMI
Dark: 0.0 0.0
0.0
8.43 f 2.30
1.0 0.0 0.01 1.0
8.18k2.14 7.78 + 2.08 8.50 + 2.08 8.78 + 2.33
0.0
7.40 + 1.36
0.1 0.1 0.1 Light: 0.0 0.0
0.1 0.1 0.1
1.0 0.0 0.01 1.0
7.61+ 1.25 63.11+0.83 67.97 f I.57 68.00 f 2.02
mesylate on cell lysis in the presence of protopor-
% Lysis after subtracting blank (Mean k SEM)
% Inhibition
55.71*0.54 60.57 f 0.20 60.60 * 0.66
0.0 -8.7 -8.8
The samples were incubated in the dark or irradiated at 37 ’ C for 90 min. The % lysis is expressed as mean*SEM. The % cell lysis for the blank in the light in the absence of PP or DM was subtracted from the % cell lysis for the samples with as well as without DM. The % inhibition was then calculated with respect to the resultant value for the control (0.1 pmol/l PP without DM).
chelator, DM. Our results show that DM inhibits the cell lysis produced by UP but not that by PP. The lack of inhibition by DM on the PP-induced cell lysis shows that the inhibitory effect of DM on the UP-induced lysis is not due to a nonspecific effect such as absorption of the radiation. Moreover since the PP-induced cell lysis has been shown to be mediated by singlet oxygen, the lack of effect of DM in this system indicates that DM does not interfere with singlet oxygen or affect the photo-induced formation of PP triplet. These findings indicate that DM may not affect the formation of similar triplet from UP or the possible singlet oxygen mediated component of cell lysis in the UP photosensitization. Our results indirectly point to the possibility that UP photosensitized cell lysis in vitro is at least partially mediated by ‘OH. If the results from this in vitro model could be extrapolated to in vivo conditions, such as clinical porphyrias, it may be concluded that the cutaneous photosensitivity due to UP, as in the case of porphyria cutanea tarda may also be at least partially mediated by ‘OH. There have been numerous studies on the role of Fe in porphyria cutanea tarda. The disease is frequently associated with increased levels of Fe in plasma and tissues, e.g. liver. It has been shown that Fe inhibits liver uroporphyrinogen decarboxylase [181. It has also been suggested that Fe could oxidize uroporphyrinogen to uroporphyrin thereby increasing the uroporphyrin levels in the blood and skin [19]. However the complete role(s) of Fe in porphyria cutanea tarda is not clear. Our results indicate that Fe, in addition to its effects on the liver, may play a role in the UP photosensitization in the skin by accelerating the formation of ‘OH,
242
which may be the major reactive species responsible for the photosensitization porphyria cutanea tarda.
in
Acknowledgements
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