I. Aravind Menon

A comparison of the phototoxicity of protoporphyrin, coproporphyrin and uroporphyrin using a cellular system in vitro

The photobiological effects of protoporphyrin (PP), coproporphyrin (CP) and uroporphyrin (UP) were investigated using an in vitro model. Suspensions of Ehrlich ascites carcinoma cells were labelled with 51Cr and irradiated in the presence of a wide range of concentrations of PP, CP and UP. It was found that PP was the most potent photosensitizer in this system; CP was less effective than PP and UP was the least potent. The cell lysis by CP was enhanced by superoxide dismutase (SOD) and inhibited by catalase; the lysis by UP was also inhibited by catalase; on the other hand, the lysis by PP was not affected by SOD or catalase. These indicate that the cell lysis by CP and UP was largely due to hydrogen peroxide produced from superoxide formed during the irradiation. The lysis produced by PP was not mediated by hydrogen peroxide. These differences in the mechanisms of the phototoxicity of the various porphyrins may have relevance in the etiology and treatment of the porphyrias.

Quantitation of hydrogen peroxide formed during UV-visible irradiation of protoporphyrin, coproporphyrin and uroporphyrin

Free porphyrins are strong photosensitizers. Previously reported findings indicate that the in vitro cell lysis induced by irradiation in the presence of coproporphyrin (CP) and uroporphyrin (UP) is mediated by H2O2 and that induced by irradiation with protoporphyrin (PP) is not mediated by H2O2. In the present study the possible role of H2O2 in the porphyrin photosensitization was investigated by direct measurement of the H2O2 formed during the irradiation of PP, CP and UP. Our results show that the amount of H2O2 formed decreased in the following order: UP, CP, PP. The amounts of H2O2 formed during irradiation of CP and PP were approximately 86% and 38% respectively in comparison to the H2O2 formed during the irradiation of UP. The formation of H2O2 was inhibited by sodium azide, a strong quencher of singlet oxygen. These observations are in good agreement with the previous report that the in vitro photolysis of Ehrlich ascites carcinoma cells by UP and CP, but not that by PP, was inhibited by catalase and clinical findings with patients with erythropoietic protoporphyria (EPP) and porphyria cutanea tarda (PCT). The patients with EPP, where the photosensitivity is due to PP, respond well to beta-carotene while beta-carotene does not protect against the photosensitivity in PCT, in which case the photosensitivity is due to uroporphyrin.

Quantitation of cutaneous inflammation induced by reactive species generated by UV-visible irradiation of Rose bengal

The present studies were undertaken to quantitate the initial inflammatory response produced by the photo-generated reactive species in rabbit skin. Rose bengal (RB), a photosensensitizer dye, was injected into the skin sites at various concentrations and exposed to UV-visible light for 30–120 min. The increase in vascular permeability and the accumulation of PMNs were investigated using125I-labeled albumin and51Cr-labeled PMNs. RB at a concentration of 1 nmol with 120-min exposure to light enhanced vascular permeability by 3.7 times and accumulation of PMNs by 3.3 times. As low as 0.01 nmol of RB produced discernible effects.β-Carofene (0.1 nmole) inhibited the inflammatory response by 75–100%, suggesting that the reactive species involved in this response was predominantly singlet oxygen. The increase in vascular permeability was inhibited by 48–70% by 25μg of chlorpheniramine maleate. It is therefore suggested that histamine plays a major role in the initial vascular response. The studies demonstrate that this rabbit model is suitable for the quantitation of photoinduced inflammatory response which is not observable by gross anatomic procedures.

A study on the sequence of phototoxic effects of rose bengal using retinal pigment epithelial cells in vitro

This paper describes a study on the sequence of the phototoxic effects of rose bengal (RB), a fluorescein derivative used as a vital stain in the diagnosis of certain external ocular diseases. Bovine melanotic RPE cells were grown in culture. These cells were labeled with [51Cr] and exposed to visible light in the presence of various concentrations of RB; the leakage of [51Cr] from the cells was used as a measure of cell lysis. Exposure to light of the cells with 0.3-10 microM RB induced approximately 13 to 43% cell lysis. The lysis progressively increased when the exposure time was varied from 10 to 30 min. A relatively short period of irradiation in the presence of RB was sufficient to produce sublytic cellular injury which could subsequently lead to complete cell lysis even in the absence of the photochemical treatment. The dark reaction was time-dependent, and reached a maximum for a given irradiation period. Our results thus show that there are two different processes that could eventually lead to the cell lysis: (a) a phototoxic effect which caused a sublytic damage and (b) a dark reaction that followed.

NON-PSORALEN TREATMENT OF VITILIGO. PART II. LESS COMMONLY USED AND EXPERIMENTAL THERAPIES

Gurrently accepted standard therapy for vitiligo bas been topical or systemic psoralens witb natural or artificial ligbt sources; bowever, not only can this tberapy be time consuming, but only approximately one tbird of patients acbieve excellent results. Tberefore, it is important to bave otber tberapies as first-line therapy or for patients who either have failed or are unsuitable for topical and/or systemic psoralens therapy. In a previous paper and in this article, we have reviewed most major studies on the therapy of vitiligo published in English in tbe last 27 years. Tberapies include expectant management (i.e., no tberapy), patient education, psychotherapy (supportive and active), cosmetic camouflage, and active therapies: medical or surgical. In a previous paper, we reviewed in detail: cosmetic camouflage and topical, intralesional, and parenteral steroids in the management of vitiligo. This paper reviews some of the other tberapies available for the treatment of vitiligo.

Role of iron in the photosensitization by uroporphyrin

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 H2O2 has a major role in the uroporphyrin induced photosensitivity. Hence we examined the hypothesis that Fe would catalyze the production of OH from H2O2 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.

Photoinduced cutaneous inflammatory response by psoralens

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA-visible irradiation (320-700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24-48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 micrograms 8-MOP cream followed by irradiation for 2 h (9.4 J cm-2) produced 3-7 times iVP, 2-4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.

Reactive Oxygen Species in The Photosensitization of Retinal Pigment Epithelial Cells by Rose Bengal

AbstractRose bengal (RB) is a fluorescein derivative used as a vital stain in certain ophthalmic diagnostic procedures. RB is a potent photo-sensitizer and has been used as a model for photosensitized reactions involving singlet oxygen. Ultraviolet (UV)-visible (UV-VIS) irradiation of RB with nitroblue tetrazolium (NBT) in the absence of glutathione (GSH) induced the reduction of NBT. However since superoxide dismutase (SOD) did not inhibit this NBT reduction it seems to be not mediated by superoxide, but due to the direct reduction of NBT by the excited state of RB. UV-VIS irradiation of RB, NBT, and 1-5 mM GSH reduced larger amounts of NBT. Moreover, SOD partially inhibited the NBT reduction under these conditions, indicating the formation of superoxide. The optimal pH for the formation of superoxide was 7-9. The superoxide formation during UV-VIS irradiation of RB and GSH was not inhibited by the singlet oxygen scavengers β-carotene, sodium azide, or 1,4-diaza-bicyclo[2.2.2]octane (DABCO), indicating t...

Photoinduced cutaneous inflammatory responses by erythrosine

AbstractErythrosine- (ERY) induced photosensitized immediate toxic reactions in rabbit skin were studied. Increase in vascular permeability (VP) and accumulation of polymorphonuclear leukocytes (PMNs) were quantitated using radiolabeled human serum albumin ([125I]HSA) and [51Cr] PMNs, respectively. ERY (10–400 nmol) injected intradermally and subsequently exposed to ultraviolet-visible radiation (320–700 nm) (UVA-VIS) for 2 hr produced skin edema in situ due to increase of VP with a dose-response relationship. This increase in VP was approximately 3.5 times when either 100 nmol of ERY or 10 nmol of rose bengal (RB) was injected separately, compared to the respective control sites (protected from UVA-VIS irradiation). The accumulation of PMNs at the sites injected with ERY and exposed to light also showed a dose-response relationship. β-Carotene (100 nmol), when injected intradermally in conjunction with ERY and exposed to light, reduced the leakage of plasma approximately 50% relative to sites without β-c...

Desensitization of rabbit skin by repeated exposure to UV-visible light of sites injected with Rose Bengal.

We have shown in a previous paper that irradiation of rabbit skin sites injected with Rose Bengal (RB) produces immediate increase in vascular permeability and accumulation of PMNs. Studies on the development of temporary tolerance and the biological parameters related to the development of such tolerant state by repeated exposure to light of RB-injected sites are reported here. The increase in VP and PMN migration induced by RB (10 nmol) are of an immediate nature, i.e., occur within the first 3 h of irradiation, and the reaction subsides gradually after 24 h. When such moderate insult is repeated, the skin becomes tolerant to subsequent exposure to light in the presence of RB. This tolerant state is temporary, i.e., the desensitized sites are fully recovered in 72 h. The loss of responsiveness of RB-injected sites previously exposed to light was not due to diffusion of the injected dye from the sites since reinjected sites also showed reduced response and the sites injected three days before but not irradiated showed normal response. The sites that were made tolerant to RB-induced phototoxic reactions, when injected with compound 48/80, an agent known to degranulate mast cells, did not show an increase in VP. This suggests that either the mast cells were depleted from the sites or the mast cells in the sites were rendered refractory by previous exposure to light. It was also found that the sites made tolerant to RB plus light were unresponsive to exogenously injected histamine. The sites tolerant to RB plus light when injected with zymosan-activated serum (ZAS) did not stimulate the migration of PMNs. This loss of chemotactic response to ZAS may have relevance to photodamage of vascular endothelium. These observations are discussed in relation to the development of the tolerant state by repeated exposures to subthreshold doses of light in solar urticaria.