Endogenous porphyrins in murine skin and transplanted PAM-212 squamous cell carcinoma tissues after injection of delta-aminolevulinic acid.

After intraperitoneal (IP) injection of delta-aminolevulinic acid (ALA), the endogenous porphyrins in murine skin and tumor tissues were determined by a method involving solvent and acid extractions. The results showed that the total amount of porphyrins in the tumor tissues after ALA injection was much higher than that in the skin from the same mice, although the amount of porphyrins in the skin from the ALA-injected mice was higher than that from the saline-injected (control) mice. The porphyrins in the tumor were mostly protoporphyrin and coproporphyrin, with only a small amount of uroporphyrin. The optimum period for porphyrin accumulation in the tumor as well as in the skin was 1 hour after the injection of ALA. As the period was extended to 3 and 6 hours, the amount of porphyrins in these tissues decreased considerably. These findings could be valuable for further application of ALA in the photodynamic therapy of skin cancer.

Depot drug delivery system for 5-fluorouracil after filtration surgery in the rabbit.

We investigated the potential value of 50:50 poly (DL glycolic acid-lactic acid) (PGLA) copolymer as a degradable depot delivery system for 5-fluorouracil (5-FU) after filtration surgery. Analysis of retrieved discs after implantation in 22 eyes of 22 pigmented rabbits showed a dual drug release profile and polymer mass loss characteristics. In a second group of pigmented rabbits implantation of PGLA discs impregnated with 5-FU (22 eyes) significantly lengthened the survival time of filtration fistulae compared with discs without 5-FU (18 eyes) or no disc (10 eyes) (p < 0.0001). Use of PGLA copolymer impregnated with 5-FU could prove valuable for patients undergoing glaucoma filtering surgery.

Ocular timolol levels after drug withdrawal: an experimental model.

Carbon 14-labelled timolol maleate was instilled into both eyes of 12 pigmented rabbits daily for 42 days. Drug levels in the aqueous humour and ocular tissues were measured up to 42 days after drug withdrawal. The results indicate that timolol concentrates mainly in melanotic tissues, with slow release. Even 42 days after withdrawal the drug was still present in pigmented ocular tissues. Timolol was detected in the aqueous up to 5 days after withdrawal. These findings explain the long-term depressant effect of topically administered timolol on aqueous production. We conclude that lower or less frequent doses of timolol should be considered in patients with glaucoma.

Fifty:fifty poly (DL glycolic acid-lactic acid) copolymer as a drug delivery system for 5-fluorouracil: a histopathological evaluation.

Fifty:fifty poly (DL glycolic acid-lactic acid) copolymer (PGLA) is a potentially useful depot drug delivery system for 5-fluorouracil (5-FU). The purpose of this study was to evaluate the fibroinflammatory reaction induced by this polymer. Polymer discs without 5-FU were inserted subconjunctivally in one eye of each of two guinea pigs and four pigmented rabbits (control group), and discs containing 20% 5-FU were inserted subconjunctivally in both eyes of nine pigmented rabbits (study group). The tissue reaction to the copolymer did not differ between rabbits and guinea pigs, with a mild mixed inflammatory reaction 1 week after implantation. At 2 weeks a thin fibrous capsule surrounded the discs, with no change in the amount of inflammation. At 4 weeks the disc had disintegrated, but residual polymer was seen within multinucleated giant cells in the episcleral tissue. Granulation tissue and inflammatory responses were mild. Less inflammation and fibrosis occurred in the study eyes, although the pattern of response was similar in the two groups. The inflammatory response to PGLA was markedly less than that to implanted collagen shields, and our findings suggest that PGLA implant is a promising ocular drug delivery system for 5-FU after filtration surgery.

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.

Quantitative determination of the melanin contents in ocular tissues from human blue and brown eyes.

This paper deals with our findings on the quantities of melanin in the tissues from blue and brown eyes. The amount of melanin in the iris, ciliary body and retinal pigment epithelium-choroid was separately determined. The results are expressed as the amount of melanin in mg tissue as well as the amount of melanin in the whole tissue. The results showed that there was no statistically significant difference between the melanin content of the iris in blue and brown eyes. However the ciliary body and retinal pigment epithelium-choroid from brown eyes had more melanin than the corresponding tissues from blue eyes. Blue and brown eyes with higher colour intensity had more melanin than the corresponding eyes with lesser intensity of colour. It is suggested that the differences between brown and blue eyes in their melanin content may have relevance to the pharmacokinetics of drugs that bind to melanin. This would mean that the larger amounts of melanin would decrease the initial levels of the drugs and would increase the drug levels after prolonged periods.

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.

Effects of hydrogen peroxide-induced oxidative damage on outflow facility and washout in pig eyes.

Previous studies show that hydrogen peroxide (H2O2) is present in the aqueous humor of many species and is capable of affecting outflow facility in animal model experiments. To study the hypothesis that oxidative damage to the outflow pathway may play a role in the pathogenesis of primary open-angle glaucoma, 3 mM H2O2 with 20 mM 3-aminotriazole and 1 mM carmustine (BCNU) in Dulbecco's phosphate-buffered saline (DPBS) was perfused into enucleated pig eyes at constant pressure. Baseline and experimental perfusions were done at two different pressures (7.5 and 30 mm Hg) to study the effect of pressure on the response to oxidative damage. Outflow facility in the baseline experiments (with DPBS only) was observed to increase nonlinearly with time during the perfusions, but could be linearized if plotted as a function of the volume perfused. Thus, a term "volumetric washout" (W) was introduced and defined as the fractional rate of change of outflow facility with respect to the volume perfused. This quantity was found to be independent of pressure in the baseline studies. Perfusion of H2O2 and inhibitors increased W at 7.5 mm Hg but decreased W at 30 mm Hg. These results indicate that oxidative damage increases outflow facility at normal pressure but decreases it at elevated pressure, suggesting that elevated pressure may increase the susceptibility of the outflow pathway to this form of insult.

Further evaluation of collagen shields as a delivery system for 5-fluorouracil: histopathological observations.

Collagen shields are a potential delivery system for antifibroblast drugs such as 5-fluorouracil after filtration surgery. To determine whether collagen shields produce histologic evidence of inflammation when implanted subconjunctivally, shields were implanted into four rabbit eyes and six guinea pig eyes and retained for 7 or 14 days. Two rabbit eyes and two guinea pig eyes served as controls. Seven days after implantation in the rabbit eyes foreign-body giant cells were present at the surface of the shield, and early deposition of connective tissue was evident around the shield. The inflammatory response at 14 days was similar but more intense. In the guinea pig eyes the collagen shields induced substantially less inflammation, and there was marked shield degradation at 14 days. The results suggest that the inflammatory response in rabbits may be species specific and that collagen shields may be of value as a drug-delivery system for antifibroblast drugs in other species.

Characterization of the pigment from homogentisic acid and urine and tissue from an alkaptonuria patient.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.

Potential value of collagen shields as a subconjunctival depot release system.

Collagen shields are fabricated from dissoluable porcine scleral tissue and have been used as an ocular drug delivery system. The aim of the present study was to determine the time and extent of shield absorption when implanted subconjunctivally, and the absorption and release of 5-fluorouracil in vitro. Thirty New Zealand white rabbit eyes were employed. BioCor 72 hour collagen shields were surgically implanted in the subconjunctival space. Rabbits were sacrificed at 7, 14 and 21 days after shield implantation, and the remaining shields removed. Remaining shields were measured by both dry weight and protein assay. The absorption and release of 5-FU from collagen shields was determined in vitro using tritiated 5-FU. The collagen shields were not fully absorbed for at least 14 days in the subconjunctival space. In vitro, 5-FU absorbed by the shields reached saturation levels at approximately 15 minutes. Nearly 100% of the 5-FU was released within 15 minutes. Although the time for subconjunctival shield absorption may be useful for antifibroblast drugs, the rate of 5-FU release from these shields is not optimal for enhancing bleb formation when shields are soaked in solutions of 5-FU.

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.

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 photosensitizer 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 using 125I-labeled albumin and 51Cr-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. beta-Carotene (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 25 micrograms 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.

Nature of the oxygen species generated by xanthine oxidase involved in secretory histamine release from mast cells.

The present studies were carried out to characterize the nature of reactive oxygen species generated by the xanthine-xanthine oxidase system involved in the release of histamine by noncytotoxic and cytotoxic mechanisms. To distinguish secretory release from lytic release, mast cells were loaded with 51Cr and the release of 51Cr into the incubation medium was used as a measure of cell lysis. The secretory release of histamine was not inhibited by superoxide dismutase or catalase alone. However, together these agents inhibited the release. This suggests that the combination of superoxide and hydrogen peroxide can evoke secretory release. The lytic release of histamine, as monitored by concomitant release of 51Cr from mast cells at higher concentration of xanthine oxidase or longer periods of incubation, seems to be related to hydrogen peroxide production since catalase inhibited the cell lysis. Since it has been reported that exogenously added hydrogen peroxide at concentrations below 10 mM did not induce cell lysis, the lytic release, although hydrogen peroxide dependent, may not be due to its direct effect on the cell surface. The cell lysis observed in the xanthine-xanthine oxidase system seems to be brought about by a complex mechanism involving the interactions of hydrogen peroxide and superoxide with cellular components. These studies indicate that the beneficial effects of superoxide dismutase seen in biological systems may partly be due to inhibition of the secretory processes stimulated by superoxide.

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.

High resolution scanning electron microscopy of rabbit corneal endothelium to show effects of UV-visible irradiation in the presence of chlorpromazine.

The ultrastructure of rabbit cornea endothelial cells was examined by scanning electron microscopy (SEM) in freeze-cleaved corneas using a Hitachi S-570 scanning electron microscope in the high resolution mode (HRSEM). In order to study phototoxic effects in vitro, rabbit corneas (experimental) were cultured as organ culture in the presence of 5 micrograms/ml chlorpromazine (CPZ) and irradiated. For comparison, control 1 corneas were not irradiated but incubated in the dark without CPZ in the medium; control 2 corneas were also kept in the dark but in the presence of CPZ; control 3 corneas were irradiated with no CPZ in the medium. Cellular damage was not seen in the three types of control corneas, but in the experimental corneas the endothelial cells showed extensive disruption of the cell membrane and some deterioration of the intracellular components. Our study confirmed that HRSEM is a satisfactory new technique for visualizing damage of the intracellular organelles of corneal endothelium.

Binding of chlorpromazine to cultured retinal pigment epithelial cells loaded with melanin.

The accumulation of chlorpromazine (CPZ) in cultured bovine amelanotic retinal pigment epithelial (RPE) cells artificially loaded with melanin was investigated. The melanin was isolated from human eye bank eyes. Suspensions of the melanin were added to the RPE cells and incubated for 3 hrs. The cells ingested the melanin which was dispersed in the cytoplasm of the cells. They were not adhering to the cell membrane. The melanin-loaded cells grew in culture, although their rate of growth was slower than that of the control RPE cells not loaded with melanin. When the melanin-loaded cells were treated with CPZ, these cells accumulated a greater amount of CPZ than the control cells. A greater amount of CPZ was released from the melanin-loaded cells than from the control cells. The results suggest that some drugs or chemicals such as CPZ could accumulate in vivo in larger quantities and for longer periods in melanotic cells than in nonmelanotic cells. These compounds may subsequently be released into the extracellular fluid, thus affecting the neighbouring cells. This phenomenon may play an important role in the activities of these drugs in the melanotic cells and in the cells adjacent to the melanotic cells. These results suggested that cultured cells loaded with melanin could be used as a suitable model for studying the mechanisms of binding of drugs to intracellular melanin, and of their subsequent release outside the cells.