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.

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.

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.

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.

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.

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.

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.

Phototoxic effects of 8-methoxypsoralen on rabbit corneal endothelium.

The phototoxic effects of 8-methoxypsoralen (8-MOP) were investigated using the rabbit corneal endothelium in organ culture. The corneas were divided into four groups: (a) irradiated with a mercury vapor lamp (emitting UVA and visible radiation) in the presence of 8-MOP (experimental), (b) irradiated without 8-MOP (control A), (c) incubated with 8-MOP (control B) and (d) incubated without 8-MOP (control C). Specular and light microscopic examination showed that the experimental corneas had greater cellular damage compared to the control corneas. The effects of 8-MOP were restricted to certain localized areas of the cornea. However there was no significant difference in the amounts of 51Cr released from the labelled experimental and control corneas. These results show phototoxic damage of the corneal endothelial cells.

Binding of timolol to iris-ciliary body and melanin: an in vitro model for assessing the kinetics and efficacy of long-acting antiglaucoma drugs.

Topical beta blockers are used to treat glaucoma patients. These drugs inhibit aqueous production for prolonged periods of time. The purpose of this study was to determine whether timolol maleate (a non-specific beta blocker) binds to human iris-ciliary body (CB) melanin and to elucidate the binding characteristics of the drug to melanin. Timolol bound to bovine iris and ciliary body by two possible mechanisms. The binding kinetics indicate that the binding is probably of a nonspecific nature. There was no statistically significant differences between the melanotic tissues (CB, iris) and the nonmelanotic tissues (lens, cornea, liver, kidney) regarding the amount of timolol bound. However significantly more timolol was bound to the isolated melanins than the whole tissues. Timolol was released from the nonmelanotic tissues at a much faster rate than from the melanotic tissues. The amount of timolol bound to iris-CB from albino and pigmented rabbits showed that the amount of timolol bound to these tissues diminished in the following order: black or gray greater than brown greater than albino. It was also found that the rate of timolol release decreased in the following order: albino greater than gray greater than brown or black. Our results demonstrate the binding of beta blocker to human, bovine and rabbit iris-CB and consequent slow release of timolol from these tissues.

Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins.

Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.