The Photoreactions of 8‐methoxypsoralen With Tryptophan and Lens Proteins*

Abstract

Abstract— We have previously demonstrated that 8‐methoxypsoralen (8‐MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri‐midine bases and 8‐MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8‐MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8‐MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8‐MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8‐MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8‐MOP. These studies strongly suggest that 8‐MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.

Dye Binding and Photodynamic Action

“…However, no chemical reaction has been observed in the absence of oxygen. It has been found very recently that Trp and ocular lens proteins photoreact with 8-MOP in the presence of oxygen [59,77]. NMR studies showed that the hydrogen atoms at the 3,4 and 4 , 5' positions of 8-MOP and at the indole C2 position of Trp were lost in the photoreactions.…”

In vivo Lens Fluorescence Photography

“…It has been shown that UV radiation (longer than 300 nm) can play a role in the generation and enhancement of nontryptophan fluorescence of lens proteins [8-11, 13, 15]. Furthermore, 8-methoxypsoralen, a known UV photosensitizer (used in treating psoriasis), can become photobound to nucleic acids and proteins within the lens resulting in its accumulation and the enhancement of lenticular fluorescence and phosphorescence [7,14,16,19,20]. Thus, UV slit lamp densitography can be used to objectively monitor one parameter of lens aging (fluorescence), as well as photosen sitized lens damage at a molecular level years before visible opacities become manifest by conventional slit lamp examination.…”

Localization of 8-Methoxypsoralen in Ocular Tissues

“…One eye from each animal was utilized for the auto radiographic studies while the second eye was utilized for measuring the radioactivity in the various ocular tissues. In selected cases, the cornea and lens were subjected to phosphores cence and EPR spectroscopy at 77°K according to methods previously reported Lerman et al, , 1980a.…”

“…It is now generally agreed that UV radiation, particularly at wavelengths longer than 300 nm, can exert a direct photochemical action on the ocular lens both in humans and in experimental animals [Lerman, 1972[Lerman, , 1976[Lerman, , 1980Zigman and Vaughn, 1974;Augusteyn, 1975;Spector et al, 1975;Kurzel et al, 1977;Lerman and Borkman, 1978]; and, we 'This work was supported by NIH Grants EY01575 and AGO1309 and V.A.Con tract No. 9436-07. have demonstrated enhanced photobiologic damage by means of photo sensitized reactions occurring within this organ Lerman et al, , 1980aThis has led to increasing concern regarding potential ocular complications in patients who are being treated with 8-methoxypsoralen (8-MOP) for psoriasis. Utilizing phosphores cence and EPR spectroscopy we have demonstrated the presence of free 8-MOP in rat and dogfish ocular lenses following a single intraperitoneal injection of this drug and in the human lens following the ingestion of a single therapeutic dose Lerman et al, 1980a], In the absence of photic stimulation it has been shown that most of the free 8-MOP diffuses out of this organ within 24 h. Autoradio graphic studies have also demonstrated labeled 8-MOP in rat and dogfish retinas for up to 24 h when these animals are maintained in the dark [ Wulf and Hart, 1978;Lermanet al, 1979] and these studies have also indicated that in the absence of photic stimulation the drug tends to leave these tissues.…”

“…9436-07. have demonstrated enhanced photobiologic damage by means of photo sensitized reactions occurring within this organ Lerman et al, , 1980aThis has led to increasing concern regarding potential ocular complications in patients who are being treated with 8-methoxypsoralen (8-MOP) for psoriasis. Utilizing phosphores cence and EPR spectroscopy we have demonstrated the presence of free 8-MOP in rat and dogfish ocular lenses following a single intraperitoneal injection of this drug and in the human lens following the ingestion of a single therapeutic dose Lerman et al, 1980a], In the absence of photic stimulation it has been shown that most of the free 8-MOP diffuses out of this organ within 24 h. Autoradio graphic studies have also demonstrated labeled 8-MOP in rat and dogfish retinas for up to 24 h when these animals are maintained in the dark [ Wulf and Hart, 1978;Lermanet al, 1979] and these studies have also indicated that in the absence of photic stimulation the drug tends to leave these tissues. However, in the presence of photic stimuli (room light as well as direct UV radiation longer than 320 nm) the free 8-MOP in these tissues can be excited, resulting in the formation of nucleic acid and proteinbound photoproducts [Lerman et al, 1980a;Megaw et al, 1980], Since the ocular lens is completely encapsulated and never sheds its cells throughout life, photobound 8-MOP would be retained in this tissue and accumulate with repeated therapy.…”