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0022-202X / 81 / 770 1-0059$02.00/ 0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, Vol. 77, No.1 76:59-64, 1981 Printed in U.S.A . COPYl'ight © 198 1 by The Williams & Wilkins Co. Phototoxicity Mechanisms: Chlorpromazine Photosensitized Damage to DNA and Cell Membranes IRENE E. KOCHEVAR, PH,D. Department of Dermatology, Columbia University, New YOI'll, New York, U.S.A. Photosensitized damage to biological molecules is the initial process in phototoxic responses. It is now recognized that many phototoxic compounds can photosensitize damage to more than one type of biological substrate. The in vitro light-initiated reactions of photo toxic compounds with DNA, soluble proteins and membrane components can be classified by their molecular mechanisms: (1) those in which an excited state of the phototoxic compound (or an unstable species derived from it) reacts directly with the biological substrate and (2) those in which a molecule derived from the phototoxic compound (a photoproduct or an activated oxygen species) reacts with the biological substrate. This paper describes the mechanisms by which chlorpromazine photosensitizes damage to membranes, protein and DNA and compares them to the mechanisms of photosensitization by psoralens, porphyrins, dyes, and other molecules. Light-induced damage to cells which is initiated by a photosensitizing compound is called phototoxicity. This phenomenon is distinguished from photoallergy in man by the fact that phototoxicity does not depend on an allergic reaction [1]. Although t h e term, phototoxicity, has a negative connotation, therapies combining chemicals and ultraviolet light (photochemotherapies) are currently used, for example, as a treatment for psoriasis. The development of more sophisticated phototherapeutic agents requires a fuller understanding of the mechanisms by which phototoxic compounds react with biological substrates. Phototoxicity can be divided into 2 processes: the initial photosensitized damage to biological compounds in vivo and the subsequent biological response to this damage. The fu'st process includes light absorption by the chemical (the photosensitizer) and t he resulting chemical reactions involving biological molecules. The molecular mechanisms for photosensitized damage to biological compounds have been studied in detail for only a few phototoxic agents. Studies are usually performed in vitro to determine if a photo toxic compound photochemically changes isolated biological molecules and, if so, what the chemical processes and products are. A significant finding emerging from this work is that many phototoxic compounds photochemically react with more than one molecule in a biological system. For instance, chlorpromazine photochemically modifies DNA, RNA, cell membranes and soluble proteins. Photo biologists are currently attempting to determine which of the multiple pathways available to a phototoxic compound (as determined in in vitro studies) is actually responsible for the in vivo phototoxicity. The second process in phototoxicity, the biological response, has been measured many ways including inactivation of viruses, lethality to bacteria and mammalian cells, mutation frequency in bacteria and erythema in human skin. Phototoxic compounds are ofte'n classified by the cellular site This work was supported by National Institutes of Health Grants GM 28843 and ES01041. Reprint requests to: Irene E. Kochevar, Ph .D ., Department of Dermatology, Columbia University, 630 West 168th Street, New York, NY 10032. 59 which they appear to damage. An excellent early study using this approach [2] demonstrated a correlation between the site of localization of several phototoxic compounds in cells (as determined by fluorescence microscopy) and photoinduced damage to these sites. Another approach described in this paper is based on the molecular mechanisms of photosensitized damage. Using this classification scheme, the mechanism of the photochemical reaction is emphasized rather than the cellular site of photobiological damage. For instance, 2 phototoxic com pounds which both react with DNA can be differentiated by t heir reaction mechanisms. In this paper, the cunent knowledge concerning phototoxicity mechanisms will be summarized using this molecular mechanistic framework. An exhaustive review of the literature is not intended. Rather, the basic chemical mechanistic scheme will be described and applied to selected phototoxic compounds. Chlorpromazine phototoxicity will be considered in detail because this compound reacts with biological molecules by several mechanisms. The photoreactions of other prominent phototoxic compounds will also be discussed. PHOTOSENSITIZA TION MECHANISMS Photosensitized damage to biological molecules is initiated by the absorption of light energy by the phototoxic compound and the subsequent excited state processes. These primary events are briefly summarized in Fig 1. Absorption of a photon by a ground state molecule produces an excited state molecule. The excited singlet state, which is usually formed fIrst by this process, may convert into a triplet excited state. Molecules in their excited states exist for only a fraction of a second before losing e~ergy by I:eturning to the ground state or by undergoing a chemIcal reactIOn. The routes for returning to the ground state include transfering energy to another molecule and giving off the energy as heat 01' light. Singlet and triplet excited state molecules are capable of basically t he same chemical reactions: cis~trans isomerization, fragmentation, ionization, ~earrange ment and intermolecular reactions. It appears that fragmentation, ionization and intermolecular reactions are important in phototoxicity mechanisms, A "quantum yield" is associated with each process the excited state undergoes. It is a measure of the probability that the process will occur and is determined experimentally. In this discussion, molecular phototoxicity mechanisms will be divided into 2 catagories: (1) Direct mechanisms in which the excited state of the photosensitizer (or an unstable species derived from it) reacts directly with the biological substrate; (2) Indi.rect mechanisms in which another molecule (a photoproduct of the phototoxic compound or an activated oxygen species) reacts with t he biological substrate (Fig 2). Direct reactions include addition of t he phototoxic compound in an excited state to a biological molecule, fragmentation to form unstable radicals which then react with the substrate and photoionization to produce cation radicals and electrons which react with the biological substrate. Because of the very short lifetimes of the excited states and radicals in these reactions, the phototoxic compound must be complexed with or be very close to the biological molecule when it absorbs light. Indirect reactions include energy transfer from the triplet state of the photosensitizer to oxygen to form excited singlet state oxygen or the 60 Vol. 77, No.1 KOCHEVAR ~s~ ~N~CI I C H2CH2CH2N(C H~}2 CH LOR PROMAZINE e nnn \ s)---4\S;.>--\S/ 8-METHOXYPSORALEN f1T~ ~ CH CH CHzNHCH~ 2 GROUND STATE MOLECULE FIG 1. Basic processes in photochemistry. The ground state molecul e absorbs a photon (step a); t he excited singlet state returns to t he ground state by releasing heat or light (step b) or undergoes a chemical reaction (step c) or converts into an excited state (step d); t he excited triplet state releases heat or light (step e) or undergoes a chemical reaction (step fl. 0(- TERTHIENYL METHYLENE BLUE ACRIDINE ORANGE Br Br EOSIN Y PROTOPORPHYRIN FIG 3. Photo toxic compounds discussed FIG 2. A classification sch eme for phototoxicity mechanisms. After light a bsorption an excited state of the phototoxic compound (or an unstable reactive species) may interact with the biological substrate (Direct Mechanism). Alternatively, a toxic photoproduct or an active oxygen species may interact with the biological s ubstrate (Indirect Mechanism). The biological response to th e altered biological substrate is observed as phototoxicity. . formation of stable photoproducts from the phototoxic compound which t hen react with the biological substrate. Since the active species in these reactions exist long enough to diffuse in a cell before reacting, they may be created a short distance fro~ th eir biological target molecules. A feature of the indirect mechanism is that several sensitizers may cause the same ch e~ical change in the biological molecule if they all generate the same active species (i.e., singlet oxygen). Direct Reactions of Phototoxic Compo unds with Biological S ubstrates Direct reaction between the excited state of a Phototoxic compound and a biological substrate often results in formation of a covalent photoaddition product. In other direct reactions, unstable reactive species are formed by photoionization (electrons and cation radicals) or fragmentation (free radicals) which damage biological molecules. The evidence that certain phototoxic compounds photosensitize damage to biological molecules by direct reactions will now be discussed. z PROTRIPTYLlNE ~n text. Chlorpromazine: Chlorpromazine (Fig 3) is a major therapeutic drug for psychiatric patients. Clinical reports of cutaneous photosensitivity to chlorpromazine appeared shortly after its introduction in the 1950's [3,4]. Phototoxic responses to chlorpromazine have been elicited in red blood cells [5,6], skin [7-9], bacteria [10], mammalian cells [5,11,12], bacteriophage [13], and viruses [14]. Chlorpromazine is also photomutagenic [15,16]. The photoch emical mechanisms responsible for these biological responses have been the topic of considerable research. Chlorpromazine has an a bsorption maximum at 305 nm in aqueous solution and absorbs more strongly at shorter wavelengths which are not of photobiologic interest. The excited singlet state converts very efficiently to the triplet state [17]. Bioch emical dam age due to direct reactions of chlorpromazine appear to be feasible since chlorpromazine fragments to form radicals [18] (Equation la) and photoionizes to form radical cations and electrons (Equation Ib) [19,20] when irradiated. These unstable reactive species may react directly with cell components. + ~s~ ~N'JlACI CI. + . e 11,1 IIb l I R P hotoaddition of chlorpromazine to double-stranded DNA has been reported [21,22]. Interestingly, in vitro the efficiency of photoaddition to single-stranded DNA and RNA is higher than to double-stranded DNA [21]. The structures of the pho- July 1981 PHOTOTOXICITY MECHANISMS toaddition products have not been elucidated. Recent studies in our laboratory indicate that photoaddition of chlorpromazine to DNA is due to reaction of a stable photoproduct of chlorpromazine rather than to a direct reaction. Consequently, these results will be discussed below in the section on indirect reactions of chlorpromazine. The in vivo and in vitro photoaddition of chlorpromazine to protein is more efficient than to double-stranded DNA [11,22]. Damage to the tail fiber proteins rather than to DNA has been cited as the cause of photoinhibition of bacteriophage by chlorpromazine [10]. The detailed mechanisms for the reaction between protein and chlorpromazine have not been well studied. Photoaddition has been attributed to direct reactions of free radicals produced by photodechlorination of chlorpromazine [23] (Equation la). Chlorpromazine photosensitizes DNA chain scission in viruses [14] and mammalian cells [12]. A stable photoproduct of chlorpromazine is not responsible for the results of the latter experiments. Since chlorpromazine associates with DNA [24], direct reactions are feasible. For example, the photoionization of chlorpromazine may initiate the observed DNA chain scission. Chlorpromazine photoionizes more efficiently as the aqueous component of the environment is reduced [20]. Consequently, irradiation of chlorpromazine complexed with DNA should produce hydrated electrons which are known to produce species which cause DNA cleavage. The chlorpromazine cation radical left after electron loss is stable when intercalated with DNA [25]. The photomutagenic effects of chlorpromazine and the induction of DNA repair processes after irradiation of mammalian cells with DNA [26] appear to result from DNA damage. Whether these forms of damage are due to direct or indirect photochemical reactions has not been determined. Psoralens: The psoralens (furocoumarins, Fig 3) are well known for their applications in psoriasis phototherapy. Their phototoxic effect on cells appears to be due to formation of photoaddition products with DNA wruch occurs by a direct mechanism. The photochemistry and photo physics of psoralens with DNA have been extensively studied and recently reviewed [27]. Prior to irradiation, psoralens form noncovalent complexes with double-stranded DNA with binding constants wruch vary with the nature of the substituents (10- 3 to 10- 5 M for 4,5',8trimethylpsoralen and 4'-aminomethyl-4, 4,5',8-trimethylpsoralen) and are influenced by the ionic environment [28]. From flow linear dichroism measurements, Tjerneld, Norden, and Ljunggren [29] concluded that planar 8-methoxypsoralen molecules intercalate between pairs of bases in native doublestranded DNA. In general, the psoralens have a long wavelength absorption maximum in the 310-320 nm range with absorption extending to about 380 nm. The singlet state of 8-methoxypsoralen converts to the triplet state with a quantum yield of 0.14 [30]. A hypochromic shift occurs when 8-methoxypsoralen intercalates with double-stranded DNA [31]. Psoralens which are intercalated with DNA photochemically add to the pyrimidine bases to form covalent monoadducts (Equation 2a). I 0=occn'" °I '" (2. ) + h H 'O'Y~O '" 0 0 HN "" I o HNy"H o ~ °!~ N H (2b) 61 The triplet state is thought to be the reactive species based on the significant triplet quantum yield for psoralens and the observation that the photoaddition of 8-methoxypsoralen to thymine [32] and to calf thymus DNA [33] are quenched by oxygen and paramagnetic ions. However, the excited state involved in photoaddition may depend on the specific ful"Ocoumarin [34-36]. Dall' Aqua et al [37] have concluded that 8methoxypsoralen adds predominately tluough its 3,4 pyrone double-bond. Based on studies with low molecular weight compounds the structures of the monoadducts appear to be cyclobutyl adducts. However, the photoproducts formed between native DNA and psoralen have not been characterized in detail yet nor has the detailed mechanism been determined. Since, in general, triplet state molecules do not undergo concerted cycloaddition processes, a stepwise mechanism for the formation of the cyclobutyl ring is expected. Spectroscopic evidence [38] indicated that the interaction of the coumarin triplet excited state with thymine may be mainly charge transfer. This process may represent the first step in cyclobutyl adduct formation. The 4',5' monoadduct in DNA has been detected by flow linear dichroism [29] because it absorbs above 320 nm. The measurements indicated that it was not intercalated with the double-stranded DNA. The formation of crosslinks at wavelengths longer than 320 nm (Equation 2b) must also involve the 4',5' monoadduct since the 3,4 monoadduct does not absorb these wavelengths [38]. The photoaddition of psoralens to DNA can be limited to the formation of monoadducts by using monofunctional psoralens, limiting the length of the light pulse [39], or irradiating psoralen with DNA in frozen solution [40]. Although photochemical reactions between psoralens and DNA are usually thought to involve pyrimidine bases, evidence that psoralens add to some adenine bases in tRNA has been reported [41]. Alpha-terthienyl: Photoaddition of alpha-terthienyl, (Fig 3) to DNA in vivo and in vitro has been reported [42]. In contrast to the psoralens used in photochemotherapy onJy adducts involving a single DNA strand were detected. A direct mechanism may be involved since a photoproduct mixture from a-terthienyl did not covalently bind to DNA. Prior complexing of a-terthienyl and DNA may account for the 20 nm red shift of the action spectrum for photoadduct forma tion from the absorption maximum of a-terthieny\. Interestingly, phototoxicity in E. coli occurred onJy in the absence of oxygen although aterthienyl inactivates enzymes by a singlet oxygen mechanism [ 43,44]' Other phototoxic compounds: Direct reactions of excited states or short-lived intermediates derived from other phototoxic compounds with biological molecules have been reported. Triplet states of dyes, such as eosin and thionine, react directly with tryptophan by an electron transfer process [45]. SimiJru'ly, hematoporphyrin in aqueous solution or in micelles sensitizes the photooxidation of tryptophan by a direct mechanism when the amino acid is in sufficiently high concentration [46]. The photoinduced covalent binding of benzo(a)pyrene to superhelical DNA [47] and of anthracene to DNA in vivo [48] have been reported. The mechanisms may involve direct photochemical reactions since polycyclic aromatic hydrocarbons non-covalently bind to DNA in the dark. Indirect Reactions of Phototoxic Compounds with Biological Substrates Photosensitized damage to DNA, proteins and cell membranes often results from reactions with species other than the light absorbing phototoxic compound itself (Indirect reactions, Fig 2). This process may occur 2 ways. First, photoproducts of the light-absorbing compound may be the active agents in the phototoxicity responses to certain agents. For example, a photoproduct may covalently bond to the biological substrate to form an adduct. The second pathway in this general mecha nism involves formation of active oxygen species (singlet oxygen, 62 Vol. 77, No.1 KOCHEVAR superoxide anion, hydroxy radical). Interaction of these species with biological substrates produces phototoxidized molecules. This mechanism appears to be general and occurs with several types of photo toxic compounds. Which biological molecule is oxidized depends upon the location of the photo toxic compound, as initially observed by Allison, Magnus, and Young [2] and recently summarized by Ito [49]. Chlo/promazine: In addition to reacting by a direct mechanism as described above, chlorpromazine also photosensitizes damage to biological molecules by the 2 indirect mechanisms: photoproduct and active oxygen. Chlorpromazine photosensitizes red blood cell hemolysis [5,?0,6]. In contrast to other membrane photosensitizers, the lysIs produced by chlorpromazine did not require the presence of oxygen (Fig 4). In addition, lysis occurred when preirradiated chlorpromazine was added to the red cell suspension [5,6]. The stable, lysis-producing photoproduct did not require oxygen for its activity and did not cause lipid oxidation in the presence of ?xygen [6]. It appeared to be acting as a detergent and perturbmg the membrane structure to cause lysis. Preirradiated chlorp~omazine is toxic to macro phages [5]. In vivo studies by L]unggren and Moeller [9] paralleled these results; injection of preuTadiated chlorpromazine solutions into guinea pig skin produced a marked cutaneous response. These results provide eVIdence that chlorpromazine may act as a phototoxic compound by a nondirect mechanism involving a stable photoproduct. In contrast, stable chlorpromazine photoproducts are not responsible for the growth inhibition in mammalian cells observed after treatment with chlorpromazine and UVA [12] and preirradiated chlorpromazine was not toxic to E. coli [22]. These contrasting results may result from differences in the photoproduct mixtures tested or in sensitivity differences between red cell membranes and fibroblast membranes. We have recently investigated the molecular basis for the photoaddition reaction of chlorpromazine to DNA [51). Prior to irradiation, chlorpromazine associates with native DNA [24]. The association complex absorbs maximally at about 30 nm longer wavelength than chlorpromazine itself and no complex is detected with denatured DNA [51]. Kahn and Davis [21] reported that chlorpromazine photoreacted more efficiently with denatured DNA than with native DNA to form a covalently bound adduct. From these results it appears that chlorpromazine molecules which are not complexed to DNA are more reactive towards photoaddition than those that are complexed to the macromolecule. In addition, preuTadiated chlorpromazine also covalently added to DNA [51]. Thus, it appears that a pho~ochemical product formed from chlorpromazme molecules whIch are not associated .with DNA reacts with DNA to form a covalent adduct. The photoproducts of chlorpromazine depend upon its environment (Eqn 3). o f"yS~ + ~N~CI ~, f"YS~ ~N~CI DIMER + POLYMER (3) I, R fj(S~ ~~~OR R .H, ALKYL R'.CH,CHP-t,N(CH,J, R' In aqueous solution in the presence of oxygen chlorpromazine sulfoxide [19,52], chlorpromazine nitroxide [53], and unidentified photoproducts are formed. In the absence of oxygen, chlorpromazine dimers and polymer [54] and 2-hydroxychlorpromazine [23] are detected. In nucleophilic organic solvents, substitution of a solvent molecule for the chlorine occurs [18,23]. In isopropanol solvent, the chlorpromazine triplet generates singlet oxygen [18]. Chlorpromazine cation radical is formed in the presence of oxygen in aqueous solution and in the absence of oxygen in organic solvents [20] (Equation Ib) . The relative roles which these products (and others) play in the indu'ect photo toxicity mechanism of chlorpromazine remains to be established. Chlorpromazine also photosensitizes the oxidation of lipid bilayers [55]. The active oxygen species has not been characterized but singlet oxygen can be generated by photoexcitation of chlorpromazine [18]. Protriptyline: The phototoxic response to protriptyline (Fig 3) may be due to the formation of a toxic, stable product [6]. Pre irradiated protriptyline elicited red blood cell lysis without initiating lipid oxidation. The photoproducts formed in the absence of oxygen were more effective than those formed in the presence of oxygen at both red cell lysis and ability to elicit erythema in guinea pig skin ,[56]. However, when protriptyline and red cells were uTadiated together in the presence and absence of oxygen, the lysis rates were nearly identical. Consequently, it appears that protriptyline photosensitizes red blood cell lysis by both indirect mechanisms: stable photo product activity and generation of a n active oxygen species. Xanthene, Thiazine and Acridine dyes: Many members of these families of dyes have been used as photosensitizers to oxidize organic substrate molecules. In fact, the concept of "photodynamic action," which is the term originally used for photosensitized processes which require oxygen, was established using dyes and porphyrins as photosensitizers. The chemical structures of the dye molecules usually contain !001,---------~~~&---~----------~~--__. 3 fused rings (Fig 3). Rose Bengal and eosin are dyes based on a xanthene ring system. The thiazine dyes (thionine, m ethylene B blue, toluidine blue and others) are based on a 3 ring system 80 containing sulfur and nitrogen in the central ring. The acridinebased dyes include acridine orange, proflavin and acriflavin. The dyes absorb in the visible region and sensitize photooxi~60 dation from theu' triplet excited states. en >Two photooxidation mechanisms (Type I and Type II) have c5 . been described and applied to biological molecules [57-59]. The ::E major pathway followed for photosensitized oxidation depends ~40 upon the dye, the substrate, the oxygen concentration and the environment. In type I photooxidation, the excited triplet state of the sensitizer reacts with the substrate by either electron or 20 hydrogen transfer (Equation 4). Thus, type I photooxidation is classified as a direct phototoxicity mechanism. Type II phoO~~__~~~L-~~~~~~~~~ tooxidation usually involves energy transfer to oxygen to form singlet oxygen (Equation 5). The reactions of singlet oxygen o 2345 12 3 4 5 IRRADIATION TIME, HR with biological molecules in simple systems has been studied in detail. The major processes are photooxidation of guanine bases FIG 4. Photohemolysis of red blood cells in t he prese nce (0 ) a nd absence (e) of oxygen sensitized by (A) chlorpromazine, 5 x 10-" M in DNA, the photooxidative loss of histidine, methionine, trypa nd (E) protoporphyrin, 1 x 10- <; M . From [6]. tophan, tyrosine and cysteine in proteins and formation of PHOTOTOXICITY MECHANISMS July 1981 hydroperoxides with unsatw·at.ed lipids. Which of t hese processes actually occurs in vivo depends upon the cellular location of the dye [49]. The xa nthe ne dyes, Eosin Y a nd Rose Bengal, w hich penetrate yeast cells but do no t bind to DNA appear to photoinactivate by a s inglet oxygen mecha nism without causing DNA damage [60]. TyPe I 63 such as e nzym e inactivation [70,71 ], do not appear to be related to lipid oxidation. Whether these effects are important in t he phototoxic response in vivo is no t known . In ma mmalian cells, water soluble porphyrins photose nsitized plasma membra ne protein crosslinks which were associated with a decrease in cell swface hydrop hobicity a nd wit h other alterations in vital membrane properties [72]. hyd r ogen 35 + sens iti ze r tripl e t RH subst rate --- CONCLUSIONS t rnnsfer ' Sf! + R' (4) elec tron transfer ts + -:RH TyPe II 35 ene rgy tr ansfe r + O2 5 + 102 (5) s inglet o xyg en T h e thiazine dyes (thionine, toluidine blue) remain mainly outside the cells a nd ap parently cause photoinactivation mainly by singlet oxygen damage to membrane components. In vivo photosensitization of gene conversion in yeast by ac ridine orange also is m a inly Type II even t hough the photosensitizer binds to DNA which is expected to favor a T ype I m echanism [60]. In fact, acridine orange forms 2 complexes with DNA, an intercalation complex a nd an aggregated complex in the outside of the DNA double h elix. The 2 complexes have different absorption spectra a nd photosensitize oxidation with different c ontributions from T yp e I and T ype II mechanisms. A dye which is located in 2 different environments can have different reactivities. For example, Bagchi and Basu [61] found t hat photodyna mic inactivation by acriflavine which was outs ide the cell made a negligible contribution compared to t he dye which had penetrated. In another example of the influence of environment, t he photosensitizing a bility of 9 xanthene dyes toward membrane damage was studied [62]. The partitioning of the dye into the membrane largely determined the dyes' reactivity in contrast to the situation in aqueous solution where t h e triplet quantum yield is the domina nt factor [63]. Porphyrins: In the presence of oxygen porphyrins photosens itize damage to cell membranes, DNA a nd soluble proteins. T he most extensively studied porphyrins ru'e protoporpnryin IX, because of its role as a skin photosensitizer in patients with erythropoietic protoporphyria, a nd h ematoporphyrin, because of its use as a tumor photochemothera peutic drug. Protoporp hyrin IX is lipid soluble whereas h ematoporphyrin is water s oluble. Consequently, different biological substrates are available to these 2 porphyrins. Porphyrins have an absorption maximum in t he 400-420 nm range (the Soret band) and a lower excitation maximum in the 500-600 nm ra nge. The initially formed excited singlet state is s hort lived and the qua ntum yield for conversion to the longerlived triplet is generally high. The phototoxic effects of porphyrins result from triplet state processes such as energy t ra nsfer to oxygen molecules to form singlet oxygen (Ty pe II photooxidation). An example of Type I photooxidation (a direct reactio n ) has been discussed above [46]. The mechanism for cell membrane disruptio n photosensitized by porphyrins has been extensively studied using red b lood cells [64,65]. Oxygen is required for protoporphyrin photosensitized red cell lysis (Fig 4). The sequence of events on a molecular level leading to red cell lysis involves singlet oxygen forme d by energy transfer from protoporphyrin triplet in th e membra nes and oxidatio n of unsaturated lipids [66,67]. Photosensitization of cell lysis by protoporphyrin also induces extensive membra ne protein crosslinking [68]. However, protein crosslinking alone does not cause lysis of resealed red blood cell ghosts [69]. Oth er effects of protoporphyrin photosensitization, Recent research concerning phototoxicity m echa nisms has produced a better understanding of t he photoch emical events result ing in damage to biological molecules. With t his new knowled ge, it is possible to classify in vitro photochemical events using a mechanistic framework. 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