Download Phototoxicity Mechanisms: Chlorpromazine

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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. The recognition t hat
each photo toxic compound can pote ntia lly dam age several different types of biological molecules is particulru'ly importa nt.
Also, t he effect of t he enviro nment of t he phototoxic compound
(i.e., association with substrate, solubility in lipid bilayer vs.
aq ueous solution) on its photose nsitizing mecha nism has been
established. This greater understanding should be applied to
cr eating better phototoxic compounds for photochemotherapy.
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3. Epstein JH, Brunsting LA, P eterso n MC, Sc hwarz BE: Study of
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4. Ki.rschba um BA, Beerma n H : P hotose nsitization due to drugs. Am
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10. Matsuo I, Ohkido M, Fujita H , S uzuki K: Chl orpromazine photosensitization: II. Failure to detect a ny evidence for invo lvement
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11. Ben-Hur E, Prager A, Green M, Rosentha l I: pH Depende nce of
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