mitochondrial toxicity of cationic photosensitizers for photochemotherapy

[CANCER RESEARCH 50, 7876-7881, December 15, 1990]

Mitochondrial Toxicity of Cationic Photosensitizers for Photochemotherapy1

Josephine S. Modica-Napolitano,2 John L. .loyal, Gulshan Ara,3 Allan R. Oseroff,4 and June R. Aprille

Mitochondrial Physiology Unit, Department of Biology, Tufts University, Medford, Massachusetts 02155 fJ. S. M-N., J. L. J., J. R. A.], and Department of Dermatology,Tufts-New England Medical Center, Boston, Massachusetts 02111 Â¡G.A., A. OJ

ABSTRACT

The triarylmethane derivative Victoria Blue-BO (VB-BO) and thechalcogenapyrylium (CP) dyes have potential for use in pholochemolher-apy, because they are taken up by the mitochondria of malignant cellsand cause cell death. To clarify the mechanism of cell killing we examinedthe phototoxic effects of VB-BO and a series of three CP dyes onbioenergetic function in isolated rat liver mitochondria. Without photoirradiation, and irrespective of the respiratory substrate used, each of thecompounds tested induced some uncoupling of oxidative phosphorylation.Visible irradiation of VB-BO produced an inhibition of mitochondria!respiration when glutamate plus malate, but not succinate, was used asthe respiratory substrate. With photoirradiation VB-BO was also shownto inhibit rotenone-sensitive NADH-cytochrome c reducÃaseactivity, butit had no effect on succinate-cytochrome c reducÃaseactivity. These dataindicate that photoactivation of VB-BO produces selective inhibition ofmitochondria! respiratory complex I. Photoirradiation of the CP dyesinhibited both complex I and complex II initiated respiratory activity.With photoirradiation, the CP dyes also inhibited both NADH- andsuccinate-cytochrome c reducÃaseactivilies, as well as olher membrane-bound enzymes, cylochrome c oxidase and succinale dehydrogenase, bulnoi Ihe milochondrial malrix enzyme, citrate synthelase, or the cylosolicenzyme, lÃ¡claledehydrogenase. a-Tocopherol prolecled bioenergelic ac-livilies againsl CP dye photodamage. These results suggesl thai milo-chondrial pholosensilizalion by CP compounds is medialed by Ihe production of membrane-damaging singlel oxygen which causes nonspecificdamage lo membranes and membrane-bound enzymes.

INTRODUCTION

There recently has been considerable interest in photochemo-therapy as a form of treatment for neoplasms of the skin, lung,breast, bladder, and brain (1-6). Cationic photosensitizers areparticularly promising since they are selectively accumulated bymalignant cells and, in combination with photoirradiation,provide a means of highly specific cell killing (4-6). Due totheir positive charge and lipophilicity, these compounds arelocalized in mitochondria in response to a negative-inside membrane potential (7, 8), and there is evidence to suggest that themitochondrion may be a primary subcellular site of damage (9).

The cationic triarylmethane derivative VB-BO5 and a series

of three CP dyes containing selenium/sulfur, selenium/oxygen,and selenium/selenium have been examined for phototoxicityin a variety of normal and carcinoma cell lines, with resultsshowing selective malignant cell killing both in vitro and in vivo(10, II).6 The purpose of this study was to investigate the effect

of these compounds on mitochondrial bioenergetic function,

Received 7/6/90; accepted 9/18/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by NIH Grants CA-45767 and CA-44205 and American CancerSociety Grant CH-400 (A. R. O.).

2To whom requests for reprints should be addressed.3Present address: Dana-Farber Cancer Institute, Jimmy Fund Building, Rm.

519, 44BinneySt., Boston, MA 02115.4 Present address: Department of Dermatology, Roswell Park Cancer Institute,

Elm and Carlton Sts., Buffalo, NY 14263.*The abbreviations used are: VB-BO, Victoria Blue-BO; CP, chalcogenapyryli

um; DCIP, 2,6-dichlorophenolindophenol; 2.4-DNP, 2,4-dinitrophenol; EDKC,JV.A"-bis(2-ethyl-l,3-dioxylene)kryptocyanine.

* Unpublished data.

with and without photoirradiation, in order to determine potential mechanism(s) of photocytotoxicity.

MATERIALS AND METHODS

Materials. VB-BO (Fig. 1) was purchased from Aldrich Chemicals(Milwaukee, WI) and used without further purification. The CP dyesselenium/selenium, selenium/sulfur, and selenium/oxygen (Fig. 2) weresynlhesized by Michael R. Detly (Easlman Kodak, Rochester, NY).The dyes were prepared al slock concentrations between 10 and 50 ftM.VB-BO slocks were dissolved in 100% elhanol, wilh Ihe final concentration of ethanol ranging from 0 to 2% (v/v) in any given assay. TheCP compounds were dissolved in phosphate buffered saline (2.7 mMKC1-1.5 mM KH2PO4-140 mivi NaCl-4.3 mivi Na2HPO4, pH 7.0).Vitamin E (a-tocopherol; Sigma Chemical Co., St. Louis, MO) wasdissolved in 100% ethanol.

Isolation of Mitochondria. Liver mitochondria were isolated frommale CD-I Sprague Dawley rats by differential centrifugaron essentially as described previously (12). Approximately 5 g of tissue wereminced and homogenized in 250 mM sucrose-1 mM Tris-HCl-1 mMEDTA (pH 7.4) and then centrifuged at 600 x g for 10 min at 4Â°C.The supernatant was centrifuged at 8000 x g for 10 min at 4Â°C.The

mitochondrial pellet was washed twice in 250 mM sucrose-1 mM Tris-HCI l mM EDTA (pH 7.4) and once in 250 mM sucrose-1 mM Tris-HC1 (pH 7.4) and then resuspended in 250 mM sucrose-1 mM Tris-HCl(pH 7.4). Protein concentration was determined by the method ofLowry el al. (13), using bovine serum albumin as a standard.

Respiration. Mitochondrial respiration was measured polarographi-cally using a Clark oxygen electrode inserted into a 1-ml water-jacketedchamber maintained at 30Â°C(14). The assay medium consisted of 225

mM sucrose, 10 mM potassium phosphate (KH2PO4/K2HPO4), 5 mMMgCl, 10 mM KC1, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Aninitial rate of oxygen consumption was recorded following the addilionof a substrate, eilher glutamale plus malate (5 mM each) or 10 mMsuccinate, and a stale 3 rate was recorded following the addilion of 100nmol of ADP. After a measurable slale 4 rale (i.e., the rate after ADPis phosphorylated) was obtained, an 80 uM concenlration of Ihe uncoupling agenl 2,4-DNP was added to obtain a rate of oxygen consumptionin the absence of coupled oxidative phosphorylation. To tesi for dyetoxicity an appropriate concentration of VB-BO, selenium/selenium,selenium/sulfur, or selenium/oxygen was introduced prior lo Ihe addilion of substrate. The dye was thus present continuously in the assaysas the initial, state 3, stale 4, and uncoupled rates were successivelydetermined.

Enzyme Assays. Succinale-cytochrome c reducÃaseaclivity was determined spectrophotometrically at 550 nm in a 1-ml volume by monitoring the increase in absorbance over time due to the reduction ofcytochrome c (15). An aliquot (20 ^g prolein) of freeze-lhawed milo-chondria was added lo 2 mM KCN, 50 mM potassium phosphate(KH2PO4/K2HPO4) (pH 7.4), and 20 mM succinate. The reaction wasinitiated with the addition of 1 mg oxidized cytochrome c. Five ^ig ofantimycin A were added to terminale Ihe assay and oblain a background(blank) rale.

Rolenone-sensilive NADH-cytochrome c reducÃaseaclivily was alsomeasured by monitoring the reduction of cytochrome c at 550 nm (16).An aliquot (20 Â¿tgprotein) of freeze-thawed mitochondria was added to2 mM KCN, 25 mM potassium phosphate (KH2PO4/K2HPO4) (pH 7.4),and 0.2 mM NADH. Oxidized cytochrome c(i mg) was added to beginthe reaction. After a measurable linear rate was observed, 2.5 ^Mrotenone was inlroduced lo Ihe assay to obtain a rotenone-insensiliverate. The rotenone-sensitive NADH-cytochrome c reducÃaserale was

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MITOCHONDRIA!. TOXICITY OF CATIONIC PHOTOSENSITIZERS

calculated by subtracting the rotenone-insensitive rate from the overall

rate.Cytochrome c oxidase activity was determined spectrophotometri-

cally at 550 nm, by monitoring the oxidation of cytochrome c (17). Thereaction was initiated by adding freeze-thawed mitochondria (3 Mgprotein) to a 1-ml assay containing 40 mivi potassium phosphate(KH2PO4/K2HPOâ€ž)(pH 7.4), and 0.7 mg reduced cytochrome c.

Succinate dehydrogenase activity was measured spectrophotometri-cally at 600 nm, by monitoring the reduction of the artificial electronacceptor, DCIP (18). The 1-ml assay volume contained freeze-thawedmitochondria (3 /ig protein), 50 mM potassium phosphate (KH2PO4/K2HPO4) (pH 7.4), 2 mM K.CN, 20 mM succinate, and 2 HIMphenazinemethosulfate. The reaction was initiated with the addition of 0.1 mMDCIP.

Activity of citrate synthetase was recorded spectrophotometrically at412 nm (19). A background rate was obtained by adding freeze-thawedmitochondria (20 ^g protein) to 0.1 mM 5,5'-dithiobis-2-nitrobenzoate

and 0.3 mM acetyl-CoA. This initial rate was subtracted from the rateobtained upon the addition of the substrate 0.1 mM oxaloacetate.

AH enzyme assays were performed at 37Â°C,except citrate synthetasewhich was performed at 25Â°C.

Photoirradiation. Photoirradiation was accomplished by using a Kodak slide projector fitted with a Corion 600 nm long pass filter,continuously delivering 25 mW/cm2 of 600-750 nm light for a periodof 5 min (7.5 J/cm2). Irradiation of the compounds was performed inthe presence of intact mitochondria or freeze-thawed mitochondria!fragments in the appropriate preincubated assay mixture and desireddye concentration. Following photoirradiation, these contents werethen transferred to cuvettes for enzyme analysis or to the polarographchamber to measure respiration, where the final addition of the initiating substrate for the particular assay was made. When vitamin E wastested for a protective effect, it was included during the irradiation step.

RESULTS

Mitochondria! Respiration. Polarographic measurements ofrespiratory activity in isolated rat liver mitochondria were madein the presence of VB-BO. Without photoirradiation, and usingglutamate plus malate as the respiratory substrate, VB-BOacted as an uncoupler of oxidative phosphorylation, exhibitinghalf-maximal uncoupling at approximately 0.3 Â¿Â¿M(Fig. 3A).The state 3 respiratory rate (the rate after addition of ADP)remained constant over a concentration range of dye in whichthe initial rate (before ADP) and state 4 rate (the rate after alladded ADP is consumed) steadily increased. Thus, the ADP-stimulated rate (state 3 rate minus initial rate) decreased inproportion to the increase in the initial rate. The inhibition of

VB-BO

(CH3CH2)2N NHCH2CH3

nme =5.0 x KT/M/cm

Fig. 1. Chemical Structure of VB-BO.

t-Bu Se/O

O >CH â€”CH=CH

t-Bu X m=660 nm t-Buâ‚¬=2.0x105/M/cm

Se/St-Bu

S >=CHâ€”CH=CH

t-Bu t-Buâ‚¬=2.0x 1Cr/M/cm

Se/Set-Bu ci- t-Bu

Se \=CH-CH = CH + Se

t-Bu\m=730 nm

e =2.0 x 105/M/cm

t-Bu

Fig. 2. Chemical structures of the chalcogenapyrylium compounds.

the 2,4-DNP-uncoupled rate was most likely due to an excessof uncoupling agents (dye plus 2,4-DNP) in the assay, whichdisrupts bilayer integrity and probably did not represent specificinhibition by the dye. With succinate as the substrate, the samepattern of respiratory uncoupling was obtained as with glutamate plus malate (data not shown). These data show thatwithout photoirradiation, VB-BO acts as a modest uncoupler.

When photoirradiated, and using glutamate plus malate as arespiratory substrate, VB-BO inhibited state 3 and uncoupledrespiratory rates in a dose-dependent manner, exhibiting 50%inhibition at approximately 0.3 and 0.35 ^M, respectively (Fig.3Ã„).A decrease in the ADP-stimulated rate (state 3 rate minusinitial rate) was attributable to the decrease in the state 3respiratory rate and a slight increase in the initial rate. Withsuccinate as the substrate, VB-BO did not inhibit respiration(data not shown). These data suggest that upon photoirradia-tion, VB-BO acts selectively as an inhibitor of mitochondrialrespiratory complex I.

The effect of the CP compounds on respiration was investigated similarly. Without photoirradiation, these dyes acted as

7877




150

0.0 0.2 0.4

DYE CONC. (uM)

150

0.6

B

0.0 0.2 04

OYE CONC. (uM)

0.6

Fig. 3. Effect of VB-BO on mitochondria! respiration using glutamate plusmalate as a respirator}' substrate. A is without, and /' is with photoirradiation(with 7.5 J/cm2, 600-750 nm light). Initial rate (LU)is that obtained following theaddition of substrate and prior to the addition of ADP; state 3 (*) is the rateobtained upon addition of ADP; ADP-stimulated rate ID) is calculated as thedifference between the state 3 and the initial rate (i.e., the rate attributed solelyto the addition of ADP); and the uncoupled rate (O) is obtained after the additionof 2,4-dinitrophenol, an uncouplerof oxidative phosphorylation.

modest uncouplers. Nonphotoactivated selenium/sulfur, withglutamate plus malate as the respiratory substrate, exhibited50% uncoupling at a concentration of 2.2 IJ.M(Fig. 4A). Theorder of potency for uncoupling by these compounds under thedescribed conditions was selenium/sulfur > selenium/selenium> selenium/oxygen with half-maximal uncoupling at 2.2, 2.6,and >3 fiM, respectively. Similar uncoupling was observed withsuccinate as the respiratory substrate (data not shown).

When the CP dyes were photoirradiated, dose-dependentinhibition of the state 3 respiratory rate was observed whenglutamate plus malate was the substrate. The order of potencyunder this set of conditions was selenium/sulfur > selenium/selenium > selenium/oxygen with 50% inhibition achieved at0.04, 0.05, and 0.11 Â¿zM,respectively. Respiratory rates in thepresence of photoirradiated selenium/sulfur are shown in Fig.4B). Comparable inhibition was obtained when succinate wasused as the respiratory substrate (data not shown). These datasuggest that photoirradiation of mitochondria in the presenceof the CP dyes inhibits both complexes I + II nonspecifically,inhibits at an electron transfer site common to the oxidationpathway of both substrates (i.e., complex III or IV), or inhibitsall sites nonspecifically. This was investigated further throughmitochondrial enzyme assays (see below).

Enzyme Assays. Upon photoirradiation in the presence ofmitochondria, VB-BO inhibited rotenone-sensitive NADH-cy-tochrome c reductase activity (which is a measure of the transferof electrons from complex I through coenzyme Q to complex

III) (Fig. 5). Photoirradiated VB-BO did not inhibit succinate-cytochrome c reductase activity (which is a measure of thetransfer of electrons from complex II through coenzyme Q tocomplex III) (Fig. 6). Neither enzyme was inhibited when VB-

200 -i

ill

O

100 -

oe

1

DYE CONC. (uM)

200 nu5KQ.

B

100-

COni_jOac

0.02 0.04 0.06

DYE CONC. (uM)Fig. 4. Effect of selenium/sulfur on mitochondrial respiration using glutamate

plus malate as a respiratory substrate. I is without and B is with photoirradiation.Comparable results were obtained in the presence of the compounds selenium/selenium and selenium/oxygen, except that higher dye concentrations were required for equivalent inhibition (see text). The rates obtained are indicated asfollows: initial (El), state 3 (â€¢),ADP-stimulated (0), and uncoupled (O), asdescribed for Fig. 3.

100

UJ

OEÃ–L

OÃE

COIII 20 -

O0.02 0.080.04 0.06

DYE CONC. (uM)Fig. 5. Rotenone-sensitive NADH-cytochrome c reductase activity in freeze-

thawed mitochondria irradiated 7.5 J/cm2, 600-750 nm light, in the presence ofincreasing concentrations of VB-BO (H), selenium/sulfur (*), selenium/selenium(D, or selenium/oxygen (O).

7878




150

Ocea 100 -

O

50 -

0.02 0.04 0.06 0.08

DYE CONC. (uM)

0.10

Fig. 6. Succinate-cytochrome c reducÃaseactivity measured in freeze-thawedmitochondria irradiated with 7.5 J/cm2, 600-750 nm light, in the presence ofincreasing concentrations of VB-BO (H), selenium/sulfur (*), selenium/selenium(D) or selenium/oxygen (O).

- 800

0.00 0.05 0.10 0.15

DYE CONC. (uM)

0.20

Fig. 7. Cytochrome c oxidase activity measured in freeze-thawed mitochondriairradiated with 7.5 J/cm2, 600-750 nm light, in the presence of increasing

concentrations of selenium/sulfur (HI),selenium/selenium (â€¢)or selenium/oxygen(D).

200 -i

Â§

100

0.0 0.2 0.80.4 0.6

DYE CONC. (uM)Fig. 8. Succinate dehydrogenase activity measured in freeze-thawed mitochon

dria irradiated with 7.5 J/cm2, 600-750 nm light, in the presence of increasingconcentrations of selenium/selenium without (El) or with 0.1 HIM(Â»)or 1 DIM(D) a-tocopherol (vitamin E).

BO was used without photoirradiation.With photoirradiation, selenium/sulfur, selenium/selenium,

and selenium/oxygen inhibited both NADH- and succinate-cytochrome c reducÃaseactivity (Figs. 5 and 6, respectively).These compounds also inhibited cytochrome c oxidase, thecomplex IV enzyme (Fig. 7), and as well as succinate dehydrogenase (Fig. 8), the complex II enzyme, indicating a general formof inhibition. Inhibition of these enzymes without photoirradia-tion was observed only when using concentrations of seleniumdye 100-fold greater than that necessary to obtain inhibitionwith photoactivation.

The relatively greater potency of selenium/sulfur and selenium/selenium compared to selenium/oxygen in the enzymeassays and the potentiating effects of irradiation were consistentwith the effect of the dyes on respiration. Dose-response curvesfor the toxicity of dye plus light were shifted to the left in theenzyme assays (Figs. 5-8) compared to the respiration assays(Figs. 3fi and 47?); this shift was ascribed to the simple massaction effect of using much less mitochondria! protein in thespectrophotometric assays compared to the polarographic assays (see "Materials and Methods").

At 1 Â¿tMselenium/selenium, a concentration 100-fold greaterthan that necessary to achieve half-maximal inhibition ofNADH- or succinate-cytochrome c reducÃaseactivities, therewas no inhibition of citrate synthetase, a mitochondrial matrixenzyme, or lactate dehydrogenase, a cytosolic enzyme, indicating that the chalcogenapyrylium dyes probably are inhibitoryfor membrane-bound enzymes only (data not shown).

Effect of Vitamin E. We examined the role of lipid per-oxidation in the generalized inhibition of membrane-boundmitochondrial enzymes by measuring the effect of selenium/selenium on succinate dehydrogenase activity in the presenceof a-tocopherol (vitamin E), a compound known to sequestermembrane-damaging oxygen radicals. With photoirradiation,100-fold more selenium/selenium was necessary to achieve half-maximal inhibition in the presence of 0.1 mM a-tocopherolthan in its absence (1.0 /Â¿Mversus 0.01 ^M selenium/selenium,respectively) (Fig. 8). This shows a protective effect of vitaminE on mitochondrial photodamage by the selenium dyes.

DISCUSSION

The mitochondrion has been implicated as an important,perhaps primary, subcellular site of damage by several photo-sensitizers which show promise for use in photochemotherapy(6, 9, 20-22). The cationic photosensitizers are concentratedby cells and into mitochondria in relation to negative insidemembrane potentials (7). The higher plasma and/or mitochondrial membrane potentials of malignant versus normal cellsaccounts for greater uptake of these compounds in tumor cellsand contributes to selective cell killing (7, 8). VB-BO and theCP dyes may be useful in photochemotherapy (9-11, 23); thusthe mechanism by which they cause photodamage to mitochondria is of interest. We used rat liver mitochondria for thesestudies, because the organization and function of potentialtarget proteins in the electron transport chain and oxidativephosphorylation is fundamentally similar among all mammalian mitochondria.

Our data show that photoirradiation of the triarylmethanederivative, VB-BO, produces a selective inhibition of mitochondrial respiratory complex I. Complex I inhibition appears to bethe mechanism of mitochondrial photodamage by another cat-ionic photosensitizer, EDKC (6). However, EDKC also inhibits

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NADH-linked mitochondrial respiration in the absence of pho-to in ad union (although to a 10-fold lesser extent), whereas thedark toxicity of VB-BO involves a dose-dependent uncouplingof oxidative phosphorylation over the same range of dye concentrations that cause inhibition of respiration with dye pluslight (Fig. 3). Therefore, while photoirradiation serves only toincrease the magnitude of the effect of EDKC on mitochondria,in the case of VB-BO it appears to alter the actual mechanismof mitochondrial toxicity.

The phototoxicity of VB-BO has been studied in vitro inhuman squamous (FaDu) and colon (CX-1) carcinoma, in human and murine melanoma (NEL, B-16) cell lines, and innonmalignant kidney epithelial cells (CV-1).7 VB-BO is pref

erentially accumulated and retained by malignant cells and isan effective tumor cell-specific photosensitizer in vitro. Thereis significant malignant cell killing at a dye and light dose whichhas no effect on CV-1 cells. In the phototoxicity studies, witha light dose of 7.5 J/cm2, a VB-BO concentration of 0.05 t*Mwas found to be sufficient to achieve 50% malignant cell killing.6In our results, light-activated 0.3 Â¿Â¿MVB-BO was required forhalf-maximal inhibition of oxidative phosphorylation in isolated rat liver mitochondria (Fig. 3B). In whole cells the plasmamembrane potential would be expected to concentrate VB-BOin the cytosol severalfold relative to the external medium (7).Therefore the concentration of VB-BO eliciting half-maximallight-induced inhibition of mitochondrial respiration using intact mitochondria is probably comparable to the cytoplasmicconcentration of VB-BO that results in significant cell death inphotocytotoxicity assays. The data presented here suggest thatmitochondrial photosensitization with selective inhibition ofrespiratory complex I is probably a principal cause of cytotox-icity by photoactivation of VB-BO.

The CP dyes are another interesting class of cationic lipo-philic compounds which are preferentially accumulated by neo-plastic cells and have absorption maxima in the red and near-IR light range. We found that the relative potency of the CPcompounds (selenium/sulfur > selenium/selenium > selenium/oxygen) for mitochondrial photosensitization observed in thisstudy was correlated with their cellular phototoxicity.6 Our data

indicate that in intact mitochondria, whereas the CP dyesintrinsically act as modest uncouplers of oxidative phosphorylation, respiratory inhibition is obtained under the condition ofdye plus light. Further, enzyme assay results show that pho-toirradiation produces a generalized inhibition of electrontransport at dye concentrations up to 100-fold less than thatnecessary to achieve similar inhibition in the dark. Photoirradiation, therefore, alters both the magnitude and mechanism ofthe effect of CP dyes on mitochondrial function. Several membrane-bound enzymes were affected whereas soluble enzymeswere not, suggesting a generalized effect of CP dyes plus light,rather than a site-specific effect.

The mitochondrion is the principal site of cellular injury dueto photosensitization by another CP dye, 8b (9). The variousconsequences of 8b photodamage include mitochondrial swelling and loss of cristae, as well as a significant reduction inelectron transport-coupled ATP synthesis. These rather generalized effects were proposed to be mediated by the productionof membrane-damaging singlet oxygen (9). In fact, the CP dyesare known to have a high yield of singlet oxygen (23). Theprotective effect of a-tocopherol against photodamage by theCP dyes we tested is consistent with singlet oxygen production

7Manuscript in preparation.

as the mechanism of photodamage by these compounds as well.Interestingly, hematoporphyrin derivative has also been shownto induce its phototoxic effect via singlet oxygen and to inhibitnonspecifically many membrane-bound enzymes including cy-tochrome c oxidase, succinate dehydrogenase, and mitochondrial ATPase (20-22). Active oxygen products of VB-BO irradiation have not been identified.

This study supports the idea that the mitochondrion is aprimary site of the phototoxicity of VB-BO and CP dyes. Forthese compounds, dark toxicity involves modest uncoupling ofoxidative phosphorylation at low dye doses. With irradiation,VB-BO becomes a selective inhibitor of respiratory complex I,whereas the CP dyes induce a more generalized inhibition ofmembrane-bound enzymes probably through production of singlet oxygen. The apparent complex I-specific action of VB-BOplus light may not be due to a special affinity of complex I forthe dye. Instead, complex I, which is the most labile of therespiratory complexes, may simply be more susceptible to thekind of lipid or protein damage induced by photoactivation ofVB-BO. This may explain why EDKC and other structurallydissimilar dyes like pyronin B (24) also appear to be specificfor complex I at low dye doses plus light, and it suggests acommon biophysical mechanism of mitochondrial injury forthese compounds. The strongly lipophilic CP dyes compriseanother class of photosensitizers. These are compounds thatcause a more generalized type of membrane damage, probablyvia singlet oxygen, that affects a wide spectrum of membranefunctions and enzymes.

ACKNOWLEDGMENTS

We thank Michael Detty for providing the chalcogenapyrylium dyesand Kalyan Wadwa and Tom Dahl for helpful conversations andunpublished data on /// vitro effects of the dye.

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1990;50:7876-7881. Cancer Res Josephine S. Modica-Napolitano, John L. Joyal, Gulshan Ara, et al. PhotochemotherapyMitochondrial Toxicity of Cationic Photosensitiziers for

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mitochondrial toxicity of cationic photosensitizers for photochemotherapy

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