[CANCER RESEARCH 43, 4191-4197, September 1983]

Photosensitization of Mitochondrial Cytochrome c Oxidase by

Hematoporphyrin Derivative and Related Porphyrinsin Vitro and in Vivo1

Scott L. Gibson and Russell Hilf2

Department of Biochemistry [S. L G., R. H.] and University of Rochester Cancer Center [R. H.], University of Rochester School of Medicine and DentistryRochester, New York 14642

ABSTRACT

Mitochondrial cytochrome c oxidase activity was inhibited byexposure to hematoporphyrin derivative followed by photoirradiation. Inhibition of enzyme activity in vitro was a dose- andtime-related event, the log of percentage inhibition being linearwith time. Exposure of mitochondria to hematoporphyrin orhydroxyethylvinyldeuteroporphyrin sensitized mitochondria tophotoirradiation, whereas protoporphyrin IX was only weaklyactive as a photosensitizer for inhibition of cytochrome c oxidase.Mitochondria from mammary glands of pregnant rats showedhematoporphyrin-induced photosensitivity similar to those fromR3230AC mammary adenocarcinomas. Mitochondria preparedfrom tumors of animals given injections of hematoporphyrinderivative in vivo and then photoirradiated in vitro demonstratedconsiderable sensitivity to light as reflected by significant inhibition of cytochrome c oxidase activity. A side by side comparisonof hematoporphyrin derivative with hematoporphyrin, using thisin vivo-in vitro experimental protocol, indicated that photosensitivity was retained for a longer time after treatment with hematoporphyrin derivative. Taken together, these data provide apotential mechanism of action, I.e., inhibition of respiration, forporphyrin-induced photosensitivity, and they offer a useful assayto investigate this family of therapeutic agents.

INTRODUCTION

HPD,3prepared from HP, is a mixture of several components,

that has demonstrable antineoplastic activity when used in conjunction with photoradiation therapy (7-11,21, 23). Most investigators have suggested that cell toxicity results from singletoxygen formation (4, 6, 26, 30). The subcellular site of cytotox-icity has not been ascertained; Kessel ef al. (20-22) demonstrated effects at the plasmamembrane, as reflected by inhibitionof certain transport activities, whereas others have reported thatDMA damage, single-strand breakage, or protein cross-linkingoccurred in response to singlet oxygen production (12,13,16,17,27).

Porphyrins were noted to have affinity for bodies high inlipoprotein content, such as mitochondria (14). Sandberg andRomslo (29) reported that the uncoupling of respiration andswelling of mitochondria caused by photoirradiation in the near-

1Supported by Contract 04-129-4109-AA from the Standard Oil Company

Research Center, Cleveland, Ohio.2Supported in part by Grant CA 11198, NIH. To whom requests for reprints

should be addressed.3The abbreviations used are: HPD, hematoporphyrin derivative; HP, hemato

porphyrin; HVDP, hydroxyethylvinyldeuteroporphyrin; PP, protoporphyrin IX; HPLC,high-pressure liquid chromatography.

Received January 24,1983; accepted June 6,1983.

UV range (340 to 380 nm) could be enhanced by the presenceof low levels of porphyrins; deuteroporphyrin and protoporphyrinwere the most active photosensitizers. HP was studied by Saletand Moreno (28), who concluded that the resulting photosensi-tization of isolated mitochondria by HP plus light at 365 nmaffected respiration, oxidative phosphorylation, and calciumtransport. Recently, Berns ef al. (2) studied several cell lines invitro after exposure to HPD; fluorescence microscopy of thesenonneoplastic cultured cells demonstrated ¡ntracellularbindingof HPD to mitochondria. Taken together, it would appear thatmitochondria may be another site of action for the antineoplasticeffects of HPD-induced photosensitization.

Studies in our laboratory, utilizing primary cultures of the well-differentiated R3230AC rat mammary adenocarcinoma, demonstrated HPD-inducedphotosensitivity (18).The resulting cytotox-icity was dose and time related, with HPD havinggreater potencythan do HP and HVDP under the conditions of the assay used.In order to explore the mechanisms producing cytotoxicity, weopted to investigate mitochondrial function as a potentially inhib-itable site of action. When consideration was accorded to samplesize and application to studies in vivo, it was decided to studycytochrome c oxidase (ferrocytochrome c:O2 oxidoreductase,EC 1.9.3.1) activity as one measure of mitochondrial function.Cytochrome c oxidase is the terminal oxidant catalyzing electrontransfer from cytochrome c to oxygen, is probably responsiblefor more than 90% of cellular oxygen consumption, and is criticalto aerobic metabolism (31). The data presented here indicatethat HPD and related porphyrins are capable of inducing aphotosensitive inhibition of cytochrome c oxidase activity inisolated mitochondria in vitro and that mitochondria, preparedfrom tumors of animals treated with HPD, are sensitive to pho-toirradiation as reflected by the inhibition of cytochrome c oxidase activity. These results suggest that inhibition of mitochondrial function may contribute to cell cytotoxicity in the antineoplastic action of HPD-induced photosensitization.

MATERIALS AND METHODS

Animals and Tissues. The R3230AC mammary adenocarcinoma wasmaintained by transplantation s.c. in the axillary region of 60- to 80-g

female Fischer rats, using the sterile trocar techniques described earlier(19). Mammary glands were obtained from pregnant (18 and 20 days)Fischer rats.

Preparation of Mitochondria. Mammary tumors or mammary glandswere excised, placed on ice in 0.9% NaCI solution, weighed, and mincedwith scissors. Two g of tissue were placed in 5 ml of cold homogenizationbuffer, containing 0.33 M sucrose, 1 mw dithiothreitol, 1 mw ethylenegly-col bis(/3-aminoethyl)-A/,A/'-tetraaceticacid, 0.03% bovine serum albumin,

and 100 HIM KCI. Tissues were homogenized twice (15-sec bursts) onice, using a Polytron (PCU-2-110; Brinkmann Industries, Westbury, N.

SEPTEMBER 1983 4191

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

S. L Gibson and R. Hilf

Y.) homogenizer, at a setting of 6. The homogenate was then centrifugeaat 500 x g for 30 min at 4°(PR-2; International Centrifuge, Needham

Heights, Mass.)- The supernatant was removed and centrifuged at15,000 x g for 30 min at 4°(J-21; Beckman Instruments, Palo Alto,

Calif.). The supernatant was discarded, and the pellet was suspended in1 ml of the homogenization buffer. This suspension was subjected tosonication for 2 min on ice (Biosonik III; Bronwill Scientific, Rochester,N. Y.), at a setting of 60. Four ml of cold homogenization buffer wereadded, and the mitochondrial suspension was mixed by vortexing. Thissuspension was centrifuged at 30,000 x g for 30 min at 4°.The resulting

supernatant was discarded, and the pellet was resuspended in anappropriate volume (from 2.0 to 5.0 ml) of the homogenization buffer toyield a final concentration of approximately 150 ^g of protein per ml ofbuffer. Aliquots (0.5 ml) of this suspension were made and were storedat -70° until used. Storage for up to 1 month had no effect on enzyme

activity (<2% change).Porphyrin Preparation. HP was obtained from Sigma Chemical Co.

(St. Louis, Mo.), HVDP, and PP were obtained from Porphyrin Products(Logan, Utah). Preparation of HPD followed the procedure of Lipson efal. (24). Prior to treatment, solutions of each porphyrin were prepared bydissolving 7 mg of the compound in 1 ml of 0.1 N NaOH, stirring for 1 hrin the dark at room temperature ("alkalinization"), and adjusting the pH

to 7.4 by dropwise addition of 0.1 N HCI; the solutions were brought toa final volume of 10 ml by addition of 4-(2-hydroxyethyl)-1-piperazine-

ethanesulfonic acid buffer, containing 5 mw glucose (pH 7.4), for a finalporphyrin concentration of 700 ¿<g/ml.

In Vivo Treatment. Eight mg of HPD were dissolved in 0.5 ml 0.1 NNaOH and stirred for 1 hr in the dark at room temperature ("alkalinization"), after which the pH was adjusted to 7.4 with 0.1 N HCI, giving a

final volume of about 1 ml. This solution was injected i.p. into 100-g

female Fischer rats bearing bilateral R3230AC tumors approximately 2cm in diameter. The rats were kept in the dark for the desired times andsacrificed by cervical dislocation. Tumors were excised, and mitochondriawere prepared in the dark as described above, frozen, and stored at-70° until analyzed. Experiments with HP, HVDP, and PP were con

ducted the same way, using an identical dose.Cytochrome c Oxidase Assay. Cytochrome c was obtained from

Sigma and dissolved in phosphate buffer (50 mw) to a final concentrationof 1.2 mM. This substrate solution was reduced by the addition of a"pinch" of NazSzO.«,followed by bubbling air through the mixture to

oxidize excess dithionite. The mitochondria to be assayed (prepared asdescribed above) were sonicated on ice (2 times for 2 min each time),and 10 n\ of the sonicated suspension were placed in a 1-ml (1-cm path

length) quartz cuvet. Immediately, 1 ml of the substrate solution containing the reduced Cytochrome c (0.1 ml 1.2 mM cytochrome c-0.9 ml 50

mM phosphate buffer, pH 7.5) was added, starting the oxidation reaction.Disappearance of reduced cytochrome c was monitored continuously at550 nm in a Gilford 2400 spectrophotometer. Rates of oxidation werelinear for at least 2 min. Activity was calculated from the slope of the lineand was expressed as jimol cytochrome c oxidized per min per mgprotein. As prepared by the above procedure, mitochondrial samplesdisplayed activities of 0.5 to 1.0 umol/mg protein.

Photoirradiation Studies in Vitro. Mitochondria were prepared fromtumors or mammary glands as described. After sonication, variousconcentrations of porphyrins were added, and the mitochondria-por-phyrin mixture was placed in separate 6- x 50-mm borosilicate test tubes

each containing an aliquot of 30 ¡A.All of these procedures wereperformed in the dark. The test tubes containing the mitochondria-porphyrin mixtures were placed 6 cm from a 14-watt fluorescent bulb atroom temperature. Incident light was measured by a power photoradi-

ometer (RX5200; Laser Precision Corp., Utica, N. Y.) equipped with anRD545 radiometer probe; the lamp provided 0.2 milliwatts/sq cm at thisdistance. Total photoradiation energy was 0.012 J/sq cm/min, and totalenergy to which mitochondria were exposed can be calculated bymultiplying the above number by the number of min of light exposure.Thus, for 1-hr light exposure, mitochondria recieved 0.72-J/sq cm dosesof this broad-spectrum (300 to 900 nm) incident light energy. Aftervarious times of photoirradiation, a 10-Ml portion of the mixture was

removed and assayed for cytochrome c oxidase activity.Photoirradiation of Mitochondria from Animals Treated in Vivo.

Mitochondrial suspensions prepared as above from tumors excised atvarious times after injection of porphyrins were placed into 6- x 50-mmborosilicate test tubes (30-^1 aliquots) and exposed to light as described

above. Due to some variation from animal to animal, the initial enzymeactivity of these mitochondrial preparations was adjusted by appropriatedilution to within 10% of one another. This procedure was used, becauseit was observed that the lower the initial enzyme activity the moredramatic was the extent of inhibition of cytochrome c oxidase activityduring exposure to light. This approach allowed for a more reproduciblecomparison of data from animal to animal and over the time of lightexposure studied.

RESULTS

Standardization of Assay for Cytochrome c Oxidase. At theoutset, it was necessary to investigate several aspects pertainingto stability of cytochrome c oxidase under the experimentalconditions to be used. For studies in vitro, mitochondrial preparations were made from pooled tissues of several tumors. Thesewere stored in aliquots at -70° for use at later times; samples

assayed for up to 28 days (assayed after 0, 3, 9, 11, 13, or 28days) of storage showed little change in enzyme activity (<2%).Initially, we examined the effects of either light alone, the presence of porphyrins alone (in the dark), or neither of these oncytochorome c oxidase activity (prior to study of photosensiti-zation). As seen from the data in Table 1, enzyme activity slowlydeclined in a comparable fashion for these 3 conditions, remaining within 10% of the starting value for the first hr of study; after2 hr, activity was slightly lower. Based on these data, anycomparisons that we have made were based on data obtainedthrough 1 hr of photoirradiation, rather than after 2 hr of lightexposure (although the data are shown in the charts). Also shown

Table 1Effects of HPD,photoirradiation, or neither on mitochondrial cytochrome c oxidase activity

The level of HPD used was 7.0 ng/ml, and the conditions of photoirradiation are described in "Materialsand Methods." Mitochondria were prepared from animalsgiven injections in vivo of 80 mg/kg body weight.

Mitochondria exposed to Enzyme activity (nmol cytochrome c oxidized/min/mg protein) at followingtimes assayed

HPD0

+0

Injected in vivoPhoto-

irradiation0

0+

0Omin484

±39* (1OOf

509 ±34 (100)519 ±43 (100)515 ±50 (100)30

min455

±27 (94)491 ±35 (96)520 ±50 (100)499 ±39 (97)60min442

±27(91)473 ±40 (93)488 ±45 (94)481 ±55 (93)120

min450

±43(93)446 ±22 (88)460 ±55 (89)461 ±34 (90)

" Mean ±S.E. from 6 to 12 experiments (these represent controls performed for each experiment).6 Numbers in parentheses, percentage of initial (Omin) activity.

4192 CANCER RESEARCH VOL. 43

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Photosensltization of Cytochrome c Oxidase by HPD

in Table 1 are cytochrome c oxidase activities of mitochondriaprepared from animals given injections of HPD and kept in thedark; the enzyme activity displayed the same behavior as for theother control conditions. Interestingly, cytochrome c oxidaseactivity was less stable in mitochondria prepared from mammaryglands of pregnant rats. Under the conditions used here, after 1hr in the dark and in the absence of HPD, cytochrome c oxidaseactivity was decreased by 23%. Even when activity was adjustedto the same initial level in preparations of tumor and normalmitochondria, less stability was observed in the mitochondriafrom normal glands. Thus, experiments comparing photosensi-

tivity for normal versus neoplastic tissues were not studiedbeyond 1 hr of photoirradiation. Addition of 0.1 mw KCN totallyinhibited cytochrome c oxidase activity in mitochondria! preparations from normal and tumor tissues, results indicating that theassay used represents cytochrome c oxidase activity.

HPO-induced Photosensitivity of Cytochrome c Oxidase in

Vitro. Chart 1 depicts the inhibition of cytochrome c oxidaseactivity in tumor mitochondria in the presence of various concentrations of HPD and exposed to light for periods of 5 min to 2 hrat room temperature. Inhibition of enzyme activity was doserelated. The latencies of inhibition to achieve a 25% reduction inactivity were 95, 23, and 12 min for HPD doses of 0.7, 3.5, and7.0 fig/m\, respectively. Linear regression analysis of the dataobtained during the first 30 min of photoirradiation provided adose relationship; the rates of inhibition (percentage of inhibition

LIGHT EXPOSURE (min)Chart 1. Effect of various concentrations of HPDIn vitro (O, 0.35 »¡g/mi;•,0.7

pg/ml; A, 3.5 *ig/ml; A, 7.0 pg/ml) to produce inhibition(inhib)of tumor mrtochondrialcytochrome c oxidase activity after various periods of photoirradiation. Data arepresented as percentage of inhibition of enzyme activity relative to controls (100%activity = 0.4 to 0.6 fimol cytochrome c oxidized per min per mg protein) in thepresenceof HPD but not exposed to light. Preparationof mitochondria and detailsof the enzyme activity are given in "Materials and Methods." Eachpoint represents

the meanof 4 separate experiments;bars, S. E. Inset, rate of inhibition(percentageof inhibition per min), calculated by linear regression analysis of the data obtainedduring the first 30 min of photoirradiation for each concentration of HPD studied.Note the linearity of the relationshipsof inhibitionand dose.

per min) were 1.93,1.24, 0.27, and 0.20 for HPD doses of 7.0,3.5, 0.7, and 0.35 ng/ml, respectively (see Chart 1, inset). Inhibition of tumor cytochrome c oxidase activity by HPD and lightwas time related (Chart 1). At the highest level of HPD (7.0 ngfml), enzyme activity was inhibited by 78% after 2 hr of photoirradiation under the conditions used. As noted above, neitherlight alone nor HPD in the absence of light had significant effectson cytochrome c oxidase activity.

Photosensitization Actions of HP, HVDP, and PP in Vitro.Several selected porphyrins were examined to assess their abilityto inhibit cytochrome c oxidase activity after photoirradiation(Chart 2). Using the same assay conditions In vitro and at similardoses (7.0 itg/ml), both HP and HVDP were comparable to HPD(at 60-min light exposures) in sensitizing mitochondria to pho-toirradiation-induced inhibition of cytochrome c oxidase activity.In contrast, PP showed significantly less potency as a photosen-

sitizer. It is interesting to note that at shorter light exposures,i.e., 5 and 15 min, both HP and HVDP sensitized the mitochondriato a greater extent than did HPD.

HPD-induced Photosensitivity of Mitochondria from Normal

Mammary Gland. To determine whether mitochondria preparedfrom normal mammary tissues were subject to HPD-inducedPhotosensitization, we obtained glands from 18- to 20-day preg

nant Fischer rats and prepared mitochondria as described above(glands from virgin rats are predominantly adipose tissue andare a less desirable control for neoplasms arising in ductal andalveolar cells). Due to the lower inherent cytochrome c oxidaseactivity in these normal mitochondria, comparative studies were

LIGHT EXPOSURE (min)

Chart 2. Effect of different porphyrins in vitro (O, HPD;•,HP; A, HVDP;A, PP)on the activity of tumor mitochondrial cytochrome c oxidase after various periodsof photoirradiation. Mitochondria were exposed to 7.0-pg/ml doses of each por-phyrin. Data are presented as percentage of inhibition of enzyme activity relativeto controls (100% activity = 0.4 to 0.6 ¿imolcytochrome c oxidized per min per mgprotein)exposed to the porphyrins but not exposed to light. Eachpoint representsthe mean of 3 separate experiments; bars, S. E.

SEPTEMBER 1983 4193

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

S. L Gibson and R. Hilf

conducted after adjusting the tumor mitochondria (dilution withhomogenization buffer) to levels of activity that were within 10%of those found in mitochondria from mammary gland (0.2 to 0.6/«molcytochrome c oxidized per min per mg protein). Thisadjustment was deemed necessary, based on the observationsthat the extent of inhibition was related to the initial enzymeactivity for a constant level of HPD; at lower enzyme activities,inhibitions of 85 to 90% were achieved within 1 hr of lightexposure. The results shown in Chart 3 indicate that mitochondria from normal mammary tissue display a degree of HPD-induced photosensitivity comparable to those from tumor tissue,as seen by a similar time course of inhibition of cytochrome coxidase activity.

Photosensitivity of Tumor Mitochondria from Porphyrin-treated Rats. The foregoingdata clearlydemonstratethe abilityof certain porphyrins to act in vitro. In order to demonstratewhether such effects could be found in vivo, tumor-bearing ratswere given i.p. injections of a single 80-mg/kg dose of HPD orHP, and individual animals were sacrificed at either 2, 24, 72, or96 hr after treatment. Mitochondria were prepared from thetumors, apportioned into 30-^1aliquots per test tube, and pho-toirradiated under the same conditions used for studies in vitro.The initial enzyme activities were adjusted (within 10% of oneanother) to account for variations from tumor to tumor. The datapresented in Chart 4 demonstrate that treatment of animals withHPD photosensitized the tumor mitochondria, measured by theinhibition of cytochrome c oxidase activity after exposure to light.At the earliest time studied, 2 hr after injection HPD, photoirra-

100

LIGHT EXPOSURE (min)

Chart 3. Effect of HPD (7.0 /ig/ml) in vitro on mammary tumor (O) or normalgland (•)mitochondria! cytochrome c oxidase activity after various periods ofphotoirradiation. Normal mammary glands were obtained from 18- to 20-daypregnant rats. The initial levels of enzyme activity were adjusted by dilution ofmitochondria from tumors to within 10% of the activity observed in mitochondriafrom normal tissue (0.2 to 0.4 (imol cytochrome c oxidized per min per mg protein).Each point represents the mean of 4 separate experiments; bars, S.E.

LIGHT EXPOSURE (min)Chart 4. Ability of HPD injected into tumor-bearing rats to sensitize mitochon-

drial cytochrome c oxidase to photoirradiation in vitro. Tumors from animals wereremoved at 2 hr (O), 24 hr (•).72 hr (A), or 96 hr (A) after a single i.p. injection ofHPD (80 mg/kg) and mitochondria were prepared as described in "Materials andMethods." Activity of cytochrome c oxidase was measured at various times after

photoirradiation. Data are presented as percentage of inhibition (control kept indark = 100% = 0.4 to 0.6 nmol cytochrome c oxidized per min per mg protein);each point represents the mean of 4 separate experiments; bars, S.E.

diation produced a modest amount of inhibition of enzyme activity, reaching 30% after 3 hr of light exposure (corrected fordecrease in cytochrome c oxidase in mitochondria not exposedto light). Mitochondria from tumors, obtained from animals sacrificed at 24, 72, or 96 hr after injection of HPD, demonstratedsimilar and significant photosensitive inhibition of cytochrome coxidase activity; after 3 hr of light exposure, enzyme activity wasinhibited by 81, 75, and 72%, respectively.

Somewhat different results were obtained for mitochondriaprepared from tumors of animals that had been given injectionsof the same dose (80 mg/kg) of HP (Chart 5). At the shortesttime after injection (2 hr), HP-induced photosensitivity of mitochondria! cytochrome c oxidase appeared to be greater thanthat seen for HPDtreatment. At 24 and 72 hr posttreatment withHP, the pattern of light-induced inhibition of enzyme activity wassimilar and comparable to that seen at these times for HPDtreatment. However, mitochondria obtained from animals 96 hrafter treatment with HP displayed a pattern of enzyme inhibitionthat more closely resembled the pattern seen in samples obtained 2 hr after treatment, results that suggest that mitochon-drial retention of HP (or its metabolites) had begun to decreasefrom the levels seen at 24 or 72 hr after treatment. These resultsimply that, based on the light-induced inhibition of cytochrome coxidase activity, HPD was retained in tumors for a longer periodof time than HP, when both were injected at equal doses.

Additional experiments were perfromed to compare the abilityof porphyrins to act in vivo. Animals were treated with a single

4194 CANCER RESEARCH VOL. 43

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Photosensitization of Cytochrome c Oxidase by HPD

LIGHT EXPOSURE (min)

Chart 5. Ability of HP injected into tumor-bearing rats to sensitize mitochondrialcytochrome c oxidase to photoirradiatton in vitro. Tumors from animals wereremoved at 2 hr (O), 24 hr (•),72 hr (A), or 96 hr (A) after a single i.p. injection ofHP (80 mg/kg) and mitochondria were prepared as described in "Materials andMethods." Activity of cytochrome c oxidase was measured at various times after

photoirradiation. Data are presented as percentage of inhibition (control kept indark = 100% = 0.4 to 0.6 ¡imo\cytochrome c oxidized per min per mg protein);each point represents the mean of 4 separate experiments; bars, S.E.

injection (80 mg/kg body weight) of HPD, HP, HVDP, or PP andsacrificed 24 hr later, tumors were removed, and mitochondriawere prepared and photoirradiated. The time courses of the light-

induced inhibition of cytochrome c oxidase activity were obtainedand are summarized in Table 2. Under these conditions, HVDPwas about as effective as HPD and HP, based on inhibition ofenzyme activity. However, PP was essentially inactive as aphotosensitizer. Thus, the photosensitizing activities of theseporphyrins were demonstrable in vivo and compared favorablywith the data obtained in the in vitro assay.

DISCUSSION

The data presented here demonstrate that mitochondria displayed a HPD-induced photosensitization, as evidenced by a

significant inhibition of cytochrome c oxidase activity. The inhibition of enzyme activity was related to the dose of HPD presentin vitro, as well as to the length of time of photoirradiation. Thelog of percentage of inhibition was linear in time. The rate ofinhibition per min (for the first 30 min of light exposure) wasdirectly related to the level of HPD added to the mitochondrialpreparation. At the same levels (7.0 /¿g/ml),inhibition of cytochrome c oxidase activity was produced by HP and HVDP in amanner similar to that seen with HPD. However, PP was relativelyweak as a photosensitizer in this in vitro assay. It should benoted that cytochrome c oxidase activity was quite stable underthese conditions, as indicated by results from assays conductedunder identical conditions in the absence of HPD or light. Even

Tabte2

Comparative photosensitivity of mitochondrial cytochrome c oxidase activity fromtumor-bearing animals 24 hr after treatment with porphyrins

Tumor mitochondria were prepared as described in 'Materials and Methods."

Values presented as percentage of cytochrome c oxidase activity remaining aftervarious times of photoirradiation using conditions described in "Materials andMethods." Data are the means from 4 separate experiments, except for protopor-

phyrin (2 experiments); standard errors were <5% for these means.

% of cytochrome c oxidase activityremaining

Light exposure(min)05

1530

60120180HPDa10097

8669563819HP100

958470554422HVDP10099

8872604530PP100

100100100100

9782

°Porphyrins were injected at 80 mg/kg body weight.

in the presence of HPD, but kept in the dark, or exposed to lightin the absence of HPD, cytochrome c oxidase activity wasmaintained with 10% of the initial value for 1 to 2 hr. This lengthof time was sufficient to produce inhibitions greater than 50%for HPD, HP, and HVDP, when 7.0 ng/m\ were added in vitro.Salet and Moreno (28) demonstrated that HP photosensitizedrat liver mitochondria, resulting in an inhibition of respiration, anuncoupling of oxidative phosphorylation, and an inhibition of Ca2+

release; they did not study HPD, however. Our results demonstrating an inhibition of cytochrome c oxidase are certainlycompatible with an inhibition of respiration, considering the essential role that this enzyme plays in oxygen consumption.Whether these effects to inhibit cytochrome c oxidase activitycontribute directly to cytotoxicity is not known and requiresfurther study to establish a cause and effect relationship. It isinteresting, however, that the time course of enzyme inhibitionresembled the time course of cytotoxicity of cultured cells exposed to HPD at 7.0 /¿g/mlfor 5 min prior to photoirradiation(16); 50% cytotoxicity was seen by 30 to 60 min of photoirradiation versus 50% inhibition of cytochrome c oxidase activityoccurring by 30 min in isolated mitochondria photoirradiatetíimmediately after exposure to HPD.

Photosensitization of mitochondria by HPD was not exclusiveto neoplastic tissue. Mitochondria prepared from the normalmammary gland, I.e., glands from pregnant animals, demonstrated sensitivity to light induced by HPD, as measured byinhibition of cytochrome c oxidase activity. It should be notedthat, when making such comparisons, awareness of the initialenzyme activity of the mitochondrial preparation is necessary.We observed that the extent of inhibition of enzyme activity wasrelated to initial enzyme activity; a lower initial activity resulted ina higher maximum inhibition, regardless of the source of tissueof the mitochondria. It is essential that multiple controls beperformed when assessing the effects of combined modalitytreatments. The fact that HPD alone in the absence of light orlight alone in the absence of a photosensitizer had little or noeffect on cytochrome c oxidase activity during the first hr ofthese experiments in vitro strengthens the usefulness of thisbiochemical parameter as an assay of photoirradiation-induced

damage by porphyrins, as do the dose and time course relationships.

In earlier reports, HP was observed to be an effective photosensitizer of mitochondrial respiration (28), and PP was found tophotosensitize the inhibition of mitochondrial succinate dehydrc-

SEPTEMBER 1983 4195

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

S. L. Gibson and R. Hilf

genäse (29). These studies were conducted with intact mitochondria prepared from normal liver of rats under conditions invitro in which a lower dose of porphyrin but a higher light doseover a shorter period of time was used compared to the studiesreported here. No data were reported by these investigators formitochondria from neoplastic tissues. Interestingly, we observedlittle or no significant effects of PP on cytochrome c oxidaseactivity in the systems studied here, and Dougherty ef al, (9)reported that PP was inactive as a photosensitizer in vivo. Thesedata suggest that different porphyrin species may possess differing photosensitizing activities depending on the mitochondrialcomponents studied and whether such studies are conducted invitro or in vivo. It will be necessary to conduct experiments withvarious porphyrins over a range of concentrations under identicalconditions in order to assess relative potency as photosensitizersof selected end points.

The demonstration here of inhibition of mitochondrial cytochrome c oxidase activity by photoirradiation of these organellesprepared from lesions of animals treated with HPD, HP, or HVDPclearly indicated that these porphyrins were taken up and retained by mitochondria in vivo. Such findings prompt us tosuggest that this approach offers a very useful assay for examining dose relationships and time courses of photosensitizers,an assay that also accounts for any inherent metabolism by thehost and/or target cell. The latter considerations are obviouslyimportant for assessment of the usefulness of this therapeuticapproach, and they obviate the justifiable criticisms raisedagainst cytotoxicity assays conducted in vitro. It should be notedthat the HPD dose used for in vivo studies was considerablyhigher than those used by others in animal studies, i.e., 5 to 10mg/kg versus the 80 mg/kg used here. However, differences inthe amount of photoirradiation should also be noted when considering the effects of HPD. For example, Dougherty ef al. (9), instudies with the SMT-F mouse mammary tumor, used 288 J of

photoirradiation per sq cm at 600 to 700 nm to produce regression of tumors after treatment with various porphyrins (up to 7.5mg per kg body weight). Thus, it is necessary to consider manyfactors, such as dose of drug used, time after drug administration, photoirradiation intensity and wavelength, tissue penetration in isolated subcellular organelles, concentration and identityof porphyrins present in cellular sites, etc., in attempting tocompare data from one laboratory to another.

Data presented here indicate that the HPD preparation (orsome of its components) was retained by tumor cells for a longerperiod of time than was HP preparation, implying that the derivative contains components that may aid in its cellular retention.Analysis by HPLC of the preparations used here demonstratedthat the "alkalinized" preparations of HP contained significant

amounts (~ 15%) of uncharacterized porphyrin species that were

more hydrophobic than was pure HP (18), and their presencecould account for the photosensitization observed here. Thismight account for the apparent discrepancy between our findingsand those of others (1, 9) who concluded that HP was inactiveas a photosensitizer in vivo. However, we have recently fractionated the HPD preparation by octyl-Sepharose column chroma-

tography and have obtained a fraction that contains only the 2diastereoisomers of HP assessed by HPLC analysis (15). ThisHP-containing fraction demonstrated photosensitizing activity for

mitochondrial cytochrome c oxidase inhibition in vitro, althoughless potent than those fractions enriched in the more hydrophobiccomponents (15); the activity of the HP-containing fraction in

vivo has not been tested to date. Interestingly, although PP isquite hydrophobic and élûtesby HPLC in the same generalregion as the unidentified HPD components (1,3,5,18, 25), PPhas little or no capacity to sensitize cytochrome c oxidase activityto photoirradiation under any of the conditions used here andwas reported to be inactive in vivo by Berenbaum et al. (1) andDougherty ef al. (9). It would appear that the in vivo treatment-

in vitro photoirradiation protocol developed here should facilitatestudies of relative potency of components of HPD, studies thatare currently under way in our laboratory.

ACKNOWLEDGMENTS

The authors are grateful to Dr. David Perlin for his expert advice on theenzymology of cytochrome c oxidase. We thank Dr. Irene Evans of the AnimalTumor Research Facility of the University of Rochester Cancer Center (GrantCA11198) for her continued assistance in the maintenance of the R3230ACmammarycarcinoma.The initial experiments were conducted by Steven J. Sollott,who receiveda Summer Oncology Fellowshipsupported by Grant T-35-HL-07496,Short Term Training for Students in Health ProfessionalSchools, NIH. We wish toacknowledge the continued collaboration of Dr. Linda K. Swofford, The StandardOil Company Research Center, who provided us with the preparation and initialanalysisof HPD used in these studies.

REFERENCES

1. Berenbaum,M. C., Bonnett, R., and Securities, P. A. In vivo biological activityof the components of hematoporphyrinderivative. Br. J. Cancer,45: 571-581,1982.

2. Berns, M. W., Dahlman,A., Johnson, F. M., Bums, R., Sperling, D., Guiltinan,M., Siemens, A., Walter, R., Wright, W., Hammer-Wilson.M., and Wile, A. Invitro cellular effects of hematoporphyrin derivative. Cancer Res., 42: 2325-2329, 1982.

3. Bonnett, R., Ridge, R. J., and Scourides, P. A. On the nature of naematopor-phyrin derivative. J. Am. Chem. Soc., 777: 3135-3140,1981.

4. Buettner, G. R., and Oberley, L. W. The apparent production of Superoxideand hydroxyl radicals by hematoporphyrinand light as seen by spin-trapping.FEBS Lett., 727: 161-164,1980.

5. Cadby, P. A., Dimitriadis, E., Grant, H. G., Ward, A. D., and Forbes, I. J.Separation and analysis of haematoporphyrin derivative components by highperformance liquid chromatography. J. Chromatog., 237: 273-281, 1982.

6. Cannistraro, S., and Van de Vorst, A. Photosensitivity by hematoporphyrin:ESR evidence for free radical induction in unsaturated fatty acids and forsingletoxygen production. Biochem.Biophys. Res.Commun., 74:1177-1185,1977.

7. Dahlman, A., Wile, A. G., Bums, R. G., Mason, G. R., Johnson, F. M., andBerns, M. W. Laser photoradiation therapy of cancer. Cancer Res., 43: 430-434,1983.

8. Dougherty, T. J. Activated dyes as antitumor agents. J. Nati. Cancer Inst., 52:1333-1336, 1974.

9. Dougherty, T. J., Boyle, D. G., Weishaupt, K. R., Henderson,B. A., Potter, W.R., Bellnier, D. A., and Wityk, K. E. Photoradiation therapy—clinicaland drugadvances.In: D. Kessel and T. J. Dougherty (eds.), Porphyrin Photosensitization, pp. 3-13. New York: Plenum PublishingCorp., 1983.

10. Dougherty, T. J„Grindey, G. B., Fiel, R., Weishaupt, K. R., and Boyle, D. G.Photoradiation therapy. II. Cure of animal tumor with hematoporphyrin andlight. J. Nati. Cancer Inst., 55:115-121,1975.

11. Dougherty, T. J., Kaufman, J. E., Goldfarb, A., Weishaupt, K. R., Boyle, D.,and Mittleman, A. Photoradiation therapy for the treatment of malignanttumors. Cancer Res., 38: 2628-2635, 1978.

12. Dubbleman, T. M. A. R., DeGoeij, A. F. P. M., and Van Stevenick, J. Photo-dynamic effects of protoporphyrins on human erythrocytes, nature of thecross-linking of membrane proteins. Biochim. Biophys. Acta, 577: 141-151,1978.

13. Evensen,J. F., and Moan,J. Photodynamicaction and chromosomal damage:a comparison of hematoporphyrinderivation (HpD)and light with X-irradiation.Br. J. Cancer, 45: 456-465, 1982.

14. Falk, J. E. Porphyrins and Metalloporphyrins, p. 24. Amsterdam: ElsevierPublishingCo., 1964.

15. Gibson, S. L., Leakey, P. B., Crute, J. J., and Hilf, R. Photosensitization ofmitochondrialcytochrome c with oxidase by hematoporphyrinderivation (HpD)in vitro and in vivo. In: D. R. Doiron (ed.),Porphyrin Localizationand Treatmentof Tumors. New York: Alan R. Liss, in press, 1983.

16. Girotti, A. W. Protoporphyrin-sensitizedphotodamage in isolated membranesof human erythrocytes. J. Am. Chem. Soc., 78: 4403-4411,1979.

17. Gomer,C. J. DNAdamage and repairs in CHO cells following hematoporphyrinphotoradiation. Cancer Lett., 77: 161-167,1980.

18. Hilf, R., Leakey, P. B., Sollott, S. J., and Gibson, S. L. Photodynamic inacti-vation of R3230AC mammary carcinoma in vitro with hematoporphyrin deriv-

4196 CANCER RESEARCH VOL. 43

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ative: effects of dose, time, and serum on uptake and phototoxicity. Photo-

chem. Photobiol., 37: in press, 1983.19. Hilf, R., Michel, I., Bell, C., Freeman, J. J., and Borman, A. Biochemical and

morphologic properties of a new lactating mammary tumor line in the rat.Cancer Res., 25: 286-299,1965.

20. Kessel, D. Effects of photoactivated porphyrins at the cell surface of leukemiaL1210 cells. Biochemistry, 76:3443-3449, 1977.

21. Kessel, D. Transport and binding of hematoporphyrin derivative and relatedporphyrins by murine leukemia L1210 cells. Cancer Res., 41: 1318-1323,

1981.22. Kohn, K., and Kessel, D. On the mode of cytotoxic action of photoactivated

porphyrins. Biochem. Pharmacol., 28: 2465-2470,1979.

23. Laws, E. R., Cortese, D. A., Kinsey, J. H., Eagan, R. T., and Anderson, R. E.Photoradiation therapy in the treatment of malignant brain tumors: a Phase I(feasibility) study. Neurosurgery, 9: 672-678,1981.

24. Lipson, R. L, Baldes, E. J., and Olsen, A. M. The use of a derivative ofhematoporphyrin in tumor detection. J. Nati. Cancer Inst, 26:1-8,1960.

25. Moan, J., Christensen, T., and Sommer, S. The main photosensitivity compo-

Photosensitizatlon of Cytochrome c Oxidase by HPD

nents of hematoporphyrin derivative. Cancer Lett., 75:161-166,1982.26. Moan, J., Pettersen, E. O., and Christensen, T. The mechanism of photody-

namic inactivation of human cells in vitro in the presence of haematoporphyrin.Br. J. Cancer, 39; 398-407,1979.

27. Moan, J., Waksvik, H., and Christensen, T. DMA single-strand breaks andsister chromatid exchange induced by treatment with hematoporphyrin andlight or by X-rays in human NHIK3025 cells. Cancer Res., 40: 2915-2918,

1980.28. Salet, S., and Moreno, G. Photodynamic effects of hematoporphyrin on respi

ration and calcium uptake in isolated mitochondria. Int. J. Radiât.Biol., Relat.Stud. Phys. Chem. Med., 39: 227-230,1981.

29. Sandberg, S., and Romslo, I. Porphyrin-sensitized photodynamic damage ofisolated rat liver mitochondria. Biochim. Biophys. Acta, 593:187-195,1980.

30. Weishaupt, K. R., Gomer, D. J., and Dougherty, T. J. Identification of singletoxygen as the cytotoxic agent in photoinactivation of a murine tumor. CancerRes., 36: 2326-2329,1976.

31. Wikstrom, M., Krab, K., and Saraste, M. Cytochrome Oxidase. A Synthesis,pp. 3-13. New York: Academic Press, Inc., 1981.

SEPTEMBER 1983 4197

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1983;43:4191-4197. Cancer Res   Scott L. Gibson and Russell Hilf 

in Vivoand in VitroHematoporphyrin Derivative and Related Porphyrins

Oxidase bycPhotosensitization of Mitochondrial Cytochrome

  Updated version

  http://cancerres.aacrjournals.org/content/43/9/4191

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/43/9/4191To request permission to re-use all or part of this article, use this link

on July 20, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from