Cancer Letters, 35 (1987) 295-302 Elsevier Scientific Publishers Ireland Ltd.

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SINGLET OXYGEN INTERMEDIACY IN THE PHOTODYNAMIC ACTION OF MEMBRANE-BOUND HEMATOPORPHYRIN DERIVATIVE

JAMES P. THOMAS”, ROBERT D. HALLb and ALBERT W. GIROTTI”

aDepartment of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 and bLaboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709(U.S.A.)

(Received 1 December 1986) (Revised version received 15 January 1987) (Accepted 17 January 1987)

SUMMARY

The cell-damaging photochemistry of hematoporphyrin derivative (HPD) has been investigated using isolated erythrocyte membranes as a test sys- tem. Irradiation of membranes in the presence of the tumor-localizing frac- tion of HPD resulted in formation of singlet molecular oxygen (‘0,) as measured by the phosphorescence at 1268 nm. The authentic product of ‘0, attack on cholesterol, 3P-hydroxy-5a-cholest-6-ene-5-hydroperoxide, was identified in this system. Relatively insignificant amounts of free radical- derived hydroperoxides were detected. These results suggest that ‘0, plays a major role in the HPD-sensitized photokilling of tumor cells in vivo.

INTRODUCTION

The photosensitizing drug, hematoporphyrin derivative (HPD) is currently undergoing clinical evaluation in conjunction with photodynamic therapy for neoplastic disease [ 11. HPD is a complex mixture of porphyrins prepared by acid/alkali treatment of hematoporphyrin (HP) [2,3]. Its monomeric con- stituents, including HP, protoporphyrin (PP) and hydroxyethylvinyldeutero- porphyrin (HVD), photosensitize well in cell-free systems, but not in vivo because they accumulate (localize) poorly in tumors [ 3,4]. The active local- izer is an aggregate of relatively hydrophobic HP dimers and oligomers, the porphyrin units of which appear to be linked by ester and/or ether bonds

Address correspondence to: A.W. Girotti.

03843835;8?:$03.50 i 1987 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland

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[5,6]. At the cellular level, plasma membranes make initial contact with the localizer and are subject to photodamage; however, translocation to nuclear and mitochondrial membranes eventually makes these sites more photosen- sitive [7]. At the molecular level, relatively little is known about the cyto- lethal events that occur or the oxidant species involved. Singlet molecular oxygen (lo,)-dependent reactions (Type II processes) have been proposed [8,9], but the evidence was based largely on the effects of putative (‘0,) traps, many of which are now known to be non-specific. In the present work, isolated erythrocyte ghosts are used as a test system for examining HPD photochemistry in a cell membrane environment. Photoactivated, mem- brane-bound localizer is shown to be a ‘0, generator by virtue of the lu- minescence observed at 1268 nm. Moreover, a definitive product of ‘0, attack is identified, the 5a-hydroperoxide of membrane cholesterol.

MATERIALS AND METHODS

Unsealed human erythrocyte ghosts were prepared as described previ- ously [lo] and stored under nitrogen in phosphate buffered saline (PBS) (25 mM sodium phosphate, 125 mM NaCl, pH 7.4) until used. HP and PP were obtained from Porphyrin Products (Logan, UT). The deuterium oxide was 98.6 at.% from Aldrich Chemical Co. (Milwaukee, WI).

Crude HPD was prepared according to Gomer and Dougherty [ll]. Chro- matography on a BioGel P-10 column, using 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Hepes) buffered saline as the eluent [12], gave 2 major fractions, PlO-A and PlO-B (in the order of increasing elution time). Chro- matography of HPD on Sephadex LH-20, using tetrahydrofuran/methanol/ 5 mM sodium phosphate, pH 7.5 (2 : 1: 1) as the eluent [ 131 produced 3 major fractions, LH-A, LH-B and LH-C (in order of elution). After solvent evapora- tion, the porphyrin residues were taken up in PBS and stored at - 20°C if not used immediately. Porphyrin concentrations were determined by their 399 nm absorbance in cetyltrimethylammonium bromide (CTAB) detergent [12]. Thin-layer chromatography (TLC) of HPD fractions was carried out on Silica Gel-60 plates, using chloroform/methanol/water (75 : 25 : 4) as the sol- vent system (141.

Stirred suspensions of HPD-sensitized membranes in PBS were irradiated with broad-band blue light at 10°C (see Ref. 10 for other details). Xanthine oxidase-catalyzed reactions were carried out as described previously [ 151. At various times, lipids were extracted with chloroform/methanol (2 : l), hy- droperoxides were reduced with NaBH,, and the resulting cholesterol prod- ucts (diols) were separated by TLC, using heptane/ethyl acetate (1: 1) as the solvent and 50% H,SO, as the color-developing spray [14,16].

Singlet oxygen luminescence in the near-infrared was measured as de- scribed previously [ 17,181, using a mercury arc source and a combination of filters that permitted maximal transmission between 500 and 650 nm. Multi- ple scans were taken between 1210 and 1330 nm at 3-nm intervals. Por-

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phyrin/membrane suspensions in 95% D,O/PBS (pD 7.4) were irradiated while passing through a flow cell at 2 ml/min. Membrane concentrations (0.25 mg protein/ml) were low enough to allow high optical transmittance at 580 nm ( > 60%).

RESULTS

Normal phase TLC of HPD fractions, prepared by aqueous and non- aqueous gel exclusion chromatography, is shown in Fig. 1. The rapidly elut- ing fractions PlO-A and LH-A contain porphyrins of relatively low mobility (note the residual fluorescence at the origin, with upward trailing). These appear to be the tumor localizers [12,13]. In PlO-A one also sees, in order of increasing mobility, significant amounts of HP, HVD, and a trace of PP. Relatively little HP, HVD or PP are seen in LH-A, confirming that this ma- terial is less contaminated with non-localizing porphyrin monomers [ 12,131. As shown previously [13], LH-B and LH-C contain progressively more of the monomers than LH-A (HP becoming predominant), while PlO-B is mostly HP. The localizing components of LH-A and PlO-A appear to be more polar in this TLC system than HP, despite the fact that their porphyrin elements

PP-

HVDf

HP-

O-

P’P I I I I I I I

HVD HP PlO-A PlO-B LH-A LH-B LH-C

Fig. 1. TLC of HPD fractions and porphyrin standards. Pooled peak fractions in P-10 effluent (PlO-A and PlO-B) and in LH-20 effluent (LH-A, LH-B, LH-C) were chromatographed by normal phase TLC and visualized by fluorescence emission (near-ultraviolet excitation). Individual por phyrins (HP, HVD, PP) were run alongside as standards. Approximately 10 pg porphyrin was applied in each lane.

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(dimers and oligomers linked by ester and/or ether bonds) have been shown to be more hydrophobic [6]. The relative hydrophilic nature of the localizer aggregates as they exist in aqueous solution may explain this observation

[131. Irradiation of fraction LH-A (approx. 9 PM in unit porphyrin) immedi-

ately after mixing with erythrocyte ghosts in D,O buffer produced a signifi- cant ‘0, emission centered at 1268 nm (Fig. 2). The magnitude of this emission was about the same as that observed with LH-A in the absence of membranes. Prolonged incubation of LH-A with membranes (2-3 days) re- sulted in a 2-fold enhancement in ‘0, emission. The Soret absorbance maxi- mum underwent a partial shift from 365 nm to 395 nm during incubation (data not shown), signifying partial dissociation of porphyrin aggregates [ 191.

I

1210 1230 12.50 1270 1290 13!0 1330

WAVELENGTH (nm>

Fig. 2. Singlet oxygen emission spectra: A stock suspension of ghosts (5.0 mg protein/ml in PBS) was incubated with fraction LH-A (106 pg porphyrin/ml) for 72 h in the dark at 4-C. For the luminescence measurements, aliquots were diluted 20.fold as follows: (B) with PBS/D,0 (25 mM sodium phosphate, 125 mM NaCl in deuterium oxide, pD 7.4); (E) with PBS/D,0 con- taining 5 mM NaN,; (F) with PBS in H,O. Other spectra represent the following situations: (C) LH-A plus ghosts in PBS/D,O, recorded < 5 min after mixing at the above concentrations; (D) LH-A alone (5 pg porphyrin/ml) in PBS/D,O; (A) LH-A (5 pg porphyrin/ml) in PBS/D,0 con- taining 10 mM CTAB (shown at l/2 the actual intensity). In order to eliminate signal spikes, each spectrum has been plotted with the r-trimmed means [25] (a = 0.16) of intensity values obtained from 12 wavelength scans.

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At a concentration of 5 mM, the scavenger azide abolished ‘0, emission without affecting background luminescence, suggesting that the latter derives from excited state porphyrin. No ‘0, emission was detected in H,O buffer (Fig. ‘2) which is consistent with the fact that ‘0, lifetime is shorter in H,O than in D,O [20]. Note that the background luminescence is also less intense in H,O compared with that in D,O. The reason for this is not cer- tain; however, deuterium effects on the luminescence of aromatic molecules have been described previously [21]. Complete disaggregation of LH-A por- phyrins in CTAB resulted in a 20-fold increase in ‘0, emission (Fig. 2). Thus, the membrane appears to promote ‘0, formation when it binds LH-A, but to a much lesser extent than detergent.

Corroborating evidence for the formation of ‘0, in HPD-sensitized mem- branes was obtained by showing that a unique photoproduct of ‘0, attack on cholesterol is generated, viz. the 5x-hydroperoxide (5a-OOH) [22]. As shown in Fig. 3A, the major cholesterol product obtained upon borohydride treatment of a lipid extract from photooxidized ghosts comigrated on TLC with material produced by rose bengal, a well-known photogenerator of ‘0, [ 231. Accordingly, the product is identified as 5a-hydroxycholesterol (5a- OH). Neither the RB- nor the HPD-sensitized formation of 5a-OOH was affected by butylated hydroxytoluene, a free radical trap, which confirms that the reaction is non-radical in nature. By way of contrast, the major cholesterol products obtained upon treating ghosts with xanthinelxanthine oxidase/iron (an oxygen radical source [15]) were the 7cl- and 7/l-hydroper- oxides (7a-OOH and 7B-OOH), known products of free radical reactions [22]. In this case identification was based on comigration with the products of 7-ketocholesterol subsequent to borohydride treatment. The trace amounts of 7a-OH and 7fl-OH in the photosensitized reactions are attributed to a radical-mediated rearrangement of 5a-OOH [22], although a small contribu- tion of Type I (free radical) photochemistry cannot be ruled out. Butylated hydroxytoluene prevented formation of these products in the enzymatic as well as the photochemical system, confirming that free radical intermediates are involved in these reactions. As shown in Fig. 3B, 5a-OOH was the major cholesterol photoproduct obtained with all the HPD fractions analyzed. The lower yield with PlO-A or LH-A compared with LH-B and LH-C may have been due to the greater fraction of aggregated porphyrins in the former cases.

DISCUSSION

We have presented unambiguous evidence that the tumor localizing por- phyrins of HPD photosensitize ‘0, formation in a natural membrane envi- ronment. The approximate doubling of the ‘0, emission intensity when the LH-A fraction was incubated with ghosts suggests a significant, albeit in- complete, ‘solubilization’ of aggregated porphyrin dimers/oligomers. Het- erogenous localization in the membranes (with competing effects of

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A

Ch -

5a-OH-

78 -OH-

O- 1

Sid 1-l

B

Ch-

5a-OH-

7B-OH- 7ci-OH-

Q

I 1

Con X0 t-1 c-1

** 3

I I

X0 RI3 (+I (-1

1 I

HPD HPD t-1 (+I

o- B

I I I I I I I I I I

Std PlO-A LH-A LH-B LH-C PlO-A LH-A LH-B LH-C Con

C- 40 min + b 165 min 4

Fig. 3. TLC of cholesterol oxidation products. (A) Comparison of photooxidation and enzymatic oxidation. Membranes (1.0 mg protein/ml in PBS) were irradiated for 90 min in the presence of unfractionated HPD (10 pg/ml) or 60 min in the presence of rose bengal, RB (5 PM). Mem- branes were also reacted with xanthine/xanthine oxidase (X0) (2 h, 37 ‘C). Reactions were car- ried out in the absence ( - ) or presence ( + ) of butylated hydroxytoluene (25 PM). (B) Photooxidation by isolated HPD constituents. Membranes (1.0 mg protein/ml in PBS) were irra- diated for 40 min and 165 min in the presence of the following fractions (10 pg porphyrin/ml): PlO-A, LH-A, LH-B, LH-C. Extracted hydroperoxides in (A) and (B) were reduced with borohy- dride before chromatography. The standard (Std) represents borohydride-reduced 7.ketocholes- terol. No cholesterol products were detected in dark or light controls (Con). Material observed at the origin (0) is primarily phospholipid. Ch, cholesterol; 5a-OH, 5a-hydroxycholesteroh 7a/7/3-OH, 7%/7/?-hydroxycholesterol. Sample load (as starting cholesterol): 0.16 mg/lane.

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enhancement and quenching, depending on the site of interaction) could have been a contributing factor in suboptimal emission. Complete solubiliza- tion into CTAB micelles resulted in a lo-fold greater enhancement in ‘0, emission, similar to that described recently by Keene et al. [24] using a time-resolved detection system. Earlier studies by Blum and Grossweiner [ 191 indicated that porphyrin binding by artificial membranes (liposomes) also increases ‘0, yields.

Identification of the reduction product of 3/l-hydroxy-5cr-cholest-6-ene-5 hydroperoxide (5~-OOH) not only confirms that ‘0, is generated by mem- brane-bound localizer, but demonstrates that it can react with a lipid target. Subcellular membranes appear to be crucial sites of HPD sensitization [7] and hence targets for ‘0, attack. Although lipid peroxidation is likely at these sites, its ultimate importance relative to other forms of membrane damage remains to be established. In this context, the question of whether IO,-initiated lipid peroxidation can be amplified by cytoplasmic reductants (e.g. ascorbate) has to be considered [IO].

ACKNOWLEDGEMENTS

This work was supported in part by grants (to A.W.G.) from the National Science Foundation (DCB-8501894), Milwaukee Regional Cancer Center, and St. Luke’s Foundation (Milwaukee). We are grateful to Dr. David Kessel for the sample of hydroxyethylvinyldeuteroporphyrin and to Gary Bachowski for assistance in the TLC separation of cholesterol products. The luminescence experiments were performed in the Laboratory of Molecular Biophysics, NIEHS.

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