Photoenhancement of Lipid Peroxidation Associated with the Generation of Reactive Oxygen Species in Hepatic Microsomes of Hematoporphyrin Derivative-treated Rats1

Hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative (HPD) undergo rapid enhancement of lipid peroxidation in the presence of solar radiation (~400 nm). Quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and 0-carotene, and inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethanol, largely protected against the enhancement of lipid peroxidation caused by HPD photosensitization. Catalase, a scavenger of hydrogen peroxide and Superoxide dismutase, a scavenger of Superoxide aniÃ3n, had little or no protective effect against HPD-photosensitized enhancement of lipid peroxidation. Our data indicate that in vitro irradiation of hepatic microsomes prepared from HPD-treated rats results in the generation of both singlet oxygen and hydroxyl radical. These reactive moitiésare associated with a rapid in crease in microsomal lipid peroxidation which may explain the unique susceptibility of membranous components of cells to this type of phototoxic injury. INTRODUCTION HPD,3 a mixture of several porphyrin moitiésprepared from hematoporphyrin, is being increasingly used as an approach to the management of malignant tumors (1, 2). It is believed that malignant cells may selectively take up porphyrins and that porphyrin photosensitization generates reactive oxygen species which can result in the oxidation of essential cellular components including proteins, lipids, amino acids, and nucleotides (3, 4). Although the subcellular site of porphyrin-induced cytotoxicity has not been ascertained, prior studies have shown that HPD photosensitization results in the destruction of mitochondria! cytochrome c oxidase (5). Furthermore, studies from our labo ratory have verified that the microsomal hemeprotein, cyto chrome P-450, is destroyed by HPD photosensitization (6). Lipid peroxidation has been broadly defined as the oxidative deterioration of polyunsaturated lipids (7). The peroxidation of lipids can be highly catastrophic to the integrity of cellular mem branes and to membrane-bound enzymes (8, 9). It is believed that lipid peroxidation of biomembranes is one mechanism whereby a foreign chemical may be toxic to cells, and this has evoked considerable interest in understanding the mechanism of this phenomenon in various biological systems (8, 9). Since lipid peroxidation is thought to be an essential marker for microsomal membrane damage, we attempted to determine whether HPD Received 1/8/85; revised 8/23/85; accepted 9/9/85. 1Supported by NIH Grants CA 34368, ES-1900, and CA-38028 and by research funds from the Veterans Administration. 1 Recipient of the Burroughs Wellcome Fund fellowship award from the Derma tology Foundation. To whom requests for reprints should be addressed, at Veterans Administration Medical Center, 10701 East Boulevard, Cleveland, OH 44106. 3The abbreviations used are: HPD, hematoporphyrin derivative; 2,5-DMF, 2,5dimethylfuran; D;.O. deuterium oxide; MDA, malonyldialdehyde. photosensitization could alter endogenous lipid peroxidation. Our studies indicate that hepatic microsomal lipid peroxidation is significantly enhanced by reactive oxygen species generated during HPD photosensitization. These results suggest that en hancement of lipid peroxidation in microsomal membranes may contribute to cell cytotoxicity evoked by HPD-induced photosen sitization. Such a response could play a role in the antineoplastic effect of photoradiation therapy with this compound. MATERIALS AND METHODS Chemicals. Histidine, 2,5-dimethylfuran, 0-carotene, Superoxide dis mutase, catalase, mannitol, benzoate, and D2O were obtained from Sigma Chemical Company, St. Louis, MO. Hematoporphyrin dihydrochloride was a product of Porphyrin Products, Logan, UT. All other chemicals were of highest purity commercially available. HPD was pre pared essentially by the method of Lipson ef a/. (10) as described earlier (6). Animals and Preparation of Hepatic Microsomes. Adult male Sprague-Dawley rats (100 ±10 g) were obtained from Holtzman Rat Farm, Madison, Wl. Rats were given injections i.p. of HPD (20, 40, 60, 80 mg/ kg of body weight). In most of the experiments performed, a HPD dose of 80 mg/kg of body weight was used as described by Gibson and Hilf (5). The control animals received an equal volume of vehicle. The animals were kept in the dark and were sacrificed by cervical dislocation 2 h after treatment with HPD. Hepatic microsomes were prepared according to the conventional method (11). The twice washed microsomal pellet was suspended in 0.1 M phosphate buffer prepared in D20 containing 10~4 M MgCI2. The protein concentration in the samples was determined ac cording to the method of Lowry ef a/. (12) using bovine serum albumin as reference standard. Exposure of Microsomes from HPD-treated Rats to UV Light. The microsomal suspension was brought to a protein concentration of 1 mg/ ml. Photooxidation was performed using a Model B-1000-A black-ray long-wave UV lamp which has an emission spectrum between 380 and 420 nm; light intensity was determined using a black-ray long-wave UV meter (Ultraviolet Products, Inc.). The HPD-treated microsomes were exposed to the UV light source at an intensity of 50 W/m2 at 0-4°C (the reaction vessel was immersed in crushed ice throughout the incubation). Assay of Lipid Peroxidation. Lipid peroxidation in hepatic microsomal preparations from control and HPD-treated rats was assessed by mea suring the formation of MDA as described by Wright ef al. (13). The following modifications were made. Lipid peroxidation was measured by incubating 0.5-0.7 mg of microsomal protein in 0.6 ml of Ca2+-free 0.1 M phosphate buffer, pH 7.4, containing 10~4 M MgCI2 for 10 min at 37°C. The reaction was terminated by the addition of 0.6 ml of 10% (w/v) trichloroacetic acid. One ml of 0.5% (w/v) 2-thiobarbituric acid was added to the reaction mixture which was then heated for 10 min in a boiling water bath. Samples were cooled under running tap water and centrifuged, and the levels of MDA were measured by estimating the formation of color at 535 nm. The concentration of MDA was calculated by using a molar extinction coefficient of 1.5 x 10* M~V All buffers used in this study were prepared in D2O unless otherwise stated. Quenchers of activated oxygen were also prepared in D2O buffer. CANCER RESEARCH VOL. 45 DECEMBER 1985