Albert W. Girotti

PHOTODYNAMIC LIPID PEROXIDATION IN BIOLOGICAL SYSTEMS

Abstract— Oxidative degradation of cell membrane lipids in the presence of molecular oxygen, a sensitizing agent and exciting light is termed photodynamic lipid peroxidation (photoperoxidation). Like other types of lipid peroxidation, photoperoxidation is detrimental to membrane structure and function, and could play a role in many of the toxic as well as therapeutic effects of photodynamic action. Recent advances in our understanding of photoperoxidation and its biomedical implications are reviewed in this article. Specific areas of interest include (a) reaction mechanisms; (b) methods of detection and quantitation; and (c) cellular defenses (enzymatic and non‐enzymatic).

Mechanisms of lipid peroxidation

This article provides an overview of how peroxidation of unsaturated lipids takes place and how it can be measured. Several different aspects of free-radical-mediated lipid peroxidation are discussed, including: the catalytic role of chelated iron and other redox metal ions; induction by reducing agents such as superoxide, ascorbate, and xenobiotic free radicals; suppression by antioxidant chemicals and enzymes; and how peroxidation that depends on pre-existing hydroperoxides (lipid hydroperoxide-dependent initiation of lipid peroxidation) can be distinguished from that which does not (lipid hydroperoxide-independent initiation of lipid peroxidation). Attention is also given to non-radical, singlet oxygen-driven peroxidation and how this can be resolved from radical-driven processes.

Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms

Unsaturated lipids in cell membranes, including phospholipids and cholesterol, are well-known targets of oxidative modification, which can be induced by a variety of stresses, including ultraviolet A (UVA)- and visible light-induced photodynamic stress. Photodynamic lipid peroxidation has been associated with pathological conditions such as skin phototoxicity and carcinogenesis, as well as therapeutic treatments such as antitumor photodynamic therapy (PDT). Lipid hydroperoxides (LOOHs), including cholesterol hydroperoxides (ChOOHs), are important non-radical intermediates of the peroxidative process which can (i) serve as in situ reporters of type I vs. type II chemistry; (ii) undergo one-electron or two-electron reductive turnover which determines whether peroxidative injury is respectively intensified or suppressed; and (iii) mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great. The purpose of this article is to review current understanding of photodynamic (UVA- or visible light-induced) lipid peroxidation with a special focus on LOOH generation and reactivity. Future goals in this area, many of which depend on continued development of state-of-the-art analytical techniques, will also be discussed.

Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothionein oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H2O2- and O2--dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn-metallothionein were preincubated with H2O2 and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein protection appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.

Enzymatic reduction of phospholipid and cholesterol hydroperoxides in artificial bilayers and lipoproteins

Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxidized liposomes underwent a burst of thiobarbituric acid-detectable lipid peroxidation which could be inhibited by prior GSH/PHGPX treatment, but not by GSH/GPX treatment. Additional experiments indicated that hydroperoxides of phosphatidylcholine, cholesterol and cholesteryl esters in low-density lipoprotein are also good substrates for PHGPX. An important role of PHGPX in cellular detoxification of a wide variety of LOOHs in membranes and internalized lipoproteins is suggested from these findings.

Inhibitory effect of zinc(II) on free radical lipid peroxidation in erythrocyte membranes

New evidence in support of zincs role as a membrane antioxidant is presented. Human erythrocyte membranes in buffered saline underwent catalase- and superoxide dismutase-inhibitable lipid peroxidation when incubated with xanthine, xanthine oxidase, and Fe(III). Free radical mediated peroxidation was measured in terms of thiobarbituric acid reactivity and iodometric determination of lipid hydroperoxides. Whereas Ca(II) had relatively little effect on lipid peroxidation, Zn(II) strongly inhibited the reaction and suppressed peroxidation-dependent lysis of resealed membranes. Inhibition of lipid peroxidation was essentially complete in the presence of 0.1 mM Zn(II), a concentration equivalent to that of added Fe(III). By contrast, Zn(II) had no effect on rose bengal-photosensitized lipid peroxidation, a predominantly nonradical, singlet oxygen-driven process. Zinc(II) also interfered with xanthine/xanthine oxidase/iron-induced peroxidation of Triton X-100-dispersed membranes, but had no effect if EDTA was present. Trivial reasons for inhibition, for example, inactivation of xanthine oxidase or complex formation with O2-, were ruled out by showing that the rate of reduction of cytochrome c by xanthine/xanthine oxidase is not affected by Zn(II). We speculate that Zn(II) acts by interfering with the redox cycling of iron, possibly by competing with the latter for membrane binding sites.

Role of lipid hydroperoxides in photo-oxidative stress signaling.

Photosensitized peroxidation of membrane lipids has been implicated in skin pathologies such as phototoxicity, premature aging, and carcinogenesis, and may play a role in the antitumor effects of photodynamic therapy. Lipid hydroperoxides (LOOHs) are prominent early products of photoperoxidation that typically arise via singlet oxygen ((1)O(2)) attack. Nascent LOOHs can have several possible fates, including (i) iron-catalyzed one-electron reduction to chain-initiating free radicals, which exacerbate peroxidative damage, (ii) selenoperoxidase-catalyzed two-electron reduction to relatively innocuous alcohols, and (iii) translocation to other membranes, where reactions noted in (i) or (ii) might take place. In addition, LOOHs, like other stress-associated lipid metabolites/peroxidation products (e.g., arachidonate, diacylglycerol, ceramide, 4-hydroxynonenal), may act as signaling molecules. Intermembrane transfer of LOOHs may greatly expand their signaling range. When photogenerated rapidly and site-specifically, e.g., in mitochondria, LOOHs may act as early mediators of apoptotic cell death. This review will focus on these various aspects, with special attention to the role of LOOHs in photooxidative signaling.

Photodynamic action of protoporphyrin IX on human erythrocytes: cross-linking of membrane proteins.

Summary When human erythrocytes (≈4% hematocrit) in the presence of 5 μM protoporphyrin IX (pH 7.4, 10°C) were exposed to ≈45 J/cm2 of blue light, photodamage occurred, which was manifested initially by

PHOTOPEROXIDATION OF CHOLESTEROL IN HOMOGENEOUS SOLUTION, ISOLATED MEMBRANES, AND CELLS: COMPARISON OF THE 5α‐ AND 6β‐HYDROPEROXIDES AS INDICATORS OF SINGLET OXYGEN INTERMEDIACY

Abstract— Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene‐addition hydroperoxides: 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide (5α‐OOH), 3β‐hydroxycholest‐4‐ene‐6α‐hydroperoxide (6α‐OOH), and 3β‐hydroxycholest‐4‐ene‐6β‐hydroperoxide (6β‐OOH). The rates of dye‐sensitized photogeneration and also the fates of 5α‐OOH and 6β‐OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5α‐OOH and 6β‐OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6β‐OOH formation was 335% that of 5α‐OOH in a variety of liposomal systems. However, after a lag, 5α‐OOH invariably decayed via allylic rearrangement to 7α‐OOH (also known to be a free radical product), whereas 6β‐OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6β‐OOH formation relative to 5α‐OOH was reduced by approximately half, with essentially no isomerization of the latter to 7α‐OOH. These results suggest that (i) environmental factors in a lipid bilayer somehow make photogeneration of 6β‐OOH more favorable than in homogeneous solution; and (ii) due to its relative stability, 6β‐OOH may be a more reliable probe of 1O2 intermediacy than 5α‐OOH.