Mitochondrial mechanisms of estrogen neuroprotection.

Mitochondria have become a primary focus in our search not only for the mechanism(s) of neuronal death but also for neuroprotective drugs and therapies that can delay or prevent Alzheimer's disease and other chronic neurodegenerative conditions. This is because mitochrondria play a central role in regulating viability and death of neurons, and mitochondrial dysfunction has been shown to contribute to neuronal death seen in neurodegenerative diseases. In this article, we review the evidence for the role of mitochondria in cell death and neurodegeneration and provide evidence that estrogens have multiple effects on mitochondria that enhance or preserve mitochondrial function during pathologic circumstances such as excitotoxicity, oxidative stress, and others. As such, estrogens and novel non-hormonal analogs have come to figure prominently in our efforts to protect neurons against both acute brain injury and chronic neurodegeneration.

A current practice for predicting ocular toxicity of systemically delivered drugs.

The ability to predict ocular side effects of systemically delivered drugs is an important issue for pharmaceutical companies. Although animal models involving standard clinical ophthalmic examinations and postmortem microscopic examinations of eyes are still used to identify ocular issues, these methods are being supplemented with additional in silico, in vitro, and in vivo techniques to identify potential safety issues and assess risk. The addition of these tests to a development plan for a potential new drug provides the opportunity to save time and money by detecting ocular issues earlier in the program. This review summarizes a current practice for minimizing the potential for systemically administered, new medicines to cause adverse effects in the eye.

Mitochondrial mechanisms of estrogen neuroprotection.

Oxidative stress, bioenergetic failure and mitochondrial dysfunction are all implicated in the etiology of neurodegenerative diseases such as Alzheimer's disease (AD). The mitochondrial involvement in neurodegenerative diseases reflects the regulatory role mitochondrial failure plays in both necrotic cell death and apoptosis. The potent feminizing hormone, 17 beta-estradiol (E2), is neuroprotective in a host of cell and animal models of stroke and neurodegenerative diseases. The discovery that 17alpha-estradiol, an isomer of E2, is equally as neuroprotective as E2 yet is >200-fold less active as a hormone, has permitted development of novel, more potent analogs where neuroprotection is independent of hormonal potency. Studies of structure-activity relationships and mitochondrial function have led to a mechanistic model in which these steroidal phenols intercalate into cell membranes where they block lipid peroxidation reactions, and are in turn recycled. Indeed, the parental estrogens and novel analogs stabilize mitochondria under Ca(2+) loading otherwise sufficient to collapse membrane potential. The neuroprotective and mitoprotective potencies for a series of estrogen analogs are significantly correlated, suggesting that these compounds prevent cell death in large measure by maintaining functionally intact mitochondria. This therapeutic strategy is germane not only to sudden mitochondrial failure in acute circumstances, such as during a stroke or myocardial infarction, but also to gradual mitochondrial dysfunction associated with chronic degenerative disorders such as AD.

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro.

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

The significance of mitochondrial toxicity testing in drug development.

Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities. Members of diverse drug classes undermine mitochondrial function, and among the most potent are drugs that have been withdrawn from the market, or have received Black Box warnings from the FDA. To avoid mitochondrial liabilities, routine screens need to be positioned within the drug-development process. Assays for mitochondrial function, cell models that better report mitochondrial impairment, and new animal models that more faithfully reflect clinical manifestations of mitochondrial dysfunction are discussed in the context of how such data can reduce late stage attrition of drug candidates and can yield safer drugs in the future.

Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants.

Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.

Novel mechanisms for estrogen-induced neuroprotection.

Estrogens are gonadal steroid hormones that are present in the circulation of both males and females and that can no longer be considered within the strict confines of reproductive function. In fact, the bone, the cardiovascular system, and extrahypothalamic regions of the brain are now well-established targets of estrogens. Among the numerous aspects of brain function regulated by estrogens are their effects on mood, cognitive function, and neuronal viability. Here, we review the supporting evidence for estrogens as neuroprotective agents and summarize the various mechanisms that may be involved in this effect, focusing particularly on the mitochondria as an important target. On the basis of this evidence, we discuss the clinical applicability of estrogens in treating various age-related disorders, including Alzheimer disease and stroke, and identify the caveats that must be considered.

Mitochondria play a central role in estrogen-induced neuroprotection.

Oxidative stress, bioenergetic impairment and mitochondrial failure have all been implicated in the etiology of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), as well as retinal degeneration in glaucoma and retinitis pigmentosa. Moreover, at least 75 debilitating, and often lethal, diseases are directly attributable to deletions or mutations in mitochondrial DNA, or in nuclear-encoded proteins destined for delivery to the mitochondria. Such widespread mitochondrial involvement in disease reflects the regulatory position mitochondrial failure plays in both acute necrotic cell death, and in the less catastrophic process of apoptosis. The potent feminizing hormone, 17 beta-estradiol (E2), has shown cytoprotective activities in a host of cell and animal models of stroke, myocardial infarct and neurodegenerative diseases. The discovery that 17alpha-estradiol, an isomer of E2, is equally as cytoprotective as E2 yet is >200-fold less active as a hormone, has permitted development of novel, more potent analogs where cytoprotection is independent of hormonal potency. Studies of structure-activity-relationships, glutathione interactions and mitochondrial function have led to a mechanistic model in which these steroidal phenols intercalate into cell membranes where they block lipid peroxidation reactions, and are in turn recycled via glutathione. Such a mechanism would be particularly germane in mitochondria where function is directly dependent on the impermeability of the inner membrane, and where glutathione levels are maintained at extraordinarily high 8-10mM concentrations. Indeed, the parental estrogens and novel analogs stabilize mitochondria under Ca(2+) loading otherwise sufficient to collapse membrane potential. The cytoprotective and mitoprotective potencies for 14 of these analogs are significantly correlated, suggesting that these compounds prevent cell death in large measure by maintaining functionally intact mitochondria. This therapeutic strategy is germane not only to sudden mitochondrial failure in acute circumstances, such as during a stroke or myocardial infarction, but also to gradual mitochondrial dysfunction associated with chronic degenerative disorders such as AD, PD and HD.

Development of 17alpha-estradiol as a neuroprotective therapeutic agent: rationale and results from a phase I clinical study.

17alpha-estradiol (17alpha-E2) differs from its isomer, the potent feminizing hormone 17beta-estradiol (17beta-E2), only in the stereochemistry at one carbon, but this is sufficient to render it at least 200-fold less active as a transactivating hormone. Despite its meager hormonal activity, 17alpha-E2 is as potent as 17beta-E2 in protecting a wide variety of cell types, including primary neurons, from a diverse array of lethal and etiologically relevant stressors, including amyloid toxicity, serum withdrawal, oxidative stress, excitotoxicity, and mitochondrial inhibition, among others. Moreover, both estradiol isomers have shown efficacy in animal models of stroke, Alzheimer's disease (AD), and Parkinson's disease (PD). Data from many labs have yielded a mechanistic model in which 17alpha-E2 intercalates into cell membranes, where it terminates lipid peroxidation chain reactions, thereby preserving membrane integrity, and where it in turn is redox cycled by glutathione or by NADPH through enzymatic coupling. Maintaining membrane integrity is critical to mitochondrial function, where loss of impermeability of the inner membrane initiates both necrotic and apoptotic pathways. Thus, by serving as a mitoprotectant, 17alpha-E2 forestalls cell death and could correspondingly provide therapeutic benefit in a host of degenerative diseases, including AD, PD, Friedreich's ataxia, and amyotrophic lateral sclerosis, while at the same time circumventing the common adverse effects elicited by more hormonally active analogues. Positive safety and pharmacokinetic data from a successful phase I clinical study with oral 17alpha-E2 (sodium sulfate conjugate) are presented here, and several options for its future clinical assessment are discussed.

Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential, ATP, and cell viability.

PURPOSE: Epidemiologic studies demonstrate a higher incidence of cataracts in estrogen-deprived postmenopausal women, but the mechanism for the increased risk of cataracts is unclear. An elevated level of H(2)O(2) in aqueous humor and whole lenses has been associated with cataractogenesis. In the present study, for the first time, the protective effect of estrogens against oxidative stress were tested in cultured human lens epithelial cells (HLECs). METHODS: To investigate the involvement of 17beta-estradiol (17beta-E(2)) in protection against oxidative stress, HLECs were exposed to insult with H(2)O(2) at a physiological level (100 microM) over a time course of several hours, with and without pretreatment with 17beta-E(2). Cell viability was measured by calcein AM assay, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to determine intracellular reactive oxygen species (ROS). Intracellular adenosine triphosphate (ATP) level was quantified with a luciferin- and luciferase-based assay and mitochondrial potential (deltapsi(m)) was monitored by a fluorescence resonance energy-transfer technique. RESULTS: H(2)O(2) caused a dose-dependent decrease in mitochondrial membrane potential, intracellular ATP levels, and cell viability. Dose-dependent increases in cell viability and intracellular ATP level were observed with pretreatment of 17beta-E(2) for 2 hours before oxidative insult. At 1 nM, 17beta-E(2) increased cell viability from 39% +/- 4% to 75% +/- 3%, and at 100 nM or higher, it increased survival to greater than 95%. The level of intracellular ATP approached normal with 17beta-E(2) at 100 nM or higher. Pretreatment with 17beta-E(2) did not diminish intracellular ROS accumulation after exposure to H(2)O(2). Moreover, two nonfeminizing estrogens, 17alpha-E(2) and ent-E(2), both of which do not bind to either estrogen receptor alpha or beta, were as effective as 17beta-E(2) in the recovery of cell viability. The estrogen receptor antagonist, ICI 182,780, did not block protection by 17beta-E(2). Both 17beta- and 17alpha-E(2) moderated the collapse of deltapsi(m) in response to either H(2)O(2) or excessive Ca(2+) loading. CONCLUSIONS: The present study indicates that both 17alpha- and 17beta-E(2) can preserve mitochondrial function, cell viability, and ATP levels in human lens cells during oxidative stress. Although the precise mechanism responsible for protection by the estradiols against oxidative stress remains to be determined, the ability of nonfeminizing estrogens, which do not bind to estrogen receptors, to protect against H(2)O(2) toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.

Photoreceptor preservation in the S334ter model of retinitis pigmentosa by a novel estradiol analog.

The cytoprotective activity of MITO-4565, a novel, non-hormonal, estradiol derivative, was evaluated in the S334ter transgenic model of retinitis pigmentosa (RP). Progressive blindness in RP is due to apoptotic death of the photoreceptors, a process mimicked by the animal models [Portera-Cailliau C, Sung C-H, Nathans J, Adler R. Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. Proc Natl Acad Sci USA 1994;91:974-8]. On postnatal day 9, 10 transgenic S334ter rats received a single intraocular injection of MITO-4565 in the left eye, and vehicle in the right eye. By postnatal day 20, the thickness of the outer nuclear layer (ONL) in the superior retina of the untreated eyes was 5.76 +/- 1.12 microm (N = 10), versus 10.72 +/- 1.52 microm (N = 10) for eyes treated with MITO-4565 (P < 0.0001, ANOVA F = 1671). Comparable cytoprotection was also observed for the inferior retina. Cytoprotection by MITO-4565 was also observed in primary cultures of rat retinal ganglion cells against NMDA excitotoxicity. Data from studies of hexose monophosphate shunt flux, mitochondrial stability, and in vitro lipid peroxidation, are in accord with previous reports [Green PS, Gridley KE, Simpkins JW. Nuclear estrogen receptor independent neuroprotection by estratrienes: a novel interaction with glutathione. Neuroscience 1997;84:7-10]; a likely mechanism of action entails moderation of membrane lipid peroxidation in a redox couple with glutathione. Such preservation of membrane integrity is particularly crucial to mitochondria, where collapse of membrane potential precipitates cell death, and where GSH is maintained at mM concentrations. Indeed, exposure to MITO-4565, but not a methoxy substituted negative control, allowed mitochondria to retain membrane potential (DeltaPsi(m)) under conditions of Ca(2+) overload that would normally induce complete mitochondrial failure. Mitochondrial interventions offer a novel therapeutic approach for RP, and other degenerative diseases of the retina.

High-throughput assessment of mitochondrial membrane potential in situ using fluorescence resonance energy transfer.

Mitochondrial dysfunction causes dozens of debilitating diseases, and is implicated in the etiology of type 2 diabetes, Parkinson's, and Alzheimer's diseases, among others. However, development of mitochondrially targeted therapeutic agents has been impeded by the lack of high-throughput screening techniques that are capable of distinguishing in intact cells the mitochondrial membrane potential (deltapsi(m)) from the plasma membrane potential, (deltapsi(p)). We report here a fluorescence resonance energy transfer (FRET) assay that specifically monitors deltapsi(m) that is not confounded by background signal arising from potentiometric dye responding to deltapsi(p). The technique relies on energy transfer between nonyl acridine orange (NAO), which stains diphosphatidyl glycerol (cardiolipin) that is indigenous to the inner mitochondrial membrane, and tetramethylrhodamine methyl ester (TMR), a potentiometric dye that is sequestered by mitochondria as a Nernstian function of deltapsi(m) and concentration. FRET occurs only when both dyes co-localize to the mitochondria, and results in quenching of NAO emission by TMR in proportion to deltapsi(m). Validation studies using compounds with well-characterized mitochondrial effects, including oligomycin, CCCP+, bongkrekic acid, cyclosporin A, nigericin, ADP, and ruthenium red, demonstrate that the FRET-based deltapsi(m) assay responds in accord with the known pharmacology. Validation studies assessing the suitability of the technique for high-throughput compound screening indicate that the assay provides a sensitive and robust assessment not only of mitochondrial integrity in situ, but also, when used in conjunction with agents such as cyclosporin A, an indicator of permeability transition.

Effect of the multitargeted tyrosine kinase inhibitors imatinib, dasatinib, sunitinib, and sorafenib on mitochondrial function in isolated rat heart mitochondria and H9c2 cells.

Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.

In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone.

Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.

Strategies to reduce late-stage drug attrition due to mitochondrial toxicity.

Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities and negative side-effect profiles. Early identification of mitochondrial liabilities for new chemical entities is therefore crucial for avoiding late-stage attrition during drug development. Limitations of traditional methods for assessing mitochondrial dysfunction have discouraged routine evaluation of mitochondrial liabilities. To circumvent this bottleneck, a high-throughput screen has been developed that measures oxygen consumption; one of the most informative parameters for the assessment of mitochondrial status. This technique has revealed that some, but not all, members of many major drug classes have mitochondrial liabilities. This dichotomy encourages optimism that efficacy can be disassociated from mitochondrial toxicity, resulting in safer drugs in the future.

Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration.

Mitochondrial impairment is increasingly implicated in the etiology of toxicity caused by some thiazolidinediones, fibrates, and statins. We examined the effects of members of these drug classes on respiration of isolated rat liver mitochondria using a phosphorescent oxygen sensitive probe and on the activity of individual oxidative phosphorylation (OXPHOS) complexes using a recently developed immunocapture technique. Of the six thiazolidinediones examined, ciglitazone, troglitazone, and darglitazone potently disrupted mitochondrial respiration. In accord with these data, ciglitazone and troglitazone were also potent inhibitors of Complexes II+III, IV, and V, while darglitazone predominantly inhibited Complex IV. Of the six statins evaluated, lovastatin, simvastatin, and cerivastatin impaired mitochondrial respiration the most, with simvastatin and lovastatin impairing multiple OXPHOS Complexes. Within the class of fibrates, gemfibrozil more potently impaired respiration than fenofibrate, clofibrate, or ciprofibrate. Gemfibrozil only modestly inhibited Complex I, fenofibrate inhibited Complexes I, II+III, and V, and clofibrate inhibited Complex V. Our findings with the two complementary methods indicate that (1) some members of each class impair mitochondrial respiration, whereas others have little or no effect, and (2) the rank order of mitochondrial impairment accords with clinical adverse events observed with these drugs. Since the statins are frequently co-prescribed with the fibrates or thiazolidinediones, various combinations of these three drug classes were also analyzed for their mitochondrial effects. In several cases, the combination additively uncoupled or inhibited respiration, suggesting that some combinations are more likely to yield clinically relevant drug-induced mitochondrial side effects than others.

Neuroprotective effects of 17beta-estradiol and nonfeminizing estrogens against H2O2 toxicity in human neuroblastoma SK-N-SH cells.

Neuroprotective effects of estrogens have been shown in various in vitro and in vivo models, but the mechanisms underlying protection by estrogen are not clear. Mounting evidence suggests antioxidant effects contribute to the neuroprotective effects of estrogens. In the present study, we assessed the protective effects of estrogens against H2O2-induced toxicity in human neuroblastoma cells and the potential mechanisms involved in this protection. We demonstrate that 17beta-estradiol (17beta-E2) increases cell survival against H2O2 toxicity in human neuroblastoma cells. 17beta-E2 effectively reduced lipid peroxidation induced by 5-min H2O2 exposure. Furthermore, 17beta-E2 exerts the protective effects by maintaining intracellular Ca2+ homeostasis, attenuating ATP depletion, ablating mitochondrial calcium overloading, and preserving mitochondrial membrane potential. Two nonfeminizing estrogens, 17alpha- and ent-estradiol, were as effective as 17beta-E2 in increasing cell survival, alleviating lipid peroxidation, preserving mitochondrial function, and maintaining intracellular glutathione levels and Ca2+ homeostasis against H2O2 insult. Moreover, the estrogen receptor antagonist fulvestrant (ICI 182,780) did not block effects of 17beta-E2, but increased cell survival and blunted intracellular Ca2+ increases. However, these estrogens failed to reduce cytosolic reactive oxygen species, even at concentrations as high as 10 microM. In conclusion, estrogens exert protective effects against oxidative stress by inhibiting lipid peroxidation and subsequently preserving Ca2+ homeostasis, mitochondrial membrane potential, and ATP levels.

PHOTOBIOLOGY OF THE SYMBIOTIC SEA ANEMONE, ANTHOPLEURA ELEGANTISSIMA: DEFENSES AGAINST PHOTODYNAMIC EFFECTS, AND SEASONAL PHOTOACCLIMATIZATION.

The sea anemone Anthopleura elegantissima, which contains photosynthetic symbionts (zooxanthellae), responds both biochemically and behaviorally to the combined environmental stresses of exposure to sunlight and photosynthetically generated hyperbaric O2. Activities of the enzymes superoxide dismutase (SOD) and catalase, which act in concert as defenses against oxygen toxicity, parallel the distribution of chlorophyll. A. elegantissima shows a finely controlled contraction behavior which shades the zooxanthellae and reduces O2 production, but which leaves the body column tissues directly exposed to sunlight. However, the body column contains disproportionately high SOD and catalase activities as defenses against photodynamic damage. This additional role of SOD is demonstrated by shade-adapted aposymbiotic anemones in which SOD and catalase activities increase by 590% and 100% respectively following a 7 day exposure to sunlight. In response to elevated levels of O2 and sunlight exposure, A. elegantissima attaches gravel and other debris to its body surface which serves as a sunscreen that effectively reduces zooxanthella expulsion during exposure to bright sunlight. Finally, anemone chlorophyll content fluctuates on a seasonal basis, varying inversely with mean solar radiation. These seasonal changes are not due to corresponding changes in the number of algal cells, but rather to changes in the chlorophyll content and chlorophyll a:c2 ratio of a fairly uniform standing crop of zooxanthellae.