In vitro study on aspects of molecular mechanisms underlying invasive aspergillosis caused by gliotoxin and fumagillin, alone and in combination.

Gliotoxin (GT) and fumagillin (FUM) are mycotoxins most abundantly produced by Aspergillus fumigatus during the early stages of infection to cause invasive aspergillosis (IA). Therefore, we hypothesized that GT and FUM could be the possible source of virulence factors, which we put to test adopting in vitro monoculture and the novel integrated multiple organ co-culture (IdMOC) of A549 and L132 cell. We found that (i) GT is more cytotoxic to lung epithelial cells than FUM, and (ii) GT and FUM act synergistically to inflict pathology to the lung epithelial cell. Reactive oxygen species (ROS) is the master regulator of the cytotoxicity of GT, FUM and GT + FUM. ROS may be produced as a sequel to mitochondrial damage and, thus, mitochondria are both the source of ROS and the target to ROS. GT-, FUM- and GT + FUM-induced DNA damage is mediated either by ROS-dependent mechanism or directly by the fungal toxins. In addition, GT, FUM and GT + FUM may induce protein accumulation. Further, it is speculated that GT and FUM inflict epithelial damage by neutrophil-mediated inflammation. With respect to multiple organ cytotoxicity, GT was found to be cytotoxic at IC50 concentration in the following order: renal epithelial cells < type II epithelial cells < hepatocytes < normal lung epithelial cells. Taken together, GT and FUM alone and in combination contribute to exacerbate the damage of lung epithelial cells and, thus, are involved in the progression of IA.

Bioavailable phytoprostanes and phytofurans from Gracilaria longissima have anti-inflammatory effects in endothelial cells.

BACKGROUND: An array of bioactive compounds with health-promoting effects has been described in several species of macroalgae. Among them, phytoprostanes (PhytoPs) and phytofurans (PhytoFs), both autoxidation products of α-linolenic acid, have been seen to exert immunomodulatory and antiinflammatory activities in vitro. The purpose of this study was to explore the bioaccesibility, bioavailability, and bioactivity of PhytoPs and PhytoFs obtained from the edible red algae Gracilaria longissima, and to gain insight into the anti-inflammatory activity of their bioavailable fraction in human endothelial cells. METHODS: The PhytoPs and PhytoFs profile and concentration of G. longissima were determined by UHPLC-QqQ-MS/MS. Algal samples were processed following a standardised digestion method including gastric, intestinal, and gastrointestinal digestion. The bioavailability of the PhytoPs and PhytoFs in the characterized fractions was assessed in a Caco-2 cell monolayer model of the intestinal barrier. The inflammation response of these prostaglandin-like compounds in human endothelial cells, after intestinal absorption, was investigated in vitro. RESULTS: Simulated digestions significantly reduced the concentration of PhytoPs and PhytoFs up to 1.17 and 0.42 µg per 100 g, respectively, on average, although permeability through the Caco-2 cell monolayer was high (up to 88.2 and 97.7%, on average, respectively). PhytoP and PhytoF-enriched extracts of raw algae impaired the expression of ICAM-1 and IL-6 inflammation markers. The inflammation markers progressed in contrast to the relative concentrations of bioactive oxylipins, suggesting pro- or anti-inflammatory activity on their part. In this aspect, the cross-reactivity of these compounds with diverse receptors, and their relative concentration could explain the diversity of the effects found in the current study. CONCLUSIONS: The results indicate that PhytoPs and PhytoFs display complex pharmacological profiles probably mediated through their different actions and affinities in the endothelium.

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications.

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.

N-acyl dopamines induce cell death in PC12 cell line via induction of nitric oxide generation and oxidative stress.

It was shown that dopamine amides of arachidonic, oleic, and docosahexaenoic acids exhibit toxicity with respect to PC12 pheochromocytoma cell line. The mechanism of realization of the cytotoxic effect of acyl dopamines is the induction of oxidative stress. This event is preceded by triggering the synthesis of nitric oxide.

Investigating the role of H2S in 4-HNE scavenging.

4-HNE (4-hydroxy-2-nonenal) is a highly reactive α,ß-unsaturated aldehyde generated from oxidation of polyunsaturated fatty acids and has been suggested to play a role in the pathogenesis of several diseases. 4-HNE can bind to amino acids, proteins, polynucleotides, and lipids and exert cytotoxicity. 4-HNE forms adducts (Michael adducts) with cysteine, lysine, as well as histidine on proteins with the thiol function as the most reactive nucleophilic moiety. Thus, detoxification strategies by 4-HNE scavenging compounds might be of interest. Recently, hydrogen sulfide (H2S) has been identified as an endogenous vascular gasotransmitter and neuromodulator. Assuming that the low-molecular thiol H2S may react with 4-HNE, methods to monitor the ability of H2S to counteract the protein-modifying and cytotoxic activity of 4-HNE are described in this chapter.

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties.

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.

Methyl substitution of a rexinoid agonist improves potency and reveals site of lipid toxicity.

(2E,4E,6Z,8E)-8-(3',4'-Dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid, 9cUAB30, is a selective rexinoid that displays substantial chemopreventive capacity with little toxicity. 4-Methyl-UAB30, an analogue of 9cUAB30, is a potent RXR agonist but caused increased lipid biosynthesis unlike 9cUAB30. To evaluate how methyl substitution influenced potency and lipid biosynthesis, we synthesized four 9cUAB30 homologues with methyl substitutions at the 5-, 6-, 7-, or 8-position of the tetralone ring. The syntheses and biological evaluations of these new analogues are reported here along with the X-ray crystal structures of each homologue bound to the ligand binding domain of hRXRα. We demonstrate that each homologue of 9cUAB30 is a more potent agonist, but only the 7-methyl-9cUAB30 caused severe hyperlipidemia in rats. On the basis of the X-ray crystal structures of these new rexinoids and bexarotene (Targretin) bound to hRXRα-LBD, we reveal that each rexinoid, which induced hyperlipidemia, had methyl groups that interacted with helix 7 residues of the LBD.

Synthesis and fungicidal activities of novel benzothiophene-substituted oxime ether strobilurins.

Twenty-one novel benzothiophene-substituted oxime ether strobilurins, which employed a benzothiophene group to stabilise the E-styryl group in Enoxastrobin (an unsaturated oxime strobilurin fungicide developed by Shenyang Research Institute of Chemical Industry, China) were designed and synthesised. The biological assay indicated that most compounds exhibited good or excellent fungicidal activities, especially against Colletotrichum lagenarium and Puccinia sorghi Schw. In addition, methyl 3-methoxypropenoate oxime ethers and N-methoxy-carbamic acid methyl esters exhibited good in vivo fungicidal activities against Erysiphe graminis, Colletotrichum lagenarium and Puccinia sorghi Schw. under the tested concentrations. Notably, (E,E)-methyl 3-methoxy-2-(2-((((6-chloro-1-(1H-benzo[b]thien-2-yl)ethylidene)amino)oxy)methyl)phenyl)propenoate (5E) exhibited more potent in vivo fungicidal activities against nearly all of the tested fungi at a concentration of 0.39 mg/L compared to Enoxastrobin.

Fumagillin: an overview of recent scientific advances and their significance for apiculture.

Fumagillin is a potent fungal metabolite first isolated from Aspergillus fumigatus. It is widely used in apiculture and human medicine against a variety of microsporidian fungal infections. It has been the subject of research in cancer treatments by employing its angiogenesis inhibitory properties. The toxicity of fumagillin has limited its use for human applications and spurred the development of analogues using structure-activity relationships relating to its angiogenesis properties. These discoveries may hold the key to the development of alternative chemical treatments for use in apiculture. The toxicity of fumagillin to humans is important for beekeeping, because any residues remaining in hive products pose a direct risk to the consumer. The analytical methods published to date measure fumagillin and its decomposition products but overlook the dicyclohexylamine counterion of the salt form widely used in apiculture.

Monounsaturated 14:1n-9 and 16:1n-9 fatty acids but not 18:1n-9 induce apoptosis and necrosis in murine HL-1 cardiomyocytes.

Patients with inborn errors of long-chain fatty acid oxidation accumulate disease-specific acylcarnitines and triacylglycerols in various tissues. Some of these patients present significant cardiac diseases such as arrhythmias and cardiomyopathy. The mechanism of how fatty acid accumulation is involved in disease pathogenesis is still unclear but apoptosis of cardiomyocytes has been suggested to be one possible mechanism of cardiomyopathy development. In this study, we measured lipid uptake and intracellular lipid accumulation after incubation of HL1 cardiomyocytes with different saturated and monounsaturated long- and medium-chain fatty acid species for various time periods and at different physiological concentrations. We assessed apoptosis induction by analyzing the mitochondrial membrane potential and TLR-4 expression as well as the composition of the accumulating triacylglycerols. We identified only 14:1 and 16:1 monounsaturated fatty acids potentially leading to an increase in TLR-4 expression and disruption of the mitochondrial membrane potential, resulting in apoptosis and necrosis in cultured cardiomyocytes. This study demonstrates significant toxicity of especially those fatty acid species in vitro that significantly accumulate in fatty acid oxidation defects presenting with cardiac disease such as very long-chain acyl-CoA dehydrogenase, carnitine acylcarnitine translocase and carnitine palmitoyl-CoA transferase deficiencies.

Synthesis of the novel (±)-2-methoxy-6-icosynoic acid--a fatty acid that induces death of neuroblastoma cells.

The first total synthesis for the novel fatty acid (±)-2-methoxy-6-icosynoic acid was accomplished in seven steps and in a 14% overall yield starting from 2-(4-bromobutoxy)-tetrahydro-2H-pyran. The title compound displayed an EC50=23±1 µM against the human SH-SY5Y neuroblastoma cell line and an EC50=26±1 µM against the human adenocarcinoma cervix cell line (HeLa) after 48 h of exposure. The corresponding non-methoxylated analog 6-icosynoic acid did not display cytotoxicity (EC50>500 µM) toward the studied cell lines as well as the 2-methoxyicosanoic acid (EC50>300 µM). The critical micelle concentration (CMC=20-30 µM) for the (±)-2-methoxy-6-icosynoic acid was also determined. It was found that α-methoxylation decreases the CMC of a fatty acid.

Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells.

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 µM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm(2). The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.

Stimulation of suicidal erythrocyte death by fumagillin.

Fumagillin, a cyclohexane isolated from fungus Aspergillus fumigatus, has anti-infective and anti-cancer potency. Fumagillin is at least partially effective by inducing suicidal death or apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide. The present study explored whether fumagillin (5-100 µM) could stimulate eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, ceramide by utilizing specific antibodies, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding and haemolysis from haemoglobin release. As a result, a 48-hr exposure to fumagillin significantly increased [Ca(2+)](i) (≥10 µM), enhanced ceramide abundance (100 µM), triggered annexin V binding (≥10 µM) and decreased forward scatter (≥10 µM). Fumagillin exposure was followed by slight but significant increase of haemolysis. Removal of extracellular Ca(2+) significantly blunted but did not abolish the effect of fumagillin (100 µM) on annexin V binding. The present observations disclose a novel effect of fumagillin, that is, stimulation of eryptosis, paralleled by Ca(2+) entry, ceramide formation, phosphatidylserine exposure and decrease of cell volume.

Chemotherapeutic sensitization of leptomycin B resistant lung cancer cells by pretreatment with doxorubicin.

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1.

Ferulic acid influences hepatic expression pattern of matrix metalloproteinases during alcohol and PUFA induced toxicity.

BACKGROUND AND OBJECTIVES: Alcoholic fibrosis and its end stage cirrhosis represent a major health problem worldwide. Liver fibrosis occurs when the rate of matrix synthesis exceeds matrix degradation. The degree of matrix remodeling depends on the ratio of active matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The objective of the present work was to study the influence of ferulic acid, a polyphenolic compound, on the expression of MMPs and TIMPs during alcohol and heated polyunsaturated fatty acid (delta PUFA) induced liver toxicity in male albino Wistar rats. MATERIALS AND METHODS: The levels of collagen, the activity of MMPs, the activity of TIMPs, the expression pattern of MMP were analyzed in liver. RESULTS: The matrix metalloproteinase expression was found to be significantly increased in alcohol as well as delta PUFA treated rats and significantly decreased in alcohol + delta PUFA treated rats. The levels of TIMPs and the collagen were significantly increased in alcohol, delta PUFA and alcohol + delta PUFA groups. Administration of ferulic acid significantly decreased the levels of collagen, TIMPs and positively modulated the expression of MMPs. CONCLUSIONS: Ferulic acid influences MMPs, TIMPs expression and effectively protects liver against alcohol and DPUFA induced liver fibrosis.

Characterization and cytotoxicity studies of the rare 21:4 n-7 acid and other polyunsaturated fatty acids from the marine opisthobranch Scaphander lignarius, isolated using bioassay guided fractionation.

The marine opisthobranch Scaphander lignarius has been analyzed in the systematic search for novel bioactive compounds in Arctic marine organisms using bioassay guided fractionation. A number of highly cytotoxic fractions were shown to contain mainly polyunsaturated fatty acids (PUFAs). Selected PUFAs were isolated and identified using both liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). It was shown that the opisthobranch contained unusual PUFAs such as several ω3 fatty acids and the ω7 heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) not isolated before. The organism was shown to be a very rich source of PUFAs and the activity of the isolated compounds against a range of human cancer cell lines (melanoma, colon carcinoma and breast carcinoma) is further reported. The ω7 PUFA was significantly more cytotoxic in comparison with reference ω6 arachidonic and ω3 eicosapentaenoic acid. A noteworthy non-selective cytotoxicity against normal lung fibroblasts was also established. The paper contains isolation protocols in addition to cytotoxicity data of the isolated compounds. The potential of marine mollusks as a source for rare PUFAs is also discussed.

Dietary n-3 polyunsaturated fatty acids and the paradox of their health benefits and potential harmful effects.

There is some evidence to support the toxicity of polyunsaturated fatty acids (PUFAs) and their oxidative products, suggesting their involvement in the pathogenesis of different chronic diseases, including cancer. It has been shown that products of PUFA oxidation may exert a carcinogenic action by forming mutagenic adducts with DNA. However, a large amount of evidence accumulated over several decades has indicated the beneficial effects of administration of n-3 PUFAs in the prevention and therapy of a series of diseases. In particular, there is much evidence that n-3 PUFAs exert anti-inflammatory and antineoplastic effects, whereas n-6 PUFAs promote inflammation and carcinogenesis. In our tissues, both of the two classes of PUFAs can be converted into bioactive products, incorporated into membrane phospholipids or bound to membrane receptors, where they may alter, often in opposite ways, transduction pathways and affect important biological processes, such as cell death and survival, inflammation, and neo-angiogenesis. In the present review, we intend to shed light on the paradox of the coexisting healthy and toxic effects of n-3 PUFAs, focusing on their possible pro-oxidant cytotoxic and carcinogenic effect, in order to understand if their increased intake, recommended by a number of health agencies worldwide and promoted by nutraceutical producers, may or may not represent a hazard to human health.

Assessment of oral toxicity and safety of 9-cis-UAB30, a potential chemopreventive agent, in rat and dog studies.

9-cis-UAB30 is a potential chemopreventative agent that has been shown to be effective on many different types of tumors. The safety and toxicity of 9-cis-UAB30 had not been previously established. These studies were conducted to evaluate the potential toxicity and pharmacokinetics in a rodent and a nonrodent species for the purpose of investigational new drug submission. Oral gavage administration of 9-cis-UAB30 at the doses 0, 3, 15, and 100 mg/kg/day to CD® rats for 28 days showed a dose-dependent (although not dose-proportional) increase in plasma drug levels in week 4. The liver was the target organ for toxicity of 9-cis-UAB30. Hepatomegaly along with increases in serum aspartate-aminotransferase and alkaline-phosphatase levels were seen in rats. Moderate hypoalbuminemia and hyperglobulinemia resulted in a decreased albumin/globulin ratio. Histopathology revealed hepatocellular change consistent with hepatic glycogen deposition. Toxicity studies in dogs did not show treatment-related toxicity at doses as high as 100 mg/kg/day (highest dose tested) administered by capsules for 28 days. No effects on the central nervous system (functional observational battery in rats) or cardiovascular function (safety pharmacology study in telemeterized dogs) were seen. The no observed adverse effect level (NOAEL) in the rat studies was 3 mg/kg/day; however, the adverse effects seen in rats receiving 15 mg/kg/day (the least observed adverse effect level) was a slight, but statistically significant, elevation in fibrinogen and decrease in prothrombin time, which may be a sign of some tendency for increased blood coagulation. The NOAEL in the dog study was at least 100 mg/kg/day.

Short-time gene expression response to valproic acid and valproic acid analogs in mouse embryonic stem cells.

Prediction of developmental toxicity in vitro could be based on short-time toxicogenomic endpoints in embryo-derived cell lines. Microarray studies in P19 mouse embryocarcinoma cells and mouse embryos have indicated that valproic acid (VPA), an inducer of neural tube defects, deregulates the expression of many genes, including those critically involved in neural tube development. In this study, we exposed undifferentiated R1 mouse embryonic stem cells to VPA and VPA analogs for 6 h and used CodeLink whole-genome expression microarrays to define VPA-responsive genes correlating with teratogenicity. Compared with the nonteratogenic analog 2-ethyl-4-methylpentanoic acid, VPA and the teratogenic VPA analog (S)-2-pentyl-4-pentynoic acid deregulated a much larger number of genes. Five genes (of ∼2500 array probes correlating with the separation) were sufficient to effectively separate teratogens from nonteratogens. A large fraction of the target genes correlating with teratogenicity are functionally related to embryonic development and morphogenesis, including neural tube formation and closure. Similar responses in R1 were found for most genes previously identified as VPA responsive in P19 and embryos. A subset of target genes was evaluated as candidate markers predictive of potential teratogenicity against a range of known teratogens using TaqMan expression arrays. These marker genes showed a positive predictive value for the teratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoic acid are known histone deacetylase (HDAC) inhibitors but not for compounds that are likely to act by other mechanisms. This indicates that HDAC inhibition may be a major mechanism by which VPA induces gene deregulation and possibly teratogenicity.

PUFAs enhance oxidative stress and apoptosis in tumour cells exposed to hypericin-mediated PDT.

Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.