Safety assessment of a newly synthesized copolymer for micellar delivery of hydrophobic caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE), a major pharmacologically active component of poplar type propolis, is known for its proapoptotic, anti-inflammatory, antioxidant, antiviral, and enzyme inhibiting activities. The aim of this study was to perform an in vitro and in vivo safety assessment of a micellar system based on a newly synthesized copolymer, consisting of polyglycidol and poly(allyl glycidyl ether) (C12-PAGE-PG) as a drug delivery platform for CAPE. The in vitro studies on HepG2 and L929 cells by MTT and LDH assays after treatment with the empty and CAPE-loaded micelles showed no cytotoxic effects of the empty micelles and retained cytotoxic activity of CAPE loaded in the micelles. No hemolysis or stimulation of mouse lymphocytes or macrophages was observed in vitro. In vivo hematological, biochemical, and histological assays on rats, treated with the empty (2580 and 5160 µg/kg) or CAPE-loaded (375 and 750 µg CAPE/kg) micelles did not reveal pathological changes of any of the parameters assayed after 14-days' treatment. In conclusion, initial toxicological data characterize C12-PAGE-PG as a non-toxic and promising copolymer for development of micellar drug delivery systems, particularly for a hydrophobic active substance as CAPE.

Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: Impact on oxidative stress and RANKL/OPG signals.

Glucocorticoid-induced osteoporosis (GIO) is one of the most common causes of secondary osteoporosis. Given that glucocorticoids are considered as a main component of the treatment protocols for a variety of inflammation and immune-mediated diseases besides its use as adjuvant to several chemotherapeutic agents, it is crucial to find ways to overcome this critical adverse effect. Caffeic acid phenethyl ester (CAPE), which is a natural compound derived from honeybee propolis displayed promising antiosteoporotic effects against mechanical bone injury in various studies. The current work aimed at investigating the potential protective effect of CAPE against GIO in vivo with emphasis on the modulation of oxidative status and receptor activator of NF-kB ligand (RANKL)/osteoprotegrin (OPG) signaling. The results showed that CAPE opposed dexamethasone (DEX)-mediated alterations in bone histology and tartarate-resistant acid phosphatase (TRAP) activity. In addition, CAPE restored oxidative balance, Runt-related transcription factor 2 (RunX2) expression and reduced caspase-3 activity in femur tissues. Co-administration of CAPE with DEX normalized RANKL/OPG ratio and Akt activation indicating a reduction in DEX-osteoclastogenesis. In conclusion, concurrent treatment of CAPE with DEX exhibited promising effects in the protection against DEX-induced osteoporosis through opposing osteoclastogenesis and protecting osteoblasts. The potent antioxidant activity of CAPE is, at least in part, involved in its anti-apoptotic effects and modulation of RunX2 and RANKL/OPG signals. The use of CAPE-enriched propolis formulas is strongly recommended for patients on chronic glucocorticoid therapy to help in the attenuation of GIO.

Hydroxytyrosol is not genotoxic in vitro.

Hydroxytyrosol (HT) is an olive-derived phenol endowed with several biological activities, some of which demonstrated in humans. Indeed, the European Food Safety Authority (EFSA) allows the health claim that HT (≥5mg/d) "protects LDL particles from oxidative damage". In terms of safety, HT has been investigated as the predominant part of raw olive mill waste water extracts that have been granted the Generally Recognized as Safe (GRAS) status. Also, a long-term toxicological study of HT proposed a NOAEL of 500mg/kg/d. As several HT-containing supplements and functional foods are entering the market, we sought to investigate the genotoxic and mutagenic potential of HT, using well-established in vitro models, i.e. the chromosomal aberration assay and the Ames test (by using the Salmonella typhimurium TA 100, TA98, TA1535, and TA1537 strains and Escherichia coli WP2(pKM101)), with and without S9-induced metabolic activation). Even though we cannot rule out that prolonged exposure to HT and its metabolites might have untoward effects, the results of this study indicate that HT is non-genotoxic and non-mutagenic at concentrations that far exceed those attainable after intake.

Dose-dependent metabolic disposition of hydroxytyrosol and formation of mercapturates in rats.

Hydroxytyrosol (HT), one of the major polyphenols present in olive oil, is known to possess a high antioxidant capacity. The aim of the present study was to investigate dose dependent (0, 1, 10 and 100 mg/kg) alterations in the metabolism of HT in rats since it has been reported that metabolites may contribute to biological effects. Special attention was paid to the activation of the semiquinone-quinone oxidative cycle and the formation of adducts with potential deleterious effects. Thus, we developed a novel analytical methodology to monitor the in vivo formation of the HT mercapturate, N-acetyl-5-S-cysteinyl-hydroxytyrosol in urine samples. Biomarkers of hepatic and renal toxicity were evaluated within the dose range tested. Following HT administration, dose-dependent effects were observed for the recovery of all the metabolites studied. At the lowest dose of 1 mg/kg, the glucuronidation pathway was the most relevant (25-30%), with lower recoveries for sulfation (14%), while at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). In addition, we report for the first time the formation of the mercapturate conjugate of HT in a dose-dependent manner. The biochemical data did not reveal significant toxic effects of HT at any of the doses studied. An increase in the GSH/GSSG ratio at the highest dose was observed indicating that the products of HT autoxidation are counteracted by glutathione, resulting in their detoxification. These results indicate that the metabolic disposition of HT is highly dependent on the dose ingested.

Oral and dermal developmental toxicity studies of phenylethyl alcohol in rats.

Phenylethyl alcohol (PEA) was tested for developmental toxicity. Pregnant rats were fed 0, 83, 266, or 799 mg/kg/d PEA on gestation days (GDs) 6 to 15; only minimal, nonsignificant effects were observed. In dermal studies, PEA (neat) was applied to the skin on GDs 6 to 15 at dosages of 0, 140, 430, or 1400 mg/kg/d and at 0, 70, 140, 280, 430, or 700 mg/kg/d in a corroborative study. Observations included maternal and embryo-fetal toxicity/abnormalities at 1400 mg/kg/d, increased incidences of rudimentary cervical ribs at ≥430 mg/kg/d, and reduced fetal body weights at ≥140 mg/kg/d. Dermal maternal and developmental no-observed-adverse-effect levels are 70 mg/kg/d, based on dermal irritation and reductions (nonsignificant) in fetal body weights. Human exposure from fragrances is 0.02 mg/kg/d, resulting in a margin of safety >2600, when marked differences in dermal absorption between rats and humans are considered. Under normal fragrance use conditions, PEA is not a developmental toxicity hazard for humans.

Cytotoxicity of arsenic trioxide is enhanced by (-)-epigallocatechin-3-gallate via suppression of ferritin in cancer cells.

Arsenic trioxide (ATO) treatment is a useful therapy against human acute promyelocytic leukemia (APL), however, it concomitantly brings potential adverse consequences including serious side effect, human carcinogenicity and possible development of resistance. This investigation revealed that those problems might be relaxed by simultaneous application with (-)-epigallocatechin-3-gallate (EGCG), one of the major components from green tea. EGCG significantly lowered down the ATO concentration required for an effective control of APL cells, HL-60. The simultaneous treatment of ATO with EGCG induced a mitochondria-dependent apoptosis in HL-60 cells significantly, which accounted for more than 70% of the cell death in the treatment. The mechanism of apoptosis induction was elucidated. EGCG in HL-60 cells acted as a pro-oxidant enhancing intracellular hydrogen peroxide significantly. ATO, on the other hand, induced heme oxygenase-1 (HO-1) to catalyze heme degradation, thereby provided ferrous iron for EGCG-induced hydrogen peroxide to precede Fenton reaction, which in turn generated deleterious reactive oxygen species to damage cell. In addition, EGCG inhibited expression of ferritin, which supposedly to sequester harmful ferrous iron, thereby augmented the occurrence of Fenton reaction. This investigation also provided evidence that ATO, since mainly acted to induce HO-1 in simultaneous treatment with EGCG, could be replaced by other HO-1 inducer with much less human toxicity. Furthermore, several of our preliminary investigations revealed that the enhanced cytotoxicity induced by combining heme degradation and Fenton reaction is selectively toxic to malignant but not non-malignant cells.

Hydroxytyrosol enhances cisplatin-induced ototoxicity: Possible relation to the alteration in the activity of JNK and AIF pathways.

Hydroxytyrosol (HT), a polyphenol widely contained as an ester in olive fruits and olive leaves, exhibits a broad spectrum of effectiveness. The present study was designed to investigate the effect of HT alone as well as in the combination with cisplatin on the House Ear Institute-Organ of Corti 1 cells (HEI-OC1) and C57BL/6 cochlear hair cells in vitro. The cell viability was measured by cell counting kit-8 (CCK8) assay. The levels of reactive oxygen species were evaluated by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. The expression of phosphorylated Jun N-terminal kinase (p-JNK) and cleaved-caspase 3 was assessed by Western blotting. The apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. The distribution of apoptosis inducing factor (AIF) was determined by immunofluorescent staining. HT alleviated the levels of reactive oxygen species in both untreated state and after cisplatin stimulus. However, HT at concentration of 100 µM decreased the cell viability of HEI-OC1 from 100 ± 17.38% in control group to 50.17 ± 1.89% and increased the expression of p-JNK and c-caspase 3 from 0.62 ± 0.10, 0.20 ± 0.050 in the control group to 1.24 ± 0.18, 0.85 ± 0.18 in the group treated with 30 µM cisplatin, as well as to 1.64 ± 0.14, 1.44 ± 0.12 in the group with 30 µM cisplatin +100 µM HT, respectively. Meanwhile, HT triggered AIF transferring to nuclei and, also, led to cochlear HCs arranging disorderly and missing. Moreover, HT elevated the expression of p-JNK and c-caspase 3 from 1.00 ± 0.27, 1.00 ± 0.26 in the control group to 2.23 ± 0.24, 22.87 ± 3.80 in the group with 30 µM cisplatin, and to 2.75 ± 0.23, 31.56 ± 3.86 in the group with 30 µM cisplatin+100 µM HT correspondingly. Taken together, data from this work reveal that HT itself possesses toxic effect on HCs mainly thorough AIF-dependent apoptosis, while, it aggravates the ototoxicity-caused by cisplatin via both JNK and AIF pathways related apoptosis. Findings from this work offer clear evidence that that HT might not be recommended to utilize for preventing cisplatin-induced ototoxicity.

Caffeic acid phenethyl ester, a coffee polyphenol, inhibits DNA methylation in vitro and in vivo.

DNA methylation represents an important epigenetic regulation of the genome. Earlier studies have suggested that dietary phenolic compounds including those contained in coffee, tea and soy products may modulate the level of DNA methylation. In this study, we first characterize the effect of caffeic acid phenethyl ester (CAPE) and other dietary phenolic compounds on DNA methylation in vitro. The IC50 values of CAPE, daidzein, isorhamnetin and genistein are 7.6, 6.9, 6.2, and 4.3 µM, respectively, in an in-vitro enzymatic assay system. Computational analysis indicates that CAPE, daidzein, isorhamnetin and genistein can bind inside the DNA substrate-binding site in human DNMT1 with a favorable binding energy. In an animal study, we find that maternal CAPE treatment shifts the coat color distribution of the 21-day-old Avy/a offspring towards the yellow phenotype, indicating that CAPE inhibits the methylation of the agouti gene promoter sequence in vivo. The results from this study may shed light on the potential epigenetic effect in the offspring resulting from maternal intake of certain coffee phenolics during pregnancy.

Acute/Subacute and Sub-Chronic Oral Toxicity of a Hidroxytyrosol-Rich Virgin Olive Oil Extract.

The objective of this study was to determine the acute (one single dose), subacute (14 days), and sub-chronic (90 days) toxicity of an aqueous virgin olive oil (VOO) extract rich in hydroxytyrosol in rats. For acute/subacute toxicity, rats were divided into three groups. The control group received distilled water (n = 9), another experimental group received a single dose of 300 mg/kg (n = 3), and a third group received one dose of 2000 mg/kg (n = 4) during 14 days. The sub-chronic study included 60rats distributed in three groups (n = 20: 10 males and 10 females) receiving daily different three doses of the VOO extract in the drinking water during 90 days: (1) 100 mg/kg, (2) 300 mg/kg, and (3) 1000 mg/kg. In parallel, a fourth additional group (n = 20: 10 males and 10 females) did not receive any extract (control group). Clinical signs, body weight, functional observations of sensory and motor reactivity, hematological and biochemical analyses, and macroscopic and microscopic histopathology were evaluated. No adverse effects were observed after the administration of the different doses of the hydroxytyrosol-rich VOO extract, which suggests that the enrichment of VOO in its phenolic compound is safe, and can be used as functional foods for the treatment of chronic degenerative diseases.

Caffeic acid phenethyl ester (CAPE)-derivatives act as selective inhibitors of acetylcholinesterase.

Unexpected inhibitory effects against eeAChE could be found for a newly synthesized class of caffeic acid phenethyl ester (CAPE) derivatives. Thus, phenethyl-(E)-3-(3,5-dimethoxy-4-phenethoxyphenyl)-acrylate (Ki = 1.97 ±â€¯0.38 µM, Ki´â€¯= 2.44 ±â€¯0.07 µM) and 4-(2-(((E)-3-(3,4-bis(benzyloxy)phenyl)acryloyl)oxy)ethyl)-1,2-phenylene (2E,2'E)-bis(3-(3,4-bis(benzyloxy)phenyl)acrylate) (Ki = 0.72 ±â€¯0.31 µM, Ki´â€¯= 1.80 ±â€¯0.21 µM) showed very good inhibition of eeAChE, while being non cytotoxic for malignant human cancer cells and non-malignant mouse fibroblasts. Also, they are weak inhibitors for BChE (from equine serum).

Modulation of LPS-induced nitric oxide production in intestinal cells by hydroxytyrosol and tyrosol metabolites: Insight into the mechanism of action.

At intestinal level, after acute or chronic exposure to iNOS-derived NO, a toxic mechanism of action leads to inflammation and degenerative diseases. The aim of this study was to investigate the effect of glucuronide and sulfate metabolites of the extra virgin olive oil phenols tyrosol (Tyr) and hydroxytyrosol (HT), in comparison with their parent compounds, on the release of NO following exposure to a pro-inflammatory stimulus, the bacterial lipopolysaccharide (LPS). Human colon adenocarcinoma cells (Caco-2), differentiated as normal enterocytes, were treated with pathological concentrations of LPS, in order to stimulate iNOS pathway, which involves NF-ĸB activation through IĸBα phosphorylation and subsequent degradation induced by Akt or MAPKs. All the tested metabolites inhibited NO release induced by LPS, acting as inhibitors of iNOS expression, with an efficacy comparable to that of the parent compounds. HT and Tyr metabolites were effective in the inhibition of IĸBα degradation. No one of the compounds was able to inhibit Akt activation, whereas they modulated p38 and ERK1/2 MAPK. Obtained data show that HT and Tyr metabolites are able to prevent a pathological NO overproduction at intestinal level, where they concentrate, thus significantly contributing to the protective activity exerted by their parent compounds against inflammation.

Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells.

The control of inflammation, which arises from complex biological responses to harmful stimuli, is an important determinant of both clinical outcomes and patient comfort. However, the side effects of many current therapies such as non-steroidal anti-inflammatory drugs mean that new safe treatments are required. We previously reported that 12.5 µg/ml hydroxytyrosol (HT) suppressed gene expression of the inducible nitric oxide (NO) synthase (iNOS) isoform and NO production, in mouse peritoneal macrophages treated with lipopolysaccharide (LPS), where nuclear factor-κB (NF-κB) gene expression was not altered. The present study evaluated the anti-inflammatory effects of various concentrations of HT in LPS-induced RAW264.7 mouse macrophages. HT suppressed NF-κB signaling and downregulated LPS-mediated expression of iNOS, cyclooxygenase-2, tumor necrosis factor alpha, and interleukin-1ß at 12.5 µg/ml, resulting in reduced production of NO and prostaglandin E2. At lower concentrations, HT seemed to act via another signaling pathway to regulate the inflammatory response. In contrast, HT did not suppress LPS-induced expression of phosphorylated p44/42 mitogen-activated protein kinase. This study showed that HT had anti-inflammatory effects on LPS-stimulated RAW264.7 cells. HT is already available as a nutritional supplement and no toxic effects have been reported. Hence, HT represents a potential novel anti-inflammatory agent.

Quantitative Structure-Cytotoxicity Relationship of Cinnamic Acid Phenetyl Esters.

BACKGROUND/AIM: Many phenolic acid phenethyl esters possess diverse biological effects including antioxidant, cytoprotective, anti-inflammation and anti-tumor activities. However, most previous antitumor studies have not considered the cytotoxicity against normal cells. Ten cinnamic acid phenetyl esters were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity and tumor-specificity, in order to find their new biological activities. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor specificity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by CC50 against tumor cells. Apoptosis markers were detected by western blot analysis. Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by force-field minimization. RESULTS: Western blot analysis demonstrated that [9] stimulated the cleavage of caspase-3, suggesting the induction of apoptosis. QSAR analysis demonstrated that TS values were correlated with shape, size and ionization potential. CONCLUSION: Chemical modification of the lead compound may be a potential choice for designing a new type of anticancer drugs.

Oxidative stress in prefrontal cortex of rat exposed to MK-801 and protective effects of CAPE.

MK-801 was shown to be one of the most neurotoxic non-competitive NMDA receptor antagonists. It is known that repeated injection of MK-801 was proposed in an animal model in psychosis. The aims of this study are to investigate the contributing effect of oxidative stress in MK-801-induced experimental psychosis model, and to show that prevention of oxidative stress may improve prognosis. Furthermore, there is evidence that oxygen free radicals play an important role in the pathophysiology of schizophrenia. In this study, Wistar Albino rats were divided into three groups: 1st group: Control, 2nd group: MK-801, 3rd group: MK-801+CAPE (Caffeic acid phenethyl ester) group. MK-801 was given intraperitoneally at the dose of 0.5 mg/kg/day for 5 days. CAPE was given to the treatment group while exposed to MK-801. In control group, saline was given intraperitoneally at the same time. After 7 days, rats were killed by decapitation. Prefrontal cortex (PFC) of rats was removed for biochemical and histological analyses. As a result, malondialdehyde (MDA), protein carbonyl (PC), nitric oxide (NO) levels and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO) and adenosine deaminase (AD) enzyme activities were found to be increased significantly in prefrontal cortex (PFC) of MK-801 group (p<0.0001) compared to control group. In CAPE treated rats, prefrontal tissue MDA, PC, NO levels and, GSH-Px, XO, AD enzyme activities were significantly decreased when compared to MK-801 groups (p<0.0001) whereas catalase (CAT) enzyme activity was not changed. Moreover, in the light of microscopic examination of MK-801 groups, a great number of apoptotic cells were observed. CAPE treatment decreased the apoptotic cell count in PFC. The results of this study showed that MK-801-induced neurotoxicity caused oxidative stress in PFC of rats. This experimental study may also provide some evidences for the new treatment strategies with antioxidants in schizophrenia.

The FEMA GRAS assessment of aromatic substituted secondary alcohols, ketones, and related esters used as flavor ingredients.

This publication is the 11th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. The list of GRAS substances has now grown to more than 2100 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. In this monograph, a detailed interpretation is presented on the renal carcinogenic potential of the aromatic secondary alcohol alpha-methylbenzyl alcohol, aromatic ketone benzophenone, and corresponding alcohol benzhydrol. The relevance of these effects to the flavor use of these substances is also discussed. The group of aromatic substituted secondary alcohols, ketones, and related esters was reaffirmed as GRAS (GRASr) based, in part, on their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential.

Caffeic acid phenethyl ester (CAPE) revisited: Covalent modulation of XPO1/CRM1 activities and implication for its mechanism of action.

Caffeic acid phenethyl ester (CAPE) is the bioactive constituent of propolis from honeybee hives and is well known for its anti-inflammatory, anticarcinogenic, antioxidant, and immunomodulatory properties. Herein, we revisited the cellular mechanism underlying the diverse biological effects of CAPE. We demonstrated that XPO1/CRM1, a major nuclear export receptor, is a cellular target of CAPE. Through nuclear export functional assay, we observed a clear shift of XPO1 cargo proteins from a cytoplasmic localization to nucleus when treated with CAPE. In particular, we showed that CAPE could specifically target the non-catalytic and conserved Cys528 of XPO1 through the means of mass spectrometric analysis. In addition, we demonstrated that the mutation of Cys528 residue in XPO1 could rescue the nuclear export defects caused by CAPE. Furthermore, we performed position-restraint molecular dynamics simulation to show that the Michael acceptor moiety of CAPE is the warhead to enable covalent binding with Cys528 residue of XPO1. The covalent modulation of nuclear export by CAPE may explain its diverse biological effects. Our findings may have general implications for further investigation of CAPE and its structural analogs.

Expanding Upon Styrene Biosynthesis to Engineer a Novel Route to 2-Phenylethanol.

2-Phenylethanol (2PE) is a key molecule used in the fragrance and food industries, as well as a potential biofuel. In contrast to its extraction from plant biomass and/or more common chemical synthesis, microbial 2PE production has been demonstrated via both native and heterologous expression of the yeast Ehrlich pathway. Here, a novel alternative to this established pathway is systematically engineered in Escherichia coli and evaluated as a more robust and efficient route. This novel pathway is constructed via the modular extension of a previously engineered styrene biosynthesis pathway, proceeding from endogenous l-phenylalanine in five steps and involving four heterologous enzymes. This "styrene-derived" pathway boasts nearly a 10-fold greater thermodynamic driving force than the Ehrlich pathway, and enables reduced accumulation of acetate byproduct. When directly compared using a host strain engineered for l-phenylalanine over-production, preservation of phosphoenolpyruvate, and reduced formation of byproduct 2-phenylacetic acid, final 2PE titers via the styrene-derived and Ehrlich pathways reached 1817 and 1164 mg L-1 , respectively, at yields of 60.6 and 38.8 mg g-1 . Following optimization of induction timing and initial glucose loading, 2PE titers by the styrene-derived pathway approached 2 g L-1 - nearly a two-fold twofold increase over prior reports for 2PE production by E. coli employing the Ehrlich pathway.

Hydroxytyrosol administration enhances atherosclerotic lesion development in apo E deficient mice.

Hydroxytyrosol is a phenol found in olive oil. To verify the effect of hydroxytyrosol on the development of atherosclerosis, two groups of apo E deficient male mice on a standard chow diet were used: the control group receiving only water, and the second group an aqueous solution of hydroxytyrosol in order to provide a dose of 10 mg/kg/day to each mouse. This treatment was maintained for 10 weeks. At the moment of sacrifice, blood was drawn and heart removed. Plasma lipids, apolipoproteins and monocyte Mac-1 expression were assayed as well as aortic atherosclerotic areas in both groups. Data showed no significant changes in HDL cholesterol, paraoxonase, apolipoprotein B or triglyceride levels. However, hydroxytyrosol administration decreased apolipoprotein A-I and increased total cholesterol, atherosclerotic lesion areas and circulating monocytes expressing Mac-1. The latter was highly correlated with lesion areas (r = 0.65, P < 0.01). These results indicate that administration of hydroxytyrosol in low cholesterol diets increases atherosclerotic lesion associated with the degree of monocyte activation and remodelling of plasma lipoproteins. Our data supports the concept that phenolic-enriched products, out of the original matrix, could be not only non useful but also harmful. Our results suggest that the formulation of possible functional foods should approximate as much as possible the natural environment in which active molecules are found.

Growth suppression and toxicity induced by caffeic acid phenethyl ester (CAPE) in type 5 adenovirus-transformed rat embryo cells correlate directly with transformation progression.

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), induces a selective growth suppressive and toxic effect toward cloned rat embryo fibroblast cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed cloned rat embryo fibroblast cells (Z-z. Su et al., Mol. Carcinog., 4: 231-242, 1991). The present study was conducted to determine whether CAPE-induced growth suppression/toxicity was a direct result of expression of the Ad5 E1A and E1B transforming genes or a consequence of the action of these genes resulting in the transformed state. For this investigation we used somatic cell hybrids and 5-azacytidine-treated Ad5-transformed rat embryo cells that display different stages of expression of the transformed phenotype. This series of cell lines has permitted us to determine whether expression of the transformed state and the stage of transformation progression regulates CAPE sensitivity. Evidence is presented indicating that sensitivity to CAPE is directly determined by the state of expression of the transformed progression phenotype, as opposed to simply the expression of the Ad5 E1A and E1B transforming genes. These results provide further evidence that CAPE may represent a unique compound that can specifically target progressed transformed cells for growth suppression and toxicity. An understanding of the mechanism underlying this selective effect of CAPE could result in the identification of important biochemical pathways mediating cellular transformation and progression of the transformed state.

Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents.

Trypanosomiasis and leishmaniasis keep being a real challenge for health and development of African countries. Existing treatments have considerable side effects and increase resistance of the parasites. We have measured antitrypanosomal and antileishmanial activity of natural phenols, tyrosol (TYR) and hydroxytyrosol (HT) and several of their esters and metabolites. We found significant IC50 values against Trypanosoma brucei for HT decanoate ester and HT dodecanoate ester (0.6 and 0.36 µM, respectively). This represents a large increase in activity with respect to HT (79 and 132 fold, respectively). Moreover, both compounds displayed a high selectivity index against MRC-5, a non-tumoral human cell line (118 and 106, respectively). Then, we synthesized a focused library of compounds to explore structure-activity. We found the ether and thiourea analogs of HT decanoate ester and HT dodecanoate ester also showed IC50 values against T. brucei in the low micromolar range. In conclusion, the di-ortho phenolic ring and medium size alkyl chain are essential for activity whereas the nature of the chemical bond among them seems less important.