Formulation and production of a blood-free and chemically defined virus production media for VERO cells.

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.

Cell line-dependent activation and antiviral activity of T-1105, the non-fluorinated analogue of T-705 (favipiravir).

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.

Quantitative Proteomics Reveals Changes in Vero Cells in Response to Porcine Epidemic Diarrhea Virus.

Outbreaks of porcine epidemic diarrhea virus (PEDV) have caused significant lethality rates in neonatal piglets, which pose a serious threat to the swine industry worldwide. Available commercial vaccines fail to protect against the emergence of high virulence of PEDV variants. Therefore, the endemic state of the PEDV infection in suckling piglets highlights the urgent need for uncovering the molecular determinants of the disease pathogenesis. In this study, stable isotope labeling by amino acids in cell culture (SILAC), combined with high-performance liquid chromatography/tandem mass spectrometry was performed to determine proteomic differences between PEDV-infected and mock-infected Vero cells at 18 h postinfection. The SILAC-based approach identified 4508 host-cell proteins, of which 120 were significantly up-regulated and 103 were significantly down-regulated at ≥95% confidence. Alterations in the expression of selected proteins were verified by Western blot. Several signaling metabolic pathways including mevalonate pathway I and the superpathway of cholesterol biosynthesis were triggered by the infection of the highly virulent strain and are linked to host innate immunity. 25-HC, an inhibitor of the mevalonate pathway, exhibited potent antiviral activity against PEDV infection. Meanwhile, the cell-cycle-related functions were significantly regulated, which may likely be responsible for the viral replication and pathogenicity of PEDV.

Genome-wide analysis of differentially expressed genes and the modulation of PEDV infection in Vero E6 cells.

PEDV remains one of the most important swine diseases that infects pigs of all ages. It causes devastating viral enteric disease in piglets with a high mortality rate, leading to significant threats and huge economic loss to the pork industry. In this study, a transcriptomic shotgun sequencing (RNA-Seq) procedure was used to study gene responses against PEDV infection. Genome-wide analysis of differentially expressed genes (DEGs) was performed in Vero E6 cells post-PEDV infection. mTOR signaling pathway activator-MHY1485, and inhibitor-PP242 were used to study the antiviral function. Results revealed that the IRF3 was significantly up-regulated post-PEDV infection. Although most of the IFN-regulatory and -related genes evaluated in this study were either down-regulated or remained unchanged, IL11 behaved significantly up-regulated, with the peak at 16 hpi. Nearly 90% of PEDV infections were suppressed in the PP242 pretreated cells whereas the reverse effect was observed in the MYH1485 pretreated cells. Results indicated that the mTOR signaling pathway played a vital role in the PEDV antiviral regulation in the Vero E6 cells. Future studies will contribute to better understand the cellular antiviral mechanism against PEDV.

M protein is sufficient for assembly and release of Peste des petits ruminants virus-like particles.

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.

iTRAQ-based comparative proteomic analysis of Vero cells infected with virulent and CV777 vaccine strain-like strains of porcine epidemic diarrhea virus.

The re-emerging porcine epidemic diarrhea virus (PEDV) variant related diarrhea has been documented in China since late 2010 and now with global distribution. Currently, a virulent PEDV CH/YNKM-8/2013 and a CV777 vaccine strain-like AH-M have been successfully isolated from the clinical samples. To dissect out the underlying pathogenic mechanism of virulent PEDV and clarify the differences between virulent and CV777 vaccine strain-like PEDV infections, we performed an iTRAQ-based comparative quantitative proteomic study of Vero cells infected with both PEDV strains. A total of 661 and 474 differentially expressed proteins were identified upon virulent and CV777 vaccine strain-like isolates infection, respectively. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in both PEDV infections. Comprehensive studies have revealed that the PEDV virulent strain suppressed protein synthesis of Vero cells through down-regulating mTOR as well as its downstream targets 4EBP1 and p70S6K activities, which were validated by immunoblotting. In addition, the virulent strain could activate NF-κB pathway more intensively than the CV777 vaccine strain-like isolate, and elicit stronger inflammatory cascades as well. These data might provide new insights for elucidating the specific pathogenesis of PEDV infection, and pave the way for the development of effective therapeutic strategies. BIOLOGICAL SIGNIFICANCE: Porcine epidemic diarrhea is now worldwide distributed and causing huge economic losses to swine industry. The immunomodulation and pathogenesis between PEDV and host, as well as the difference between virulent and attenuated strains of PEDV infections are still largely unknown. In this study, we presented for the first application of proteomic analysis to compare whole cellular protein alterations induced by virulent and CV777 vaccine strain-like PEDV infections, which might contribute to understand the pathogenesis of PEDV and anti-viral strategy development.

Proteome analysis of porcine epidemic diarrhea virus (PEDV)-infected Vero cells.

Porcine epidemic diarrhea virus (PEDV) causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs. A large-scale outbreak of PED occurred in China in 2010, with PEDV emerging in the United States in 2013 and spreading rapidly, posing significant economic and public health concerns. In this study, LC-MS/MS coupled to iTRAQ labeling was used to quantitatively identify differentially expressed cellular proteins in PEDV-infected Vero cells. We identified 49 differentially expressed cellular proteins, of which 8 were upregulated and 41 downregulated. These differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. Based on these differentially expressed proteins, we propose that PEDV might utilize apoptosis and extracellular signal regulated kinases pathways for maximum viral replication. Our study is the first attempt to analyze the protein profile of PEDV-infected cells by quantitative proteomics, and we believe our findings provide valuable information with respect to better understanding the host response to PEDV infection.

Limited type I interferons and plasmacytoid dendritic cells during neonatal respiratory syncytial virus infection permit immunopathogenesis upon reinfection.

UNLABELLED: Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action. IMPORTANCE: Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

Herpes simplex virus type 1 induces nuclear accumulation of hyperphosphorylated tau in neuronal cells.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that remains latent in host neurons. Viral DNA replication is a highly structured process in which the redistribution of nuclear proteins plays an important role. Although tau is most widely known as a microtubule-associated protein found in a hyperphosphorylated state in the brains of patients with Alzheimer's disease (AD), this protein has also been detected at other sites such as the nucleolus. Here, we establish that HSV-1 infection gives rise to an increase in tau phosphorylation and that hyperphosphorylated tau accumulates in the nucleus, forming defined structures in HSV-1-infected neuronal cells reminiscent of the common sites of viral DNA replication. When tau expression in human neuroblastoma cells was specifically inhibited using an adenoviral vector expressing a short hairpin RNA to tau, viral DNA replication was not affected, indicating that tau is not required for HSV-1 growth in neuronal cells. Given that HSV-1 is considered a risk factor for AD, our results suggest a new way in which to understand the relationships between HSV-1 infection and the pathogenic mechanisms leading to AD.

Potential toxic effects of iron oxide nanoparticles in in vivo and in vitro experiments.

The aim of this study was to determine the potential toxic effects of iron(II,III)oxide nanoparticles (IONPs). In in vivo experiments, the toxic effects of IONPs were monitored in adult male Wistar rats by morphological methods after a single intratracheal instillation. For the control group 1 ml of physiological saline per animal was given, and the treatment group received the same volume of a suspension containing 1 and 5 mg kg⁻¹ body weight IONPs. Lungs and internal organs underwent histopathological examination after 1, 3, 7, 14 and 30 days. The mutagenic effect of these nanoparticles was evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and on Escherichia coli WP2uvrA strain, in the presence and absence of the mammalian metabolic activation system S9. The in vitro cytotoxic effect of IONPs was also examined in Vero cells after short-term (4 h) and long-term (24 h) exposure. There were no pathological changes in examined internal organs, except a very weak pulmonary fibrosis developing by the end of the first month in the treated rats. While in vitro MTT assay showed a moderate cytotoxic effect, IONPs proved to be devoid of mutagenic effect in the bacterial systems tested. The results may be a useful extension of our knowledge on the safety of magnetite nanoparticles in view of their possible medical applications, such as in hyperthermia and magnetic resonance imaging.

Peptide pheromone plantaricin a produced by Lactobacillus plantarum permeabilizes liver and kidney cells.

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.

Development of an animal-component free medium for vero cells culture.

This work describes the development of an animal-component free medium (IPT-AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum-free medium (SFM) referred as IPT-SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT-SFM was further improved to obtain an animal-component free medium named IPT-AFM. IPT-AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue-culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non-animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT-AFM were investigated in T-flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 +/- 0.18 and a specific growth rate micro 0.019 +/- 0.003 h(-1) were achieved in IPT-AFM.

Retrograde Shiga toxin trafficking is regulated by ARHGAP21 and Cdc42.

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.

Characterization of a persistent respiratory syncytial virus showing a low-fusogenic activity associated to an impaired F protein.

In this work we have characterized the virus (RSV(48)) present in passage 48 of a respiratory syncytial virus persistently infected murine macrophage-like cell culture. This virus was noncytopathic in macrophages and had a low-fusogenic activity in RSV-permissive cell lines, although the level of this activity varied among the different cell lines tested. The fusogenic activity of RSV(48) in Vero cells, as evaluated by the number and size (nuclei per syncytium) of syncytia, was lower than that shown in cells H358. However, the syncytia formed by RSV(48) in Vero cells increased significantly when the virus was treated with trypsin previous to cell infection and the protease was left in the medium during the development of polykarions. Moreover, the fusogenic activity of RSV(48) was increased by a brief acidic pH treatment of infected cells. These results imply that the RSV(48) F protein was inefficiently activated by intracellular proteases in Vero cells and exposure to low pH favours membrane fusion. Analysis of the nucleotide and the deduced amino acid sequences of the RSV(48) F protein showed nine amino acid residue differences with respect to the RSV(wt) sequence, some of which mapped to positions that suggest they might be responsible for the low-fusogenic activity observed for the RSV(48) F protein.

In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.

Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.

Bimolecular fluorescence complementation (BiFC) is a recently developed technique for detection of protein-protein interactions in living cells. In this study, a new red BiFC system was developed by splitting mCherry, a mutant monomeric red fluorescent protein, into two fragments between amino acids 159-160 and was verified using a pair of interacting proteins, SV40 large T antigen (LTag), and human p53 protein. By combined use of the mCherry-based red BiFC system with a Venus-based yellow BiFC system, the interaction between LTag and p53 as well as the interaction between sp100 and promyelocytic leukemia protein (PML), were detected simultaneously in Vero cells. The brilliant redness, short maturation time, and the long excitation and emission wavelengths (587/610 nm) of mCherry make the new BiFC system an excellent candidate for analyzing protein-protein interactions in living cells and for studying multiple protein-protein interactions when coupled with other BiFC systems.

Expression of L protein of Hantaan virus 84FLi strain and its application for recovery of minigenomes.

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the family Baculoviridae, is an insect-specific virus that can enter a variety of mammalian cells. The potential of this versatile virus for protein expression or gene therapy in mammalian cells has become the focus of many studies. In most mammalian cells, transduced AcMNPV genes are either not expressed or expressed at an extremely low level. Here, we studied the effects of the two major AcMNPV trans-activators, IE1 and IE2, on the activation of AcMNPV genome in Vero E6 cells. Microarray analysis showed that when IE1 was overexpressed, it significantly activated genes gp64 and pe38, and upregulated ie2, he65, pcna, orf16, orf17 and orf25. Although, there were only two genes, pe38 and orf17, that were activated by IE2, we discovered interestingly that the combination of IE1 and IE2 factors had a synergistic effect on activation of the AcMNPV genome in mammalian cells, and activated around 38 %, or 59 out of the 155 genes placed on the microarray. This is the first detailed study of baculoviral transcription regulation in mammalian cells, and it shows that the baculoviral genome can be activated in a mammalian system, and also that the two major trans-activators, IE1 and IE2, play a central role in this activation.

Evaluation of the antioxidant and cytotoxic activity of arzanol, a prenylated alpha-pyrone-phloroglucinol etherodimer from Helichrysum italicum subsp.microphyllum.

Various phenolics and (mero)terpenoids from Helichrysum italicum subsp. microphyllum, a plant endemic to Sardinia, were investigated for their capacity to inhibit non-enzymatic lipid peroxidation. These compounds were studied in simple in vitro systems, under conditions of autoxidation and of iron (EDTA)-mediated oxidation of linoleic acid at 37 degrees C. Arzanol, a pyrone-phloroglucinol etherodimer, and helipyrone, a dimeric pyrone, showed antioxidant activity, and could protect linoleic acid against free radical attack in assays of autoxidation and EDTA-mediated oxidation. Methylarzanol, as well as the sesquiterpene alcohol rosifoliol, showed a decreased, but still significant, protective effect against linoleic acid oxidation. Arzanol and helipyrone were also tested in an assay of thermal (140 degrees C) autoxidation of cholesterol, where arzanol showed significant antioxidant activity. The cytotoxicity of arzanol was further evaluated in VERO cells, a line of fibroblasts derived from monkey kidney. Arzanol, at non-cytotoxic concentrations, showed a strong inhibition of TBH-induced oxidative stress in VERO cells. The results of the present work suggest that the natural compound arzanol exerts useful antioxidant properties in different in vitro systems of lipid peroxidation.