The outcome of 5-ALA-mediated photodynamic treatment in melanoma cells is influenced by vitamin C and heme oxygenase-1

Photodynamic therapy (PDT) is an important clinical approach for cancer treatment. It involves the administration of a photosensitizer, followed by its activation with light and induction of cell death. The underlying mechanism is an increased production of reactive oxygen species (ROS) leading to oxidative stress, which is followed by cell death. However, effectiveness of PDT is limited due to an initiation of endogenous rescue response systems like heme oxygenase-1 (HO-1) in tumor cells. In recent years, consuming of antioxidant supplements has become widespread, but the effect of exogenously applied antioxidants on cancer therapy outcome remains unclear. Thus, this study was aimed to investigate if exogenous antioxidants might decrease ROS-induced cytotoxicity in photodynamic treatment. Lycopene, β-carotene, vitamin C, N-acetylcysteine, trolox, and N-tert-butyl-α-phenylnitrone in different doses were administered to human melanoma cells prior exposure to photodynamic treatment. Supplementation with vitamin C resulted in a significant decrease of the cell death rate, whereas the other tested antioxidants had no effect on cell viability and oxidative stress markers. The simultaneous application of vitamin C with the HO-1 activity inhibitor zinc protoporphyrine IX (ZnPPIX) caused a considerable decrease of photodynamic treatment-induced cytotoxicity compared to ZnPPIX alone. It can be summarized that exogenously applied antioxidants do not have a leading role in the protective response against photodynamic treatment. However, further studies are necessary to investigate more antioxidants and other substances, which might affect the outcome of photodynamic treatment in cancer therapy.     

Rapid screening and characterisation of antioxidants of Cosmos caudatus using liquid chromatography coupled with mass spectrometry

Ulam raja (Cosmos caudatus) is used traditionally for improving blood circulation. In this study, it was found that ulam raja had extremely high antioxidant capacity of about 2,400 mg l-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. Antioxidant peaks in extract of ulam raja were firstly characterized using free radical spiking test through high performance liquid chromatography coupled with mass spectrometry (MS). Upon reaction with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, intensities of antioxidant peaks will be significantly reduced. HPLC/MS(n) was further applied to elucidate the chemical structures of antioxidant peaks characterized in the spiking test. More than twenty antioxidants were identified in ulam raja, and their chemical structures were proposed. The major antioxidants in ulam raja were attributed to a number of proanthocyanidins that existed as dimers through hexamers, quercetin glycosides, chlorogenic, neo-chlorogenic, crypto-chlorogenic acid and (+)-catching. High content of antioxidants antioxidants contained in ulam raja could be partly responsible for its ability to reduce oxidative stress.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Phenolic compounds and antioxidant activity from red grape marc extracts

The aims of this work were to determine the amounts of the different classes of phenolic compounds in an ethanolic extract from red grape marc and its components, peels and seeds, and to evaluate their antioxidant activities by the beta-carotene bleaching test for their utilization as natural antioxidants. The results showed that red grape marc was rich in polyphenol compounds with a clear antioxidant activity. The extracts, in fact, at very low concentration (20 ppm) in total phenols showed an antioxidant activity (AA) higher than 43% on average, while at higher concentration (80-160 ppm) all the fractions had an AA comparable to that of butlylated-hydroxytoluene. Grape seeds seemed to give the highest contribution to such AA, as they contained high quantities of proanthocyanidines, a type of flavonoid known for its high antioxidant properties.     

Cell-protective and antioxidant activity of two groups of synthetic amphiphilic compounds — Phenolics and amine<Emphasis Type="Italic">N</Emphasis>-oxides

Two classes of newly synthesized amphiphilic compounds, phenolic antioxidants ("phenolics") and N-oxides exert in vivo antioxidant effects on live S. cerevisiae cells. Both groups have low toxicity, phenolics being more toxic than N-oxides and compounds with a longer alkyl chain having higher toxicity than those with a shorter alkyl chain. Phenolic antioxidants protect yeast cells exposed to the superoxide producer paraquat and peroxyl generator tert-butylhydroperoxide better than N-oxides at 3-fold higher concentration. Both types of antioxidants enhance the survival of pro-oxidant-exposed cells of S. cerevisiae mutants deficient in cytosolic and/or mitochondrial superoxide dismutase and could be good compounds which mimic the role of superoxide dismutases. The results of measurement of antioxidant activity in an in vitro chemiluminescence test differ from the results obtained in vivo with S. cerevisiae superoxide dismutase mutants. In contrast to their action on live cells, phenolics are less effective than N-oxides in preventing lipid peroxidation of an emulsion of lipids isolated from S. cerevisiae membranes.     

Zeaxanthin in combination with ascorbic acid or alpha-tocopherol protects ARPE-19 cells against photosensitized peroxidation of lipids

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.     

Herbicidal and antioxidant responses of transgenic rice overexpressing Myxococcus xanthus protoporphyrinogen oxidase.

We analyzed the herbicidal and antioxidant defense responses of transgenic rice plants that overexpressed the Myxococcus xanthus protoporphyrinogen oxidase gene. Leaf squares of the wild-type incubated with oxyfluorfen were characterized by necrotic leaf lesions and increases in conductivity and malonyldialdehyde levels, whereas transgenic lines M4 and M7 did not show any change with up to 100 microM oxyfluorfen. The wild-type had decreased F(v)/F(m) and produced a high level of H(2)O(2) at 18 h after foliar application of oxyfluorfen, whereas transgenic lines M4 and M7 were unaffected. In response to oxyfluorfen, violaxanthin, beta-carotene, and chlorophylls (Chls) decreased in wild-type plants, whereas antheraxanthin and zeaxanthin increased. Only a slight decline in Chls was observed in transgenic lines at 48 h after oxyfluorfen treatment. Noticeable increases of cytosolic Cu/Zn-superoxide dismutase, peroxidase isozymes 1 and 2, and catalase were observed after at 48 h of oxyfluorfen treatment in the wild-type. Non-enzymatic antioxidants appeared to respond faster to oxyfluorfen-induced photodynamic stress than did enzymatic antioxidants. Protective responses for the detoxification of active oxygen species were induced to counteract photodynamic stress in oxyfluorfen-treated, wild-type plants. However, oxyfluorfen-treated, transgenic plants suffered less oxidative stress, confirming increased herbicidal resistance resulted from dual expression of M. xanthus Protox in chloroplasts and mitochondria.     

Probucol prevents oxidative injury to endothelial cells

We evaluated the effect of the antioxidant, probucol, on the cytotoxic effects of oxidized low density lipoprotein (OX-LDL) or of cumene hydroperoxide (CumOOH) on cultured bovine endothelial cells (EC). The addition of CumOOH to EC caused the release of lactate dehydrogenase and the accumulation of thiobarbituric acid-reacting substances (TBARS), effects that were protected against by preincubation with either probucol or tocopherol. Similarly, preincubation of EC with those antioxidants protected against OX-LDL toxicity and the accumulation of TBARS. The content of probucol in EC measured by high performance liquid chromatography was directly correlated with the extent of protection against OX-LDL toxicity. We also found that treatment of EC with serum from patients receiving treatment with probucol resulted in the detection of probucol in the cells. We conclude that probucol is transported and incorporated into EC membranes to act as a radical-trapping antioxidant, protecting the EC against oxidative stress. Our results also indicate that lipid peroxidation in cellular membranes involves cell injury inflicted by OX-LDL.     

Role of reactive oxygen species in extracellular signal-regulated protein kinase phosphorylation and 6-hydroxydopamine cytotoxicity

A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants ('SOD mimetics') on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.     

Can antioxidants be beneficial in the treatment of lead poisoning?

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species, reducing the antioxidant defense system of cells via depleting glutathione, inhibiting sulfhydryl-dependent enzymes, interfering with some essential metals needed for antioxidant enzyme activities, and/or increasing susceptibility of cells to oxidative attack by altering the membrane integrity and fatty acid composition. Consequently, it is plausible that impaired oxidant/antioxidant balance can be partially responsible for the toxic effects of lead. Where enhanced oxidative stress contributes to lead-induced toxicity, restoration of a cell's antioxidant capacity appears to provide a partial remedy. Several studies are underway to determine the effect of antioxidant supplementation following lead exposure. Data suggest that antioxidants may play an important role in abating some hazards of lead. To explain the importance of using antioxidants in treating lead poisoning the following topics are addressed: (i) Oxidative damage caused by lead poisoning; (ii) conventional treatment of lead poisoning and its side effects; and (iii) possible protective effects of antioxidants in lead toxicity.     

Natural antioxidants synergistically enhance the anticancer potential of AP9-cd, a novel lignan composition from Cedrus deodara in human leukemia HL-60 cells

Antioxidants have been used as adjuvant with anticancer therapy to synergize the potential of the anti-neoplastic therapeutics. Based on the fact, we have studied the effect of three natural antioxidants curcumin, silymarin and acteoside on AP9-cd (standardized lignan composition from Cedrus deodara) induced cytotoxicity in human leukemia HL-60 cells. The antioxidant potential of individual test compounds was first evaluated with ferric reducing antioxidant power (FRAP) test, which revealed that all four molecules behave as antioxidants. The apoptotic potential of AP9-cd was significantly enhanced in HL-60 cells in the presence of curcumin, silymarin and acteoside. It was confirmed by using various models like MTT assay, DNA fragmentation, nuclei condensation, sub-Go DNA population, Annexin-V-FITC binding, ROS depletion and immunoblotting in HL-60 cells. AP9-cd and individual antioxidants alone at low doses (10μg and 10μM, respectively) have meager or no cytotoxicity in HL-60 cells, whereas in mutual combinations, there were 2-3 times enhancement in Annexin-V-FITC and sub-Go DNA population. Moreover, prominent DNA ladders were observed at low doses of AP9-cd in combinations with various antioxidants. The Hoechst staining of the nucleus also revealed the same results for the HL-60 cells treated with AP9-cd and different antioxidants. The molecular diagnostics revealed that the combinations induced a strong antioxidant effect which was correlated with the downregulation of NF-κB expression in the nucleus. Out of the three antioxidants, curcumin was found to be more potent than acteoside and silymarin in terms of enhancing the apoptotic potential of AP9-cd. These results propose an important role of natural antioxidant as adjuvant to enhance the anticancer potential of AP9-cd and more likely other anti-neoplastic therapeutics.     

An evaluation of the effects of photoactivation of bithionol, amiodarone and chlorpromazine on human keratinocytes in vitro

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which existed prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.     

Stress, metabolism, and antioxidants in two wild passerine bird species

Antioxidants protect against free-radical damage, and free radicals, in turn, are thought to underlie aging. Thus, measuring antioxidants may aid field ecologists in understanding the physiological mechanisms that underlie life-history trade-offs. Antioxidant levels are known to vary markedly in response to the stress of capture in many birds. These changes in antioxidants could result from regulation (e.g., by stress-related hormones) or consumption (e.g., by an increase in free radicals due to increased metabolic rate). Here we experimentally test the effect of increased metabolic rate on circulating antioxidant and corticosterone concentrations in two wild passerine bird species, house sparrows (Passer domesticus) and gray catbirds (Dumetella carolinensis). We increased metabolic rate via exposure to low ambient temperatures overnight in captivity and measured circulating antioxidant capacity, uric acid, corticosterone, and oxygen consumption in cold-exposed and control individuals. Both species showed increases rather than decreases in all antioxidant parameters overnight, contradicting a consumption-by-energy-expenditure hypothesis. Both positive and negative correlations between antioxidant response and corticosterone response were occasionally but not consistently present, refuting a generalized regulation-by-corticosterone hypothesis. High baseline uric acid predicted diminished response of corticosterone and all antioxidants. Thus, high uric acid may reflect recent stress, poor condition, or a compensatory response. Relationships among metabolic rate, antioxidants, and corticosterone differed qualitatively between the species.     

Total antioxidant levels are low during active TB and rise with anti-tuberculosis therapy

In tuberculosis, oxidative stress is a result of tissue inflammation, poor dietary intake of micronutrients due to illness, free radical burst from activated macrophages, and anti-tuberculosis drugs. These free radicals may in turn contribute towards pulmonary inflammation if not neutralized by antioxidants. The total antioxidant status (TAS) of individuals is a function of dietary, enzymatic, and other systemic antioxidants and is therefore an indicator of the free radical load. Our aim was to evaluate the TAS of healthy and M. tuberculosis-infected persons from a high TB incidence community, as well as tuberculosis patients at various stages of antituberculosis drug treatment and to correlate results with plasma micronutrient levels. Blood plasma samples from TB infected patients and following antituberculosis drug treatment were assayed for TAS, vitamins A, E and Zinc. Statistical analysis of results was by one-way ANOVA and the Tukey multiple comparison post test. Active TB patients showed a significantly lower TAS (P < 0.001) compared to the community controls. We also show that TAS values increase during therapy. Results correlated with micronutrients vitamin A and zinc but vitamin E remained unaffected. We suggest that total antioxidant status of TB patients should be considered for more effective disease control and that diets low in antioxidants may render individuals susceptible to tuberculosis.     

Oxidative stress,circulating antioxidants,and dietary preferences in songbirds

Oxidative stress is an unavoidable consequence of metabolism and increases during intensive exercise. This is especially problematic for migratory birds that metabolize fat to fuel long-distance flight. Birds can mitigate damage by increasing endogenous antioxidants (e.g. uric acid) or by consuming dietary antioxidants (e.g. tocopherol). During flight, birds may increase protein catabolism of lean tissue which may increase circulating uric acid and many birds also consume an antioxidant-rich frugivorous diet during autumn migration. We evaluated three related hypotheses in a migratory passerine: (1) protein consumption is positively related to circulating antioxidants, (2) a dietary oxidative stressor [i.e. polyunsaturated fatty acid (PUFA)] influences antioxidant capacity and oxidative damage, and (3) oxidative stress influences dietary antioxidant preferences. White-throated Sparrows (Zonotrichia albicollis) consuming a high protein diet increased circulating uric acid; however, uric acid, antioxidant capacity, and oxidative stress did not differ between birds consuming a high PUFA versus a low PUFA diet, despite increased oxidative damage in high PUFA birds. Birds did not prefer antioxidant-rich diets even when fed high PUFA, low protein. We conclude that White-throated Sparrows successfully mitigated oxidative damage associated with a high PUFA diet and mounted an endogenous antioxidant response independent of uric acid, other circulating antioxidants, and dietary antioxidants.     

Lag-time measurement of antioxidant capacity using myoglobin and 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid): rationale, application, and limitation.

The application of a simple lag-time assay for antioxidant capacity using myoglobin and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ABTS has been studied for its general application conditions. In the presence of an antioxidant, the ABTS(*+) radical-cation-forming chromogenic reaction, catalyzed by myoglobin and initiated by hydrogen peroxide (H(2)O(2)), has a lag period, and its duration is linearly correlated to the concentration of that antioxidant. The high linearity between the lag time and the antioxidant concentration remained unchanged regardless of the assay conditions. It was also found that the linearity was better for antioxidants at lower concentrations. The change of assay condition could significantly affect the relative antioxidant value of a chemical to the standard (ascorbic acid), although not to a large extent. Most of antioxidants investigated were found suitable to be assayed using this method. Some antioxidants, e.g., genistein, however, were not, probably due to their low reactivity toward ferrylmyoglobin or ABTS(*+). In conclusion, the lag-time assay is a reliable method for measuring the antioxidant capacity, provided caution is taken for antioxidants that mainly act through lowering the rate of the chromogenic reaction.     

Superoxide radical-scavenging effects from polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds

Synthetic organic selenium compounds such as 2-phenyl-1,2-benzisoselenazol-3(2H)-one may show glutathione peroxidase-like antioxidant activity. Recently, we synthesized new organic selenium compounds that are thought to be effective antioxidants. To study their possible applications as antioxidants, we evaluated two selenoureas, N,N-dimethylselenourea and 1-selenocarbamoylpyrrolidine, and two tertiary selenoamides, N-(phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide, for their superoxide radical (O2-)-scavenging effects and toxicity. We measured (O2-)-scavenging effects in polymorphonuclear leukocytes (PMNs) with a specific, sensitive and real-time kinetic chemiluminescence method. Furthermore, the toxicity of these compounds was measured in some human cell lines and PMNs using the tetrazolium method. Hydrogen peroxide was measured by a scopoletin method. Finally, translocation of an NADPH oxidase component, p47 phagocyte oxidase, to the cell membrane was investigated by confocal laser scanning microscopy. N,N-Dimethylselenourea and 1-selenocarbamoylpyrrolidine effectively scavenged (O2-) released from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs, and the 50% inhibitory concentrations were 6.8 +/- 2.2 and 6.5 +/- 2.5 microm, respectively. N-(Phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide also effectively scavenged (O2-) from PMNs, and the 50% inhibitory concentrations were 11.3 +/- 4.8 and 20.3 +/- 6.4 microm, respectively. Selenoureas showed very low toxicity in human cell lines and PMNs, even at high concentrations, whereas tertiary selenoamides were cytotoxic. These compounds did not produce significant amounts of hydrogen peroxide from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs. None of the compounds significantly affected the translocation of p47 phagocyte oxidase. Selenoureas acted as effective antioxidants and showed low toxicity in some human cells. Thus, these compounds might be new candidates as antioxidative substances.     

Inhibition of carbon tetrachloride-induced lipid peroxidation by novel antioxidants in rat hepatic microsomes: dissociation from hepatoprotective effects in vivo

The ability of two novel antioxidants, U-74,006F and U-78,517G, as well as the known antioxidant N,N'-diphenyl-p-phenylenediamine to inhibit lipid peroxidation induced by carbon tetrachloride (CCl4) was investigated in Aroclor 1254-induced rat hepatic microsomes. All three compounds completely inhibited lipid peroxidation in microsomes as measured by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of lipid peroxidation was not a function of decreased bioactivation of CCl4, as the compounds did not substantially inhibit benzphetamine N-demethylase activity or covalent binding of [14-C]CCl4 to lipid or protein. Parallel studies examined the hepatoprotective effects of the compounds in vivo. Rats were pretreated with antioxidant or vehicle prior to administration of CCl4 (300 or 600 microL/kg i.p.). Sera were collected 24 h postadministration of CCl4 and analyzed for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and total bilirubin. Administration of CCl4 produced elevations in ALT, moderate changes in bilirubin, and no change in ALP activities. Histological examination of CCl4-treated livers revealed lipidosis and centrilobular necrosis. The antioxidants partially improved the clinical chemistry parameters, but had minimal effects on the histological lesion. In contrast to the complete inhibition of lipid peroxidation observed in the in vitro studies, none of the antioxidants markedly protected against CCl4-induced toxicity in vivo.     

Antioxidant and photosynthetic response of a purple-leaved and a green-leaved cultivar of sweet basil (Ocimum basilicum) to boron excess

Boron (B) toxicity induces oxidative stress and alterations in the photosynthetic process. The occurrence of visible symptoms depends on plant species and even on cultivar. However, limited information is available concerning antioxidant responses to B toxicity; therefore a study was carried out to assess the role of antioxidants in hydroponically grown sweet basil submitted to B excess. Two cultivars were compared: the purple-leaved ‘Red Rubin’ that shown scarce symptoms of B-induced toxicity and the green-leaved ‘Tigullio’ in which they were evident.Sweet basil plants were grown in “floating raft system” for 20 days with 0.2 (control), 2 and 20 mg L⁻¹ of B in the nutrient solution. At the end of treatments visible symptoms of damage were evaluated and some parameters were measured: growth, leaf B accumulation, gas exchange and chlorophyll fluorescence, pigment, phenols and malondialdehyde (MDA) concentrations, total non-enzymatic antioxidant ability, antioxidant molecules and enzymes.B excess negatively affected growth and photosynthesis in both cultivars but differential mechanisms were recorded. ‘Tigullio’ exhibited a larger B accumulation in leaves as compared to ‘Red Rubin’. Moreover, in ‘Red Rubin’ plants a greater constitutive content of ascorbic acid, glutathione, anthocyanins and, consequently, a stronger antioxidant ability than ‘Tigullio’ were recorded. MDA test confirmed that the extent of oxidative stress was larger in ‘Tigullio’ than in ‘Red Rubin’. A general stimulation of antioxidant enzymes occurred by increasing B concentration in the growth medium. Notable, anthocyanins were likely involved in the B tolerance shown by ‘Red Rubin’ in consideration of their antioxidant properties and because of the role of these compounds in photoprotection. This paper represents a contribution to understanding the role of antioxidant compounds in plant tolerance to B toxicity.     

Do spin traps also act as classical chain-breaking antioxidants? A quantitative kinetic study of phenyl tert-butylnitrone (PBN) in solution and in liposomes

Free radical spin traps such as phenyl tert-butylnitrone (PBN) are often reported to provide protection of the central nervous system of animal models against free radical damage, and the effects are attributed to its "antioxidant activity." The effects of PBN and p-CH(3)O-PBN were compared with known antioxidants, alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), in quantitative kinetic studies of lipid peroxidation thermally initiated under controlled conditions. Results obtained on the spin traps in organic solvents and in dilinoleoyl phosphatidylcholine (DLPC) bilayers indicated that the spin traps do not act as peroxyl radical trapping antioxidants but rather act only as moderate "retarders" of oxygen uptake at relatively high concentration. At low oxygen partial pressures, e.g., 14 torr, which better reflect oxygen partial pressures in biological systems, PBN provides a more significant reduction in oxygen uptake (up to 50%) by DLPC bilayers but still did not act as a typical antioxidant. However, at low partial pressures, PBN does act cooperatively with PMHC. It is suggested that its role in biological fluids and tissues may be to extend the suppressed oxidation by natural antioxidants expected to be present. The combination of antioxidant/spin trap, alpha-(3, 5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone did not exhibit any enhanced antioxidant efficiency compared with the related hindered phenol, 2,6-di-tert-butyl-4-methoxyphenol.