Chromium (VI) Can Activate and Impair Antioxidant Defense System

The changes in glutathione-dependent cycle enzymes and catalase activities under Cr(VI)-induced oxidative stress were investigated in two distinct cell lines: L-41−human epithelial-like cells and HLF−fetal human diploid lung fibroblasts, which differ in tissue origin, proliferation, and antioxidant enzymes activities. The chromium concentrations from 1 to 5 μM cause nontoxic effects and activate antioxidant enzymes to overcome oxidative stress. In spite of some differences in the endogenous antioxidant activities, both cell lines reveal the same range of toxic concentrations (20–30 μM). The irreversible inhibition of glutathione-dependent antioxidant enzymes develops under toxic concentrations and serves as a marker of toxicity. The endogenous antioxidant activity influences time-dependent expression of Cr(VI) toxicity and the dynamics of antioxidant enzymes activity under nontoxic conditions. The cell antioxidant defense system is an important marker of the cell adaptive capacity under nontoxic and toxic conditions.     

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o-diphenols to o-quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a -COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3-dihydro-1,4-benzodioxin type (3, 6-8), 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-2,3-dicarboxy-1,2-dihydro naphthaline (1) 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-3-carboxynaphthaline (5) and 2,6-bis-(3',4'-dihydroxyphenyl)-1-carboxy-3-oxacyclo-(3,0)-pent an-2-on-1-ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.     

The Oxyradical-Scavenging Activity of Azelaic Acid in Biological Systems

We have previously shown that azelaic acid, a C₉ dicarboxylic acid, as disodium salt (C₉2Na) is capable of inhibiting significantly the hydroxylation of aromatic compounds and the peroxidation of arachidonic acid due to reactive hydroxyl radicals (HO'). In this paper we have investigated the ability of C₉2Na to inhibit the oxyradical induced toxicity towards two tumoral cell lines (Raji and IRE 1) and normal human fibroblasts (HF). Oxyradicals were generated either by the addition of polyphenols to the medium, or by direct irradiation of phosphate buffered-saline in which cells were incubated from 15min prior to incubation in normal medium. The effects of C₉2Na were compared with those obtained by mannitol (MAN), superoxide dismutase (SOD) and catalase (CAT). C₉2Na, MAN, SOD and CAT significantly decreased the polyphenol toxicity towards cell lines cultured up to 24 h. After 48 h of incubation the above compounds lost the capability of protecting cells from polyphenol toxicity. This suggests that the toxic role of oxyradicals (O₂^-, H₂O₂, HO^.) persists for about 24h and, subsequently other toxic mechanisms must be involved, which are not affected by oxyradical scavengers. SOD and CAT did not show any protective effect on UV induced cytotoxicity, while both C₉2Na and MAN were capable of reducing significantly the UV damage towards cell lines, even after 48 h incubation. This can be explained by the fact that UV cytotoxicity depends mainly on the generation of HO', that can be “scavenged” by C₉2Na or MAN, but not by SOD or CAT. C₉2Na and MAN were not significantly degraded in the period during which they afford protection against HO^..     

Comparison of the characteristics of fungal and plant tyrosinases

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, -casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein -casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink -casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink -casein. In the reaction conditions studied, TrT showed the best ability to crosslink -casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.     

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.     

Effect of phenolic compounds on symbiotic nitrogen fixation in pigeonpea(Cajanus cajan (L.) Millsp.)

The effect of different phenolic compounds:p-hydroxybenzoic acid, resorcinol and chlorogenic acid (mono-, di- and polyphenol) was studied on nodulation and related metabolic processes in pigeonpea (Cajanus cajan L. cv. Al-15). Nitrogenease activity, leghaemoglobin and ascorbic acid content of the nodules increased with the application of phenols. Phenols increase the contents of amino acids, proteins and total soluble carbohydrates in the nodules as reserve food materials.     

Cell signaling and receptors with resorcinols and flavonoids: redox, reactive oxygen species, and physiological effects

There have been appreciable numbers of reviews on monophenols, catechols, and hydroquinones. However, the resorcinol class has received less attention. This review deals with resorcinols and flavonoids. Emphasis is on cell signaling in addition to antioxidant (AO) properties and pro-oxidant effects. The apparent dichotomy is rationalized. There is extensive literature dealing with various aspects of cell signaling in addition to receptor involvement. The physiological responses are provided along with integration into the unifying mechanistic theme of electron transfer (ET)-reactive oxygen species (ROS)-oxidative stress (OS). The multifaceted approach involving redox processes and cell signaling is unique in providing novel insight.     

Chemical and toxic evaluation of a biological treatment for olive-oil mill wastewater using commercial microbial formulations

Olive-oil-mill wastewater (OMW) has significant polluting properties due to its high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and phenols. In the present study, different commercial bacterial formulations were used in the biological treatment of OMW. COD and toxicity testing using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna) were employed to evaluate abatement of the organic load and reduction of the toxic potential. In addition, the four most active formulations were tested mixed pair-wise on the basis of their unique characteristics in order to evaluate the improvement of treatment. The effect of treatment was assessed by measuring COD removal, reduction of total phenols, and decreased toxicity. The results obtained with the mixed formulations showed that the maximum removal of the organic load was about 85%, whereas phenols were reduced by about 67%. The toxicity for rotifers decreased by 43% and for crustaceans by about 83%.     

Cell signaling and receptors with resorcinols and flavonoids: redox,reactive oxygen species,and physiological effects

There have been appreciable numbers of reviews on monophenols, catechols, and hydroquinones. However, the resorcinol class has received less attention. This review deals with resorcinols and flavonoids. Emphasis is on cell signaling in addition to antioxidant (AO) properties and pro-oxidant effects. The apparent dichotomy is rationalized. There is extensive literature dealing with various aspects of cell signaling in addition to receptor involvement. The physiological responses are provided along with integration into the unifying mechanistic theme of electron transfer (ET)-reactive oxygen species (ROS)-oxidative stress (OS). The multifaceted approach involving redox processes and cell signaling is unique in providing novel insight.     

Toxicity of oxalysine and oxalysine-containing peptides against Candida albicans: regulation of peptide transport by amino acids.

A lysine antimetabolite, L-4-oxalysine [H2NCH2CH2OCH2CH(NH2)COOH], and oxalysine-containing di-, tri-, tetra- and pentapeptides inhibited growth of Candida albicans H317. Micromolar amounts of amino acids were found to overcome ammonium repression of the di- and tripeptide transport system(s) in strain H317. Several amino acids increased the toxicity of oxalysine-containing di- and tripeptides for C. albicans with little or no increase in toxicity of oxalysine or oxalysine-containing tetra- and pentapeptides. L-Lysine completely reversed the toxicity of oxalysine by competing with the transport of oxalysine into the cells. In contrast, L-lysine increased the toxicity of oxalysine-containing di- and tripeptides, but had no effect on the toxicity of oxalysine-containing tetra- and pentapeptides. Incubation of cells with L-lysine for 4 h resulted in a 15-fold increase in the rate of transport of radiolabelled dileucine, indicating that increased sensitivity of C. albicans to some toxic peptides in the presence of L-lysine may be attributed to an increased rate of transport of these peptides. Our results indicate that the dipeptide and tripeptide transport system(s) of C. albicans are regulated by micromolar amounts of amino acids in a similar fashion to the regulation of peptide transport in Saccharomyces cerevisiae and that multiple peptide transport systems differentially regulated by various nitrogen sources and amino acids exist in C. albicans.     

Kinetic cooperativity of tyrosinase. A general mechanism

Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).     

Relation of the antimutagenic activity of simple phenols to the number of hydroxyl groups

The influence of mono-phenol, di-resorcinol and tri-pyrogallol hydroxyl groups of simple unsubstituted phenols on the mutagenic potentials of benzo(a)pyrene was studied in vivo (micronuclear test on bone marrow polychromatic erythrocytes) and in vitro (test of direct point mutations at V79/HGPRT system induced by metabolic activation by mouse liver microsomal enzymes). The phenols decreased the mutagenic activity of benzo(a)pyrene in in vivo tests, with pyrogallol being the most active, it followed by resorcinol and phenol. The mixtures of benzo(a) pyrene + pyrogallol and benzo(a)pyrene + resorcinol were significantly less mutagenic in in vitro tests than benzo(a)pyrene and benzo(a)pyrene + phenol.     

The toxicity of adenosine analogues against Babesia bovis in vitro.

The toxicities of 20 analogues of deoxyadenosine or adenosine were tested in vitro against the intraerythrocytic parasite Babesia bovis. IC37 values (the concentration of compound required to reduce cell survival to 37%) were determined for each compound. Tubercidin (7-deaza-adenosine), 2-bromo-adenosine, 8-bromo-3-ribosyl adenine and 6-phenylamino-deoxyadenosine were shown to be the most toxic towards B. bovis. Comparison of the toxicity results for these compounds in B. bovis with those in human melanoma cell lines indicated a differential toxicity, in that many of the compounds were toxic towards B. bovis but were relatively non-toxic towards human melanoma cell lines and vice versa. These results suggest that the mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids, may vary significantly between B. bovis and mammalian cells, allowing such drugs to be considered for parasite chemotherapy.     

Nitric oxide promotes strong cytotoxicity of phenolic compounds against Escherichia coli: the influence of antioxidant defenses

The induction of mutagenic and cytotoxic effects by simple phenolics, including catechol (CAT), 3,4-dihydroxyphenylacetic acid (DOPAC), hydroquinone (HQ), and 2,5-dihydroxyphenylacetic (homogentisic) acid (HGA), appears to occur through an oxidative mechanism based on the ability of these compounds to undergo autoxidation, leading to quinone formation with the production of reactive oxygen species. This is supported by the detection of such adverse effects in plate assays using Escherichia coli tester strains deficient in the OxyR function, but not in OxyR(+) strains. The OxyR protein is a redox-sensitive regulator of genes encoding antioxidant enzymes including catalase and alkyl hydroperoxide reductase, which would eliminate hydrogen peroxide. Methyl-substituted phenolics such as 4-methylcatechol (MCAT) and methylhydroquinone (MHQ) produced, in addition to oxidative toxicity, marked cytotoxic effects against OxyR(+) cells, thus revealing a mechanism of toxicity not mediated by hydrogen peroxide that could involve quinones and quinone methides arising from MCAT and MHQ oxidation. Quinone compounds could also be responsible for the enhanced cytotoxicity of certain phenolics when combined with a nitric oxide (NO(*)) donor such as diethylamine/NO (DEA/NO). Phenolics scavenge NO(*) and, in turn, NO(*) oxidizes phenolics to form their quinone derivatives. In OxyR(+) cells, where the oxidative toxicity is inhibited, DEA/NO promoted exceptional increases in the cytotoxicity of CAT and 3,4-dihydroxycinnamic (caffeic) acid (CAF), which both exhibited very low oxidative cytotoxicity, as well as in that of MCAT, HQ, and MHQ. In contrast, DEA/NO failed to promote toxicity by DOPAC and HGA, probably due to their ability to undergo oxidative polymerization, leading to the formation of melanins. Spectroscopic studies demonstrated quinone generation from the oxidation of CAF, HQ, and MHQ by DEA/NO. The o-quinone derived from CAF was rather unstable and decomposed during its isolation. For the generation of toxic quinones, e.g., to be used as therapeutic agents producing antitumor or antibacterial effects, the isolation step could be avoided with the method proposed. It combines quinone precursors, i.e. phenolic compounds, with an oxidant such as NO(*).     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

Changes in free polyamine level and di- and polyamine oxidase activity in cucumber roots under allelochemical stress conditions

Seven-day-old seedlings of the cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.5 mM solutions of phenols (p-coumaric, ferulic, p-hydroxybenzoic and vanillic acids) as stress factors. The level of free polyamines as well as activities of their catabolic enzymes, i.e. di- and polyamine oxidases (DAO - EC 1.4.34 and PAO - EC 1.4.36), were estimated for the first three hours of the stress. Cucumber roots were found to have only the presence of putrescine and spermidine. Root treatment with phenols caused a violent decrease of both amine contents during the first hour of the stress. These changes were associated with the increase of amine oxidase activity.     

ANTIALGAL ACTIVITY OF SOME SIMPLE PHENOLS1

The antialgal activity of a number of simple phenols was examined for their effect on the growth of 7 species of unicellular marine algae. The 3 knoiun algal phenols, 5-bromo-3,4-dihydroxybenzaldehyde; 2,3-dibromo-4,5-dihydroxybenzylalcohol, and 3,4-dihy-droxyphenylethylamine, were highly toxic as were other ortho dihydroxy compounds. Monohydroxy compounds were notably less toxic. Skeletonema costatum and Olisthodiscus sp. were the most sensitive organisms examined and Dunaliella tertiolecta was the most resistant. Possible ecological implications of these results are discussed.     

Use and improvement of microbial redox enzymes for environmental purposes

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.     

Effect of n-hexane on the disposition and toxicity of the 1,3-butadiene metabolite 3-butene-1,2-diol

3-Butene-1,2-diol (butenediol), a major metabolite of 1,3-butadiene (butadiene), can undergo either detoxification or biotransformation to potentially toxic metabolites, including 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone (HMVK). Butadiene exposure can occur concomitantly with hexanes, which share common biotransformation pathways with butadiene. To determine the potential influence of hexane co-exposure on butadiene toxicity, the present study examined the effect of n-hexane on butenediol disposition [as measured by urinary excretion of (N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine) (MI level)] and genotoxicity (as measured by the frequency of bone marrow micronucleated erythrocytes) and acute toxicity (as measured by body weight changes) in the rat. The results show that butenediol was not genotoxic to adult or immature rats but was acutely toxic to adult but not immature rats. The results also suggest that n-hexane co-exposure may attenuate the acute toxicity by butenediol in adult rats and that immature rats may be less sensitive than adults to the acute toxicity.     

Effects of tyrosinase activity on the cytotoxicity of 3,4-dihydroxybenzylamine and buthionine sulfoximine in human melanoma cells

The rationale for melanoma specific dihydroxybenzene containing antitumor agents is based in part upon the ability of the enzyme tyrosinase to oxidize these pro drugs to toxic intermediates. In situ tyrosinase activity was demonstrated to be affected by both cell density and time from plating in pigmented melanoma cells. Phenylthiourea, which completely inhibited tyrosinase activity with minimal cytotoxicity was found to block the growth inhibitory activity of the antitumor dopamine analog 3,4-dihydroxybenzylamine (3,4-DHBA) (NSC 263475). The antioxidant dithioerythritol was also found to inhibit tyrosinase activity and to block the growth inhibitory effects of 3,4-DHBA in pigmented melanoma cell lines. Buthionine sulfoximine (BSO) was shown to be cytotoxic to melanoma cells and its growth inhibitory effects appears to correlate with tyrosinase levels. Furthermore, BSO was shown to potentiate the growth inhibitory effects of 3,4-DHBA on marginally pigmented human melanoma cell lines.