Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.     

Changes in the antigenic properties of proteins of laboratory mice during oxidative stress

Changes in the level of oxidative damage to proteins in CD1 outbred mice γ irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to γ radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.     

Fluorometric analysis of iodinated aflatoxin in minicultures ofAspergillus flavus andAspergillus parasiticus

Summary A convenient miniassay for aflatoxin has been developed for cultures ofAspergillus flavus andA. parasiticus grown for 3–10 days in 10 ml of a coconut extract medium. The sensitivity of the assay, as measured by photofluorometry (365 nm maximum excitation; 445 nm maximum emission), is of the order of 0.01 M (3.12 ng/ml) for aflatoxin B₁ dissolved in aqueous iodine (0.26 mM). High performance liquid chromatography, monitored by fluorometric analysis of both an aflatoxin B₁ standard and selected culture filtrates, confirmed the sensitivity of the assay and indicated specificity for iodine-enhanced fluorescence of aflatoxin in the coconut extract medium. Thin layer chromatography further confirmed the aflatoxin titers and the specificity for enhancement of aflatoxins B₁ and G₁ in culture filtrates.Alabama Agricultural Experiment Station Journal No. 6-871297.     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

A versiconal hemiacetal acetate converting enzyme in aflatoxin biosynthesis

Conversion of the aflatoxin biosynthetic intermediate versiconal hemiacetal acetate (VHA) in a cell free extract ofAspergillus parasiticus ATCC 15517 is investigated. The enzymatic reaction is monitored by a method using high performance liquid chromatography (HPLC). The major product of the enzymatic reaction is a water soluble compound not chloroform-extractable at pH 7.5. The product becomes chloroform extractable upon acidification of the reaction medium and is separated and quantitated by reversed-phase HPLC. It is tentatively identified as versiconal hemiacetal alcohol, which is converted to versicolorin C (VC) upon acid treatment.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B₁.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B₁ at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 10¹ to 7 × 10⁵ cfu g^–1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B₁. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.This revised version was published online in October 2005 with corrections to the Cover Date.     

Isolation of aflatoxigenic strains of Aspergillus and detection of aflatoxin B1 from feeds in India

In a preliminary study, 256 feed samples collected from different parts of Northern India were examined for the presence of aflatoxigenic strains of Aspergillus flavus/parasiticus and for detection of Aflatoxin B₁ (AFB₁). Out of 198 A. flavus and 15 A. parasiticus strains isolated, 76% and 86% respectively, were found to be toxigenic. Aflatoxin B₁ content of these feeds, as estimated by thin layer chromatography (TLC) and enzyme linked immunosorbent assay (ELISA) were very high (average 0.412 ± 0.154 ppm) in comparison to the permissible Indian regulation level (0.03 ppm). Seasonal variation of incidence and level of toxin in feed was recorded and it was high during monsoon/post monsoon period.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.     

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.     

Metabolic defence against oxidative stress: the road less travelled so far

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly‐reactive chalcogen—oxygen (O₂). They are known to evoke intricate strategies to defend themselves from the reactive by‐products of oxygen—reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)‐cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH—the reductive engine of many ROS‐combating enzymes—is maintained by metabolic enzymes including, but not exclusively, glucose‐6 phosphate dehydrogenase (G6PDH) and NADP‐dependent isocitrate dehydrogenase (ICDH‐NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro‐oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids—moieties capable of non‐enzymatic decarboxylation in the presence of oxidants—as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid‐mediated detoxification of the ROS will be commented on within the context of oxidative stress.     

Carotenoids, oxidative stress and female mating preference for longer lived males

Some of the most spectacular exaggerated sexual ornaments are carotenoid dependent. It has been suggested that such ornaments have evolved because carotenoid pigments are limiting for both signal expression and in their role as antioxidants and immunostimulants. An implicit assumption of this hypothesis is that males which can afford to produce more elaborate carotenoid-dependent displays are signalling their enhanced ability to resist parasites, disease or oxidative stress and hence would be predicted to live longer. Therefore, in species with carotenoid-dependent ornaments where a parent's future longevity is crucial for determining offspring survival, there should be a mating preference for partners that present the lowest risk of mortality during the breeding attempt, as signalled by the ability to allocate carotenoids to sexual displays. In an experimental study using three-spined sticklebacks (Gasterosteus aculeatus), we show that when dietary carotenoid intake is limited, males attempt to maintain their sexual ornament at the expense of body carotenoids and hence suffer from reduced reproductive investment and a shorter lifespan. These males also suffer from an increased susceptibility to oxidative stress, suggesting that this may constitute the mechanism underlying the increased rate of ageing. Furthermore, in pairwise mate-choice trials, females preferred males that had a greater access to carotenoids and chance of surviving the breeding season, suggesting that females can make adaptive mate choice decisions based on a male's carotenoid status and potential future longevity.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

Chilling, oxidative stress and antioxidant responses in shoot cultures of rice

Chilling of shoot cultures from Oryza sativa L. cv. Taipei 309, to 4 °C leads to conditions of oxidative stress. Tissue H₂O₂ was observed to increase more than fourfold by 8 d of chilling, and levels of reduced glutathione, which normally rise in growing shoot cultures at 25 °C, were considerably repressed in chilled cultures. Whilst the activity of ascorbate peroxidase in chilled shoots remained similar to the activities in control cultures at 25 °C, the most notable effects of chilling to 4 °C were the very significant loss of catalase and glutathione reductase activity. Although prior exposure of shoot cultures to abscisic acid (ABA) at 25 °C increased levels of catalase activity, such increased levels were not sustained when the pre-treated cultures were placed at 4 °C. Moreover such pre-treatment with ABA did not increase the subsequent ability of shoot cultures to grow at 4 °C.Abbreviations GSH reduced glutathione - GSSG oxidised glutathione - ABA cis-abscisic acid This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Acetyl coenzyme A carboxylase in species of Triticum of different ploidy

The cellular amounts and cellular activities of acetyl CoA carboxylase (ACC; EC 6.4.1.2.) were determined in the first leaves of diploid, tetraploid and hexaploid species of Triticum (wheat). Per leaf the ACC activities were very similar in T. monococcum (2 ), T. dicoccum (4 ) and T. aestivum (6 ). The ACC activity per chloroplast also showed little variation between species of different ploidy but since chloroplast number increases with ploidy, the ACC activities and ACC amounts per cell also increased with ploidy. These cellular increases in ACC amounts associated with increases in gene dosage were highly co-ordinated in the diploids T. monococcum and T. tauschii and their respective autotetraploids so the specific activity of ACC was highly conserved in these plants. The relevance of these findings to attempts to genetically manipulate lipid biosynthesis in chloroplasts is discussed.Abbreviation ACC acetyl CoA carboxylase We are very grateful to Dr. Kevin Pyke and Miss Jo Marrison for many helpful discussions and to Dr. Collin Law for the generous gift of seeds.     

Role of oxidative stress in the pathogenesis of alcohol-induced liver disease

Abstract

Chronic alcohol consumption is a well-known risk factor for liver disease, which represents a major cause of morbidity and mortality worldwide. The pathological process of alcohol-induced liver disease is characterized by a broad spectrum of morphological changes ranging from steatosis with minimal injury to more advanced liver damage, including steato-hepatitis and fibrosis/cirrhosis. Experimental and clinical studies increasingly show that the oxidative damage induced by ethanol contribute in many ways to the pathogenesis of alcohol hepatotoxicity. This article describes the contribution of oxidative mechanisms to liver damage by alcohol.     

Roles of sirtuins in the regulation of antioxidant defense and bioenergetic function of mitochondria under oxidative stress

Abstract

In addition to serving as the power house of mammalian cells, mitochondria are crucial for the maintenance of cellular homeostasis in response to physiological or environmental changes. Several lines of evidence suggest that posttranslational modification (PTM) of proteins plays a pivotal role in the regulation of the bioenergetic function of mitochondria. Among them, reversible lysine acetylation of mitochondrial proteins has been established as one of the key mechanisms in cellular response to energy demand by modulating the flux of a number of key metabolic pathways. In this article, we focus on the role of Sirt3-mediated deacetylation in: (1) flexibility of energy metabolism, (2) activation of antioxidant defense, and (3) maintenance of cellular redox status in response to dietary challenge and oxidative stress. We suggest that oxidative stress-elicited down-regulation of Sirt3 plays a role in the pathophysiology of diabetes, cardiac hypotrophy, mitochondrial diseases, and age-related diseases. Besides, the physiological role of newly identified lysine acylation mediated by Sirt5 and its biochemical effects on oxidative metabolism are also discussed. Moreover, we have integrated the regulatory function of several protein kinases that are involved in the phosphorylation of mitochondrial enzymes during oxidative stress. Finally, the functional consequence of the synergistic regulation through diverse protein modifications is emphasized on the maintenance of the bioenergetic homeostasis and metabolic adaptation of the animal and human cells. Together, we have provided an updated review of PTM in mitochondrial biology and their implications in aging and human diseases through an intricate regulation of energy metabolism under oxidative stress.     

Antioxidative properties of ginsenoside Ro against UV-B-induced oxidative stress in human dermal fibroblasts

Ginsenoside Ro (Ro), an oleanolic acid-type ginsenoside, exhibited suppressive activities on reactive oxygen species (ROS) and matrix metalloproteinase-2 (MMP-2) elevation in UV-B-irradiated fibroblasts. Ro could overcome the reduction of the total glutathione (GSH) contents in UV-B-irradiated fibroblasts. Ro could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Collectively, Ro possesses a potential skin anti-photoaging property against UV-B radiation in fibroblasts.