Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the ␤-oxidation of fatty acids and their enzymatic or nonenzymatic byproducts (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus. To better understand the role of ROS formation in toxin production, we generated a mutant (⌬ApyapA) having the ApyapA gene deleted, given that ApyapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the ⌬ApyapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of ApyapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.Reactive oxygen species (ROS), such as superoxide anionand lipoperoxides (LOOH), which are formed from unsaturated fatty acids and can be produced in the cell during metabolic processes, can be overproduced following the action of oxidative stressors present in the environment (32,49,57). To counteract the potentially dangerous accumulation of ROS, cells have evolved strategies (49, 61) based on enzymatic or nonenzymatic systems (28,45). The main antioxidant enzymes in cells involved in ROS removal are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). If H 2 O 2 exceeds the cell-scavenging capacity, it can generate highly reactive HO ⅐ through a Fenton reaction, which initiates the formation of LOOH in the membrane lipids (32). When ROS accumulation occurs, the oxidant/antioxidant balance is perturbed, which can damage the cell membrane and cell metabolism (free-radical theory of aging) (26). ROS produced at certain time points during the cell's life cycle and at low physiological concentrations play a crucial role in the organism's homeostasis and cell functions. As second messengers, ROS take part in the plant's developmental processes (18,24,31) and in the defense mechanisms against pathogens and abiotic stress (5, 24, 52, 62). Similar effects have been shown in mammals, where ROS at proper levels stimulate antioxidant reactions, immune system modulation, and regulation of cell proliferation (3,4,55,59,65). One of the major objectives of studying the biology of stress is to identify the key factors that control the switch from cytoprotective responses to cell dysfunction following oxidative insult (11)....

Mycotoxins are harmful secondary metabolites produced by a range of widespread fungi belonging in the main to Fusarium, Aspergillus and Penicillium genera. But why should fungi produce toxins? And how is the biosynthesis of these toxins regulated? Several separate factors are now known to be capable of modulating mycotoxin synthesis; however, in this study, focussing just on mycotoxins whose regulatory mechanisms have already been established, we introduce a further factor based on a novel consideration. Various different mycotoxin biosynthetic pathways appear to share a common factor in that they are all susceptible to the influence of reactive oxygen species. In fact, when a fungus receives an external stimulus, it reacts by activating, through a well-defined signal cascade, a profound change in its lifestyle. This change usually leads to the activation of global gene regulators and, in particular, of transcription factors which modulate mycotoxin gene cluster expression. Some mycotoxins have a clear-cut role both in generating a pathogenetic process, i.e. fumonisins and some trichothecenes, and in competing with other organisms, i.e. patulin. In other cases, such as aflatoxins, more than one role can be hypothesised. In this review, we suggest an "oxidative stress theory of mycotoxin biosynthesis" to explain the role and the regulation of some of the above mentioned toxins.

Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and beta-glucan content. Mushroom beta-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with beta-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, beta-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the "oxidative message" to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the ApyapA gene, a homologue of the yeast Yap-1, disables the fungus's capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer AoloxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Aoyap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus.

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.

A close correlation between lipoperoxide formation in cells ofAspergillus parasiticus and aflatoxin biosynthesis has been established in rich and poor media in which oxidative stress was induced by addition of cumene hydroperoxide, a lipoperoxidation inducer. The presence of hydroperoxides of linoleic acid inA. parasiticus mycelia was analysed by liquid chromatography-mass spectrometry (LC-MS). This relation appears to be driven by activation of certain oxidative stress related transcription factors, such asyap1-like,skn7-like andhsf2-like. Activation of these factors then leads to the promotion of transcription of genes encoding antioxidant-related enzymes, such as superoxide dismutase, catalase and glutathione peroxidase.The incomplete seavenging of intracellular oxidation inA. parasiticus cells can lead to aflatoxin biosynthesis. The relationship between oxidative stress and aflatoxin biosynthesis is indicated by the high correlation among increased activity of lipoperoxidation and the antioxidant defence system with formation of aflatoxins.With regard to the relationship of oxidative stress and aflatoxin biosynthesis, the mechanism of action of butylated hydroxyl anisole (BHA), an antioxidant compound, in the control of aflatoxin biosynthesis was also investigated. Results indicate this compound can act,per se, by inhibiting lipoperoxidation and by inducing antioxidative defence responses of the fungal cell.

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