Glycation modulates superoxide dismutase 1 aggregation and toxicity in models of sporadic amyotrophic lateral sclerosis.

Different SOD1 proteoforms are implicated## in both familial and sporadic cases of Amyotrophic Lateral Sclerosis (ALS), an aging-associated disease that affects motor neurons. SOD1 is crucial to neuronal metabolism and health, regulating the oxidative stress response and the shift between oxidative-fermentative metabolism, which is important for astrocyte-neuron metabolic cooperation. Neurons have a limited capacity to metabolize methylglyoxal (MGO), a potentially toxic side product of glycolysis. MGO is highly reactive and can readily posttranslationally modify proteins, in a reaction known as glycation, impacting their normal biology. Here, we aimed to investigate the effect of glycation on the aggregation and toxicity of human SOD1WT (hSOD1WT). Cells with deficiency in MGO metabolism showed increased levels of hSOD1WT inclusions, displaying also reduced hSOD1WT activity and viability. Strikingly, we also found that the presence of hSOD1WT in stress granules increased upon MGO treatment. The treatment of recombinant hSOD1WT with MGO resulted in the formation of SDS-stable oligomers, specially trimers, and thioflavin-T positive aggregates, which can promote cell toxicity and TDP-43 pathology. Together, our results suggest that glycation may play a still underappreciated role on hSOD1WT and TDP-43 pathologies in sporadic ALS, which could open novel perspectives for therapeutic intervention.

Hexokinase 2: The preferential target of trehalose-6-phosphate over hexokinase 1.

Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.

Understanding xylose isomerase from Burkholderia cenocepacia: insights into structure and functionality for ethanol production.

The inability of the yeast Saccharomyces cerevisiae to produce ethanol from xylose has hampered the biofuel production from lignocellulosic biomass. However, prior studies reveal that functional expression of xylose isomerase (XI) from Burkholderia cenocepacia (XylABc) in S. cerevisiae has remarkably improved xylose consumption and ethanol productivity. Yet, little is known about kinetic and structural properties of this enzyme. Hereby, a purified recombinant XylA was assayed in vitro, showing optimal enzyme activity at 37 °C and pH 7.2. The Km of XylA for D-xylose was at least threefold lower than the Km results for any XI published to date (e.g. XylA from Piromyces sp.). In addition, oligomerization behavior as a tetramer was observed for XylA in solution. Functional and structural comparative analyses amongst three microbial XIs were further performed as theoretical models, showing that xylose orientation at the active site was highly conserved among the XIs. Mg2+ ions anchor the sugar and guide its pyranoside oxygen towards a histidine residue present at the active site, allowing an acid-base reaction, linearizing xylose. Electrostatic surface analyses showed that small variations in the net charge distribution and dipole moment could directly affect the way the substrate interacts with the protein, thus altering its kinetic properties. Accordingly, in silico modeling suggested the tetramer may be the major functional form. These analyses and the resulting model promote a better understanding of this protein family and pave the way to further protein engineering and application of XylA in the ethanol industry.

The trehalose protective mechanism during thermal stress in Saccharomyces cerevisiae: the roles of Ath1 and Agt1.

Trehalose on both sides of the bilayer is a requirement for full protection of membranes against stress. It was not known yet how trehalose, synthesized in the cytosol when dividing Saccharomyces cerevisiae cells are shifted from 28°C to 40°C, is transported to the outside and degraded when cells return to 28°C. According to our results, the lack of Agt1, a trehalose transporter, although had not affected trehalose synthesis, reduced cell tolerance to 51°C and increased lipid peroxidation. The damage was reversed when external trehalose was added during 40°C adaptation, confirming that the reason for the agt1Δ sensitivity is the absence of trehalose at the outside of the lipid bilayer. The 40-28°C condition caused cytosolic trehalase (Nth1) activation, reducing intracellular trehalose and, consequently, the survival rates after 51°C. Although lower than nth1Δ strain, cells deficient in acid trehalase (ath1Δ)  maintained increased trehalose levels after 40°C-28°C shift, which conferred protection against 51°C. Both Ath1 and Agt1 were found into vesicles near to plasma membrane in response to stress. This suggests that Agt1 containing vesicles would fuse with the membrane under 40°C to transport part of the cytosolic trehalose to the outside. By a similar mechanism, Ath1 would reach the cell surface to hydrolyze the external trehalose but only when the stress would be over. Corroborating this conclusion, Ath1 activity in soluble cell-free extracts increased after 40°C adaptation but decreased when cells returned to 28°C. During 40°C, Ath1 is confined into vesicles, avoiding the cleavage of the outside trehalose.

The second International Symposium on Fungal Stress: ISFUS.

The topic of 'fungal stress' is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.

Improvement of Brazilian bioethanol production - Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process.

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15-25 % of wet weight/v) and high sugar concentration (18-22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.

Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models.

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

The PWWP domain of the human oncogene WHSC1L1/NSD3 induces a metabolic shift toward fermentation.

WHSC1L1/NSD3, one of the most aggressive human oncogenes, has two isoforms derived from alternative splicing. Overexpression of long or short NSD3 is capable of transforming a healthy into a cancer cell. NSD3s, the short isoform, contains only a PWWP domain, a histone methyl-lysine reader involved in epigenetic regulation of gene expression. With the aim of understanding the NSD3s PWWP domain role in tumorigenesis, we used Saccharomyces cerevisiae as an experimental model. We identified the yeast protein Pdp3 that contains a PWWP domain that closely resembles NSD3s PWWP. Our results indicate that the yeast protein Pdp3 and human NSD3s seem to play similar roles in energy metabolism, leading to a metabolic shift toward fermentation. The swapping domain experiments suggested that the PWWP domain of NSD3s functionally substitutes that of yeast Pdp3, whose W21 is essential for its metabolic function.

Quantitative proteomic analysis of the Saccharomyces cerevisiae industrial strains CAT-1 and PE-2.

Brazilian ethanol fermentation process commonly uses baker's yeast as inoculum. In recent years, wild type yeast strains have been widely adopted. The two more successful examples are PE-2 and CAT-1, currently employed in Brazilian distilleries. In the present study, we analyzed how these strains compete for nutrients in the same environment and compared the potential characteristics which affect their performance by applying quantitative proteomics methods. Through the use of isobaric tagging, it was possible to compare protein abundances between both strains during the fermentation process. Our results revealed a better fermentation performance and robustness of CAT-1 strain. The proteomic results demonstrated many possible features that may be linked to the improved fermentation traits of the CAT-1. Proteins involved in response to oxidative stress (Sod1 and Trx1) and trehalose synthesis (Tps3) were more abundant in CAT-1 than in PE-2 after a fermentation batch. Tolerance to oxidative stress and trehalose accumulation were subsequently demonstrated to be enhanced for CAT-1, corroborating the comparative proteomic results. The importance of trehalose and the antioxidant system was confirmed by using mutant stains deleted in Sod1, Trx1 or Tps3. These deletions impaired fermentation performance, strengthening the idea that the abilities of accumulating high levels of trehalose and coping with oxidative stress are crucial for improving fermentation. SIGNIFICANCE: The importance of the present works emerges from the necessity to better understand the peculiar biological features from two important bioethanol industrial strains of Saccharomyces cerevisiae during batch fermentation. We applied an iTRAQ-based quantitative proteomics analysis to compare these two important strains during batch fermentation and identified possible features involved in the fermentation performance. The results provided by this work will serve as an initial framework for future investigations on the biology of both strains.

Trehalose-6-Phosphate as a Potential Lead Candidate for the Development of Tps1 Inhibitors: Insights from the Trehalose Biosynthesis Pathway in Diverse Yeast Species.

In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 µM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.

Oxidative Stress and Amyloid Toxicity: Insights From Yeast.

Alzheimer's disease is the most common neurodegenerative disorder. One of the factors that promotes neurodegeneration is the accumulation of senile plaques formed by Aß peptide. In this paper, it was analyzed that if oxidative stress is cause or consequence of amyloid cascade and the role of antioxidant defense system in this process, using S. cerevisiae (with a multicopy plasmid containing the Aß1-42 sequence) as experimental model. Cells grown on glycerol were more tolerant than when grown on glucose, strengthening the role of the antioxidant defense system against Aß accumulation. Antioxidant defense deficiency did not change the pattern of amyloid aggregation. On the other hand, the presence of Aß increased the level of intracellular oxidation and induced the activity of catalase, superoxide dismutase, and aconitase. Peroxissomal catalase deficient cells (Δcta1), were more sensitive to Aß toxicity than the wild type strain, while mitochondrial superoxide dismutase (Sod2) deficient cells displayed the highest frequency of petites. Besides, Aß alters the oxygen consumption and the activity of complex III and IV. Taken together, our results point out that the Aß toxicity mechanism involves an oxidative stress induction by increasing ROS production into the mitochondria, where Cta1 and Sod2 play a crucial role in the regulation of the redox balance. J. Cell. Biochem. 118: 1442-1452, 2017. © 2016 Wiley Periodicals, Inc.

PWWP domains and their modes of sensing DNA and histone methylated lysines.

Chromatin plays an important role in gene transcription control, cell cycle progression, recombination, DNA replication and repair. The fundamental unit of chromatin, the nucleosome, is formed by a DNA duplex wrapped around an octamer of histones. Histones are susceptible to various post-translational modifications, covalent alterations that change the chromatin status. Lysine methylation is one of the major post-translational modifications involved in the regulation of chromatin function. The PWWP domain is a member of the Royal superfamily that functions as a chromatin methylation reader by recognizing both DNA and histone methylated lysines. The PWWP domain three-dimensional structure is based on an N-terminal hydrophobic ß-barrel responsible for histone methyl-lysine binding, and a C-terminal α-helical domain. In this review, we set out to discuss the most recent literature on PWWP domains, focusing on their structural features and the mechanisms by which they specifically recognize DNA and histone methylated lysines at the level of the nucleosome.

Structure Elucidation, Antimicrobial and Cytotoxic Activities of a Halimane Isolated from Vellozia kolbekii Alves (Velloziaceae).

A new halimane diterpene was isolated from Vellozia kolbekii Alves (Velloziaceae) and identified as (5R,8R,9S,13R)-halim-1,10-ene-15,16-diol (1). It showed cytotoxicity against three human cancer cell lines, SF-295 (glioblastoma), MDA-MB-435 (melanoma), and HCT-8 (colon adenocarcinoma). In the mechanism of cytotoxic action, halimane 1 interferes in two major phases of the cell cycle: in S phase, in which DNA synthesis occurs and where it is very sensitive to damage, and G2M phase which is the phase of preparation for mitosis and mitosis itself, showing apoptosis-inducing properties. Antimicrobial activity towards Gram-positive and Gram-negative bacteria was studied and, against Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, a MIC value of 0.025 µM was observed for halimane 1, which is more active than the positive control chloramphenicol.

Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms.

Fermentation products can chaotropically disorder macromolecular systems and induce oxidative stress, thus inhibiting biofuel production. Recently, the chaotropic activities of ethanol, butanol and vanillin have been quantified (5.93, 37.4, 174kJ kg(-1)m(-1) respectively). Use of low temperatures and/or stabilizing (kosmotropic) substances, and other approaches, can reduce, neutralize or circumvent product-chaotropicity. However, there may be limits to the alcohol concentrations that cells can tolerate; e.g. for ethanol tolerance in the most robust Saccharomyces cerevisiae strains, these are close to both the solubility limit (<25%, w/v ethanol) and the water-activity limit of the most xerotolerant strains (0.880). Nevertheless, knowledge-based strategies to mitigate or neutralize chaotropicity could lead to major improvements in rates of product formation and yields, and also therefore in the economics of biofuel production.

Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation.

BACKGROUND: Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control. METHODS: To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry. RESULTS: Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg(2+) in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant. CONCLUSION: Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1. GENERAL SIGNIFICANCE: A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.

Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress

Propolis is a natural product widely used for humans. Due to its complex composition, a number of applications (antimicrobial, antiinflammatory, anesthetic, cytostatic and antioxidant) have been attributed to this substance. Using Saccharomyces cerevisiae as a eukaryotic model we investigated the mechanisms underlying the antioxidant effect of propolis from Guarapari against oxidative stress. Submitting a wild type (BY4741) and antioxidant deficient strains (ctt1∆, sod1∆, gsh1∆, gtt1∆ and gtt2∆) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1∆, acquired tolerance when previously treated with 25 µg/mL of alcoholic propolis extract. Such a treatment reduced the levels of ROS generation and of lipid peroxidation, after oxidative stress. The increase in Cu/Zn-Sod activity by propolis suggests that the protection might be acting synergistically with Cu/Zn-Sod.

The involvement of GSH in the activation of human Sod1 linked to FALS in chronologically aged yeast cells.

Mutations in Cu, Zn-superoxide dismutase (Sod1) have been associated with familial amyotrophic lateral sclerosis, an age-related disease. Because several studies suggest that oxidative stress plays a central role in neurodegeneration, we aimed to investigate the role of the antioxidant glutathione (GSH) in the activation of human A4V Sod1 during chronological aging. Transformation of wild-type and A4V hSod1 into a gsh null mutant and in its parental strain of Saccharomyces cerevisiae indicated that during aging, the number of viable cells was strongly influenced by A4V hSod1 mainly in cells lacking GSH. Activity of hSod1 increased in response to aging, although the increase observed in A4V hSod1 was almost 60% lower. Activation of hSod1 (A4V and WT) did not occur after aging, in cells lacking GSH, but could still be observed in the absence of Ccs1. Furthermore, no increase in activity could be seen in grx1 and grx2 null mutants, suggesting that glutathionylation is essential for hSod1 activation. The A4V mutation as well as the absence of GSH, reduced hSod1 activity, and increased oxidative damage after aging. In conclusion, our results point to a GSH requirement for hSod1 Ccs1-independent activation as well as for protection of hSod1 during the aging process.

In vitro and in vivo activity of a new unsymmetrical dinuclear copper complex containing a derivative ligand of 1,4,7-triazacyclononane: catalytic promiscuity of [Cu2(L)Cl3].

Here we present the synthesis of the dinuclear complex [Cu(II)2(L)Cl3] (1), where L is the deprotonated form of the 3-[(4,7-diisopropyl-1,4,7-triazacyclononan-1-yl)methyl]-2-hydroxy-5-methylbenzaldehyde ligand. The complex was characterized by single crystal X-ray diffraction, potentiometric titration, mass spectrometry, electrochemical and magnetic measurements, EPR, UV-Vis and IR. Complex 1 is able to increase the hydrolysis rate of the diester bis-(2,4-dinitrophenyl)phosphate (2,4-BDNPP) by a factor of 2700, and also to promote the plasmidial DNA cleavage at pH 6 and to inhibit the formazan chromophore formation in redox processes at pH 7. Using Saccharomyces cerevisiae (BY4741) as a eukaryotic cellular model, we observed that 1 presents reduced cytotoxicity. In addition, treatment of wild-type and mutant cells lacking Cu/Zn-superoxide dismutase (Sod1) and cytoplasmic catalase (Ctt1) with 1 promotes increased survival after H2O2 or menadione (O2˙(-) generator) stress, indicating that 1 might act as a Sod1 and Ctt1 mimetic. Considered together, these results support considerations regarding the dynamic behaviour of an unsymmetrical dinuclear copper(II) complex in solid state and in aqueous pH-dependent solution.

Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress.

Propolis is a natural product widely used for humans. Due to its complex composition, a number of applications (antimicrobial, antiinflammatory, anesthetic, cytostatic and antioxidant) have been attributed to this substance. Using Saccharomyces cerevisiae as a eukaryotic model we investigated the mechanisms underlying the antioxidant effect of propolis from Guarapari against oxidative stress. Submitting a wild type (BY4741) and antioxidant deficient strains (ctt1Δ, sod1Δ, gsh1Δ, gtt1Δ and gtt2Δ) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1Δ, acquired tolerance when previously treated with 25 µg/mL of alcoholic propolis extract. Such a treatment reduced the levels of ROS generation and of lipid peroxidation, after oxidative stress. The increase in Cu/Zn-Sod activity by propolis suggests that the protection might be acting synergistically with Cu/Zn-Sod.

Evaluation of heavy metal toxicity in eukaryotes using a simple functional assay.

Although carcinogenesis caused by metals has been intensively investigated, the mechanisms of action, especially at the molecular level, are still unclear. This work aimed to investigate Cd(2+), Cu(2+), Ni(2+), Cr(3+), and Zn(2+) mutagenicity and its relationship with oxidative stress. We have applied the Functional Assay for the Separation of Alleles in Yeast (FASAY) with only minor modifications to detect p53 defects caused by metals. In this method, human p53-coding gene (TP53) expressed in Saccharomyces cerevisiae activates transcription of the ADE2 reporter gene. Yeast cells, expressing p53, were exposed to increased concentrations of metals and, then, plated on media supplemented or not with adenine. Yeast colonies containing functional p53 grow independently of adenine supplementation and colonies containing nonfunctional p53 are dependent on this nutrient. Mutations in the TP53 are implicated in the pathogenesis of half of all human tumors. According to our results, Cd(2+) was found to be the most toxic metal and produced the highest oxidative damage to lipids and proteins. At low concentrations (40 µM), this metal decreased viability and completely inhibited cell growth, while higher concentrations were necessary to produce the same toxic effect by Cu(2+), Cr(3+), and Ni(2+). Zn(2+) showed no significant toxicity. Cd(2+) strongly induced damages and altered the function of p53, while Cu(2+), followed by Cr(3+), showed lower percentages of p53-mutant colonies. Our results point towards a relationship between the loss of functional p53 protein and oxidative stress, a mechanism that can be associated with tumor formation induced by heavy metals in mammalian cells. By this adaptation of FASAY developed by us it is possible to easily and rapidly detect mutations caused by metals or other stresses.