Detection of DNA Adduct Formation in Rat Lungs by a Micro-HPLC/MS/MS Approach.

Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

BACKGROUND: In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. RESULTS: Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml-1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. CONCLUSIONS: We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.

Brazilian Ironstone Plant Communities as Reservoirs of Culturable Bacteria With Diverse Biotechnological Potential.

Extensive mineral extractivism in the Brazilian Iron Quadrangle (IQ) region has destroyed large areas of land, decimating plant species, and their associated microbiota. Very little is known about the microbiota of the region; hence, cultivable bacteria associated with plants of its soils were investigated for their biotechnological potential. Samples were collected from nine plant species and six soils, and 65 cultivable bacterial isolates were obtained. These represent predominantly gram-positive bacilli (70%) capable of producing amylases (55%), proteases (63%), cellulases (47%), indole acetic acid (IAA) (46%), siderophores (26%), and to solubilize phosphate (9%). In addition, 65% of these were resistant to ampicillin, 100% were sensitive to tetracycline, and 97% were tolerant to high arsenic concentrations. Three isolates were studied further: the isolate FOB3 (Rosenbergiella sp.) produced high concentrations of IAA in vitro in the absence of tryptophan - shown by the significant improvement in plant germination and growth rate where the isolate was present. For isolates C25 (Acinetobacter sp.) and FG3 (Serratia sp.), plasmids were purified and inserted into Escherichia coli cells where they modified the physiological profile of the transformed strains. The E. coli::pFG3B strain showed the highest capacity for biofilm production, as well as an increase in the replication rate, arsenic tolerance and catalase activity. Moreover, this strain increased DNA integrity in the presence of arsenic, compared to the wild-type strain. These results help to explain the importance of bacteria in maintaining plant survival in ferruginous, rocky soils, acting as plant growth promoters, and to highlight the biotechnological potential of these bacteria. IMPORTANCE  The Iron Quadrangle region is responsible for ∼60% of all Brazilian iron production and, at the same time, is responsible for housing a wide diversity of landscapes, and consequently, a series of endemic plant species and dozens of rare species - all of which have been poorly studied. Studies exploring the microbiota associated with these plant species are limited and in the face of the continuous pressure of extractive action, some species along with their microbiota are being decimated. To understand the potential of this microbiota, we discovered that cultivable bacterial isolates obtained from plants in the ferruginous rocky soil of the Iron Quadrangle region have diverse biotechnological potential, revealing a genetic ancestry still unknown.

DNA Adduct Formation in the Lungs and Brain of Rats Exposed to Low Concentrations of [13C2]-Acetaldehyde.

Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [13C2]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N2-propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [13C4]-1, N2-propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI+-HRMS3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

Intermittent fasting results in tissue-specific changes in bioenergetics and redox state.

Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart.

Quantification of DNA adducts in lungs, liver and brain of rats exposed to acetaldehyde.

Air pollution is a major risk for human health. Acetaldehyde is an environmental pollutant present in tobacco smoke, vehicle exhaust and several food products. Formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2'-deoxyguanosine in DNA to primarily form N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dGuo). The subsequent reaction of N(2)-ethylidene-dGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2´-deoxyguanosine (1,N(2)-propanodGuo). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of 1,N(2)-propanodGuo and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo) in tissues of rats exposed to 12 ppb, 33 ppb and 96 ppb acetaldehyde in atmospheric air for 50 days. A significant increase in the levels of 1,N(2)-propanodGuo was observed in lung tissues of rats exposed to 12 ppb (7.8/10(8) dGuo); 33 ppb (8.9/10(8) dGuo) and 96 ppb (11.6/10(8) dGuo) compared to controls (4.2/10(8) dGuo). For comparative purposes, the levels of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo), which is produced from a,b-unsaturated aldehydes formed during the lipid peroxidation process were also measured. Elevated levels of 1,N(2)-edGuo were observed only in lung tissues of animals exposed to 96 ppb acetaldehyde. 1,N(2)-propanodGuo also differed quantitatively in liver but not in brain. The monitoring of 1,N(2)-propanodGuo levels in tissues provides important information on acetaldehyde genotoxicity and may contribute to the elucidation of the mechanisms associated with acetaldehyde exposure and cancer risk. Supported byFAPESP:2011/10048-5, CAPES, INCT Redoxoma:573530/2008-4,NAP Redoxoma: 2011.1.9352.1.8, CEPID Redoxoma:2013/07937-8.

Elevated α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine levels in urinary samples from individuals exposed to urban air pollution.

Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.