Influence of polystyrene nanoparticles on the toxicity of tetrabromobisphenol A in human intestinal cell lines.

Research and regulatory efforts in toxicology are increasingly focused on the development of suitable non-animal methodologies for human health risk assessment. In this work we used human intestinal Caco-2 and HT29/MTX cell lines to address the potential risks of mixtures of the emerging contaminants tetrabromobisphenol A (TBBPA) and commercial polystyrene nanoparticles (PSNPs). We employed different in vitro settings to evaluate basal cytotoxicity through three complementary endpoints (metabolic activity, plasmatic, and lysosomal membrane integrity) and the induction of the oxidative stress and DNA damage responses with specific endpoints. Although no clear pattern was observed, our findings highlight the predominant impact of TBBPA in the combined exposures under subcytotoxic conditions and a differential behavior of the Caco-2 and HT29/MTX co-culture system. Distinctive outcomes detected with the mixture treatments include reactive oxygen species (ROS) increases, disturbances of mitochondrial inner membrane potential, generation of alkali-sensitive sites in DNA, as well as significant changes in the expression levels of relevant DNA and oxidative stress related genes.

Evaluating the Influence of Water Scarcity on the Host Response of Garlic to the Stem and Bulb Nematode Ditylenchus dipsaci.

Ditylenchus dipsaci is a plant-parasitic nematode with a great economic impact on bulbous crops, including garlic (Allium sativum L.), and is distributed worldwide, particularly in the Mediterranean region. Traditionally, garlic was a rainfed crop in Spain, but irrigated areas have increased during the last few decades. However, the expected climatic conditions, with longer and more intense droughts, will make it necessary to reduce the water supply to garlic crops. This poses the urgent need to select garlic cultivars more tolerant to water scarcity and that are also more resistant to plant pathogenic organisms. The aim of this work was to analyze the influence of water stress on the host response of garlic plants to D. dipsaci. The specific objectives were to evaluate the level of nematode infestation in plants from four garlic genotypes treated with a reduced irrigation regime and compare them with those of control plants not subjected to water stress. The observed results were correlated with changes in the bulb and root development, as well as in the physiological parameters (total chlorophyll concentration and proline accumulation). The effects were different depending on whether the plants were subjected to water stress before or after nematode inoculation, as well as whether the water stress was continuous or discontinuous. Garlic inter-cultivar variability also affected the obtained results.

Whole-Genome Sequence of Toxic Freshwater Cyanobacterium Chrysosporum ovalisporum Strain UAM-MAO.

Here, we report the complete nucleotide sequence of Chrysosporum ovalisporum UAM-MAO, a filamentous, cylindrospermopsin-producing cyanobacterium involved in bloom forming in freshwater systems worldwide. It was isolated from an artificial pond in Madrid, Spain. The genome sequence contains 336 contigs, consisting of 7,478,035 bp and 2,851 putative protein-coding genes.

Influence of Glycine and Arginine on Cylindrospermopsin Production and aoa Gene Expression in Aphanizomenon ovalisporum.

Arginine (Arg) and glycine (Gly) seem to be the only substrates accepted by the amidinotransferase that catalyze the first step of the synthesis pathway of the cyanotoxin cylindrospermopsin (CYN), leading to guanidinoacetate (GAA). Here, the effect of these amino acids on the production of CYN in cultures of the cylindrospermopsin-producing strain, Aphanizomenon ovalisporum UAM-MAO, has been studied. Arg clearly increased CYN content, the increment appearing triphasic along the culture. On the contrary, Gly caused a decrease of CYN, observable from the first day on. Interestingly, the transcript of the gene ntcA, key in nitrogen metabolism control, was also enhanced in the presence of Arg and/or Gly, the trend of the transcript oscillations being like that of aoa/cyr. The inhibitory effect of Gly in CYN production seems not to result from diminishing the activity of genes considered involved in CYN synthesis, since Gly, as Arg, enhance the transcription of genes aoaA-C and cyrJ. On the other hand, culture growth is affected by Arg and Gly in a similar way to CYN production, with Arg stimulating and Gly impairing it. Taken together, our data show that the influence of both Arg and Gly on CYN changes seems not to be due to a specific effect on the first step of CYN synthesis; it rather appears to be the result of changes in the physiological cell status.

First evidence of accumulation in cyanobacteria of guanidinoacetate, a precursor of the toxin cylindrospermopsin.

Guanidinoacetate (GAA) is one of the most extensively studied toxic guanidine compounds. Changes in GAA can affect the nervous system and induce hyperhomocysteinemia, representing a risk factor for cardiovascular diseases. In cyanobacteria, GAA is thought to be an intermediate in the synthesis of the toxin cylindrospermopsin (CYN), one of the most common known cyanotoxins that affects multiple organs and functions in animals and plants. In spite of the evidence supporting GAA toxicity and its role in CYN synthesis, no data have been reported on the accumulation of GAA in any cyanobacterium. We have analyzed and compared the content of GAA in cultures of diverse cyanobacteria types, both cylindrospermopsin producing (CYN(+)) and not producing (CYN(-)). The results obtained show that GAA accumulates in the majority of the strains tested, although the highest content was found in one of the CYN(+) strain, Aphanizomenon ovalisporum UAM-MAO. In this strain, both GAA and CYN can be located within and out the cells. In conclusion, GAA appears to be a general cyanobacterial metabolite that due to its proven toxic should be considered when studying and managing cyanobacteria toxicity.

Characterization of Aphanizomenon ovalisporum amidinotransferase involved in cylindrospermopsin synthesis.

An increasing abundance of Aphanizomenon ovalisporum in water bodies from diverse world regions has been reported in the last few years, with the majority of the isolated strains producing the toxin cylindrospermopsin (CYN), leading to a rise in ecological and health risks. The understanding of CYN synthesis is crucial in the control of CYN production. An amidinotransferase (AMDT) seems to be the first enzyme involved in the synthesis of CYN. In this study, we have cloned and overexpressed the aoaA gene from the constitutive CYN producer A. ovalisporum UAM-MAO. The recombinant purified AoaA was characterized, confirming that it is an l-arginine:glycine AMDT. It shows an optimal activity between 32 and 37°C, at pH from 8 to 9. The activity exhibits a mixed (ping-pong/sequential) kinetic mechanism, and is inhibited by the reaction product guanidine acetate (GAA) in a noncompetitive manner. Mg(2+) stimulates AoaA activity while Co(2+) and Mn(2+) inhibit it. AoaA conserves the critical residues of the catalytic site and substrate specificity of AMDTs, as the previously reported AMDT from Cylindrospermopsis raciborskii Cyr. Both proteins can be included in a new group of prokaryotic AMDTs involved in CYN production.

Structure and evolution of the Cinful retrotransposon family of maize.

A maize cDNA clone was isolated by virtue of its intense hybridization to total maize genomic DNA, indicating homology to highly repetitive sequences. Genomic homologues were identified and subcloned from an adh1-bearing maize yeast artificial chromosome (YAC). Sequencing revealed that the expressed sequence was part of a Ty3-gypsy-type retrotransposon. We discovered and sequenced two complete retrotransposons of this family, and named them Cinful elements because they are members of a family of maize retrotransposons including Zeon-1 and the first plant transposable element sequenced, the solo long terminal repeat (LTR) called Cin1. All are defective, as Cinful-1 and Cinful-2 elements lack gag and Zeon-1 lacks pol homology. Despite the apparent lack of an intact "autonomous" element, the Cinful family has expanded to a copy number of about 18 000, representing just under 9% of the maize genome. Both point mutations and major rearrangements, including possible gene acquisition, differentiate members of the Cinful family. Cinful family members were found to have an unusual feature that we also observed in two other Ty3-class retrotransposons of teosinte and tobacco: related tandem repeats that separate their internal domains with a gag- or pol-containing homology from a 3' segment of unknown function. The conserved and variable features identified provide insights into the origin, mutational history, and functional components of this major constituent of the maize genome.