Mariangela Prati

Effects of population density on seabass (Dicentrarchus labrax, L.) gene expression

Abstract The issue of animal welfare in aquaculture is growing of interest and there is an increasing consumer demand for documentation of safe and ethically defendable food production. In this context, we have looked for molecular markers among those genes whose expression results modified by the different farming conditions. We have compared gene expression of seabass farmed at different population densities by differential display. We have obtained six bands differentially expressed whose sequences we have deposited in the public databases; two of them resulted suppressed by high population density, while four were induced by the treatment. Population density has, therefore, an effect at gene level by repressing or enhancing the expression of different genes. These genes can be used as biomarkers to rapidly detect, by polymerase chain reaction (PCR), the occurred exposure of the fish to the stressor. We are certain that the new molecular techniques will find their place in the everyday management of fish farming; on the other hand, we are also aware that the scarcity of the genomic resources for some fish species, in spite of their economical interest, will retard the beneficial effects that modern biotechnology could bring to aquaculture industry. Therefore, an effort should be made to reduce, as far as it concerns genomics resources, the gap that separates farming species from “model organisms’’.

Ecotoxicological soil evaluation by FETAX

The frog embryo teratogenesis assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran Xenopus laevis. FETAX satisfies the requirements of low cost, reliability and reproducibility and, thanks to its three endpoints (i.e., mortality, teratogenicity and growth inhibition) can detect the xenobiotics that affect embryonic development. In this paper, we have used FETAX to evaluate samples of soils collected in an oil-contaminated area. Embryos were exposed directly to the soil to be tested. Particular attention was devoted to provide a statistical procedure for analysing mortality and malformation data as well as growth retardation.

Developmental toxicity, uptake and distribution of sodium chromate assayed by frog embryo teratogenesis assay-Xenopus (FETAX)

The embryotoxicity and teratogenicity of Cr(VI) on the survival and morphology of the anuran Xenopus laevis have been assessed by frog embryo teratogenesis assay-Xenopus (FETAX). The lethal median (LC(50)) and teratogenic median (TC(50)) concentration values of Cr(VI) were 890 microM and 260 microM, respectively. The calculated teratogenic index (TI) value was 3.42, suggesting that hexavalent chromium has a teratogenic potential. Malformations of embryos included lifting of the body, coiling of the tail and body oedema. Furthermore, the chromium salt caused significant growth retardation at 25 microM exposure concentrations. The use of radiolabelled (51)Cr(VI) allowed the determination of the time course uptake of Cr in Xenopus exposed to concentrations ranging from 0.025 to 500 microM. The evaluation of its distribution into the body (head-abdomen-tail) was evaluated at different exposure times. Chromium is taken up at 24 h by Xenopus embryos for all concentrations tested. At 48 h post fertilization (stage of larva) the amount of Cr accumulated by the two-day-old larva ranged from 0.42 to 580 pg mg(-1) wet weight at 0.025 and 500 microM respectively. These amounts were lower than those at 24 h (2.77 to 11016 pg mg(-1) wet weight embryo) reaching values of the same order of magnitude at 120 h (five-days-old larva). Since at 48 h Xenopus development leads to a swimming embryo, the observed uptake at 24 h could be the result of the binding of Cr to jelly coat compounds surrounding the embryo body as confirmed by gel filtration experiments on (51)Cr-jelly coat. The interaction of Cr with jelly coat is in agreement with the role of jelly coat in protecting the embryo against pathogen and chemical toxins to ensure fertilization. This work further supports the hypothesis that Cr contamination of surface waters could contribute to explain the reported worldwide depletion of frog population.

Biological effect of the Planktothrix sp. FP1 cyanobacterial extract.

Cyanobacteria are common and potentially harmful inhabitants of freshwater and marine environments worldwide. Some waterbloom-forming cyanobacteria are toxic and they may cause animal death and adversely affect human health. A filamentous freshwater cyanobacterium, Planktothrix sp. FP1, was found to be responsible for a toxic algal bloom in Lake Varese (Italy) during August of 1997. In the present study, the biological effects of the Planktothrix sp. FP1 cell extract on Xenopus embryos and on human Peripheral Blood Mononuclear Cells (PBMC) were investigated. FETAX (Frog Embryo Teratogenesis Assay-Xenopus) showed that the cyanobacterial extract had no teratogenic potential, though embryotoxicity was detected (LC(50) 2.944g/l wet weight). The same extract inhibited the proliferation of PBMC stimulated in vitro by phytohemagglutinin (PHA), and strongly interfered with the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma).

Selective Pressure on the Allantoicase Gene During Vertebrate Evolution

During vertebrate evolution, the uric acid degradation pathway has been modified and several enzymes have been lost. Consequently, the end product of purine catabolism varies from species to species. In the past few years, we have focused our attention on vertebrate allantoicase (an uricolytic pathway enzyme), whose activity is present in certain fish and amphibians only, but whose mRNA we detected also in mammals. As allantoicase activity disappeared in amniotes, we wonder why these sequences not only remain present in the mammalian genome, but are still transcribed. To elucidate this issue, we have cloned and analyzed comparable cDNA sequences of different organisms from ascidians to mammals. The analysis of the nonsynonymous–synonymous substitution rate that we performed on the coding region comprising exons 3 to 8 by means of maximum likelihood suggested that a certain amount of purifying selection is acting on the allantoicase sequences. Some implications of the preservation of an apparently unnecessary gene in higher vertebrates are discussed.

Property comparison of recombinant amphibian and mammalian allantoicases.

Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional allantoicase, therefore we expressed the Xenopus and mouse allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon

BACKGROUND Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON-induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250  µM) at different stages and examined after 48 hr of culture. The sensitive window for FON-induced abnormalities was during the hours E9 h8.00 PM-E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS FON was able to affect the expression of some genes in a stage-specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON-induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA-related signals.

Gene expression in nanotoxicology: A search for biomarkers of exposure to cobalt particles and ions

Despite the wide use of nanoscale materials in several industrial applications as well as in biology and medicine, very little research has been carried out on the potential toxicity of nanoparticles. We had previously obtained 10 differentially expressed mRNAs in BALB3T3 fibroblasts exposed to different forms of cobalt, i.e., microparticles, nanoparticles, and ions. Those genes represented candidate biomarkers for indicating specific cellular effects after cobalt nanoparticle exposure. In the present paper, we have further evaluated the expression of those genes by real-time RT-PCR after exposure to different forms of cobalt. Moreover, we also tested some genes associated with cobalt toxicity, such as VEGF, HIF-1α, and Bnip3. We identified biomarkers that are sensitive to cobalt ions that we think to be the reactive form. Our data, in fact, are consistent with the possibility that Co-nano, due to their large surface area, once inside the cell dissolve and act as ions.

Genomic organization and chromosome localization of the murine and human allantoicase gene.

Allantoicase is one of the enzymes involved in uricolysis. The enzymes of this catabolic pathway (i.e. allantoinase, allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. In mammals, as well as in birds and reptiles, the activity of allantoicase is absent; notwithstanding, we recently cloned human and mouse cDNA sequences with high similarity with previously characterized allantoicases. In the present paper, we report the genomic organization of the allantoicase gene in mouse and in man. Both genes are constituted by 11 exons that appear to be very conserved; introns are more variable in length while maintain the same phase but for intron 4. We have also detected a second transcript of the human allantoicase gene in which exon 1 is absent. Moreover, the mouse gene maps in chromosome 12 at 13.0 cM from the centromere.

FETAX, a versatile tool in toxicology, can be conveniently integrated with molecular biology techniques

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay for developmental toxicants that makes use of the embryos of the anuran Xenopus laevis. FETAX, thanks to its three endpoints (i.e., mortality, malformation, and growth inhibition), can also detect the xenobiotics that affect embryonic development, a weak link in the life cycle of an organism. The FETAX protocol, however, is amenable to modification in several ways and to being integrated with molecular biology techniques that considerably increase the capability of the test. As exposure to xenobiotics may alter gene expression and therefore mRNA and protein patterns, transcriptome and proteome modifications can be studied with the aim to obtain new insights on the mechanisms of embryotoxicity and teratogenesis or simply to obtain molecular markers of exposure useful in the early diagnosis of environmental stress.