High level of γH2AX phosphorylation in the cord-blood cells of large-for-gestational-age (LGA) newborns.

Newborns can experience adverse effects as a consequence of maternal or in utero exposure, altered growth of the fetus, or placental dysfunctions. Accurate characterization of gestational age allows monitoring of fetal growth, identification of deviations from the normal growth trajectory, and classification of babies as adapted, small, or large for gestational age (AGA, SGA, or LGA). The aim of this work was to evaluate nuclear and oxidative damage in umbilical cord-blood cells of newborns (sampled at birth), by applying the γH2AX assay and the fluorescent probe BODIPY581/591 C11, to detect DNA DSB and cell membrane oxidation, respectively. No statistically significant differences were observed in the proportion of oxidized cord-blood cells among the groups of newborns, although the LGA group showed the highest value. With regard to genome damage, elevated levels of γH2AX foci were detected in the cell nuclei from LGA newborns as compared to AGA or SGA babies, whose values did not differ from each other. Considering that the observed DNA damage, although still repairable, can represent a risk factor for obesity, metabolic diseases, or other pathologies, monitoring genome and cell integrity at birth can provide useful information for prevention of diseases later in life.

UCTD and SLE patients show increased levels of oxidative and DNA damage together with an altered kinetics of DSB repair.

Immunological tolerance is a critical feature of the immune system; its loss might lead to an abnormal response of lymphocytes causing autoimmune diseases. One of the most important groups belonging to autoimmune disorders is the connective tissue diseases (CTD). CTD are classified among systemic rheumatic diseases and include pathologies such as systemic lupus erythematosus (SLE), and undifferentiated CTD (UCTD). In this study, we evaluated oxidative and genome damage in peripheral blood lymphocytes from patients with SLE and UCTD, further classified on the basis of disease activity and the presence/absence of a serological profile. Oxidative damage was evaluated in cell membrane using the fluorescent fatty acid analogue BODIPY581/591 C11. The percentage of oxidised lymphocytes in both SLE and UCTD patients was higher than in the control group, and the oxidative stress correlated positively with both disease activity and autoantibody profile. The γH2AX focus assay was used to quantify the presence of spontaneous double strand breaks (DSBs), and to assess the abilities of DSBs repair system after T cells were treated with mitomycin C (MMC). Subjects with these autoimmune disorders showed a higher number of γH2AX foci than healthy controls, but no correlation with diseases activity and presence of serological profile was observed. In addition, patients displayed an altered response to MMC-induced DSBs, which led their peripheral cells to greatly increase apoptosis. Taken together our results confirmed an interplay among oxidative stress, DNA damage and impaired DNA repair, which are directly correlated to the aggressiveness and clinical progression of the diseases. We propose the evaluation of these molecular markers to better characterise SLE and UCTD, aiming to improve the treatment plan and the quality of the patients' life.

Assessing the cytotoxic/genotoxic activity and estrogenic/antiestrogenic potential of essential oils from seven aromatic plants.

Alternative therapies with new drugs are needed because the clinical efficacy of conventional chemotherapy is often reduced due to collateral effects. Many natural products of plant origin, including essential oils (EOs) have proved to be effective in prevention and therapy of several diseases such as bacterial infections, chronic diseases and cancer. In the present study, we investigated some biological activities of EOs extracted from seven plants: Rosmarinus officinalis, Salvia somalensis, Thymus vulgaris, Achillea millefolium, Helichrysum italicum, Pistacia lentiscus, Myrtus communis. In particular, we evaluated the cytotoxic and genotoxic activity using the cytochalasin B-blocked micronucleus assay (CBMN) in human peripheral lymphocytes, cytotoxicity in a human ovarian carcinoma cell line (A2780), and the estrogenic/antiestrogenic activity using a yeast strain expressing the human estrogen receptor alpha (ERα). Our results show that most EOs can have a strong cytotoxic and a slight/moderate genotoxic effect on human peripheral lymphocytes, and also a pronounced cytotoxic effect in A2780 cells. In addition, some EOs seem to have a marked antiestrogenic activity that could potentially perturb the estrogen-dependent tissues.

Could age modify the effect of genetic variants in IL6 and TNF-α genes in multiple myeloma?

Cytokines play a central role in multiple myeloma (MM) pathogenesis thus genetic variations within cytokines coding genes could influence MM susceptibility and therapy outcome. We investigated the impact of 8 SNPs in these genes in 202 MM cases and 235 controls also evaluating their impact on therapy outcome in a subset of 91 patients. Despite the overall negative findings, we found a significant age-modified effect of IL6 and TNF-α SNPs, on MM risk and therapy outcome, respectively. Therefore, this observation suggests that genetic variation in inflammation-related genes could be an important mediator of the complex interplay between ageing and cancer.

Tolerance of benzalkonium chloride, formalin, malachite green, and potassium permanganate in goldfish and zebrafish.

OBJECTIVE: To determine tolerance of goldfish and zebrafish to benzalkonium chloride, formalin, malachite green, and potassium permanganate. DESIGN: Tolerance study. ANIMALS: Adult goldfish (Carassius auratus) and zebrafish (Danio rerio). PROCEDURES: Groups of fish (n = 10/group) were exposed to each disinfectant at the therapeutic dosage; at 0.25, 0.5, 3, and 5 times the concentration used for the therapeutic dosage; and at the concentration used for the therapeutic dosage but for 3 or 5 times the recommended exposure time. RESULTS: In both species, exposure to malachite green at the therapeutic dosage resulted in toxic effects, including death. Exposure to formalin at the therapeutic dosage resulted in toxic effects in goldfish, but not zebrafish, and exposure to potassium permanganate resulted in toxic effects in zebrafish, but not goldfish. On the basis of the ratio of therapeutic dosage to median lethal dosage, in goldfish, formalin was more toxic than benzalkonium chloride, which was more toxic than malachite green, which was more toxic than potassium permanganate. In zebrafish, potassium permanganate was more toxic than formalin and benzalkonium chloride, which were approximately equally toxic and more toxic than malachite green. Extending treatment time increased the toxicity of potassium permanganate in zebrafish and the toxicity of formalin and malachite green in goldfish, but did not alter the toxicity of the other disinfectants. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was no consistency between zebrafish and goldfish in their tolerance to disinfectants, and that therapeutic dosages reported in the literature for these disinfectants were not always safe.

Presence and inducibility by beta-naphthoflavone of CYP1A1, CYP1B1 and phase II enzymes in Trematomus bernacchii, an Antarctic fish.

This study investigated some aspects of xenobiotic metabolism in the Nototheniidae Trematomus bernacchii, a key sentinel species for monitoring Antarctic ecosystems. After laboratory exposure to beta-naphthoflavone (betaNF), basal levels and time-course induction of CYP1A, CYP1B and CYP3A were measured as enzymatic activities, immunoreactive protein content and mRNA expression in liver, gills, intestine and heart. Additional analyses in the liver included enzymatic activities of testosterone hydroxylase, (omega)- and (omega-1)-lauric acid hydroxylase and some phase II enzymes related to the AhR battery genes, DT-diaphorase, glutathione S-transferases and UDP-glucuronyl transferases. Responsiveness of hepatic CYP1A1 after exposure to betaNF demonstrated an higher sensitivity of MEROD than EROD activity and long lasting expression of mRNA still induced after 20 days from the treatment. Testosterone metabolism, oxidation of lauric acid and activities of phase II enzymes were not affected by betaNF indicating that their modulation is not mediated by Ah receptor. Induction of CYP1A was more limited in gills and absent in intestine and heart. The first nucleotide sequence for CYP1B1 in an Antarctic fish has been obtained, revealing a homology of 89% and 72% respectively to CYP1B1 of plaice and CYP1B2 of carp. Constitutive expression of CYP1B1 was restricted to gills where it was also induced by betaNF. Obtained results represent an additional contribution to the ecotoxicological characterization of T. bernacchii and further support the use of biomarkers for early detection of chemical pollution in Antarctica.

Genotypic versus phenotypic identification of staphylococcal species of canine origin with special reference to Staphylococcus schleiferi subsp. coagulans.

A comparative study was performed to examine the respective accuracy of 16S rDNA sequencing and of the commercial biochemical assay ID32 STAPH (bioMérieux, Marcy l'Etoile, France) in the identification of 232 staphylococcal samples representing 20 species and subspecies isolated from 367 dogs. Notable differences in species distribution were observed by comparing genotypic and phenotypic data. Partial sequencing of 16S rDNA resulted in an unambiguous identification of 226 (97.4%) of the isolates, whereas the phenotypic approach resulted in a correct diagnosis of 162 (69.8%) of the isolates. Statistical agreement between genotypic and phenotypic identification of staphylococci was substantial (Kappa coefficient of 0.6-0.8) for Staphylococcus aureus, S. hominis, S. warneri, S. cohnii subsp. urealyticus, and S. simulans, and "almost perfect" (Kappa coefficient of 0.8-1) for S. intermedius, S. epidermidis, S. equorum, S. haemolyticus, S. sciuri, and S. kloosi. No agreement above that expected by chance (Kappa coefficient=0) was observed for S. schleiferi subsp. coagulans, which was either confounded with S. intermedius and S. capitis, or categorized as unacceptable by the biochemical assay. Given the growing importance of this pathogen in veterinary medicine and its frequent misidentification with related staphylococci, a PCR-RFLP approach producing a S. schleiferi-specific restriction profile was developed. This fast and reliable assay represents a valuable tool in assisting in the monitoring of this pathogen.

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis.

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.

Effects of 17beta-estradiol, 4-nonylphenol and PCB 126 on the estrogenic activity and phase 1 and 2 biotransformation enzymes in male sea bass (Dicentrarchus labrax).

The endocrine system of wildlife is exposed to a wide variety of natural and man-made chemicals which may lead to damage to the reproductive system and other adverse effects, including alteration of drug-metabolizing enzymes. In the present study, the effects of in vivo exposure to a natural (17beta-estradiol: E2) or a xenoestrogen (4-nonylphenol: NP) estrogen or an anti-estrogen (3,3',4,4',5-pentachlorobiphenyl: PCB 126) upon vitellogenin (VTG) synthesis and hepatic phase 1 and 2 enzymes have been investigated in adult male sea bass. By means of ELISA analysis with the use of polyclonal antibodies prepared against VTG purified from E2-treated sea bass, we assessed the time course and sensitivity of VTG induction in the plasma of sea bass treated with E2 at 0.1, 0.5, 2.5 and 5.0 mg/kg doses or NP at 5.0 or 50 mg/kg doses, respectively. Sea bass sensitivity to this induction was found to be similar to that of other fish species, but with a delay in maximal response. E2 treatment also caused a selective time- and dose-dependent inhibition of hepatic CYP1A-linked EROD and phase 2 glutathione S-transferase (GST) activities, without affecting the activity of CYP3A-linked 6beta-testosterone hydroxylase, (omega)- and (omega-1)-lauric acid hydroxylases or phase 2 DT-diaphorase. A similar selective inhibition on CYP1A was also observed in fish treated with 50 mg/kg NP. The results regarding CYP1A and CYP3A were also confirmed by Western blot analysis. When the sea bass were treated with either 10 or 100 microg/kg PCB 126, an AhR ligand not yet tested in vivo in fish to assess its anti-estrogenicity, a modest and selective induction of EROD and DT-diaphorase activities was observed. Interestingly, both these activities were recovered to their control levels in sea bass co-treated with 0.5 mg/kg E2 and 10 or 100 microg/kg PCB 126, probably through a cross-talk mechanism between the estrogen receptor and AhR or other transcription factors that regulate the expression of these enzymes. Furthermore, it was demonstrated that PCB 126 possesses a potent anti-estrogenic activity in the sea bass in vivo as it inhibited the E2-induced VTG synthesis with an IC50 of 28 microg/kg. The results of this study suggest that the exposure of fish to xenoestrogens or anti-estrogens may alter, in addition to various physiological processes, the expression of specific CYPs and phase 2 enzymes, thereby reducing the capability of their detoxication system.

Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR.

The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture.