A pharmacogenetic-driven approach for controlled ovarian hyperstimulation by FSH treatment.

AIM: The aim of this study was to develop a pharmacogenetic- (PGx) driven approach for a controlled ovarian hyperstimulation (COH) treatment protocol used for in vitro fertilization procedures. The enrolled patients were genotyped for a single nucleotide polymorphism (SNP) N680S, within the FSHR. METHODS: Seventy-eight women, who had previously received at least two COH cycles without positive fertilization with FSH and AMH values <10 mUI/mL and >0.3 ng/mL respectively were enrolled. They were genotyped for N680S and then categorized in high (HR), intermediate (IR), and poor responders (PR). Each subgroup received a tailored FSH treatment of 100, 225, and 400 UI/mL, respectively. The response was evaluated considering differences with previous COH cycle in terms of number of follicles (FR), oocytes (OR), and embryos produced (EMB). RESULTS: With regards to the endpoint considered comparing the non-PGx with the PGx approach, for what regards the FR a statistically significant increase of their numbers was observed with the PGx-tailored approach (HR P<0.0001; IR P=0.00892; PR P=0.0032). Similar statistical significant results were also achieved for OR but only for HR (P<0.0001) and IR (P=0.00169). Last but not least for the EMB (HR P<0.001; IR P=0.00670 and PR P<0.0001) all the different genotype considered achieved a statistical significance. CONCLUSION: This study, although with a limited number of enrolled patients, showed that a FSH treatment with a PGx-driven approach might have the potential to improve COH clinical outcome.

Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro.

Owing to the increasing development of nanotechnology, there is a need to assess how engineered nanomaterials can interact with living cells. The main purpose of the present study was to assess whether metal cobalt nanoparticles (CoNP 100-500 nm) are genotoxic compared to cobalt ions (Co(2+)). Uptake experiments were carried out by incubating peripheral blood leukocytes (PBLs) with (57)Co(2+) (added to stable Co(2+) 10(-2) M to obtain concentrations in the range of 10(-5) to 10(-4) M) or with (60)CoNP for 24 and 48 h. Whereas intracellular Co(2+) showed slight or no variations over the baseline levels, CoNP were taken up efficiently leading to intracellular CoNP concentrations of 485 +/- 106.1 and 970 +/- 99 fg per cell after 24 and 48 h, respectively. The genotoxicity end points considered in this study were the frequency of binucleated micronucleated (BNMN) cells and the percentage of tail DNA (% Tail DNA) fragmentation by means of the comet assay. Genotoxic effects were evaluated by incubating PBLs of three healthy donors with subtoxic concentrations (10(-5) to 8 x 10(-5)M) of Co(2+) in the form of cobalt chloride, CoNP and 'washed' CoNP, the latter to exclude any interference by Co(2+). On a group basis, Co(2+) induced a clear trend in the increase of the BNMN frequency, whereas CoNP showed only minor changes. Moreover, we observed a high variability among donors in the induction of micronuclei. The comet assay showed a statistically significant dose-related increase in % Tail DNA for CoNP (P < 0,001), whereas Co(2+) did not induce significant changes over control values. These findings suggest that nanosized Co can be internalized by human leukocytes and can interact with DNA leading to the observed genotoxic effects, which are, however, modulated both by donor's characteristics and/or by Co(2+) release.

Genotoxicity induced by arsenic compounds in peripheral human lymphocytes analysed by cytokinesis-block micronucleus assay.

This work focuses on the analysis of genotoxic effects on human peripheral lymphocytes exposed in vitro to different arsenic (As) compounds by means of the cytokinesis-block micronucleus assay. The study was carried out by challenging peripheral human lymphocytes with six As compounds in trivalent or pentavalent forms such as arsenite (As(III)) and arsenate (As(V)) and organoarsenic species such as monomethylarsonous acid (MMAs(III)), monomethylarsonic acid (MMAs(V)), dimethylarsinic acid (DMAs(V)) and trymethylarsine oxide (TMAO(V)). For As(III) and As(V) at concentrations of 4 and 32 microM, respectively, an increase of micronuclei (MN) frequency was found. MMAs(III) and MMAs(V) induced a statistically significant increase of MN frequency at the dose of 2 and 500 microM, respectively. For DMAs(V), no significant increase of MN was observed, although a decrease of the nuclear division index (NDI) was evident, indicating a cytotoxic effect. The genotoxic mechanism of action of MMAs(III) was further evaluated by means of fluorescence in situ hybridization analysis. Due to a higher percentage of centromere-positive MN, MMAs(III) showed a clear aneuploidogenic property. Finally, for TMAO(V) no genotoxicity was observed up to 1 mM. These results show how speciation is important in determining the genotoxic and cytotoxic effects of As compounds in human peripheral lymphocytes and support the emerging hypothesis that the induction of aneuploidy could be a mechanism by which As exerts its carcinogenic properties.

An in vitro study on the free radical scavenging capacity of ergothioneine: comparison with reduced glutathione, uric acid and trolox.

BACKGROUND: Treatment of oxidative stress-related pathologies is a possible therapeutical strategy for the future. Natural product with antioxidant properties could trigger this goal. The aim of this in vitro study was to assess the antioxidant activity of the natural product ergothioneine (EGT), a compound of plant origin, which is assimilated and conserved by mammals in erythrocytes, kidney, seminal fluid and liver. METHODS: We measured the antioxidant activity of EGT as its ability to antagonize the oxidation of alpha-keto-gamma-methiolbutyric acid (KMBA) by hydroxyl radical, peroxyl radicals and peroxynitrite. The results are expressed as total oxyradical scavenging capacity (TOSC) units. Glutathione (GSH), uric acid and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), the water-soluble analog of vitamin E, were used as the reference antioxidants. RESULTS: EGT was the most active scavenger of free radicals as compared to classic antioxidants as GSH, uric acid and trolox. In particular, the highest antioxidant capacity exhibited by EGT vs. peroxyl radicals (5.53 +/- 1.27 units) resulted 25% higher than the value obtained with the reference antioxidant trolox (4.4 +/- 0.6 units, P < 0.01). The scavenging capacity of EGT towards hydroxyl radicals (0.34 +/- 0.09 units) was 60% higher, as compared to uric acid (0.21 +/- 0.04 units, P < 0.001), which represent the reference antioxidant vs. hydroxyl radicals. Finally, EGT showed the highest antioxidant activity also towards peroxynitrite (5.2 +/- 1.0 units), with a scavenging capacity 10% higher than that of uric acid (4.7 +/- 0.9 units, P < 0.05). CONCLUSIONS: This study showed that EGT has potent intrinsic anti-hydroxyl, anti-peroxyl and anti-peroxynitrite radicals antioxidant activity, as compared to classic molecules with antioxidant capacity as GSH, trolox and uric acid. This appears of interest, given the increasing use of non-vitamins cocktails for therapeutical approaches to many oxidative-induced pathologies.

Relationship between genotoxicity biomarkers in somatic and germ cells: findings from a biomonitoring study.

A biomonitoring study to evaluate chromosome and DNA damage respectively in somatic and germ cells of a group of male workers exposed to styrene by using biomarkers of genotoxicity was carried out. Styrene-exposed workers from three different areas of Tuscany and healthy subjects, of comparable mean age, sex and lifestyle characteristics, as a control group not exposed to chemicals, have been enrolled. In addition to previous reports [L. Migliore, A. Naccarati, A. Zanello, R. Scarpato, L. Bramanti and M. Mariani (2002) Hum. Reprod., 17, 2912-2918; L. Migliore, A. Naccarati, F. Coppedè et al. (2006) Pharmacogenet. Genomics, 16, 87-99] we present now data on a cross-sectional investigation involving a homogeneous group of subjects for which data on both somatic and germ cells have been obtained from individuals (42 exposed and 25 controls). Somatic cell genotoxicity was assessed by analysing the frequency of micronucleated binucleated cells (MNBN) in blood lymphocytes. The micronucleus assay was coupled with centromeric fluorescence in situ hybridization (FISH) analysis. Primary DNA damage in germ cells was evaluated by alkaline single-cell gel electrophoresis (Comet assay) and the percentage of the tail DNA (%TD) was used as parameter of Comet evaluation. Moreover, to investigate the frequencies of aneuploidy and diploidy in sperm, we performed multicolour FISH, using DNA probes specific for the centromeric regions of sex chromosomes and chromosome 2, in decondensed sperm nuclei of samples with normal semen parameters in a subgroup of individuals. Mandelic and phenylglyoxylic acids (MAPGA) in end of shift samples were determined as biomarkers of internal dose. MAPGA excretion was consistent with an exposure to styrene above the threshold limit value-time weighted average concentration of 20 p.p.m. Styrene workers showed significantly higher frequency of MNBN as compared to controls (13.8 +/- 5.2 versus 6.2 +/- 5.1; P < 0.001), due to higher proportions of both micronuclei (MN) arising from chromosomal breakage (C-MN) and harbouring whole chromosomes (C+MN). DNA damage in sperm cells was also higher among styrene-exposed, the %TD being 11.02 +/- 2.99 versus 7.42 +/- 2.30 in controls (P < 0.001). The incidence of aneuploidy and diploidy for the tested chromosomes in sperm did not show a statistically significant difference between workers and controls. However, a positive correlation was found between genotoxic damage detected in somatic and in germ cells, even after removing the effect of age (r = 0.475; P < 0.001). Although cytogenetic biomarkers detected both in somatic and germ cells were interrelated, no relationships were apparent with exposure parameters. Styrene exposure may increase the likelihood of both chromosome and DNA damage in somatic and germ cells, thus supporting the hypothesis of an interference on reproductive capacity among exposed workers. This is the first time that a field study shows a correlation between two biomarkers of genotoxicity evaluated at the same time in somatic and germ cells.

Susceptibility to chromosome malsegregation in lymphocytes of women who had a Down syndrome child in young age.

Recent findings seem to converge towards a unified hypothesis trying to relate Down's syndrome (DS), trisomy 21 and Alzheimer's disease (AD). The majority of DS individuals develop neuropathological characteristics of AD by the age of 40. Previous cytogenetic studies performed by us showed an increased frequency of aneuploidy in peripheral lymphocytes and fibroblasts of AD patients and a preferential occurrence of chromosome 21 in malsegregation events. An increased frequency of AD among young mothers of individuals with DS (MDS) is reported. This study investigates the cytogenetic characteristics and the predisposition to chromosome malsegregation of peripheral blood lymphocytes in a group of women (n = 35) who had a Down syndrome child in young age (<35 years) and in a control group (n = 30). We applied the micronucleus assay and the dual-color FISH in order to assess the susceptibility to malsegregation events. The results indicate a higher frequency of binucleated micronucleated cells in MDS in respect to the control group (16.1+/-9.1 per thousand versus 8.7+/-5.4 per thousand). Moreover, our data reveal that peripheral lymphocytes of MDS are more prone to chromosome non-disjunction with both chromosomes, 13 and 21, equally involved.

Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients.

It is well established that oxidative stress plays a key role in the degenerative neuronal death and progression of Alzheimer's disease (AD), although it is not clear if it is the primary triggering event in the pathogenesis of this disorder. Mild cognitive impairment (MCI) is a clinical condition between normal aging and AD, characterized by a memory deficit without loss of general cognitive and functional abilities. We performed this study by a comet assay analysis to evaluate the level of primary and oxidative DNA damage in two groups of MCI and AD patients, compared to healthy controls. Data showed a significantly higher level of primary DNA damage in leukocytes of AD and also of MCI patients compared to control individuals (average: 2.09+/-0.79 and 2.47+/-1.01, respectively for AD and MCI, versus 1.04+/-0.31 in controls). Moreover, the amount of oxidised DNA bases (both purines and pyrimidines) was significatively higher in the two groups of patients (AD and MCI) compared to controls. Our results give a further indication that oxidative stress, at least at the DNA level, is an earlier event in the pathogenesis of AD.

Searching for the role and the most suitable biomarkers of oxidative stress in Alzheimer's disease and in other neurodegenerative diseases.

The contribution of oxidative stress to neurodegeneration is not peculiar of a specific neurodegenerative disease, oxidative stress has been found implicated in a number of neurodegenerative disorders among which Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS). Even increasing are studies dealing with the search for peripheral biomarkers of oxidative stress in biological fluids or even in peripheral tissues themselves such as fibroblasts or blood cells. The application of the modified version of the comet assay for the detection of oxidised purines and pyrimidines in peripheral blood leukocytes results particularly useful if the study requires repeated blood drawn from the same individual, for instance if a clinical trial is performed with a preventive therapy. Likely damage occurs to every category of biological macromolecules and we consider, in the context of neurodegenerative diseases, particularly critical the proteic level. The identification of subjects at risk to develop AD or with pre-pathogenic conditions, the possibility to use "a battery of assays" for the detection of oxidative damage at peripheral level, together with recent advances in brain imaging, will allow to better address studies aimed not only to therapeutic purposes but also mainly to primary prevention.