Francesco Marchetti

Candidate protein biodosimeters of human exposure to ionizing radiation

Purpose: To conduct a literature review of candidate protein biomarkers for individual radiation biodosimetry of exposure to ionizing radiation. Materials and methods: Reviewed ∼300 publications (1973 – April 2006) that reported protein effects in mammalian systems after either in vivo or in vitro radiation exposure. Results: We found 261 radiation-responsive proteins including 173 human proteins. Most of the studies used high doses of ionizing radiation (>4 Gy) and had no information on dose- or time-responses. The majority of the proteins showed increased amounts or changes in phosphorylation states within 24 h after exposure (range: 1.5- to 10-fold). Of the 47 proteins that are responsive at doses of 1 Gy and below, 6 showed phosphorylation changes at doses below 10 cGy. Proteins were assigned to 9 groups based on consistency of response across species, dose- and time-response information and known role in the radiation damage response. Conclusions: ATM (Ataxia telengiectasia mutated), H2AX (histone 2AX), CDKN1A (Cyclin-dependent kinase inhibitor 1A), and TP53 (tumor protein 53) are top candidate radiation protein biomarkers. Furthermore, we recommend a panel of protein biomarkers, each with different dose and time optima, to improve individual radiation biodosimetry for discriminating between low-, moderate-, and high-dose exposures. Our findings have applications for early triage and follow-up medical assessments.

The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men

BACKGROUNDnLittle is known about the effect of paternal nutrition on aneuploidy in sperm. We investigated the association of normal dietary and supplement intake of folate, zinc and antioxidants (vitamin C, vitamin E and beta-carotene) with the frequency of aneuploidy in human sperm.nnnMETHODSnSperm samples from 89 healthy, non-smoking men from a non-clinical setting were analysed for aneuploidy using fluorescent in situ hybridization with probes for chromosomes X, Y and 21. Daily total intake (diet and supplements) for zinc, folate, vitamin C, vitamin E and beta-carotene was derived from a food frequency questionnaire. Potential confounders were obtained from a self-administered questionnaire.nnnRESULTSnAfter adjusting for covariates, men with high folate intake (>75th percentile) had lower frequencies of sperm with disomies X, 21, sex nullisomy, and a lower aggregate measure of sperm aneuploidy (P <or= 0.04) compared with men with lower intake. In adjusted continuous analyses, total folate intake was inversely associated with aggregate sperm aneuploidy (-3.6% change/100 microg folate; 95% CI: -6.3, -0. 8) and results were similar for disomies X, 21 and sex nullisomy. No consistent associations were found between antioxidant or zinc intakes and sperm aneuploidy.nnnCONCLUSIONSnMen with high folate intake had lower overall frequencies of several types of aneuploid sperm.

Paternally Transmitted Chromosomal Aberrations in Mouse Zygotes Determine Their Embryonic Fate

Abstract The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.

DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

The postmeiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the 3 weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization (dbf)), late spermatids (14-8dbf) and sperm (7-1dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than 2 weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of postmeiotic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e., smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

Mechanisms and targets involved in maternal and paternal age effects on numerical aneuploidy

Trisomy in the human appears to be predominantly associated with maternal age. The maternal‐age effect, however, shows considerable variability across affected chromosomes. Chromosome‐specific variation has been reported in the shapes of the maternal‐age‐effect curves, including very small effects for the large chromosomes (groups A and B), linear increases (chromosome 16), and exponential increases (chromosome 21). There is also variation among chromosomes in whether the segregation errors occur predominantly at maternal meiosis I, meiosis II, and/or postfertilization mitotic divisions. There is also limited epidemiological evidence for a paternal‐age effect, which was recently supported by the findings of age‐related increases in sperm aneuploidy using fluorescence in situ hybridization methods. The paternal‐age effect is considerably smaller than the maternal and is more likely to involve meiotic II errors of the sex chromosomes, whereas the maternal‐age effect is more likely to arise from meiotic I errors producing autosomal trisomies. These and other differences suggest that constitutional aneuploidy arises by multiple mechanisms that may affect (1) the nature and timing of an initiating lesion affecting the oocyte or sperm; (2) the cellular physiology at the time of the nondisjunction event at meiosis I, II, or postfertilization; and (3) the selection against specific chromosomal aneuploidies during embryonic development. Multidisciplinary research is needed to understand the maternal‐ and paternal‐age effects on aneuploidy, to (1) identify and characterize the genes that control meiosis, recombination, and segregation; (2) identify the microenvironmental factors around the oocyte and male germ cells that are involved in the age effects; (3) develop a laboratory animal model for the age effects; (4) characterize the role of genetics, physiology, and environmental toxicology for the parental‐age effects; and (5) identify cohorts of men and women of differing ages who have been exposed to high doses of candidate aneugens and conduct epidemiological investigations of aneuploidies transmitted to their offspring.

Induction of chromosomal aberrations in mouse zygotes by acrylamide treatment of male germ cells and their correlation with dominant lethality and heritable translocations

The objectives of this research were: 1) to investigate the time course of the cytogenetic defects induced by acrylamide (AA) treatment (5 × 50 mg/kg) of male germ cells in first‐cleavage zygote metaphases using PAINT/DAPI analysis, and 2) to characterize the correlation between chromosomal aberrations at first cleavage, dominant lethality, and heritable translocations. PAINT/DAPI analysis employs multicolor fluorescence in situ hybridization painting plus DAPI staining to detect both stable and unstable chromosomal aberrations at first‐cleavage metaphase of the zygote. High levels of chromosomally defective zygotes were detected after mating at all postmeiotic stages (20–190‐fold, P < 0.001). Early spermatozoa (6.5 d post‐treatment) were the most sensitive, with 76% of the zygotes carrying cytogenetic defects. A significant 10‐fold increase was also detected 27.5 d post‐treatment, indicating that AA had a cytogenetic effect on meiotic stages. PAINT/DAPI analysis revealed that: 1) AA‐induced chromosomal breaks occurred at random, and 2) the frequencies of symmetrical and asymmetrical exchanges were similar at all mating days, except 9.5 d after AA treatment, where significantly (P < 0.02) more asymmetrical aberrations were found. Furthermore, the proportions of zygotes carrying unstable and stable chromosomal aberrations followed a similar post‐treatment time course as the proportions of dominant lethality among embryos and heritable translocations among offspring. These findings indicate that PAINT/DAPI analysis of zygotic metaphases is a promising method for detecting male germ cell mutagens capable of inducing chromosomal aberrations and for evaluating the associated risks for embryonic loss and balanced translocations at birth. Environ. Mol. Mutagen. 30:410–417, 1997

Micronutrients intake is associated with improved sperm DNA quality in older men

OBJECTIVEnTo investigate whether lifestyle factors such as increased dietary intake of micronutrients reduce the risks of sperm DNA damage, and whether older men benefit more than younger men.nnnDESIGNnCross-sectional study design with equalized assignments into age groups.nnnSETTINGnNational laboratory and university.nnnPATIENT(S)nNonclinical group of 22-80-year-old nonsmoking men (n = 80) who reported no fertility problems.nnnMAIN OUTCOME MEASURE(S)nSperm DNA damage measured by alkaline and neutral DNA electrophoresis (i.e., sperm Comet assay).nnnRESULT(S)nSociodemographics, occupational exposures, medical and reproductive histories, and lifestyle habits were determined by questionnaire. The average daily dietary and supplement intake of micronutrients (vitamin C, vitamin E, b-carotene, zinc, and folate) was determined using the 100-item Modified Block Food Frequency Questionnaire (FFQ). Men with the highest intake of vitamin C had approximately 16% less sperm DNA damage (alkaline sperm Comet) than men with the lowest intake, with similar findings for vitamin E, folate, and zinc (but not β-carotene). Older men (>44 years) with the highest vitamin C intake had approximately 20% less sperm DNA damage compared with older men with the lowest intake, with similar findings for vitamin E and zinc. The older men with the highest intake of these micronutrients showed levels of sperm damage that were similar to those of the younger men. However, younger men (<44 years) did not benefit from higher intakes of the micronutrients surveyed.nnnCONCLUSION(S)nMen with higher dietary and supplement intake of certain micronutrients may produce sperm with less DNA damage, especially among older men. This raises the broader question of how lifestyle factors, including higher intakes of antioxidants and micronutrients, might protect somatic as well as germ cells against age-associated genomic damage.

Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolour fluorescencein situ hybridization painting

We describe a fluorescencein situ hybridization (FISH) procedure for assessing zygotic risk of paternal exposure to endogenous or exogenous agents. The procedure employs multicolour FISH with chromosome-specific DNA painting probes plus DAPI staining for detecting both balanced and unbalanced chromosomal aberrations in mouse first-cleavage (1-Cl) zygote metaphases. Four composite probes specific for chromosomes 1, 2, 3 or X, each labelled with biotin, plus a composite probe specific for chromosome Y labelled with digoxigenin, were used. We applied this method to evaluate the effects of paternal exposure to acrylamide, a model germ cell clastogen. First-cleavage zygote metaphases, collected from untreated females mated to males whose sperm or late spermatids were treated with acrylamide, were scored for the induction of structural aberrations using bothchromosome painting (PAINT analysis) andDAPI analysis. Structural chromosomal aberrations were observed in the sperm-deried, but not in the eggderived, pronuclei. While 59.4% of the zygotes had structural aberrations by DAPI analysis, 94.1% of the same zygotes had structural aberrations by PAINT analysis (P<0.001), illustrating the increased sensitivity for detecting translocations and insertions obtained by adding chromosome painting. These findings show that FISH painting of mouse 1-Cl zygotes when used in conjunction with DAPI analysis is a powerful model for investigating the cytogenetic defects transmitted from father to offspring.

The expression of Troponin T1 gene is induced by ketamine in adult mouse brain.

The glutamatergic system has been implicated in neuropsychiatric disorders, such as schizophrenia, bipolar disorder and Alzheimers disease, which also have a high prevalence of metabolic syndrome. Treatment with ketamine, a non-competitive glutamate N-methyl-d-aspartic acid (NMDA) receptor antagonist, is known to have paradoxical effects of neuroprotection and neurotoxicity. We investigated gene expression in brain tissue of adult mice treated with ketamine to characterize the expression profiles and to identify the affected metabolic pathways. Adult male mice were treated by a single intraperitoneal (i.p.) injection of either s(+)ketamine (80 mg/kg) or distilled water (as the control). Fifty genes were differentially expressed in ketamine-treated mouse brains compared with control mice using oligonucleotide microarray analysis, and the expression of Troponin T1 (Tnnt1) gene was consistently elevated (2- to 4-fold) (p<0.001). Ketamine-induced Tnnt1 expression was confirmed and characterized using RNA in situ hybridization techniques in paraffin embedded brain tissue sections. Tnnt1 expression was induced in the granule layer of the hippocampus, amygdala, hypothalamus, Purkinje cells of cerebellum (p<0.0001), and cerebral cortex. Tnnt1 gene is known to interact directly with FoxO1, which is involved in multiple peripheral metabolic pathways and central energy homeostasis. Our findings suggest that the induction of Tnnt1 gene expression in adult mouse brains by ketamine may illustrate the genes involved in the metabolic syndromes observed in neuropsychiatric disorders.

Elemental composition of human semen is associated with motility and genomic sperm defects among older men

BACKGROUNDnOlder men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes.nnnMETHODSnWe employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65-80 years old) and 10 younger men (22-28 years old) who were concurrently assayed for sperm function and genomicly defective sperm.nnnRESULTSnThe older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01).nnnCONCLUSIONSnWe identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects.