FISH in analysis of gamma ray-induced micronuclei formation in barley

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity

Targeting the epigenome: effects of epigenetic treatment strategies on genomic stability in healthy human cells

Epigenetic treatment concepts have long been ascribed as being tumour-selective. Over the last decade, it has become evident that epigenetic mechanisms are essential for a wide range of intracellular functions in healthy cells as well. Evaluation of possible side-effects and their underlying mechanisms in healthy human cells is necessary in order to improve not only patient safety, but also to support future drug development. Since epigenetic regulation directly interacts with genomic and chromosomal packaging density, increasing genomic instability may be a result subsequent to drug-induced epigenetic modifications. This review highlights past and current research efforts on the influence of epigenetic modification on genomic stability in healthy human cells

DNMT3L Is a Regulator of X Chromosome Compaction and Post-Meiotic Gene Transcription

Previous studies on the epigenetic regulator DNA methyltransferase 3-Like (DNMT3L), have demonstrated it is an essential regulator of paternal imprinting and early male meiosis. Dnmt3L is also a paternal effect gene, i.e., wild type offspring of heterozygous mutant sires display abnormal phenotypes suggesting the inheritance of aberrant epigenetic marks on the paternal chromosomes. In order to reveal the mechanisms underlying these paternal effects, we have assessed X chromosome meiotic compaction, XY chromosome aneuploidy rates and global transcription in meiotic and haploid germ cells from male mice heterozygous for Dnmt3L. XY bodies from Dnmt3L heterozygous males were significantly longer than those from wild types, and were associated with a three-fold increase in XY bearing sperm. Loss of a Dnmt3L allele resulted in deregulated expression of a large number of both X-linked and autosomal genes within meiotic cells, but more prominently in haploid germ cells. Data demonstrate that similar to embryonic stem cells, DNMT3L is involved in an auto-regulatory loop in germ cells wherein the loss of a Dnmt3L allele resulted in increased transcription from the remaining wild type allele. In contrast, however, within round spermatids, this auto-regulatory loop incorporated the alternative non-coding alternative transcripts. Consistent with the mRNA data, we have localized DNMT3L within spermatids and sperm and shown that the loss of a Dnmt3L allele results in a decreased DNMT3L content within sperm. These data demonstrate previously unrecognised roles for DNMT3L in late meiosis and in the transcriptional regulation of meiotic and post-meiotic germ cells. These data provide a potential mechanism for some cases of human Klinefelter's and Turner's syndromes

Normal and Cut-Off Values of the Cytokinesis-Block Micronucleus Assay on Peripheral Blood Lymphocytes in the Croatian General Population

Mikronukleus (MN) test na limfocitima periferne krvi jedna je od najvažnijih metoda koje se primjenjuju u citogenetičkom nadzoru. Osnovni preduvjet za primjenu nekog testa u svrhu nadzora profesionalno izloženih populacija jest poznavanje normalnih vrijednosti promatranoga biološkog pokazatelja (biomarkera) u kontrolnoj populaciji. Baze podataka na razini opće populacije moraju se redovito obnavljati novim podacima. Cilj ovog istraživanja bio je utvrditi normalne i granične vrijednosti MN-testa na limfocitima periferne krvi 200 zdravih ispitanika obaju spolova iz opće populacije Republike Hrvatske te ispitati koji čimbenici pridonose spontanom nastanku MN. Na razini istražene populacije utvrđeno je prosječno (6,90±3,32) MN (medijan 7 MN), dok je raspon pojedinačnih vrijednosti iznosio 0 do 18 MN u 1000 binuklearnih stanica. Gornja granična vrijednost dobivena izračunavanjem 95. percentila za cjelokupnu promatranu populaciju iznosi 12,5 MN na 1000 limfocita. Utvrđeno je da na spontani nastanak MN utječu spol, dob i navika pušenja. Žene u prosjeku imaju više vrijednosti svih parametara MN-testa od muškaraca, a u njih je bio i naglašeniji porast vrijednosti citogenetičkog nalaza zbog navike pušenja. Kako su literaturni podaci o utjecaju pušenja cigareta na nastanak MN kontradiktorni, planiran je nastavak istraživanja radi razjašnjavanja utjecaja dnevno utrošenog broja cigareta i ukupnog trajanja pušačkog staža na vrijednosti parametara MN-testa. Usporedba rezultata s literaturnim podacima potvrdila je da su dobivene vrijednosti u skladu s vrijednostima MN-testa zabilježenim na općoj populaciji u drugim svjetskim laboratorijima. Normalne i granične vrijednosti MN-testa utvrđene u ovome istraživanju poslužit će kao osnova za usporedbu i tumačenje nalaza MN-testa u ispitanika izloženih populacija te daljnju nadogradnju laboratorijske baze podataka.The cytokinesis-block micronucleus (CBMN) assay on peripheral blood lymphocytes is one of the most important methods employed in cytogenetic biomonitoring. For the purposes of biological dosimetry, it is important to kno the spontaneous frequency of a biomarker and its normal values in general population. These values are used for population databases, which should be updated regularly. In this study, MN levels were investigated in cytokinesis-blocked lymphocytes of 200 healthy male and female blood donors selected at random from the general population of Croatia. The aim was to assess the variability and determine possible infl uences of external and/or internal factors on the background levels of MN and to establish the cut-off value for the CBMN assay. The background frequency of MN was (6.90±3.32) MN (median 7 MN) and the range was 0 to 18 MN per 1000 binuclear lymphocytes. The cut-off value, which corresponds to 95th percentile of the distribution of 200 individual values, was 12.5 MN. Spontaneous formation of MN was infl uenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking signifi cantly increased the values of all parameters evaluated by the CBMN assay. Since the existing literature data on smoking-related formation of MN are contradictory, we will continue these investigations to resolve how the number of cigarettes smoked per day and the duration of smoking in years infl uence the results of the CBMN assay. Our results are consistent with the background MN frequencies reported by other cytogenetic laboratories worldwide. Normal and cut-off values estimated in this study will be used to update the current general population data and as reference for occupationally or accidental exposure