Structural Chromosome Damage Research Articles

Safety assessment of a solid lipid curcumin particle preparation: In vitro and in vivo genotoxicity studies.

Curcumin, one of the three principal curcuminoids found within turmeric rhizomes, has long been associated with numerous physiologically beneficial effects; however, its efficacy is limited by its inherently low bioavailability. Several novel formulations of curcumin extracts have been prepared in recent years to increase the systemic availability of curcumin; Longvida®, a solid lipid curcumin particle preparation, is one such formulation that has shown enhanced bioavailability compared with standard curcuminoid extracts. As part of a safety assessment of Longvida® for use as a food ingredient, a bacterial reverse mutation test (OECD TG 471) and mammalian cell erythrocyte micronucleus test (OECD TG 474) were conducted to assess its genotoxic potential. In the bacterial reverse mutation test, Longvida® did not induce base-pair or frame-shift mutations at the histidine locus in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537, in the presence or absence of exogenous metabolic activation. Additionally, two gavage doses (24 h apart) of Longvida® to Swiss albino mice at 500, 1000, or 2000-mg/kg body weight/day did not cause structural or numerical chromosomal damage in somatic cells in the mammalian erythrocyte micronucleus test. It was therefore concluded that Longvida® is non-genotoxic.

Vorinostat is genotoxic and epigenotoxic in the mouse bone marrow cells at the human equivalent doses

Vorinostat was approved as the first histone deacetylase inhibitor for the management of cutaneous T cell lymphoma. However, it’s in vivo genetic and epigenetic effects on non-cancerous cells remain poorly understood. As genetic and epigenetic changes play a critical role in the pathogenesis of carcinogenesis, we investigated whether vorinostat induces genetic and epigenetic alterations in mouse bone marrow cells. Bone marrow cells were isolated 24 h following the last oral administration of vorinostat at the doses of 25, 50, or 100 mg/kg/day for five days (approximately equal to the recommended human doses). The cells were then used to assess clastogenicity and aneugenicity by the micronucleus test complemented by fluorescence in situ hybridization assay; DNA strand breaks, oxidative DNA strand breaks, and DNA methylation by the modified comet assay; apoptosis by annexin V/PI staining analysis and the occurrence of the hypodiploid DNA content; and DNA damage/repair gene expression by polymerase chain reaction (PCR) Array. The expression of the mRNA transcripts were also confirmed by real-time PCR and western blot analysis. Vorinostat caused structural chromosomal damage, numerical chromosomal abnormalities, DNA strand breaks, oxidative DNA strand breaks, DNA hypomethylation, and programed cell death in a dose-dependent manner. Furthermore, the expression of numerous genes implicated in DNA damage/repair were altered after vorinostat treatment. Accordingly, the genetic/epigenetic mechanism(s) of action of vorinostat may play a role in its carcinogenicity and support the continued study and development of new compounds with lower toxicity.

Safety of use of Monk fruit extract as a food additive in different food categories.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of Monk fruit extract proposed for use as a new food additive in different food categories. Monk fruit extracts are prepared by water extraction of the fruits of Siraitia grosvenorii. Cucurbitane glycosides, mogrosides, are the main components of the S. grosvenorii fruit and mogroside V is the main mogroside in the Monk fruit extract. Mogroside V is absorbed to some extent and is systemically bioavailable. Monk fruit extract containing 25% and 55% mogroside V were negative in the bacterial reverse mutation assay and did not induce structural and/or numerical chromosomal damage. However, the Panel noted that the in vitro toxicity studies including study with metabolic activation were not sufficiently informative to evaluate the genotoxic potential of the metabolites generated after microbial metabolism, including the aglycone. The effects on the testis observed in a 90‐day study with monk fruit extract‐52% mogroside V cannot be dismissed and the adversity of these effects cannot be ruled out. No effects on parental, reproductive or development toxicity were observed in a reproductive and developmental screening study in rats. For male animals, the time of exposure did not cover the full length of spermatogenesis and, therefore, a longer term study at higher doses would be needed to clarify the effects on testes observed in the 90‐day study. No maternal and developmental toxicity was observed. Considering the systemic availability of mogroside V, the effects observed in the rat subchronic study and following the principles of EFSA Guidance on food additives evaluation, data from chronic/carcinogenicity toxicity testing would have been warranted. Exposure to mogroside V was calculated based on the proposed use levels. The Panel concluded that toxicity database on Monk fruit extract is insufficient to conclude on the safety of the use of Monk fruit extract as a food additive.

Genetic and epigenetic alterations induced by the small-molecule panobinostat: A mechanistic study at the chromosome and gene levels

Increasing evidence supports the role of genetic and epigenetic alterations in a wide variety of human diseases, including cancer. Assessment of these alterations is hence essential for estimating the hazardous effects of human exposure to medications. Panobinostat received US Food and Drug Administration’s approval in 2015 for treatment of certain tumors and its usefulness as part of a strategy to treat other diseases, such as human immunodeficiency virus infection, is currently investigated. Nevertheless, no data on in vivo genotoxical and epigenotoxical effects of panobinostat are available. The aim of the current study was to assess the genotoxical and epigenotoxical properties of panobinostat in murine bone marrow cells. Molecular mechanisms underlying these alterations were also evaluated. We show that mice treated with panobinostat doses recommended for human developed numerical chromosomal abnormalities, structural chromosomal damage, oxidative DNA damage, and DNA hypomethylation. These effects were dose-dependent. Further, panobinostat altered the expression of 23 genes implicated in DNA damage, as determined by RT² Profiler polymerase chain reaction (PCR) array, and confirmed by quantitative real-time PCR and western blotting. Collectively, these findings indicate that panobinostat exposure induces aneugenicity, clastogenicity, oxidative DNA damage, DNA hypomethylation, and down-regulation of repair gene expression, which may be responsible for panobinostat-induced genotoxical and epigenotoxical effects. Considering the potential toxicity of panobinostat, the medicinal use of panobinostat must be weighed against the risk of tumorigenesis and the demonstrated toxicity profile of panobinostat may support further development of chemotherapeutic treatments with reduced toxicity. Diminishing the metabolic liabilities associated with panobinostat exposure, and simultaneous use of panobinostat with DNA repair enhancers, are examples of strategies for drug design to reduce panobinostat carcinogenicity.

A 90-day toxicity and genotoxicity study with high-purity phenylcapsaicin

To evaluate the safety of the synthetic capsaicin analogue phenylcapsaicin (PheCap; 7-phenylhept-6-yne-acid-hydroxy-3-mathoxylbenzylamide, CAS no 848127-67-3), a 90-day repeated dose oral gavage of 0, 30, 100 or 250 mg/kg body weight (bw)/day toxicity study with a 28-day recovery period was conducted using Wistar rats. Examinations of clinical signs, body and organ weight, haematology, urinalysis, clinical chemistry, food consumption and macroscopic, as well as histopathological tissue examinations were carried out for signs of toxicity. Degenerative, but reversible changes in the liver at 250 mg/kg bw/day, and local irritating effects in the stomach at 100 and 250 mg/kg bw/day were found. These findings were associated with test item-related clinical symptoms, that is, diarrhoea, salivation and moving of bedding material. PheCap did neither cause gene mutations by base pair changes or frame shifts in the genome of the tester stains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 or TA 102 nor induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, it can be concluded that PheCap is not genotoxic. The No Observed Adverse Effect Level (NOAEL) of PheCap for systemic toxicity is considered to be at 100 mg/kg bw/day which is based on degenerative changes in the liver. Due to irritating effects in the stomach, the NOAEL for local effects was established at 30 mg/kg bw/day.

Marker chromosomes can arise from chromothripsis and predict adverse prognosis in acute myeloid leukemia

AbstractMetaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non–core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

Marker Chromosomes Can Arise from Chromothripsis and Predict Adverse Prognosis in Acute Myeloid Leukemia

Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event.Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome.Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p<.001 each). MC were associated with a poorer prognosis compared to other non-CBF aberrant karyotypes as well as with lower remission rates (CR+CRi; 36.0% vs. 55.8% in AML96 ≤60 years, p=0.01; 14.3% vs. 44.1% in AML2003, p<0.001), inferior event-free survival (2.24 vs. 6.54 months, p<0.001; 3.45 vs. 8.03 months, p<0.001) and overall survival (5.72 vs. 11.87 months, p<0.001; 8.68 vs. 20.78, p=0.01). In multivariate analysis with co-variables age, prior MDS, therapy-related AML and adverse-risk cytogenetics according to MRC criteria, MC independently predicted poor prognosis in AML96 ≤60 years but not in AML2003 with its higher allogeneic transplantation rate.As detected by array-CGH, in about one third of MC karyotypes (18/49, 36.7%, including 3 cases with 8 or 9 copy number switches) MC had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of complex aberrant karyotypes without MC (1/34) (p<0.001). Chromothripsis in MC karyotypes typically involved one single chromosome (n=11), with two or three chromosomes affected in 5 and 2 patients, respectively. There was no predilection for a particular chromosome. MC karyotypes positive for chromothripsis were characterized by a particularly high degree of karyotype complexity as compared to those that were negative for chromothripsis (complex aberrant 100% vs. 64.5% p<0.01; abnl(17p) 50.0% vs. 16.1%, p=0.01). In 12/18 (66.7%) cases, at least one of the chromothriptic chromosomes was reported as loss in the karyotype formula, suggesting that the grouping of a chromothriptic chromosome as a marker is paralleled by a putative loss of the affected chromosome. The chromothripsis positive MC karyotype subgroup had a particularly dismal prognosis with a combined CR+CRi rate of 2/16 vs. 10/31 (p=0.14). It also displayed inferior event-free and overall survival, though statistical significance was not reached for either endpoint, likely due to the already poor prognosis of the entire MC positive group.Conclusion: This is the first study showing that MC are a frequent finding predominantly in adverse-risk AML and associated with particularly poor prognosis. Our data provide evidence that a substantial portion of MC arise from chromothripsis. DisclosuresThiede:AgenDix: Employment, Other: Ownership.

CYTOGENETIC EFFECTS OF LOW NEUTRON DOSES IN MAMMALIAN CELLS

Актуальность. В связи с облучением больших групп людей в малых дозах при лечебных и диагностических процедурах и потребностью прогнозирования канцерогенного риска облучения представляется актуальным изучение закономерностей биологических эффектов малых доз радиации. Относительно мало исследованы особенности биологического действия нейтронного излучения и его эффекты на цитогенетическом уровне в области малых доз, при этом они могут быть важны при проведении нейтронной терапии и разработке нормативных документов по радиационной защите на ускорителях ионов, в том числе терапевтического назначения.Цель – изучение закономерностей образования структурных повреждений хромосом в клетках млекопитающих при действии быстрых нейтронов в области доз до 1 Гр.Материал и методы. Цитогенетическую эффективность нейтронов с энергией 14,5 МэВ исследовали на лимфоцитах крови человека и клетках китайского хомячка СНО-К1. Диапазон доз нейтронов составлял 0,1–1,2 Гр, мощностей доз – 0,3–60 мГр/мин, частота следования импульсов – 50, 5, и 0,017 Гц. Приготовление препаратов хромосом проводили по общепринятым стандартным методикам. При анализе учитывали весь спектр аберраций хромосом. Результаты. В исследованном диапазоне доз нейтронов для обеих клеточных культур наблюдалась одинаковая закономерность: резкий подъем выхода аберраций хромосом при дозах 0,12–0,15 Гр, замедление роста числа аберраций с увеличением дозы нейтронов до 0,3–0,5 Гр, далее стандартная дозовая зависимость. Указанная закономерность выявлена как по суммарной частоте аберраций хромосом, так и по частоте дицентриков (лимфоциты) и парных фрагментов (СНО-К1). Заключение. Полученные результаты свидетельствуют о проявлении феномена гиперчувствительности и индуцированной радиорезистентности в клетках лимфоцитов человека и китайского хомячка при действии нейтронов с энергией 14,5 МэВ.

Chromosomal instability, aneuploidy, and cancer.

The link between aneuploidy and cancer has been recognized overa century ago (1). Abnormalities in chromosome copy numbersarise from persistent errors in chromosome segregation dur-ing cell division, a process known as chromosomal instability(CIN) (2). CIN is a principal contributor to genetic heterogene-ity in cancer (3) and is an important determinant of clinicalprognosis and therapeutic resistance (4, 5). Over the past twodecades, our understanding of the mechanisms that lead to CINas well as our appreciation of its consequences on cellular via-bility and tumor evolution have grown considerably (4, 6). Sohas our recognition of the multitude of questions that remainunanswered.The papers in this Research Topic broadly address recentadvances in our understanding of CIN in cancer. They also illus-trate the diverse experimental approaches and model organismsused in studying genomic instability. This topic is divided intotwo major categories: the first five papers address the genesis ofCIN in cancer by summarizing the cell biological mechanisms thatunderlie chromosome missegregation. They also venture into thepoorly understood area of the genetic basis of CIN,while develop-ing an experimental model system amenable to high-throughputgenetic analysis. The remaining papers address the consequencesof imbalance in chromosome number on the cellular fitness andadaptation.Multiple mechanisms have been shown to lead to CIN – inits numerical and structural forms. They include defects in thespindle assembly checkpoint (7), sister chromatid cohesion (8),the regulation of microtubule attachments to chromosomes atkinetochores (9, 10), centrosome duplication (11, 12), telom-ere maintenance (13), and pre-mitotic replication stress (14).Herein, German Pihan (15) reviews the regulation of the cen-trosome duplication cycle and how it is intricately synchronizedwith the cell division cycle. The complexity of this regulatorynetwork might explain pervasiveness of centrosome dysfunctionin human tumors, but it also provides multiple attractive phar-macological targets that have the potential to induce mitoticcatastrophe. Yokoyama and Gruss (16) further discuss how chro-mosomes take on part of the responsibility to ensure the fidelity oftheir own segregation. Chromatin-associated factors – beyond theRan GTPase – have now been shown necessary for a properlyfunctioning mitotic spindle. Interestingly, many of these fac-tors localize to the Nuclear Pore Complex (NPC), highlightingan incipient spatiotemporal relationship between the interphasenuclear structure and the mitotic spindle (17). Thus the processof faithful chromosome segregation starts well before the onset ofmitosis.While many of the cellular events that underlie CIN have nowbeen uncovered, the genetic basis of chromosome missegregationand aneuploidy remains elusive. A growing number of geneticperturbations have been shown capable of inducing CIN in oth-erwise normal mammalian cell lines (6). Yet, it remains unknownwhether these experimental conditions mimic naturally occur-ring genetic events that lead to CIN during tumor progression.Further complicating this matter is the self-propagating natureof CIN (18), which can mask initial instigating genetic triggers.Herein,Rao andYamada (19) review the linear progression modelof colon carcinogenesis from adenoma to carcinoma. They dis-cuss how many of the sequential genetic events that occur dur-ing carcinogenesis have the potential to compromise the fidelityof chromosome segregation. More generally, Orr and Compton(20) discuss the intimate relationship governing CIN and knownoncogenic pathways. Given what we now know, they argue thatalmost every major oncogenic pathway can be implicated in somemanner in the genesis of CIN. Yet this relationship is almostcertainly bidirectional as chromosome missegregation has beenshown to generate downstream structural chromosomal damage,whichcaninturnindependentlyactivateoncogenicpathways(21–23). This complex relationship highlights the need for appropriategenetic models to better understand CIN. To this end, Salemiet al. (24) develop a chromosome segregation error correctionassay, using the

Phenolic antioxidant 2,6-di-tert-butyl-p-cresol (vitamin E synthetic analogue) does not inhibit 1,1’-dimetyl-4,4’-bipyridium dichloride (paraquat)-induced structural chromosomal damage in cultured leukocytes of the dark-spotted-frog Pelophylax (Rana) nigr

Pro-oxidative effect of phenolic antioxidant (vitamin E) in combination with the initiators on human low-density lipoprotein is known. Recently I reported that oxidative stress induced by vitamin E in combination with the herbicide paraquat enhances structural chromosomal damage in cultured anuran leukocytes. In the present study, the phenolic antioxidant vitamin E-synthetic-analogue 2,6-di-tert-butyl-p-cresol (BHT) in combination with paraquat was found to enhance structural chromosomal damage in cultured Pelophylax (Rana) nigromaculatus leukocytes more than paraquat only and paraquat plus nicotinamido adenine dinucleotido phosphate served as positive control, although BHT only had no effect on induction of structural chromosomal damage. Paraquat plus BHT-enhanced structural chromosomal damage was inhibited by combination of the superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin and the hydrogen peroxide scavenger catalase. In test based on reduction of paraquat cation, BHT was found to reduce paraquat cation chemically to paraquat monocation radical. These results suggest that BHT functions in chemically donating electron to paraquat and thereby induces an acute accumulation of reactive oxygen species, resulting in increase in chromosomal damage.

Persistent genomic instability in peripheral blood lymphocytes from hodgkin lymphoma survivors

Advances in cancer treatment have led to an increase in patient survival. However, exposure to genotoxic chemotherapeutic agents and ionizing radiation may induce persistent genetic damage in cancer survivors. In this study, we detected genomic instability in chromosomes of peripheral blood lymphocytes from Hodgkin lymphoma patients, 2-17 years after MOPP (nitrogen mustard, Oncovin, procarbazine, and prednisone) chemotherapy with or without radiotherapy. Samples were obtained from 11 healthy individuals, 5 pretreatment patients, and 20 posttreatment patients. Cytogenetic analysis with GTG banding was performed on 1,000 lymphocyte metaphases per donor to identify genomic instability, including numerical and structural chromosomal aberrations, at a resolution of 10 Mb across the entire genome. Our results showed that anticancer treatment did not induce significant differences in the frequency of aneuploidy among the three study groups. However, 1 of the 11 healthy individuals, and 13 of the 20 posttreatment patients had a high frequency of chromosomal breaks and gross chromosomal rearrangements. The types of aberrations observed were random and complex, consistent with persistent genomic instability that was induced by cancer treatment. Clonal expansion of cells with chromosomal lesions was observed in one posttreatment patient only. These findings show that anticancer treatments induce persistent genomic instability, but not aneuploidy. Chemotherapy may affect genes with a role in DNA damage surveillance or repair, which in turn allows the accumulation of nontargeted structural chromosomal damage in future generations of cells. This genomic instability may facilitate the development of second malignancies in Hodgkin lymphoma survivors.

Dl-α-tocopherol enhances the herbicide 1,1’-dimetyl-4,4’-bipyridium dichloride (paraquat, PQ) genotoxicity in cultured anuran leukocytes

This cytogenetic and pharmacological study attempts to clarify genotoxicity-enhancement-effect of dl-α-tocopherol (one form of vitamin E) in combination with the herbicide 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat, PQ) on cultured anuran leukocytes using the superoxide dismutase-mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (Mn(III)TMpyP), the hydrogen peroxide-scavenger catalase and the electron donor nicotinamide adenine dinucleotido phosphate (NADPH). PQ only was found to induce structural chromosomal damage in cultured anuran leukocytes in a dose-dependent manner. PQ plus NADPH, which served as positive control, enhanced the genotoxic effect of PQ. Dl-α-tocopherol only did not induce any structural chromosomal damage in the leukocytes. PQ plus dl-α-tocopherol, however, enhanced the genotoxic effect of PQ. PQ plus Mn(III)TMpyP, PQ plus catalase and PQ plus Mn(III)TMpyP plus catalse suppressed the genotoxic effect of PQ. Furthermore, PQ plus dl-α-tocopherol-enhanced chromosomal damage was also inhibited by Mn(III)TMpyP plus catalase. These results suggest that dl-α-tocopherol in combination with PQ functions as an electron donor to PQ.

An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay

The aims of this study were to establish whether individual differences exist in the frequency and size of vacuoles found in human sperm and to ascertain whether such vacuoles are involved in causing DNA damage. Morphologically normal sperm were obtained from 15 IVF and 2 ICSI patients and 3 fertile donors. (i) Sperm heads were analyzed for the presence of vacuoles under a 1000× differential interference contrast microscope. (ii) In three patients and two donor samples, structural chromosomal damage was evaluated in normal sperm containing large vacuoles and selected at 1000× magnification for injection into mouse oocytes. (iii) In 10 patients and two donor samples, confocal laser microscopy detected DNA damage in sperm-exhibiting large vacuoles and stained with an in situ cell death detection kit. (i) Vacuoles were observed in almost all normal sperm from patient and donor ejaculates and were mainly located at the tip or middle area of the sperm heads. However, average incidence of normal sperm exhibiting large vacuoles was 4.6 and 4.2% in the patient and donor groups, respectively. (ii) Sperm chromosome assays did not reveal any differences in the incidence of structural chromosome aberrations between sperm exhibiting large vacuoles and those without them (9.1 versus 4.1%). (iii) No significant difference in frequency of TUNEL-positive cells was found between normal sperm with large vacuoles and those without them in the samples examined. Among 227 sperm exhibiting large vacuoles, only 7 cells were TUNEL positive. The results showed that large vacuoles were not responsible for DNA damage, suggesting that intra-cytoplasmic injection of morphologically selected sperm may not be required for patients who produce high-quality semen.

Aneugenic effect of ionizing radiation in mammalian and human somatic cells

Aneuploidy is among the most serious impairments of hereditary material in somatic and germline cells of living organisms. Chromosome loss or the appearance of an extra homolog in the chromosome set can result in either cell death or the development of various neoplasms with high probability of malignancy. It was traditionally believed that ionizing radiation produces primarily a clastogenic effect. However, there is apparently an aneugenic component of radiation, with mechanism different from that of structural chromosome damage. The present review focuses on the evidence for the existence of the aneugenic effect of ionizing radiation in mammalian and human somatic cells.

Relationship between micronuclei formation and p53 induction

Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei ( p = 0.0001, r 2 = 0.9372), indicating that the level of p53 is associated with chromosomal damage.

In vivochromosome damaging effects of an inosine monophosphate dehydrogenase inhibitor: Ribavirin in mice

OBJECTIVE: To investigate the <i>in vivo </i>mutagenic effects of ribavirin in mice.<br>METHODS: Mice were injected (i.p.) 20, 100, or 200 mg/kg ribavirin (single exposure) for bone marrow micronucleus, peripheral blood micronucleus and bone marrow chromosome aberration tests. Five treatments of 200 mg/kg ribavirin was given (i.p.) for sperm morphology test. The tests were performed as per the standard procedures. <br>RESULTS: Ribavirin induced significant number of micronucleated polychromatic erythrocytes (MNPCEs) at 24, 48 and 72 h following the exposure with more effects at 24 h (p<0.05-0.001). Micronucleated PCEs were more at 48 h in lower dose-levels and at 72 h in highest dose-level in the peripheral blood (p<0.05-0.001). Ribavirin induced structural chromosomal damage hence producing the fragments for the micronucleus formation. Ribavirin decreased the PCE%, P/N ratio and the mitotic index indicating that it prevents cell division in mouse bone marrow. Ribavirin also decreased the testis weight and induced the formation of abnormal sperms. <br>CONCLUSION: Ribavirin is a potent mutagen and cytotoxic agent in mice <i>in vivo</i>. Further, it also induces point mutations in germ cells yielding abnormal sperms. The genotoxic effects of ribavirin are not exerted in a dose-dependent pattern in mouse.

Clastogenicity of atrazine assessed with the Allium cepa test.

Atrazine is classified as a restricted use pesticide and it is currently included in an international revision program for re-evaluating the human and ecological (non-human populations) health risks associated with its release into the environment. The present study was undertaken to add new data on the genotoxic potential of atrazine using the Allium cepa chromosome aberration test. The test concentrations were based on the Maximum Contaminant Levels in water intended for human consumption set by European and US regulations. Atrazine produced a concentration-related increase in the number of total somatic chromosome aberrations, although this increase was statistically significant (p<0.05) only at the highest test concentration (5 microg/L). Analysis of the categories of structural chromosome damage indicated that breaks were the predominant lesion induced; the percent of cells per bulb with breaks also increased in a concentration-related manner, and the increase was statistically significant at the two highest test concentrations (1 and 5 microg/L) (p<0.05). The Allium cepa plant assay detected the clastogenicity of atrazine at concentrations that are likely to be encountered in water, a common site of atrazine contamination.

Detection by dual color fluorescence in situ hybridization of in vivo chromosome damage in rat hepatocyte nuclei.

Our previous study using a multicolor fluorescence in situ hybridization (FISH) technique revealed that region-specific DNA probes for rat chromosome 1 enabled the detection of structural chromosome damage in rat interphase nuclei from peripheral blood and bone marrow cells. In the present study, this FISH technique was modified for application to a non-hematopoietic organ (liver) and the usefulness of the system was tested using diethylnitrosamine (DEN) as a model hepatocarcinogen. Male Sprague-Dawley rats were orally treated once with DEN at 200 mg/kg. Their livers were removed at 4, 7 or 14 days after treatment and homogenized with a tissue grinder to isolate hepatocyte nuclei. The nucleus suspension was fixed in methanol:acetic acid and air dried. Dual color FISH with two probes, one labeled with tetramethylrhodamine and one with digoxigenin, demonstrated that the maximum increase in the frequency of nuclei with spatially abnormal signals was observed 7 days after treatment. A dose-response relationship for induction of abnormal nuclei was observed. This improved dual color FISH system is potentially valuable for assessing in vivo clastogenicity in all rat organs.

Cytogenetic alterations in lymphocytes of Alzheimer's disease and Parkinson's disease patients.

We investigated the presence of cytogenetic alterations in peripheral blood lymphocytes of Alzheimer's disease (AD) and Parkinson's disease (PD) patients. Detection of spontaneous structural and/or numerical chromosome damage has been assessed by micronucleus (MN) assay coupled with fluorescence in situ hybridization (FISH). The cytogenetic investigation was performed on 22 AD patients, 18 PD patients, and 20 controls. The spontaneous frequencies of micronuclei (MN) in human lymphocytes of both AD and PD patients were significantly higher than in controls. The majority of MN was composed of whole chromosomes in AD patients, while a prevalence of MN arising from chromosome breakage was observed in PD patients. Different molecular mechanisms underlie cytogenetic alterations observed in peripheral lymphocytes of AD and PD patients.

The relationship between radiation-induced G(1)arrest and chromosome aberrations in Li-Fraumeni fibroblasts with or without germline TP53 mutations.

We previously showed that cultured fibroblasts from patients with the cancer-prone Li-Fraumeni (LF) syndrome, having heterozygous germline TP53 mutations, sustain less ionizing radiation-induced permanent G1 arrest than normal fibroblasts. In contrast, fibroblast strains from LF patients without TP53 mutations showed normal G1 arrest. We have now investigated the relationship between the extent of G1 arrest and the level of structural chromosome damage (mainly dicentrics, rings and acentric fragments) in cells at their first mitosis after G1 irradiation, in 9 LF strains with TP53 mutations, 6 without TP53 mutations and 7 normal strains. Average levels of damage in the mutant strains were 50% higher than in normals, whereas in non-mutant LF strains they were 100% higher. DNA double strand breaks (dsb) are known to act as a signal for p53-dependent G1 arrest and to be the lesions from which chromosome aberrations arise. These results suggest that a minimal level of dsb is required before the signal for arrest is activated and that p53-defective cells have a higher signal threshold than p53-proficient cells. Dsb that do not cause G1 blockage can progress to mitosis and appear as simple deletions or interact to form exchange aberrations. The elevated levels in the non-mutant strains may arise from defects in the extent or accuracy of dsb repair. In LF cells with or without TP53 mutations, the reduced capacity to eliminate or repair chromosomal damage of the type induced by ionising radiation, may contribute to cancer predisposition in this syndrome. © 2001 Cancer Research Campaign http://www.bjcancer.com