cytogenetic and germ cell effects of phosphine inhalation by rodents: II. subacute exposures to rats and mice

Phosphine (PH₃) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH₃, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH₃ for 6 hr/day for 9 days over an 11 -day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH₃ up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11 -day period as measured by several cytogenetic assays. To evaluate the effects of PH₃ on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH₃ for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH₃ by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm. © 1994 Wiley-Liss, Inc.     

Assessment of 1,2-dimethylhydrazine in bone marrow micronucleus assay: variations in protocol and response.

The effect of variations in experimental protocol on the assessment of the genotoxicity of 1,2-dimethylhydrazine (DMH) in the bone marrow micronucleus assay was determined. The incidence of micronuclei (MN) in the bone marrow of CBA mice treated with DMH (either intraperitoneally (i.p.) or orally) was found to be significantly greater than that observed in C57B1/6J mice using the same dose and dosing regimen. With i.p. injection, DMH, at doses of 20 and 50 mg/kg, was found to be positive in the bone marrow MN test in CBA mice only. In C57B1/6J mice, DMH (i.p.) was found to be positive at only the 50 mg/kg dose. With oral administration, DMH was positive in the MN test only at the 50 mg/kg dose and only in CBA mice. No significant difference in the percentage of MN was observed when 300, 500, or 1,000 polychromatic erythrocytes (PCEs) were scored following a single treatment of DMH. Cyclophosphamide (CY) was found to induce a dose-dependent increase in the percentage of MN observed in the bone marrow of C57B1/6J mice. DMH tested positive in the colon nuclear aberration (NA) assay in both strains of mice using both i.p. and oral routes of administration, although C57B1/6J mice were found to be more sensitive than CBA mice. No significant difference was observed regarding the percentage of NAs observed in the colon between mice injected i.p. or orally gavaged.     

Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells

The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN −) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. We also applied the assay to exfoliated bladder cells of 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 μg As/L) and 18 matched controls (average level, 16 μg As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN −) and those containing whole chromosomes (MN+) both increased (1.65-fold, P = 0.07, and 1.37-fold, P = 0.15, respectively), suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN-frequency increased 2.06-fold (P = 0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN − and MN+ were positively associated with urinary arsenic and its metabolites. However, the association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can now be determined in epithelial tissues. © 1996 Wiley-Liss, Inc.     

Hypermethylation of CpG islands is associated with increasing chromosomal damage in chinese lead‐exposed workers

Lead is a widely existing environmental pollutant with potential carcinogenicity. To investigate the association of blood lead level (B‐Pb) with potential chromosomal damage and cancer, we analyzed micronucleus (MN) frequency of peripheral blood lymphocytes (PBLs) and the methylation status of six human tumor suppressor genes (TSGs) post lead exposure. In the study, 147 lead‐exposed workers were divided into two groups according to their B‐Pb P₅₀ value, with other 50 lead‐unexposed workers as a control group. The cytokinesis‐blocked micronucleus (CBMN) assay was performed to detect chromosomal damage of PBLs of both lead‐exposed and ‐unexposed workers. The methylation‐specific polymerase chain reaction (MSP‐PCR) was further used to examine the methylation status of six TSGs (GSTP1, hMLH1, MGMT, p14, p15, and p16). Results showed that MN frequencies of high B‐Pb workers 8.1 ± 3.1‰ and low B‐Pb workers 5.7 ± 2.3‰ were significantly higher than that of control group 2.8 ± 1.9‰ (P < 0.01), while the MN frequency of high B‐Pb workers was also higher than that of the low B‐Pb workers (P < 0.01). The MN frequency in PBLs of lead‐exposed group with the methylated TSGs was significantly higher than that in PBLs with the unmethylated TSGs (P < 0.05). Notably, the CpG island methylator phenotype (CIMP) correlated with chromosome damage (P < 0.05). Additionally, workers with high B‐Pb had higher chromosome damage than those with low B‐Pb (P < 0.05). Taken altogether, the results suggest that lead‐exposed workers with CIMP positive and high B‐Pb have a higher risk of being vulnerable to tumorigenesis. Environ. Mol. Mutagen. 59:549–556, 2018. © 2018 Wiley Periodicals, Inc.     

Evaluation of a suitable DNA damage biomarker for human biomonitoring of exposed workers

The objective of this study was to identify a sensitive and noninvasive biomarker of early genotoxic effects, for health risk assessment of workers exposed to mixtures of low doses of xenobiotics. We studied 30 workers exposed to antineoplastic drugs, 57 workers exposed to different mixtures of polycyclic aromatic hydrocarbons (PAHs) (41 airport workers and 16 paving workers) and 76 controls. Comet and micronucleus (MN) tests were performed on lymphocytes and exfoliated buccal cells. The MN assay on lymphocytes did not show significant differences between exposed and controls, while the MN assay on exfoliated buccal cells showed higher values in workers exposed to antineoplastics as compared with controls (0.85 vs. 0.48, P = 0.042). The comet assay on lymphocytes showed a higher comet percentage value (18.11 vs. 11.24 in controls, P = 0.001) and mean tail moment (TM) value (21.84 vs. 16.72 in controls, P = 0.003) in individuals exposed to PAHs as compared with controls; no significant differences were found in exposed to antineoplastics. The comet assay on exfoliated buccal cells did not show significant differences between exposed and control groups for comet percentages, whereas the TM value was higher in workers exposed to PAHs (55.1 vs. 32.31 for controls, P < 0.001). These results show that exfoliated buccal cells, obtained by a noninvasive procedure, represent robust target cells to assess the occupational exposure to inhalable mixture of chemicals at low doses. The comet assay seems to be suitable to promptly evaluate the genotoxic effects of PAHs mixtures that also contain volatile substances. The MN test is suitable to evaluate the effects of antineoplastics. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Micronucleus induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-irradiation and cyclophosphamide

A cytokinesis-block micronucleus (MN) method for the simultaneous but separate measurement of chromosome damage in erythroid and myeloid bone marrow cells is described. MN induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-ray or cyclophosphamide (CP) was investigated. Immediately after whole body irradiation with acute doses of either 0, 1, 2 or 4 Gy x-rays, or 2 hr after treatment with either 0, 12.5, 25, or 50 mg CP/kg body weight, bone marrow cells were collected and then cultured in medium supplemented with 3.0 μ/ml cytochalasin B for 24 hr. The binucleated cells were scored in erythroid, myeloid, lymphoid and other cells. The myeloid/erythroid (M/E) ratio was decreased by x-irradiation or CP treatment in a dose-dependent manner. The dividing index (DI; binucleated cells/binucleated + mononucleated cells; %) was decreased in both eryth-roid and myeloid cells in the same manner. Dose-dependent increases in MN frequency were observed following x-irradiation in both erythroid and myeloid cells. A similar dose-dependent MN induction was observed with CP. The MN frequency in myeloid cells was much greater than in erythroid cells (about 4-fold following 4 Gy exposure, and more than 10-fold after 50 mg/kg CP). Lymphoid and other cells were not suitable for scoring DI and MN frequency because of insufficient numbers of binucleated cells. These results suggest that micronuclei can be identified in both myeloid and erythroid cells and that myeloid cells are more susceptible to x-ray or CP-induced chromosomal damage than erythroid cells as expressed by MN induction. © 1994 Wiley-Liss, Inc.     

Evaluation of the mutagenicity of the anti-inflammatory drug salicylazosulfapyridine (SASP)

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.     

Genotoxicity of hydrochlorothiazide in cultured human lymphocytes. I. Evaluation of chromosome delay and chromosome breakage

Hypertension is often treated with diuretics, like hydrochlorothiazide (HCTZ). Previous results on the in vitro genotoxicity of HCTZ are equivocal. In the present study, we have evaluated the genotoxicity of HCTZ in cultured human lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. In addition, micronucleus (MN) induction was analyzed by Fluorescence In Situ Hybridization (FISH) with an alpha-satellite DNA centromeric probe to distinguish between clastogenic and aneugenic effects. Lymphocyte cultures from 32 healthy adults were exposed to 5 and 40 microg/ml HCTZ. Age, gender, and smoking were evaluated as factors affecting the MN analysis. We found that HCTZ increased MN frequencies. FISH analysis revealed that HCTZ exerts its genotoxicity more strongly at the 40 microg/ml concentration, and principally through chromosome delay (aneugenicity). Multiregression analysis of our results confirmed the known effect of age and gender on MN induction in human lymphocytes. Smoking was also a confounding factor for MN induction, especially for centromere-negative MN frequencies. Under the experimental conditions used, only age had a clear positive effect on the response of lymphocytes to HCTZ. These data indicate that HCTZ produces micronuclei in cultured human lymphocytes by a mechanism that involves chromosome delay and to a lesser extent through chromosome breakage.     

Micronuclei assay and FISH analysis in human lymphocytes treated with six metal salts

The capability of some metal compounds for inducing micronuclei (MN) in human lymphocytes was studied. In this investigation, Al (III), Cd (II), Hg (II), Sb (V), Te (VI), and Tl (I) salts were considered. The FISH (fluorescence in situ hybridization) technique with a centromeric probe was coupled with the MN assay in binucleated cells in order to detect both centromere-positive MN (C+ MN) due to malsegregation phenomena and centromere-negative MN (C- MN) due to chromosome breakage. The blood of two young nonsmoking male donors was employed for all experiments. In both donors, all the tested metal compounds, with the exception of Tl(2)SO(4), showed a statistically significant increase of MN compared to controls, at least at one dose. FISH analysis revealed an increase in the fraction of C+ MN for Al, Cd, and Hg compounds, and of C- MN for the Sb salt; however, this was not a statistically significant increase. A different efficiency was observed for the different metal compounds, in particular, KSbO(3) and CH(3)HgCl, which were highly genotoxic, whereas the others showed minimal effects.     

Detection of chromosome malsegregation to the daughter nuclei in cytokinesis-blocked transgenic mouse splenocytes

Most of the recently developed tests for detecting aneugenic activity of chemicals are based on the induction of micronuclei (MN) in cytokinesis-blocked (CB) binucleated cells. In such a test, aneugens can be discriminated from clastogens by checking for the presence of centromeres in the MN, indicating the loss of whole chromosomes. Tracing particular chromosomes in interphase nuclei using fluorescencein situ hybridization (FISH) with chromosome-specific DNA probes is another method used for detecting numerical chromosome aberrations. Here, we describe a method using a cytokinesis-blocked MN assay in combination with identifying specific chromosomes of mice. For this purpose transgenic mice with foreign DNA inserted in three pairs of their chromosomes were generated. Splenocytes of these mice were cultured and treatedin vitro with vinblastine (VBL) or X-rays, followed by recovery in medium containing cytochalasin B. By tracing the marker chromosomes in binucleated splenocytes, reciprocal products of chromosome malsegregation to the daughter nuclei could be easily traced. The results showed that besides clastogenic activity, X-rays also exhibited aneugenic activity. Treatment with vinblastine showed a close relationship between micronuclei induction and chromosome malsegregation, although at higher doses malsegregation processes became more prominent. Simultaneous malsegregation of more than one chromosome was observed frequently, but the three marker chromosomes were found to be randomly involved in this process.     

Dietary folate deficiency enhances induction of micronuclei by arsenic in mice

Folate deficiency increases background levels of DNA damage and can enhance the genotoxicity of chemical agents. Arsenic, a known human carcinogen present in drinking water supplies around the world, induces chromosomal and DNA damage. The effect of dietary folate deficiency on arsenic genotoxicity was evaluated using a mouse peripheral blood micronucleus (MN) assay. In duplicate experiments, male C57Bl/6J mice were fed folate-deficient or folate-sufficient diets for 7 weeks. During week 7, mice on each diet were given four consecutive daily doses of sodium arsenite (0, 2.5, 5, or 10 mg/kg) via oral gavage. Over the course of the study the folate-deficient diet produced an approximate 60% depletion of red blood cell folate. Folate deficiency by itself was associated with small but significant increases in MN in normochromatic erythrocytes (NCEs) and polychromatic erythrocytes (PCEs). Arsenic exposure was associated with significant increases in MN-PCEs in both folate-deficient and folate-sufficient mice. MN-PCE frequencies at the 10 mg/kg dose of arsenic were increased 4.5-fold over vehicle control in folate-deficient mice and 2.1-fold over control in folate-sufficient mice. At the 5 and 10 mg/kg doses of arsenic, MN-PCE levels were significantly higher (1.3-fold and 2.4-fold, respectively) in folate-deficient mice compared to folate-sufficient mice. Very few MN from either control or treated animals in either experiment exhibited kinetochore immunostaining, suggesting that the MN were derived from chromosome breakage rather than from whole chromosome loss. These results indicate that folate deficiency enhances arsenic-induced clastogenesis at doses of 5 mg/kg and higher.     

Ozone inhalation leads to a dose‐dependent increase of cytogenetic damage in human lymphocytes

Ozone is an important constituent of ambient air pollution and represents a major public health concern. Oxidative injury due to ozone inhalation causes the generation of reactive oxygen species and can be genotoxic. To determine whether ozone exposure causes genetic damage in peripheral blood lymphocytes, we used a well‐validated cytokinesis‐block micronucleus Cytome assay. Frequencies of micronuclei (MN) and nucleoplasmic bridges (NB) were used as indicators of cytogenetic damage. Samples were obtained from 22 non‐smoking healthy subjects immediately before and 24‐hr after controlled 4‐hr exposures to filtered air, 100 ppb, and 200 ppb ozone while exercising in a repeated‐measure study design. Inhalation of ozone at different exposure levels was associated with a significant dose‐dependent increase in MN frequency (P < 0.0001) and in the number of cells with more than one MN per cell (P < 0.0005). Inhalation of ozone also caused an increase in the number of apoptotic cells (P = 0.002). Airway neutrophilia was associated with an increase in MN frequency (P = 0.033) independent of the direct effects of ozone exposure (P < 0.0001). We also observed significant increases in both MN and NB frequencies after exercise in filtered air, suggesting that physical activity is also an important inducer of oxidative stress. These results corroborate our previous findings that cytogenetic damage is associated with ozone exposure, and show that damage is dose‐dependent. Further study of ozone‐induced cytogenetic damage in airway epithelial cells could provide evidence for the role of oxidative injury in lung carcinogenesis, and help to address the potential public health implications of exposures to oxidant environments. Environ. Mol. Mutagen. 56:378–387, 2015. © 2014 Wiley Periodicals, Inc.     

The effect of combined therapy with ritodrine,erythromycin and verapamil on the frequency of micronuclei in peripheral blood lymphocytes of pregnant women

Abstract The main aim of this study was to investigate the genotoxic effect of combined pharmacotherapy applied in post-operative treatment of cervical cerclage in pregnant women over six days. This study included 19 phenotypically healthy pregnant women in mid-trimester with a diagnosis of cervical insufficiency, mean age 28±5.33. The frequency of micronuclei (MN) was estimated in peripheral blood lymphocytes of patients before surgical intervention and after the end of applied pharmacotherapy by application of cytokinesis block micronucleus (CBMN) test. Mean value of baseline MN frequency was 6.84±2.91 MN/1000 binucleated cells, and after the end of the applied therapy, 10.32±4.27 MN/1000 binucleated cells (P<0.001) were found. The data of cell proliferation index showed that the combined therapy did not induce significant difference in cell kinetics (P>0.05). Our results showed that combined pharmacotherapy treatment over six days significantly increased the frequency of MN in peripheral blood lymphocytes of pregnant women.     

Genotoxicity of styrene–acrylonitrile trimer in brain,liver, and blood cells of weanling F344 rats

Styrene–acrylonitrile Trimer (SAN Trimer), a by‐product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2‐year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN‐RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose‐related increases (P < 0.0001) in MN‐RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical‐related bone marrow toxicity. Results of the Comet assay showed significant, dose‐related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical‐related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Diabetes and chronic nitrate therapy as co-determinants of somatic DNA damage in patients with coronary artery disease

Somatic DNA damage has been linked to coronary artery disease (CAD). However, whether genetic instability is linked to CAD per se or to concomitant potentially genotoxic metabolic and pharmacological factors remains still unclear. The aim of this study was to evaluate the determinants of somatic DNA damage in a large population of patients undergoing coronary angiography. A total of 278 in-hospital patients (215 men, age 61.8±0.7 years) were studied by using micronucleus assay (MN) in human lymphocytes, which is one of the most commonly used biomarker for somatic DNA damage. Significant CAD (>50% diameter stenosis) was present in 210 patients (179 men, age 62.3±0.7 years). Normal coronary arteries were observed in 68 patients (35 men, age 60.2±1.7 years). There were no significant differences between patients with and without CAD, but patients with multivessel disease had the highest MN levels (P=0.01). MN frequency was also found significantly higher in presence of type 2 diabetes (P<0.0001), dyslipidemia (P=0.048) and nitrate therapy (P=0.0002). A significant additive effect was also observed between diabetes and nitrate therapy (P=0.02). On multivariate logistic regression analysis, diabetes [odds ratio =6.8 (95% confidence interval, 3.2–14.5), P<0.0001] and nitrate therapy [odds ratio =2.4 (95% confidence interval, 1.3–4.7), P=0.01] remained the only significant determinants for the 50th percentile of MN (>12). These results indicated that diabetes and, to a lesser extent, chronic nitrate therapy are major determinants of somatic DNA instability in patients with CAD. DNA damage might represent an additional pathogenetic dimension and a possible therapeutic target in the still challenging management of coronary artery disease concerning diabetics.     

Effect of sodium molybdate on microbial fixation of nitrogen

The effect of 0, 100, 200, 300, 400, and 500 μM concentrations of sodium molybdate in the culture media of the five Azotobacter species isolated from Allahabad soil was studied. It was observed that the presence of molybdenum in the culture media of all the bacterial samples increased the fixation of nitrogen indicating that molybdenum is essential for increasing the efficiency of the nitrogen fixers for nitrogen fixation.     

Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points

The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9-120 micro g/ml) in the presence of either cytochalasin B (CYB) (3 micro g/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 micro M). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific alpha-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents.     

Increased cytogenetic damage detected by FISH analysis on micronuclei in peripheral lymphocytes from alcoholics

Alcohol abuse greatly increases the risk of various malignancies, including cancer of the liver and digestive tract. Although it is thought that this may be due, at least partially, to the mutagenic properties of ethanol, little is known about the genotoxic effects of ethanol in humans. We investigated the chromosomal damage in lymphocytes from 20 alcoholics and 20 controls using the micronucleus (MN) assay combined with fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe capable of differentiating centromere positive (C+) from centromere negative (C-) MN. The frequency of MN in binucleate lymphocytes was significantly higher in alcoholics than in controls (12.0 +/- 5.4 and 7.6 +/- 1.6, respectively; P: < 0.05). FISH revealed significantly higher frequencies of C+ MN in alcoholics than in controls (8.2 +/- 4.8 and 3.4 +/- 1.4, respectively; P: < 0.05). In the alcoholics, no association was found between years of alcohol abuse and frequency of MN or C+ MN. However, age influenced MN and C+ MN frequency both in alcoholics and controls. These results indicate that alcohol abuse may well induce chromosome loss in humans, suggesting a possible aneugenic mechanism of alcohol. This effect could contribute to the health hazards related to alcoholism such as cancer risk.     

Carbon nanotubes induce oxidative DNA damage in RAW 264.7 cells

The induction of DNA and chromosome damage following in vitro exposure to carbon nanotubes (CNT) was assessed on the murine macrophage cell line RAW 264.7 by means of the micronucleus (MN) and the comet assays. Exposures to two CNT preparations (single‐walled CNT (SWCNT > 90%) and multiwalled CNT (MWCNT > 90%) were performed in increasing mass concentrations (0.01–100 μg/ml). The frequency of micronuclei was significantly increased in cells treated with SWCNT (at doses above 0.1 μg/ml), whereas MWCNT had the same effect at higher concentrations (1 μg/ml) (P < 0.05). The results of the comet assay revealed that the effects of treatment with SWCNT were detectable at all concentrations tested (1–100 μg/ml); oxidized purines increased significantly, whereas pyrimidines showed a significant increase (P < 0.001) only at the highest concentration (100 μg/ml). In cells treated with MWCNT, an increase in DNA migration due to the oxidative damage to purines was observed at a concentration of 1 and 10 μg/ml, whereas pyrimidines showed a significant increase only at the highest mass concentration tested. However, both SWCNT and MWCNT induced a statistically significant cytotoxic effect at the highest concentrations tested (P < 0.001). These findings suggest that both the MN and comet assays can reliably detect small amount of damaged DNA at both chromosome and nuclear levels in RAW 264.7 cells. Moreover, the modified version of the comet assay allows the specific detection of the induction of oxidative damage to DNA, which may be the underlying mechanism involved in the CNT‐associated genotoxicity. Environ. Mol. Mutagen., 2010. © 2010 Wiley‐Liss, Inc.     

Genotoxicity of doxorubicin in F344 rats by combining the comet assay,flow‐cytometric peripheral blood micronucleus test,and pathway‐focused gene expression profiling

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7‐week‐old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes‐modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non‐neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species. Environ. Mol. Mutagen. 55:24–34, 2014. © 2013 Wiley Periodicals, Inc.^?