Percentage Of Aberrant Cells Research Articles

Improved Detection of Chromosomal Abnormalities in CLL by Conventional Cytogenetics Using CpG Oligonucleotide and Interleukin-2 Stimulation. A Belgian Multicentric Study

Numerous studies have shown that the presence, number and type of chromosomal aberrations represent an independent predictor of prognosis in B-cell chronic lymphocytic leukemia (CLL). Consequently, cytogenetic analysis is routinely performed in this disease. However, CLL lymphocytes have a poor mitotic index, generating only 40–50% of abnormal karyotypes. The rate of detection can be increased to 80% by interphase FISH analysis. Since some aberrations, i.e. those not involving the classical regions (13q, 12, 11q, 17p, and 6q), can escape FISH detection, there has been great interest in improved culturing methods with an immunostimulatory CpG oligonucleotide (CpG). We performed a multicentric cytogenetic study to assess the impact of 2 different culturing procedures on the detection of clonal abnormalities in 159 consecutive unselected cases with CLL referred to our centers for routine analysis from October 2007 to July 2008. Dual 72 hours cultures of bone marrow or peripheral blood were set up with the addition of either a conventional B cell mitogen (TPA) or CpG and interleukin-2 (IL2). Cytogenetic analysis was performed on both cultures. FISH analysis using a CLL probe panel analyzing 1–6 regions (13q, centromere 12, 11q, 17p, 6q and 14q32) was also applied on uncultured material, on CpG and/or on TPA culture in 146 cases. Quality of banding and proliferation capacity were assessed in 38 cases. The quality was good in 17 (CpG) and 8 (TPA), intermediate in 12 (CpG) and 18 (TPA), and poor in 9 (CpG) and 12 (TPA) cases. The mitotic index was slightly higher in CpG cultures: <15 mitoses/slide were seen in 12 (CpG) and 15 (TPA) cases, 15–20 mitoses/slide in 13 (CpG) and 14 (TPA) cases, and >20 mitoses/slide in 13 (CpG) and 9 (TPA) cases, respectively. Clonal abnormalities were identified in 89 cases (56%). In 56 cases, the aberrant clone was detected in both cultures. Of these, the percentage of aberrant metaphases was similar in both cultures in 13, higher in CpG culture in 32 and higher in TPA culture in 11 cases. In 29 and 4 additional cases, a clonal abnormality was detected only in CpG or TPA culture, respectively. Thus, the percentage of abnormal karyotypes with CpG and TPA was 53 and 38%, respectively (p=0,005). “Typical” aberrations, including del(13q), +12, del(11q), del(17p) and del(6q), were detected in 16, 26, 15, 9, and 7 (CpG) cases, and in 10, 20, 16, 8, and 3 (TPA) cases, respectively. Translocations (balanced and unbalanced) were observed in 46 (CpG) and 27 (TPA) cases, whereas aberrations involving 14q32 were seen in 5 (CpG) and 4 (TPA) cases, respectively. Interphase FISH detected del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 53, 1, 5, 0, 0 and 1 cases, respectively, in which cytogenetic analysis was either normal or abnormal but did not show the specific change. Conversely, FISH did not detect del(13q), +12, del(11q), del(17p), del(6q) and del(14q) in 1, 1, 4, 1, 2 and 0 cases in which the specific change was visible at karyotypic level. FISH was performed on both CpG and TPA cultures in 9 selected cases harbouring an aberration for which a specific commercial probe was available (+12, n=3; del(13q), n=4; del(11q), n=1; and del(17p), n=1) and showing different percentages of aberrant cells in both cultures. In 3 of these cases, fresh uncultured material was also analyzed. The highest percentage of abnormal nuclei was observed in TPA culture in all cases. In the 3 uncultured samples the percentage of aberrant nuclei was similar to the CpG culture. In conclusion, our results confirm an increased detection rate of abnormalities in CLL by using CpG/IL2 stimulation. Interphase FISH can further increase the detection rate of recurrent abnormalities. However, neither cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques and the necessity of performing both.

Human Embryonic Stem Cells May Display Higher Resistance to Genotoxic Stress as Compared to Primary Explanted Somatic Cells

The use of human embryonic stem (hES) cells in genotoxicity screening can potentially overcome the deficiencies associated with using immortalized cell lines, primary explanted somatic cells, and live animal models. Hence this study sought to compare the responses of hES cells and primary explanted somatic cells (IMR-90 cells, human fetal lung fibroblasts) to genotoxic stress, to evaluate whether hES cells can accurately reflect the normal physiology of human somatic cells. The effects of mitomycin C (MMC) on the chromosomal stability of hESC and IMR-90 was assayed and compared by fluorescence in situ hybridization (FISH) with telomere-specific peptide nucleic acid and multicolor (m) FISH techniques. The results showed that, the percentage of aberrant cells increased from 6% in the untreated control to 57.5% at the higher dose of 0.06 microg/ml MMC (9.6-fold increase) group in the case of IMR-90 cells, whereas hES cells displayed a corresponding increase from 6% to 28% (4.6-fold increase). Telomere FISH ascertained that the main types of damage induced by MMC are chromosomal breaks and the loss of telomeric signals. No fusions were observed in all samples analyzed. This was further confirmed by mFISH, which showed that fusions and translocations were not the type of aberration induced by MMC, with no such aberrations being observed in all samples analyzed. Hence, hES cells of the H1 line are apparently more resistant to MMC-induced DNA damage, as compared to the IMR-90 cells. These results highlight possible intrinsic differences in response to damaging agents between hES cells and normal somatic cells.

Chromosomal aberrations by fluorescence in situ hybridization (FISH)—Biomarker of exposure to carcinogenic PAHs

The fluorescence in situ hybridization (FISH) technique with whole chromosome painting for chromosomes #1 and #4 was used to study the impact of air pollution containing higher concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in three European cities, Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). In each site were followed an exposed group, who were police officers or bus drivers who work usually through busy streets for at least 8 h, and a reference group, who spent more than 90% of their daily time indoors. In Prague, a significant increase was observed in percentage of aberrant cells (% AB.C.) in the police officers compared to the reference group (0.33 ± 0.25 versus 0.24 ± 0.18, p < 0.05). In Kosice, the exposed group differed from reference in the endpoints F G/100 1.52 ± 1.18 versus 1.12 ± 1.30, p < 0.05; % AB.C. 0.30 ± 0.19 versus 0.21 ± 0.20, p < 0.05; t/1000 3.91 ± 3.18 versus 2.84 ± 3.10, p < 0.05. In Sofia were followed two exposed groups: police officers and bus drivers. All FISH endpoints were significantly higher in police officers compared to reference group ( F G/100 1.60 ± 0.99 versus 0.82 ± 0.79, p < 0.01; % AB.C. 0.25 ± 0.14 versus 0.13 ± 0.13, p < 0.01; t/1000 4.19 ± 2.65 versus 2.13 ± 2.05, p < 0.05; rcp 1.46 ± 1.07 versus 0.70 ± 0.76, p < 0.05). In bus drivers compared to reference there was an increase in % AB.C. (0.25 ± 0.18 versus 0.13 ± 0.13, p < 0.05). This is the first study when FISH method was used to analyze the impact of environmental air pollution. According to the original hypothesis it is expected that the most important group of chemicals responsible for the biological activity of air pollution represent c-PAHs.

Fluorescence in situ Hybridization Reveals Closely Correlated Results in Cytological and Histological Specimens of Hematological Neoplasias Compared to Conventional Cytogenetics

Objectives: Fluorescence in situ hybridization (FISH) has become a useful tool to identify chromosomal aberrations in non-dividing cells. Numerous studies have compared chromosomal banding analysis (CBA) and FISH on fixed cultured bone marrow cells. However, up to now, there has been no study comparing two main sources of diagnostic material, i.e. bone marrow aspirates and trephine biopsies. We therefore analyzed these materials by FISH in comparison with CBA. Methods: CBA revealed chromosomal aberrations in 18 patients suffering from myelodysplastic syndrome (n = 13), acute myeloid leukemia (n = 3), or chronic myeloproliferative syndrome (n = 2). FISH was performed on fixed cultured bone marrow cells, aspirates and trephine biopsies from each patient. Results: Percentages of aberrant cells in the different materials correlated highly with Pearson values of 0.909 for biopsy/fixed cultured cells (p < 0.001), 0.830 for biopsy/aspirate (p < 0.001) and 0.768 for aspirate/fixed cultured cells (p < 0.001). Moreover, in bone marrow biopsies peritrabecular and central intertrabecular areas yielded very similar FISH results with a high correlation (r = 0.968, p < 0.001). FISH revealed a lower proportion of aberrant cells than CBA in 90% of the specimens. Conclusions: In summary, the different materials available for the FISH examination are comparable in sensitivity and show similar quantitative results. Therefore, the use of biopsy sections for the routine FISH examination of chromosomal abnormalities is a valid method.

Polymorphism in the ERCC2 codon 751 is associated with arsenic-induced premalignant hyperkeratosis and significant chromosome aberrations

In West Bengal, India more than 6 million people are exposed to high levels of arsenic through drinking water. Since, only 15-20% of the exposed individuals show arsenic-induced skin lesions, it is assumed that genetic variation might play an important role in arsenic toxicity and carcinogenicity. Arsenic exposure often leads to the development of hyperkeratosis, the precursor of arsenic-induced skin cancer. ERCC2 (excision repair cross-complementing rodent repair deficiency, complementation group 2) is a nucleotide excision repair pathway gene, and its SNPs have been implicated in several types of epithelial cancers. We investigated the possible association of ERCC2 codon 751 A-->C polymorphism (lysine to glutamine) with arsenic-induced hyperkeratosis and correlated ERCC2 genotypes with increased frequencies of chromosomal aberration to ascertain whether any genotype leads to sub-optimal DNA repair. For this association study, 318 unrelated arsenic exposed subjects (165 with hyperkeratosis and 153 without any arsenic-induced skin lesions), drinking water contaminated with arsenic to a similar extent, were recruited. Genotyping was done through PCR-RFLP procedure. Lys/Lys genotype was significantly over-represented in the arsenic-induced hyperkeratosis-exhibiting group [odds ratio (OR) = 4.77, 95% confidence interval (CI) = 2.75-8.23]. A statistically significant increase in both CA/cell and percentage of aberrant cells was observed in the individuals with AA genotype compared to those with AC or CC genotype combined (P < 0.01) in each of the two study groups, as also, in the total study population. This study indicates that ERCC2 codon 751 Lys/Lys genotype is significantly associated with arsenic-induced premalignant hyperkeratosis and is possibly due to sub-optimal DNA repair capacity of the ERCC2 codon 751 Lys/Lys genotype.

Plasma Antioxidant Activity and Chromosome Aberrations in Humans Exposed to Long-Term Low-Dose γ-Irradiation

Plasma antioxidant activity was evaluated in the lecithin-Fe(2+) model system and cytogenetic studies were carried out in 16 men. A relationship between elevated antioxidant activity of the plasma and increased level of chromatid aberrations and with percentage of aberrant cells was detected in the experimental group, but no correlation was found between antioxidant activity and the integral dose of gamma-irradiation and rate of its accumulation. An assumption was made about exhaustion of the buffer capacity of the antioxidant defense system resulting from long-term exposure to unidentified factors of technogenic nature increasing plasma antioxidant activity and level of chromosome aberrations. In subjects with high level of antioxidant defense, naturally low antioxidant activity and low percentage of chromatid aberrations were probably maintained.

A preliminary cytogenetic and hematological study of photocopying machine operators

The incidences of chromosomal aberrations(CAs) as well as sister chromatid exchange frequencies (SCEs) was evaluated from 12 photocopying machine operators working on an average 8-9 hours per day for more than five years. A complete blood picture of each individual was assessed with an automatic particle cell counter. Additionally, blood pressure was measured at the time of blood collection from all photocopying machine operators. For comparison, the control group included another 12 individuals matched according to age, sex, socioeconomic conditions as well as other personal habits. The observations of the present study are indicators of health hazard for, although small, there was a significant increase in the percentage of aberrant cells (P<0.05), total aberrations (P<0.01) as well as total aberrations excluding chromatid gaps (P<0.01) among photocopying machine operators when compared to controls. However, results on SCE analysis of photocopying operators revealed no significant difference from the controls. At the same time all photocopying operators exhibited normal hematological parameters as well as blood pressure values.

Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization.

The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G(2) and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to gamma rays and allowed to repair at 37 degrees C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to gamma rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

The radioenhancement of two human head and neck squamous cell carcinomas by 2′-2′ difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

Purpose: The present study aimed at investigating if 2′-2′ difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. Methods: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 μM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. Results: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. Conclusion: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.

High-dose clastogenic activity of aniline in the rat bone marrow and its relationship to the carcinogenicity in the spleen of rats.

To clarify the question of clastogenicity of aniline in rats two studies were performed: a bone marrow micronucleus test and a bone marrow metaphase test. In the micronucleus test aniline (as aniline hydrochloride) was administered to groups of seven male PVG rats at single oral doses of 0, 300, 400, or 500 mg/kg body weight. Bone marrow was obtained 24 and 48 h after oral treatment. Smears of bone marrow were stained with acridine orange and erythrocytes were examined for the presence of micronuclei. Animals receiving cyclophosphamide (1x7.5 mg/kg) served as positive controls. Clinical signs observed in animals dosed at 300 mg/kg and above included cyanosis, light brown coloured urine and cold to touch. Small, but statistically significant and dose-related increases in the incidence of micronulei over the vehicle control values were observed at the 24-h sampling time only. Cyclophosphamide induced a significant and comparably much higher increase in micronuclei than aniline. In the bone marrow metaphase test aniline (as aniline hydrochloride) was administered to groups of seven male PVG rats at single oral dose levels of 0, 300, 400, or 500 mg/kg body weight. Bone marrow was sampled 18 and 30 h after dosing. A group treated with cyclophosphamide (1x40 mg/kg) served as positive control. A small increase in the percentage of aberrant cells above solvent control values was recorded in one rat at 400 mg/kg and four rats at 500 mg/kg at the 18-h sampling time only. The positive control cyclophosphamide induced a much higher rate of aberrant cells in all animals. Several lines of evidences are presented against a causal relationship between the clastogenic activity in male PVG rats at 400 and 500 mg/kg and the carcinogenicity in the spleen of Fischer 344 rats starting at 30 mg/kg in males. Among these are the dose-response relationship of the tumour incidence, the close correlation between degree of spleen damage and tumour induction, the lack of carcinogenic effects in mice even at higher dose levels, or in rats at dose levels inducing only slight haematotoxicity and spleen toxicity, and the available data on the mode of action of other chemicals inducing spleen tumours.

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number ( 2n♂=23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers’ testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose–effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.

Apoptosis can be a confusing factor in in vitro clastogenic assays.

Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

Enhancement of bone marrow radioprotection and reduction of WR-2721 toxicity by Ocimum sanctum

The radioprotective effect of the leaf extract of Ocimum sanctum (OE) in combination with WR-2721 (WR) was investigated on mouse bone marrow. Adult Swiss mice were injected intraperitoneally (i.p.) with OE (10 mg/kg on 5 consecutive days), or 100–400 mg/kg WR (single dose) or combination of the two or double-distilled water (DDW) and whole-body exposed to 4.5 Gy γ-irradiation (RT). Metaphase plates were prepared from femur bone marrow on days 1, 2, 7 and 14 post-treatment and chromosomal aberrations were scored. The maximum number of aberrant cells was observed at 24 h after irradiation in all the groups. However, pretreatment with OE or WR individually resulted in a significant decrease in aberrant cells as well as different types of aberrations. The combination of the two further enhanced this effect; resulting in a 2-fold increase in the protection factor (PF=6.68) compared to 400 mg/kg WR alone. The percent aberrant cells decreased linear-quadratically with WR dose when given individually, while in the OE+WR pretreatment animals the values showed a linear dose response. Combination of OE with WR doses above 200 mg/kg completely eliminated rings, polyploidy and pulverization of chromosomes. Percent aberrant cells decreased with time in all groups, though the values remained higher than normal even on day 14 in the RT alone as well as those treated with single agent+RT. WR doses above 200 mg/kg before RT resulted in significantly higher frequency of aberrant cells compared to RT and OE+RT groups on day 14, suggesting delayed WR toxicity; but combination of OE with WR brought down these values to normal level, indicating that OE combination, in addition to enhancing WR protection, may also act as a detoxifier. The protective effect of OE and WR is also reflected in the enhancement of bone marrow CFU survival. Both OE and WR possessed significant free radical scavenging activity in vitro. The combination of the two further enhanced this effect, suggesting that the enhanced free radical scavenging activity by combining the two protectors results in the higher bone marrow cell protection. The significant elevation in chromosome protection obtained by combining OE with WR, with reduction in the latter's toxicity at higher doses, suggests that the combination may have promise for radioprotection in humans.

Protection against radiation-induced chromosome damage in mouse bone marrow by Ocimum sanctum

The radioprotective effect of the leaf extract of Ocimum sanctum (Ocimum extract, OE) was investigated by taking chromosome aberrations as the end point. Adult Swiss mice were whole-body exposed to 1–6 Gy of gamma radiation with/without pretreatment with 10 mg/kg b.wt. of OE intraperitoneally for 5 consecutive days. Radiation was given 30 min after the last injection. Metaphase plates were prepared from femur marrow on days 1, 2, 7 and 14 post-treatment and the frequency of aberrant cells and individual aberrations were scored. OE alone did not have any significant effect on the chromosomes. Maximum percent of aberrant cells was observed at 24 h in all the exposed groups. The percent aberrant cells showed a linear quadratic increase with radiation dose, in both radiation alone (RT) and OE + RT-treated groups. Exchange (dicentrics and rings) and multiple (pulverized and severely damaged cells) aberrations also showed a similar response. However, the slopes of OE + RT was significantly shallower than RT groups ( p<0.05). A dose-modifying factor of 2.63 was obtained taking percent aberrant cells for 2 Gy as the base. Progressive decline in the percent aberrant cells as well as the number of aberrations with time after irradiation was observed in both RT and OE + RT groups. OE treatment resulted in a faster recovery compared to RT alone group. At doses below 3 Gy, OE pretreatment almost completely eliminated the exchange aberrations from the cell population by day 2. Studies on a chemical system demonstrated that OE significantly reduced the generation of hydroxyl radical; a lower dose of OE (1 mg/ml) was more effective than 5 mg/ml and this effect was more pronounced than that produced by DMSO. These results show that OE affords in vivo protection against radiation-induced cytogenetic damage. Free radical scavenging is a likely mechanism of OE protection.

Inhibition of methotrexate‐induced chromosomal damage by vanillin and chlorophyllin in V79 cells

Methotrexate (MTX), a chemotherapeutic agent used to treat cancer, produces cytogenetic damage and has a cytostatic effect in a variety of test systems. Several antigenotoxic agents have been studied in various in vitro and in vivo systems. However, data are limited regarding their ability to modulate MTX-induced genotoxicity. In the present study, vanillin (VA) and chlorophyllin (CHL) were used as antigenotoxic agents to study their ability to minimize the DNA damage caused by MTX. Exponentially growing V79 Chinese hamster lung cells were treated with MTX at five different concentrations (5–100 μg/ml) with S9 activation for 6 h and post-treated with two concentrations of either VA (50 or 100 μg/ml) or CHL (50 or 100 μg/ml) for 40 h. Cytochalasin B was added for the micronucleus (MN) assay along with antigenotoxic agents to evaluate MN in binucleated cells. Chromosomal aberrations were also evaluated in parallel cultures. Results indicate that MTX alone induced a dose-dependent decrease in the nuclear division index (NDI) and the mitotic index (MI). A significant increase in percent micronucleated binucleated cells (MNBN) and percent aberrant cells (Abs) was observed. Studies using VA as an antigenotoxic agent showed a decrease in the number of MNBN (26.3–83.1%) and Abs (16.0–87.5%) with the addition of either 50 or 100 μg VA/ml. The addition of CHL also significantly reduced the number of MNBN (53.0–91.5%) at both concentrations tested. Chromosomal aberrations were also significantly reduced (41.0–83.0). These studies indicate that both VA and CHL are capable of effectively minimizing MTX-induced chromosomal damage. Teratogenesis Carcinog. Mutagen. 17:313–326, 1997/98. © 1998 Wiley-Liss, Inc.

Inhibition of methotrexate-induced chromosomal damage by vanillin and chlorophyllin in V79 cells

Methotrexate (MTX), a chemotherapeutic agent used to treat cancer, produces cytogenetic damage and has a cytostatic effect in a variety of test systems. Several antigenotoxic agents have been studied in various in vitro and in vivo systems. However, data are limited regarding their ability to modulate MTX-induced genotoxicity. In the present study, vanillin (VA) and chlorophyllin (CHL) were used as antigenotoxic agents to study their ability to minimize the DNA damage caused by MTX. Exponentially growing V79 Chinese hamster lung cells were treated with MTX at five different concentrations (5-100 micrograms/ml) with S9 activation for 6 h and post-treated with two concentrations of either VA (50 or 100 micrograms/ml) or CHL (50 or 100 micrograms/ml) for 40 h. Cytochalasin B was added for the micronucleus (MN) assay along with antigenotoxic agents to evaluate MN in binucleated cells. Chromosomal aberrations were also evaluated in parallel cultures. Results indicate that MTX alone induced a dose-dependent decrease in the nuclear division index (NDI) and the mitotic index (MI). A significant increase in percent micronucleated binucleated cells (MNBN) and percent aberrant cells (Abs) was observed. Studies using VA as an antigenotoxic agent showed a decrease in the number of MNBN (26.3-83.1%) and Abs (16.0-87.5%) with the addition of either 50 or 100 micrograms VA/ml. The addition of CHL also significantly reduced the number of MNBN (53.0-91.5%) at both concentrations tested. Chromosomal aberrations were also significantly reduced (41.0-83.0). These studies indicate that both VA and CHL are capable of effectively minimizing MTX-induced chromosomal damage.

Biological effect monitoring in industrial workers from the Czech Republic exposed to low levels of butadiene

Blood samples were collected twice (in 1993 and 1994) from 19 workers exposed to 1,3-butadiene and 19 matched controls. Three exposed and three control subjects were the same in 1993 and 1994. Personal passive dosimetry was performed in 1993 and twice in 1994 on the day preceding blood sampling. Mean exposure level in 1994 was 1.76 ± 4.20 ppm (S.D.) and individual exposure levels ranged between 0.012 ppm (detection limit) and 19.77 ppm. Using the clonal assay, geometric mean of hprt mutant frequencies adjusted for cloning efficiency, age and smoking were, respectively, 7.85 (± 7.09) × 10 −6 and 10.14 (± 9.16) × 10 −6 in pooled (1993 plus 1994) exposed and control subjects. The difference was not statistically significant indicating that 1,3-butadiene did not induce a detectable increase in mutations at the hprt locus. A similar result was obtained for the 1994 subjects alone. There was no difference between adjusted geometric mean mutant frequencies of exposed and unexposed non-smokers or between exposed and unexposed smokers. Analysis of chromosomal aberrations in lymphocytes from 1994 subjects indicated that the percentage of aberrant cells was significantly enhanced in exposed subjects. In 1993 (data not shown), it was impossible to demonstrate a significant increase of aberrant cells in subjects exposed to 1,3-butadiene. Frequencies of micronuclei in cytochalasin-B blocked binucleate lymphocytes in exposed and unexposed 1994 subjects were not significantly different. This was also the case for earlier samples analyzed in the same plant. Using the comet assay for 1994 subjects, no statistically significant difference was found between the whole group of exposed and unexposed subjects. This was true for both the comet tail length and the percentage of DNA in the tail. In exposed smokers, however, the comet tail length was significantly longer than in unexposed smokers. Unexpectedly, in unexposed smokers the tail length was significantly shorter than in unexposed non-smokers. It was also unexpected that the percentage of DNA in the comet tail was significantly lower in exposed non-smokers than in unexposed non-smokers.

Inhibitory effect of folinic acid on radiation-induced micronuclei and chromosomal aberrations in V79 cells

Folinic acid (FA), clinically called leucovorin, has been widely used as a nutrient supplement in dietary intake and is capable of inhibiting cytotoxicity and chromosomal damage induced by chemicals. However, data on its antigenotoxic effect on radiation-induced chromosomal damage are limited. The present study was, therefore, performed to investigate the effect of FA on radiation-induced (X-rays and UV radiation) micronuclei (MN) and structural chromosomal aberrations (SCA) concurrently in V79 Chinese hamster lung cells. Exponentially growing cells were exposed to five doses of X-rays (1–12 Gy) and UV radiation (50–800 μ × 10 2/cm 2) and post-treated with 5 or 50 μg FA/ml of culture medium for 16 h. The slides were analyzed for the presence of MN and SCA using standard procedures. The results showed that X-ray treatment alone produced dose-related cytotoxicity as measured by nuclear division index (NDI) and mitotic index (MI). X-rays produced a clear dose-related clastogenicity as measured by percent of micronucleated binucleated cells (MNBN) (5–79%) and percent of aberrant cells (11–92%). FA at 5 μg/ml slightly decreased X-ray induced chromosomal damage in both assays; however, the inhibition was significant (12–46% of MNBN, 14–48% in aberrant cells) only when X-ray-treated cultures were post-treated with 50 μg FA/ml. Post-treatment of FA had no effect on X-ray induced cytotoxicity as measured by NDI and MI. A similar a dose-related increase in % MNBN (0.5–10.3%) and percent aberrant cells (6–35%) was produced by UV radiation treatment alone. There were significant percentages of MNBN and aberrant cell inhibitions at both 5 and 50 μg/ml in both assays. As in the case of X-ray-treated cells, there was a clear dose-related cytotoxicity in UV-treated cells alone. No reduction in NDI or MI was found when UV-exposed cells were post-treated with 5 or 50 μg of FA. These data demonstrate the beneficial effect of FA in decreasing radiation-induced chromosomal damage.

Biological monitoring of workers exposed to emissions from petroleum plants.

This paper presents some of the results from the Commission of the European Communities collaborative research program (contract number EV5V-CT92-0221), whose aim is to investigate the relationship between exposure to petroleum emissions, benzene, and induction of genetic damage in human cells. Twenty-four workers from petroleum plants in Poland and 35 unexposed controls were examined for cytogenetic effects and ras oncoprotein levels and their relationship to confounding factors (e.g., smoking habit, sex family cancer history, and seasonal influence). Preliminary data of chromosome aberrations (CA) and sister chromatid exchanges (SCE) showed differences among sampling subgroups. In this present study, the levels of ras p21 proteins were determined and further analyses of CA, SCE, high frequency cells (HFC), and proliferative rate index (PRI) have been undertaken. Results show that the exposed group has statistically significant increases in CA, and percent of aberrant cells. There were no differences between exposed and unexposed groups in SCE, HFC, PRI, or the levels of ras p21 proteins. Smoking was found to statistically significantly affect levels of CA, percent of aberrant cells, SCE, HFC, and ras proteins. Sister chromatid exchanges were also statistically significantly sex dependent (7.5 breaks/cells for females and 6.8 breaks/cell for males). There were no statistically significant differences for CA, percent aberrant cells, SCE, HFC, or ras p21 protein levels in subgroups characterized according to cancer cases reported in the immediate family. A seasonal variability was shown with statistically significant increases in various biomarkers in the winter. Unexposed groups also showed increases due to smoking and season. The nonsmoking group individuals also showed statistically significant increases in cytogenetic damage with exposure.

Modification of radiation-induced chromosome damage and micronucleus induction in mouse bone marrow by misonidazole and hyperthermia.

The effect of misonidazole (MISO), local hyperthermia (HT) and their combination on radiation-induced chromosome damage and micronucleus (MN) induction was studied in mouse bone marrow cells. It was found that MISO treatment did not enhance the clastogenic effect of radiation, which indicates a lack of radiosensitization of bone marrow chromosomes. But post-irradiation HT increased the frequency of aberrant cells and MN. A combination of MISO and HT produced a significant increase in the frequency of radiation-induced aberrant cells and MN at all the radiation doses as compared to radiation alone. The percentage of aberrant cells as well as the percentage of MN showed a linear quadratic increase with radiation dose in all the treatment groups. At higher radiation doses, cells with > 1 MN increased quadratically with a pronounced increase in cells bearing > 2 MN and severely damaged cells (SDCs) at radiation doses above 3.0 Gy in the HT and MISO+HT treated groups. Our results indicate that though MISO itself may not have a radiosensitizing effect on mouse chromosomes, a combination of MISO with HT can enhance the radiation damage in normal bone marrow.