Local genotoxic effects of formaldehyde in humans measured by the micronucleus test with exfoliated epithelial cells

Formaldehyde (FA) is genotoxic in vitro in cultured mammalian cells. When FA reaches the nuclear DNA, it forms DNA-protein cross-links (DPX). Incomplete repair of DPX can lead to the formation of mutations, in particular chromosome mutations and micronuclei (MN) in proliferating cells. Due to its high reactivity, FA leads primarily to local genotoxic effects at the site of contact. In humans, local genotoxic effects of FA have been studied with the micronucleus test (MNT) in exfoliated nasal and buccal mucosa cells. This approach is considered to be highly relevant because these tissues are the actual targets of FA, and MN are a sensitive indicator for the mutagenic action of FA. The published studies suggest that inhalation of FA leads to increased MN frequencies in nasal and/or buccal mucosa cells. However, a critical review of the data reveals that the effects are not consistent, and the studies should be interpreted with caution. One problem is the lack of standardization of the MNT with exfoliated cells and the high assay variability. Another problem concerns the quality of published studies indicating local genotoxic effects of FA in humans. Incomplete information on study design, exposure, and confounding factors frequently limit the interpretation of these studies. On the basis of the available data, it is not yet possible to assess the local genotoxicity of FA in humans and to draw meaningful conclusions with regard to a dose-effect relationship for risk estimation.     

The micronucleus assay in exfoliated human cells: A mini-review of papers from the CIS

A critical analysis of data from micronucleus assays in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and the shortcomings of several of them are discussed. These results are compared whenever possible with literature data. The aim of this mini-review is a criticism of the shortcomings of these papers in order to stimulate improvement of the presentation of micronucleus assay data, which will allow the comparison of these results with data presented by foreign investigators. Published in Russian in Tsitologiya i Genetika, 2007, Vol. 41, No. 2, pp. 56–66. The text was submitted by the authors in English.     

The micronucleus assay in exfoliated human cells: a mini-review of papers from the CIS

The critical analysis of the data concerning micronucleus assay in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and shortcomings of some of them are discussed. Presented results are compared whenever possible with literature data. The aim of the mini-review is a criticism of shortcomings of the papersforfurther improvement of the presentation of the data on micronucleus assay which will give the possibility to compare the results with the data presented by foreign investigators.     

Biomonitoring of low levels of mercurial derivatives in water and soil by Allium micronucleus assay

The Allium micronucleus (MNC) assay was developed to monitor low levels of mercury in aquatic and terrestrial environments. Four mercurial derivatives namely mercuric chloride (MC), methyl mercuric chloride (MMC), phenyl mercuric acetate (PMA) and a methoxy ethyl mercuric chloride based fungicide, Emisan-6, were tested to assess the sensitivity and versatility of the Allium MNC assay. Allium bulbs were set directly on water and soil contaminated with known levels of mercurial derivatives (0.0001-10.00 ppm). On the 5th day the endpoints measured were root length, mitoses with spindle abnormality and cells with MNC in root meristems. The effective concentrations of the test chemicals that cause 50% of root length as compared to control (EC50) were determined from dose-response curves so obtained. The lowest effective concentration tested (LECT) and highest ineffective concentration tested (HICT) for each of the mercurial derivatives for the induction of spindle malfunction and MNC were determined. It was found that EC50, LECT and HICT values for mercurial derivatives in soil were higher than those in water. The frequencies of cells with MNC and mitoses with spindle abnormality were highly correlated indicating that MNC is a good parameter of spindle malfunction. The present approach increased the sensitivity of the Allium assay by 10-fold, the detection limit being 0.001-0.1 ppm and 0.1-1.0 ppm in aquatic and terrestrial environments respectively, depending on the species of mercury.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinirrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the saine compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Detection of the cytogenetic effect of inhaled aerosols by the micronucleus test

The induction of micronuclei in mice exposed to aerosols of the following 6 genotoxic chemicals by inhalation was examined: cyclophosphamide (CP), methyl methanesulfonate (MMS), mitomycin C (MMC), dimethylnitrosamine (DMN), ethylcarbamate and colchicine. Exposure of mice to CP aerosols at a theoretical concentration of 2426 mg/m3 for 29, 81 and 139 min induced 0.6, 1.0 and 2.3% micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow 24 h after the termination of exposure. The other chemicals except for DMN showed a similar exposure-response relationship following in vivo exposures to their aerosols. The results obtained in this study suggest that the cytogenetic effect of inhaled aerosols can be detected by the micronucleus test, and the method described in the present report is useful as a rapid in vivo test for atmospheric aerosols.     

Use of the micronucleus assay with exfoliated epithelial cells as a biomarker for monitoring individuals at elevated risk of genetic damage and in chemoprevention trials.

This review summarises the current database on the micronucleus (MN) assay with exfoliated cells (MEC assay) and evaluates the predictive value of this model for the detection of human cancer risks. The MEC test is a cost effective, non-invasive method, in which the formation of MN in exfoliated cells from different organs, such as oral and nasal cavity, bladder, cervix, and oesophagus is used as an endpoint to detect endogenous, lifestyle, occupational and environmental exposures to genotoxins as well as chemoprotection of various compounds in intervention studies. The results suggest that the MN assay might be a useful approach to identify antimutagens which are protective in humans. Based on the comparison of the data from MN experiments with results from epidemiological cancer studies, we conclude that the MEC assay is a useful biomarker for the detection of human cancer risk in organs to which the MEC test can be applied. However, the current data base is not sufficient to draw a firm conclusion on the specificity of this approach.     

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay.

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinitrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the same compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.     

Increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers

Hairdressers are exposed daily to chemical substances, such as dyes, chemical straighteners and curling chemicals, which can be absorbed, inhaled or possibly ingested. We analyzed the frequency of micronuclei (MNC) in exfoliated cells of the buccal mucosa of 50 hairdressers and 50 controls in Pelotas, RS, Brazil. An assessment was carried out on the incidence of MNC, binucleated cells (BNC), broken egg cells (BEC), budding cells (BC), and the sum of anomalies (SA), in 2000 cells per individual. The data were analyzed with SPSS, using the Mann-Whitney U-test, α = 0.05. The mean number of anomalies in hairdressers was 2.02 ± 3.60 MNC; 8.50 ± 5.07 BNC; 9.06 ± 3.83 BEC; 0.32 ± 0.62 BC, and 19.90 ± 9.61 SA; in controls it was 0.36 ± 1.06 MNC; 5.20 ± 4.73 BNC; 5.92 ± 2.67 BEC; 0.10 ± 0.36 BC, and 11.58 ± 6.67 SA; the differences for all parameters were significant. The non-occupational factors did not significantly influence the alterations. A significant increase of BEC (P = 0.003) was observed in the hairdressers and SA (P = 0.033) in females. The lowest income level influenced MNC (P = 0.044), and the habit of not smoking influenced SA (P = 0.020). We concluded that exposure to substances used by hairdressers is genotoxic for men.     

The micronucleus assay in exfoliated buccal cells: application to occupational exposure to polycyclic aromatic hydrocarbons.

Many polycyclic aromatic hydrocarbons (PAHs) have been identified as cancer-inducing chemicals for animals and/or humans. Also, there is sufficient evidence that exposures in the occupational settings are carcinogenic or probably carcinogenic to human. Engine exhaust and used engine oils are major PAH sources in engine repair workshops and traffic. Analysis of micronucleus (MN) in exfoliated buccal cells is a sensitive method for monitoring genetic damage in human populations. In our study, we used three different occupational groups (Group 1; engine repair workers, Group 2; taxi drivers, Group 3; traffic police) and two controls (Control I for Group 1 and Control II for Group 2 and Group 3) for the exposed groups. We analysed MN frequencies in exfoliated buccal cells and compared the exposed groups (Group 1; n=34, Group 2; n=17, Group 3; n=15) and subjects not occupationally exposed to PAH (Control I; n=28, Control II; n=20). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 1 and Control I were 0.07+/-0.05 and 0. 05+/-0.04, respectively (p>0.05; Table 2). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 2, 3 and Control II were 0.12+/-0.05, 0.10+/-0.05 and 0.03+/-0.03, respectively (p<0. 0001, p<0.05; Table 2) Smokers and nonsmokers do not differ with respect to the incidence of MN in all groups.     

Screening for interindividual differences in radiosensitivity by means of the micronucleus assay in human lymphocytes

The response of unstimulated peripheral lymphocytes to a single dose of 3 Gy of 137Cs gamma rays was analysed in blood samples from 30 donors by a conventional micronucleus assay and from 14 donors by the cytokinesis-block (CB) method. Significant interindividual variations could be detected for the baseline levels and for induced levels of micronuclei. An age effect could be demonstrated with the conventional method for the number of spontaneous MN, but not with the CB method. The corresponding numerical estimate was 3.4 +/- 1.3% increase per year. No such increase was apparent for induced frequencies. Provided that cell proliferation kinetics is reliably taken into account the micronucleus assay could be helpful for diagnosing potential radiosensitive individuals.     

A survey on the cytogenetic status of the Croatian general population by use of the cytokinesis-block micronucleus assay

The cytokinesis-block micronucleus assay (CBMN) was used to assess the variability and determine possible influences of external and internal factors on the background levels of cytogenetic damage in peripheral blood lymphocytes (PBL) of 50 healthy volunteers selected at random from the general population of Croatia. The mean MN frequency for all subjects was 4.74+/-0.31 per 1000 cells and the mean cytokinesis-block proliferation index (CBPI) was 1.82+/-0.01. The mean frequency of nucleoplasmic bridges (NPB) for all subjects was 0.06+/-0.04 and of nuclear buds (NB) 0.12+/-0.05. The canonical correlation analyses indicate a positive non-significant correlation between the MN frequency and age, gender and smoking habits. Results of factor structure and canonical weights showed that age and gender rather than smoking habits control the incidence of MN in PBL of healthy volunteers. The lowest median value of MN was observed in subjects younger than 30 years (both smoking and non-smoking). Generally, non-smokers had lower median values of MN compared to smokers. In non-smokers, males showed lower micronucleus incidence than females. Within the non-smokers smaller differences in the median values of MN between subgroups (male and female; age subgroups) were observed. Among smokers, females had a two-fold higher median value of MN frequency than males, but this difference was not significant (p=0.2643, Mann-Whitney U test). Canonical correlation analyses indicate a strong and significant correlation between cell proliferation parameters (M1-M4 and CBPI) and age, gender and smoking habits. The most sensitive parameters were M3 and M4. Age had the strongest effect on M3, while M4 was highly influenced by smoking habits. Gender had an equal non-significant effect on both parameters. The usefulness of the new criteria for the cytokinesis-block MN assay measuring DNA damage as a sensitive biomarker in biomonitoring studies is confirmed.     

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.     

Non-invasive exploration of neonatal gastric epithelium by using exfoliated epithelial cells

Background & Aims

In preterm infants, exfoliated gastric epithelial cells can be retrieved from aspirates sampled through the naso-gastric feeding tube. Our aims were to determine (1) whether the recovery of exfoliated cells is feasible at any time from birth through the removal of the nasogastric tube, (2) whether they can be grown in culture in vitro, and (3) whether the physiological state of exfoliated cells expressing H+/K+ -ATPases reflects that of their counterparts remaining in situ at the surface of the gastric epithelium in neonatal rat pups.

Methods

In infants, gastric fluid aspirates were collected weekly after birth or every 3 hours over 24-h periods, and related to clinical parameters (Biocollection PROG/09/18). In rat pups submitted to a single fasting/refeeding cycle, we explored circadian exfoliation with the cellular counter-parts in the gland. All samples were analyzed by confocal imaging and Enzyme-Linked Immunosorbent Assay.

Results

Epithelial cells were identified by microscopy using membrane-bound anti-H+/K+ ATPases antibody, assessed for nucleus integrity, and the expression of selected proteins (autophagy, circadian clock). On 34 infants, the H+/K+ -ATPase-positive cells were consistently found quiescent, regardless of gestational age and feeding schedule from day-5 of life to the day of removal of the naso-gastric tube. By logistic regression analysis, we did find a positive correlation between the intensity of exfoliation (cellular loss per sample) and the postnatal age (p<0.001). The H+/K+ ATPase-positive cells established in culture retained the expression of a biomarker of progenitor status (Pouf5F1-Oct4). In rat pups, the expression pattern of Survivin in H+/K+ ATPase-positive exfoliated cells paralleled that observed in cells remaining at the surface of the gastric gland.

Conclusions

Tracking parietal cells can improve clinical monitoring and understanding of the autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway.     

The micronucleus assay in human exfoliated cells of the nose and mouth: application to occupational exposures to chromic acid and ethylene oxide

We have applied the micronucleus (MN) assay to exfoliated cells of buccal and nasal cavities to monitor the genotoxic risk in a group of workers exposed to chromic acid and in another group exposed to ethylene oxide (EtO). The first group comprised 16 subjects working in a 'hard' type chrome-plating factory showing increased chromium absorption and chromium-induced rhinopathy. The second group comprised 9 subjects working in a sterilization unit, exposed to EtO concentrations lower than 0.38 ppm as timed weighted average (TWA) for a working shift; 3 of them were involved in a acute exposure too. The frequency of MN in buccal mucosa was within the norm for exposure both to chromium and to EtO. The MN frequency in nasal mucosa was not altered in chromium platers, whereas a significant increase (p less than 0.01) in MN was found in 2 out of 3 subjects involved in the accidental EtO leakage and a non-significant increase in MN was found in the group chronically exposed to EtO.     

Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay

The in vitro genotoxicity of PLGA-PEO (poly-lactic-co-glycolic acid-polyethylene oxide copolymer) nanoparticles was assessed in TK6 cells using the comet assay as well as cytokinesis-block micronucleus (CBMN) assay. The cells were exposed to 0.12-75μg/cm2 of PLGA-PEO nanoparticles during 2 and 24h for analysis in the comet assay, and to 3-75μg/cm2 of these nanoparticles during 4, 24, 48 and 72h, respectively, for analysis in the CBMN assay. Two different protocols for treatment with cytochalasin B were used. We found that PLGA-PEO was neither cytotoxic (measured by relative cell growth activity and cytokinesis-block proliferation index (CBPI)), nor did it induce DNA strand-breaks (detected by the comet assay) or oxidative DNA lesions (measured by the comet assay modified with lesion-specific enzyme formamidopyrimidine-DNA-glycosylase). There were no statistically significant differences in the frequencies of micronucleated binucleated cells (MNBNCs) between untreated and treated cells in either of the conditions used. This suggests that PLGA-PEO did not have potential genotoxicity. However, using two experimental protocols of the micronucleus assay, PLGA-PEO nanoparticles showed a weak but significant increase in the level of MN in mononucleated cells, in cells treated for 48h with PLGA-PEO nanoparticles when cytochalasin B was added for the last 24h (1st protocol), and in cells treated for 24h with PLGA-PEO nanoparticles followed by washing of NPs and addition of cytochalasin B for another 24h (2nd protocol). It remains unclear whether the increase of MNMNC after treatment with PLGA-PEO nanoparticles is the effect of a possible, weak aneugenic potential or early effect of these particles, or due to another reason. These results suggest that aneugenicity in addition to clastogenicity may be considered as an important biomarker when assessing the genotoxic potential of polymeric nanoparticles.     

Application of the micronucleus test to exfoliated epithelial cells from the oral cavity of beedi smokers, a high-risk group for oral cancer

The primary sites for occurrence of oral cancer include the buccal mucosa, tongue, alveolus, palate, lip and the floor of the mouth. In this study, an attempt was made to estimate the cytogenetic damage in different regions of the oral mucosa in people habituated to smoking beedi,which is one of the major forms of tobacco consumption in India and believed to be a major risk factor for oral cancer. By using the micronucleus assay on exfoliated cells from the buccal mucosa, palate and tongue of beedi smokers, we examined an early cellular response to the effect of beedi smoking. A total number of 50 randomly selected male subjects were included in the study. Case and control groups (smokers and non-smokers, respectively) comprised 25 subjects each. The difference in mean micronucleated cell count between cases and controls was significant (P <0.01) for buccal mucosa and palate, but not for tongue. The correlation between age and micronucleus cell count was weak for both cases (r=0.27) and controls (r=0.36).     

Assessment of local genotoxic effects of formaldehyde in humans measured by the micronucleus test with exfoliated buccal mucosa cells

Volunteers (10 women, 11 men) were exposed to formaldehyde (FA) vapors for 4h per day over a period of 10 working days under strictly controlled conditions. Exposure varied randomly each day from constant 0.15 ppm up to 0.5 ppm with four peaks of 1.0 ppm for 15 min each (13.5 ppm h cumulative exposure over 10 working days). FA was masked on four days by co-exposure to ethyl acetate. During exposure, subjects had to perform bicycle exercises (about 80 W) three times for 15 min. Buccal smears were prepared 1 week before the start of the study (control 1), at the start of the study before the first exposure (control 2), at the end of the exposure period of 10 days and 7, 14 and 21 days thereafter. Two thousand cells per data point were analyzed for the presence of micronuclei (MN) and the frequency of MN per 1000 cells was determined on slides coded by an independent quality-assurance unit. No significant increase in the frequency of MN was measured at any time point after the end of the exposure. Twenty-one days after the end of the exposure MN frequencies were significantly lower in comparison with control 1. This study, which was performed under GLP-like conditions, clearly indicates that FA does not induce MN in buccal mucosa cells after peak exposures up to 1 ppm and a cumulative exposure of 13.5 ppm h over 2 weeks.     

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.