Mutagenesis, Vol. 16, No. 5, 449-452,
September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
The micronucleus assay in human exfoliated urothelial cells: application in a genotoxicity study of workers exposed to a mineral jelly containing sodium nitrite and N-phenyl-1-naphthylamine
Institut de Médecine du Travail, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cedex, 1 INSERM, EMIU 00-10, Faculté de Médecine, 2 bis Boulevard Tonnelé, Tours and 2 Service d'Épidémiologie, Économie de Santé et Prévention, CHU, Hôtel Dieu, Boulevard Léon Malfreyt, 63000 Clermont-Ferrand, France
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Abstract |
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Exposure to certain chemical agents in occupational settings has been identified as carcinogenic to the human bladder. Micronucleus (MN) analysis in exfoliated urothelial cells is an interesting method for biomonitoring genetic damage in human populations. However, few studies have been performed in an occupational context. The aim of this study was to examine whether the occupational use of a mineral jelly induced a genotoxic risk for workers employed at a single factory producing bearings using the MN test on exfoliated urothelial cells. The prevalence of micronucleated exfoliated urothelial cells (MNC) was determined in 35 female workers with dermal exposure to the jelly and 41 female controls. The mean percentage of MNC (expressed as percent cells with MN per 1000 cells scored) observed in the exposed worker group was 0.46 ± 0.11% (range 0–2.8) and in the control group 0.14 ± 0.03% (range 0–0.8). There is a significant job effect (P = 0.0018, MANCOVA) on the prevalence of MNC, whereas age and smoking habit had no significant effect (P = 0.90 and 0.91, respectively). There is no interaction between job and smoking habit (P = 0.4421). Exposure to the mineral jelly appeared to be the main factor inducing the increased prevalence of MNC. This may be due to the presence of mutagens/carcinogens in the jelly: an aromatic amine, N-phenyl-1-naphthylamine (CAS no. 90-30-2), which is carcinogenic in mice, or sodium nitrite (CAS no. 7632-00-0), which is genotoxic in human cell systems. In conclusion, these results suggest that use of the mineral jelly could present a genotoxic risk for workers. We think that the MN assay on exfoliated cells could be valuable for biological monitoring purposes in occupational contexts as a marker of significant exposure to bladder mutagenic/carcinogenic agents.
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Introduction |
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Several occupations or industries appear to be associated with increased risk of bladder cancer (Risch et al., 1988
; Steineck et al., 1990). Some chemical agents, such as polycyclic aromatic hydrocarbons, some aromatic amines, N-nitroso compounds and their metabolites, have been reported to be potent bladder carcinogens in animals and/or humans (International Agency for Research on Cancer, 1987). The carcinogenicity of these chemicals may operate via genotoxic mechanisms (Sasaki et al., 1999). The micronucleus (MN) assay using human cells has been proposed as a biomarker for detecting genotoxic damage from exposure to mutagenic/carcinogenic substances.
MN are microscopically visible, round or oval cytoplasmic bodies next to the nucleus and separated from it. They are formed by acentric chromosome fragments or whole chromosomes which are not incorporated into daughter nuclei during cell division. The MN assay measures the frequency of MN, usually reported as the number of cells containing MN per total cells counted. This assay was first used in exfoliated cells by Stich et al. (1983). Some investigators have in recent years explored the benefits of performing this test in exfoliated urothelial cells in urine samples as a biological marker of the genotoxic effects of bladder mutagenic/carcinogenic agents (Ribeiro et al., 1990; Cid et al., 1991; Rosin and Anwar, 1992; Moore et al., 1993; Lehucher-Michel et al., 1995; Burgaz et al., 1995; Murray and Edwards, 1999).
To our knowledge, very few studies have been conducted using this biomarker in exfoliated urothelial cells to evaluate the relationship between occupational exposure and genotoxic effects in the human bladder (Ribeiro et al., 1990; Cid et al., 1991; Murray and Edwards, 1999). It was therefore considered interesting to examine whether the occupational use of a mineral jelly containing sodium nitrite (CAS no. 7632-00-0) and N-phenyl-1-naphthylamine (CAS no. 90-30-2) could present a genotoxic risk for workers employed at a factory producing bearings using the MN assay on exfoliated urothelial cells. N-phenyl-1-naphthylamine may be carcinogenic in mice (Wang et al., 1984) and sodium nitrite is genotoxic in human cell systems (Luca et al., 1987; Routledge et al., 1994).
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Materials and methods |
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Subjects
The study was conducted on an exposed group of 35 healthy females and a control group of 41 healthy females from the same area after obtaining informed consent. Information regarding medication, smoking status and medical and occupational histories was obtained by questionnaires.
The exposed subjects were employed at a single factory producing bearings and had been dermally exposed to the mineral jelly for 20 years. On the basis of work histories the control subjects were believed not to be exposed to known bladder-specific mutagenic/carcinogenic agents. None of the subjects had received any medical treatment (in particular non-steroidal anti-inflammatory drugs, anti-cancer therapy or vitamin supplements) or pelvic or abdominal radiography for at least 1 year.
The absence of urinary tract disease was medically verified by paper reagent strips and urinary cytology.
Urine sampling and slide preparation
Urine samples (300–500 ml) were collected in plastic bottles containing a fixative solution (methanol/acetic acid, 3:1) from morning to afternoon and stored at 4°C until preparation of slides. The first void of the day was not collected because degradation of exfoliated bladder cells occurs when the cells have been in contact with urine overnight. Exfoliated cells were isolated from urine samples by centrifugation and slides prepared according the method previously described by Lehucher-Michel et al. (1995, 1996). Exfoliated cells from the bladder were concentrated by centrifugation for 10 min (400 g). Following a 20 min storage period at 4°C, cells were submitted to a second 10 min centrifugation and fixed in a fixative solution (methanol/acetic acid, 3:1). The fixative solution was partially but not completely removed, the pellet was resuspended, dropped onto pre-cleaned microscope slides and dried for 24 h at room temperature.
MN scoring procedure
Slides were first examined under low magnification to determine the quality of the slides and the presence of inflammatory signs (bacteria, leukocytes). Infections need to be recognized because they may cause irritation in the bladder and lead to an increased number of MNC (Rosin and Anwar, 1992; Moore et al., 1993)
A fluorescent dye (propidium iodide at 1 µg/ml in antifade solution) was used to counterstain the DNA. A microscope equipped with epifluorescent illumination was used to examine the slides. Coded slides were read at 400x magnification to detect MN and confirmation was performed at 1000x (Burgaz et al., 1995). Only cells that were not smeared or overlapped and that contained intact nuclei were recorded. Micronucleated cells (MNC) were scored according to criteria proposed by Stich et al. (1983). All questionable MN were cross-checked by two readers and were disregarded according to strict adherence to the scoring criteria. For each individual, 1000 intact urothelial cells were examined for the presence of MN.
Statistical analysis
Differences between exposed worker and control characteristics were examined using the 
2 and exact tests. The prevalence of MNC was first calculated for each individual and MNC prevalences were then averaged to obtain the MNC frequency values for each group: exposed workers and control subjects. The results are expressed as percentage MNC. We applied a multivariate analysis of covariance (MANCOVA) on MNC counts taking into account age as a covariate and job (2 classes: workers and controls) and smoking habit (3 classes: non-smokers, moderate smokers, <10 cigarettes/day, and heavy smokers, 
10 cigarettes/day) as factors. A significance level of 0.05 was chosen. Statistical analyses were performed using the PC SAS software (SAS Institute).
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Results |
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Individual characteristics of exposed workers and controls are reported in Tables I and II
, respectively. Both groups were similar for age (exposed workers, 41.3 ± 1.6 years; controls, 42.8 ± 1.4 years), smoking habit (exposed workers, 16/35 smokers, 45.7%; controls, 15/41 smokers, 36.6%) and alcohol consumption (exposed workers, 8/35 consumers; controls, 9/41 consumers).
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The mean percentage of MNC observed in the exposed worker group was 0.46 ± 0.11% (range 0–2.8) and in the control group 0.14 ± 0.03% (range 0–0.8) (Table III
). There is a significant job effect (P = 0.0018) on the prevalence of MNC, whereas age and smoking habit had no significant effect (P = 0.90 and 0.91, respectively) (Table III). There is no interaction between job and smoking habit (P = 0.4421).
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For the MANCOVA we did not take into account alcohol consumption as a factor because the two groups are similar and there were few consumers in each group but mainly because alcohol consumption is not known to be a carcinogen for the bladder nor to induce an increase in the prevalence of micronucleated urothelial cells.
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Discussion |
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Application of the MN assay to exfoliated cells of various human tissues provides evidence of genotoxic damage associated with exposure to environmental mutagens or carcinogens. (Ribeiro et al., 1990
; Cid et al., 1991; Rosin and Anwar, 1992; Moore et al., 1993; Lehucher-Michel et al., 1995; Burgaz et al., 1995; Murray and Edwards, 1999). MN in exfoliated bladder cells result from genotoxic damage in basal cells of the urothelium (Vine, 1990). Epithelial cells are replaced in the bladder every 1–3 weeks (Stich et al., 1983; Moore et al., 1997a,b). Because epithelial cells are derived from basal cells, recent genetic damage to the basal layer can be reflected in the presence of micronucleated exfoliated cells (Moore et al., 1997a,b).
In this study the MN assay was applied to exfoliated epithelial cells of the bladder from workers with dermal exposure to a mineral jelly to evaluate the genotoxic risk. The exposed workers had significantly higher mean levels of MNC than controls (Table III
). There is still a difference in the absence of the main confounding effect of smoking (Table III). These results may represent damage to urothelial cells associated with use of the jelly in the weeks prior to urine collection.
Most mutagens and carcinogens act via genotoxic mechanisms and thus mutagenic or carcinogenic substances might be present in the jelly. The genotoxic damage may be due to one or both of the jelly compounds, i.e. sodium nitrite (CAS no. 7632-00-0) and N-phenyl-1-naphthylamine (CAS no. 90-30-2). Sodium nitrite can be genotoxic by itself in animal and human cellular systems (Luca et al., 1987; Routledge et al., 1994). Morever, in the presence of amines it can lead to formation of carcinogenic N-nitroso compounds (Spieglhalder and Preussmann, 1983). A metabolite of N-phenyl-1-naphthylamine, 
-naphthylamine (CAS no. 134-32-7) is mutagenic to Salmonella but does not induce MN in mice (Sasaki et al., 1999). The alkaline single cell gel electrophoresis assay (Comet assay), which detects DNA lesions, produces a positive response in the bladders of mice treated with -naphthylamine. According to Naito et al. (1995) it has probably no carcinogenic effect on animals and little, if any, carcinogenic effect on humans. The latter effect can be attributed to the presence of a second compound (an impurity), such as an isomer of -naphthylamine, ß-naphthylamine, which is carcinogenic (Naito et al., 1995). Nevertheless Wang et al. (1984) suggested that both technical grade and pure N-phenyl-1-naphthylamine (CAS no. 90-30-2) may be carcinogenic in mice following repeated s.c. injection. Furthermore, commercial N-phenyl-1-naphthylamine can contain ß-naphthylamine as an impurity.
Cigarette smoking is known to be a risk factor for bladder cancer. The carcinogens in cigarette smoke include polycyclic aromatic hydrocarbons, aromatic amines and nitrosamines (International Agency for Research on Cancer, 1986). DNA adducts identified as the arylamine 4-aminobiphenyl are present in exfoliated bladder cells of smokers and carcinogen–DNA adducts have been associated with mutation and chromosome damage in vitro and in vivo (Talaska et al., 1991a,b). Some studies have compared the frequency of MN in exfoliated urothelial cells from smokers and from non-smokers (Reali et al., 1987; Lehucher-Michel et al., 1995; Burgaz et al., 1997). Significantly higher levels were found in smokers than in non-smokers. Furthermore, Rosin and Anwar (1992) observed that individuals infected with Schistosoma haematobium who smoke may have more chromosome breakage in the bladder. Finally, a synergistic effect of cigarette smoking and alcohol consumption on the frequency of MN in buccal mucosa cells has been observed (Stich et al., 1982). The last two groups suggest that tobacco smoke could have an additive or synergistic effect with other factors inducing MNC. We did not observe such an effect in our study.
The development of screening and biological monitoring methods is highly desirable for the assessment and management of the risk to human health. The MN assay on urothelial cells might be pertinent for these purposes. Besides the general advantages of urinary biomarkers which can be used in an occupational context, i.e. non-invasive, ready accessibility and self-collection, the MN assay can be applied to the target cells of bladder carcinogens. However, few studies have been performed in occupational settings. This may be due to technical problems, for example related to the poor quality of urothelial cells in urine and to difficulties in identifying MN (,Lehucher-Michel et al.1995).
Ribeiro et al. (1990) performed a cytogenic study on workers occupationally exposed to a mixture of aromatic amines and other compounds in a product plant and Cid et al. (1991) studied leather tanning workers. They did not find a significant difference between exposed and control individuals in either study, although the mean levels of MNC in the exposed group were slightly higher than in the control group. They concluded that occupational exposure did not produce genotoxic effects in the populations studied. Murray and Edwards (1999) used the MN assay to assess the risk of cytogenotoxic damage in workers exposed to 4,4'-methylene bis(2-chloroaniline) (MOCA). The MOCA workers (n = 12) had a significantly higher mean number of MNC than controls (9.69 ± 0.32 versus 5.18 ± 0.11 MNC per 1000 cells). They concluded that it may be prudent to reduce the current exposure to MOCA and that the measurement of MN in exfoliated urothelial cells may be useful as a preliminary screen to identify groups of workers with higher exposure and in whom further analysis may be advisable.
As did these authors, we think that the MN assay on exfoliated cells could be valuable for biological monitoring purposes in occupational contexts as a marker of significant exposure to bladder mutagenic/carcinogenic agents.
In conclusion, the current results led to primary preventive action in the factory to avoid dermal contact with the mineral jelly. Further study of workers after avoidance of dermal contact must be conducted to confirm the role of the mineral jelly in increased MNC prevalence and to explore the relevance of the MN test in biological monitoring of workers exposed to bladder mutagens or carcinogens.
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Acknowledgments |
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This study was supported by grants from the Ligue Contre le Cancer, Comité Départemental du Puy de Dôme, the CRAM Centre and CRAM Auvergne.
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Notes |
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3 To whom correspondence should be addressed. Tel: +33 473 608 036; Fax: +33 473 274 649
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References |
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Received on October 9, 2000; accepted on June 6, 2001.
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