Mutagenesis, Vol. 17, No. 1, 89-93,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o-phenylphenol
Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA and 1 Department of Biology, Oakwood College, Huntsville, AL 35896, USA
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Abstract |
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o-Phenylphenol (OPP), a widely used fungicide and antibacterial agent, has been considered to be among the top 10 home and garden pesticides used in the USA. Earlier studies have consistently shown that the sodium salt of OPP (SOPP) causes bladder cancer in male Fischer 344 (F344) rats, whereas OPP has produced variable results. This difference has been attributed to the presence of the sodium salt. To determine cellular and genetic alterations in the rat bladder and the influence of the sodium salt, F344 rats were administered 2% OPP, 2% NaCl and 2% NaCl + 2% OPP in their diet for 14 days. Twenty-four hours before being killed the animals were administered 5-bromo-2'-deoxyuridine (BrdU) by i.p. injection. Bladder cells were isolated, stained with DAPI and scored for the presence of micronuclei and incorporation of BrdU into replicating cells. To determine changes in chromosome number, we used fluorescence in situ hybridization (FISH) with a DNA probe for rat chromosome 4. Significant increases in the frequency of micronuclei and BrdU incorporation were seen in bladder cells of rats from all treatment groups. In contrast, the frequency of hyperdiploidy/polyploidy in treated animals was not increased over that seen in controls. A high control frequency of cells with three or more hybridization signals was seen, probably due to the presence of polyploid cells in the bladder. The presence of polyploid cells combined with cytotoxicity and compensatory cell proliferation makes it difficult to determine whether OPP is capable of inducing aneuploidy in the rat urothelium. In summary, these studies show that OPP can cause cellular and chromosomal alterations in rat bladder cells in the absence of the sodium salt. These results also indicate that at high concentrations the sodium salt can enhance chromosomal damage in the rat urothelium.
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Introduction |
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o-Phenylphenol (OPP) and its sodium salt sodium o-phenylphenate (SOPP) are broad spectrum fungicides and antibacterial agents, which are extensively used in a variety of agricultural and non-agricultural applications. SOPP is commonly used for post-harvest treatment of citrus fruits and vegetables for the prevention of mold, whereas OPP is employed as a hospital and household disinfectant (Allan, 1991
). The National Occupational Exposure Survey, conducted by the National Institute of Occupational Safety and Health, has estimated that >650 000 non-agricultural US workers may be occupationally exposed to OPP and SOPP (National Institute for Occupational Safety and Health, 1999). In addition, OPP has been considered to be among the top 10 home and garden pesticides in the USA (Grossman, 1995). This widespread use of OPP suggests the potential for substantial consumer and occupational exposure.
Due to their widespread usage, these compounds have undergone extensive testing. OPP and SOPP have generally been reported as inactive or weakly active in in vitro and in vivo short-term standard genotoxicity tests (for a review see Stouten, 1998). In vivo assays, like the dominant lethal assay in mice and the bone marrow micronucleus assay in mice and rats, have also yielded negative results (Stouten, 1998). Following the administration of OPP to rats, significant increases in protein binding have been seen in the rat bladder (Reitz et al., 1983, 1984). Negative or mixed results have been seen for DNA binding in the rat bladder, using liquid scintillation counting or the 32P-post-labeling technique (Pathak and Roy, 1992; Ushiyama et al., 1992; Smith et al., 1998). Recently, the highly sensitive accelerator mass spectrometry (AMS) was used to quantitate macromolecular binding occurring in the bladder of OPP-treated rats. No increases in DNA binding were detected, whereas dose-related protein binding in the bladder was confirmed using this technique (Kwok et al., 1999).
SOPP has been consistently shown to induce tumors in the urinary tract of male F344 rats (Hiraga and Fujii, 1981; Fukushima et al., 1989; Hasegawa et al., 1991), whereas OPP has produced more variable results (Hiraga and Fujii, 1984; Fukushima et al., 1985, 1989; Wahle and Christenson, 1996; Wahle et al., 1997). Tumors in SOPP-fed rats occurred at lower comparable doses, with shorter latency periods and were more malignant than those that occurred in the positive studies of OPP-treated rats. These findings and pathological observations (Fukushima et al., 1985) have led researchers to propose that the carcinogenic strength of SOPP was greater than that of OPP and that this effect can be attributed primarily to the sodium salt. Other researchers contend that the difference in potency between OPP and SOPP is due to the difference in urinary pH of rats administered the two chemicals (Fujii et al., 1987).
Sodium salts of several acids, such as ascorbate, glutamate, aspartate, citrate, erythorbate, bicarbonate and, to a limited extent, chloride, have produced effects on urothelial proliferation and tumorigenesis in the rat bladder (Cohen et al., 1995). It has also been shown that high concentrations of sodium appear to be critical for the urothelial effects of some of these compounds (Cohen et al., 1995). Acid saccharin had no effect on urinary bladder epithelial proliferation, whereas the greatest effect was seen with the sodium salt, followed by the potassium and calcium salts (Hasegawa and Cohen, 1986). These observations have led researchers to hypothesize that an elevation in urinary pH and Na+ concentration act as promoters in the development of urinary bladder cancers in the rat.
Because of its inactivity in standard genotoxicity assays and the general absence of DNA binding, OPP has been considered to act via non-genotoxic mechanisms. However, few genotoxicity assays have been conducted in the rat bladder, the target organ of OPP carcinogenesis. The aim of this study was to determine cellular and genetic alterations in the rat bladder following treatment with OPP and to investigate the influence of the sodium salt on the tumorigenicity of OPP.
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Materials and methods |
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Test chemicals
2-Phenylphenol (OPP) (CAS no. 90-43-7) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Sodium chloride was obtained from Sigma Chemical Co. (St Louis, MO).
Animals
Male F344 rats (~16 weeks old) were obtained from Harlan Sprague–Dawley (Indianapolis, IN). All animals were acclimated for 7 days prior to use. The animals were housed in polycarbonate cages in an environmentally controlled room (maintained at 22°C and 50% relative humidity on a 12 h photocycle) and fed a diet of Formulab rat chow (PMI Feeds, St Louis, MO). The rats were randomly divided into four groups. Food and water were available ad libitum throughout the experiment. Results for 5–9 animals per group pooled from three separate experiments are presented in this study.
Experimental procedure
Diets containing 2% OPP, 2% NaCl and 2% NaCl + 2% OPP were prepared by mixing the compound with water and Formulab rat chow. The animals were fed the diet for 14 days. Body weights of the animals were monitored daily. The rats were killed on day 14. Twenty-four hours before being killed they were administered 5-bromo-2'-deoxyuridine (BrdU) (100 mg/kg) in DMSO/saline (1:2) by i.p. injection. For each animal, the bladder was removed, rinsed in ice-cold saline and inverted over PE160 tubing. The bladder was then tied securely with 00 silk thread and inflated with 0.9% NaCl using an 18 gauge needle and a 1 ml syringe. The epithelial cells were scraped off the exposed luminal surface of the inflated bladders, using a 22 mm2 no. 2 glass coverslip, into a clean Petri dish containing ice-cold saline. The cells were transferred to a 15 ml screw cap centrifuge tube with a Pasteur pipet and stored on ice. The Petri dish was rinsed once with ~5 ml of saline, which was then added to the tube containing the cells. The bladder cells were centrifuged at 300 g for 5 min at room temperature, the supernatant aspirated off and the cells resuspended in 1 ml of saline. Single cell preparations were made by vigorously pipetting the cell suspension with a Pasteur pipet and by vortexing. The cell suspension was transferred to glass slides using a Cytospin 2 cytocentrifuge (Shandon, Pittsburg, PA) at 600 r.p.m. for 5 min at room temperature. The slides were air dried, fixed in 100% methanol for 30 min and stored under nitrogen in the presence of anhydrous calcium sulfate at –20°C until use.
BrdU labeling and micronucleus assay
Replicating cells were studied using BrdU incorporation and determining the labeling index. BrdU labeling was conducted by denaturing the cells in 0.07 N NaOH followed by neutralization with phosphate-buffered saline (PBS). The slides were then incubated with an anti-BrdU antibody (Becton Dickinson Immunocytometry Systems, CA), diluted in 0.5% Tween-20 in PBS, in a humidified chamber for 30 min. The antibody was then detected by incubation with Texas Red-conjugated goat anti-mouse IgG (10 µg/ml; Molecular Probes, Eugene, OR) in a humidified chamber for 30 min. After washing the slides in PBS, the DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1 µg/ml) in antifade mounting medium.
Fluorescence in situ hybridization
The chromosome 4-specific DNA probe (Hoebee and de Stoppelaar, 1996) was a generous gift from Dr Barbara Hoebee (Bilthoven, The Netherlands). It was amplified and labeled with digoxigenin-dUTP (Boehringer-Mannheim, Indianapolis, IN) by nick translation in our laboratory. Previously described methods were used to perform the FISH experiments (Eastmond and Pinkel, 1990; Trask and Pinkel, 1990). To facilitate penetration of the FISH probe into aged slides, cells were incubated in 0.1% saponin (0.05 g saponin, 0.019 g EGTA in 50 ml ddH2O) for 30 min at room temperature, transferred to a pepsin solution (1 µg/ml in 0.01 N HCl) at room temperature for 30 min and incubated with 100 µl of a proteinase K solution (1 µg/ml in 10 mM Tris, pH 7.5, 10 mM EDTA, 150 mM NaCl) at 37°C in a humidified chamber for 10 min. Lastly, the slides were treated with 2% paraformaldehyde for 1 min at 4°C. The slides were washed in PBS in between each of the above steps. The target DNA was denatured in 70% formamide, 2x SSC (72°C), the hybridization mixture added and the target and probe incubated overnight at 37°C. The denatured hybridization cocktail consisted of 1 µl of digoxigenin-labeled probe, 1 µl sheared herring sperm DNA (1 mg/ml; Sigma), 1 µl ddH2O and 7 µl of MM2.1 hybridization mix (to give a final concentration of 55% formamide, 1x SSC, 10% dextran sulfate) (see Trask and Pinkel, 1990, for additional details). Post-hybridization washes were performed in 0.1x SSC, pH 7.0, three times for 5 min at 65°C. The slides were then rinsed three times in PN buffer (0.1 M phosphate buffer, pH 8.0, containing 0.5% NP-40) at room temperature. The digoxigenin-labeled probe was detected using a FITC-conjugated sheep anti-digoxigenin antibody (20 µg/ml in PN buffer with 5% non-fat dry milk supernatant; Boehringer-Mannheim). DAPI (0.5 µg/ml) in diphenylenediamine antifade solution) was used to counterstain the DNA.
Microscopy and scoring criteria
A Nikon fluorescence microscope equipped with a FITC/DAPI/Texas Red filter (Chroma Technology Corp., Brattleboro, VT) was used to visualize the fluorescent signals at 1250x magnification. All slides were randomly coded prior to scoring. 2000 cells for each bladder were scored for micronuclei. Only micronuclei clearly delineated from the main nucleus were scored as micronucleated cells (Countryman and Heddle, 1976). It should be noted that due to the presence of binucleated cells and the difficulty of distinguishing the membrane, individual intact nuclei were scored as cells and these nuclei are referred to as cells throughout the text and in the figures. In some circumstances, where the membrane was not clearly visible, a micronucleus was scored based on proximity to a main nucleus.
For BrdU labeling, the DNA in the nucleus was scored as completely labeled if the entire nucleus was deep red or uniformly labeled and partially incorporated if half or less of the nucleus was labeled with Texas Red. Only intact nuclei were scored. 2000 nuclei for each bladder were counted to determine the percentage of nuclei incorporating BrdU into their DNA. The replication index was calculated as the number of nuclei incorporating BrdU divided by the total number of nuclei counted.
Previously described criteria were used for scoring hyperdiploidy in bladder cells (Eastmond and Pinkel, 1990). The frequency of hybridization regions per nucleus was determined from coded slides by scoring a minimum of 1000 cells/rat. A nucleus containing three or more chromosome 4 hybridization regions was considered as a hyperdiploid cell.
Statistical analyses
The frequencies of micronuclei, total BrdU labeling and hyperdiploidy in rat bladder cells were compared using the non-parametric Kruskal–Wallis ANOVA with the Mann–Whitney U-test used as a post hoc test. Critical values were determined using a 0.05 probability of Type I error.
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Results |
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To determine cellular and genetic alterations induced by OPP, the frequencies of micronuclei, BrdU incorporation and hyperdiploidy occurring in rat bladder epithelial cells were studied following treatment of male F344 rats with a control diet and diets containing 2% NaCl, 2% OPP and 2% OPP + 2% NaCl.
Body weights
Body weights of the rats were recorded on a daily basis (data not presented). The rats fed the control diet and 2% NaCl diet gained weight daily but the animals on 2% OPP and 2% NaCl + 2% OPP lost weight during the initial 2–3 days of treatment. After day 4, all animals gained weight at a similar rate regardless of dietary treatment.
Micronuclei
The micronucleus assay was used to quantitate chromosomal breakage or loss occurring in rat bladder epithelial cells and the results are presented in Figure 1. The frequency of micronuclei in the controls averaged 0.27%, whereas the frequencies in the 2% NaCl-treated rats increased to 0.72%, a significant elevation (P = 0.0047) above the control value. OPP-treated rats showed a 4-fold increase in the occurrence of micronuclei over the controls (1.05%) (P = 0.0005). Rats treated with 2% NaCl + 2% OPP showed the highest frequency of micronuclei (1.6%), which was significantly higher (P = 0.0031) than that seen in the controls, the 2% OPP- and 2% NaCl-treated rats.
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BrdU incorporation
To examine cell proliferation in rat bladder epithelial cells, BrdU labeling was employed. Total incorporation of BrdU in the 2% OPP-treated animals (13.1%) was ~40-fold that of the controls (0.31%, P = 0.0003), while the total labeling seen in the 2% NaCl-treated rats (1.29%) was also significantly elevated (P = 0.0005) over the controls (Figure 2
). Labeling of cells with BrdU in the 2% NaCl + 2% OPP treatment (4.1%) was significantly lower (P = 0.0041) than that in the 2% OPP-treated rats, but significantly elevated (P = 0.005) over the controls.
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Hyperdiploidy/polyploidy
Induction of numerical chromosomal changes in rat bladder epithelial cells was studied using FISH with a chromosome 4-specific probe. The frequency of hyperdiploidy/polyploidy in the controls was 0.32%, whereas the frequency in the 2% NaCl-treated rats was 0.46%, which did not differ significantly from the controls (P = 0.482) (Table I
). The frequency in the 2% OPP-treated rats (0.37%) was also not significantly elevated (P = 0.817) over the controls. The frequency of hyperdiploid/polyploid cells in the 2% NaCl + 2% OPP group was 0.5%, which was slightly higher (P = 0.047) than that of the 2% OPP-treated rats, but did not differ significantly from the controls.
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Discussion |
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In this study we have attempted to investigate cellular and genetic end-points in the rat which are relevant to OPP-induced bladder carcinogenesis. The three end-points assayed in the bladder were the micronucleus assay, FISH with rat chromosome-specific DNA probes and cell proliferation using BrdU incorporation.
The OPP-treated rats exhibited a modest but significant increase in micronucleated bladder cells over that seen in the controls. This contrasts with previous studies of OPP in rat and mouse bone marrow (Stouten, 1998), in which no increases in micronuclei were seen. This highlights the importance of assessing genotoxicity in the target organ, rat bladder epithelial cells. In addition to the increase seen in the 2% OPP-treated rats, the frequency of micronuclei was increased ~3-fold in 2% NaCl-treated rats and ~6-fold in 2% NaCl + 2% OPP-treated animals. This indicates not only that NaCl can have a genotoxic effect in the rat bladder but also that the combination of NaCl and OPP shows an additive genotoxic effect. Micronuclei can be formed either by chromosomal breakage or chromosomal loss. At this point it is not known whether micronuclei were formed as a result of chromosome loss or breakage. Previous studies have indicated that in the presence of metabolic activation OPP and its metabolites can induce the formation of sister chromatid exchanges and chromosomal aberrations in CHO-K1 cells (Tayama-Nawai et al., 1984; Tayama et al., 1989). The formation of centromere-containing micronuclei in vitro has also been reported in arachidonic acid-supplemented V79 cells treated with phenylhydroquinone (PHQ), a primary metabolite of OPP (Lambert and Eastmond, 1994). Micronuclei can also arise as a result of oxidative damage. The generation of 8-hydroxydeoxyguanosine in calf thymus DNA (Nagai et al., 1995), in CHO-K1 cells (Nakagawa and Tayama, 1996) and in V79 cells (Henschke et al., 2000) treated with PHQ has also been reported. Recent observations that OPP does not bind to DNA (Smith et al., 1998; Kwok et al., 1999) suggest that micronuclei may also be formed through an indirect mechanism, such as interference with the mitotic spindle, inhibiting enzymes important in DNA replication or by initiating oxidative stress (van Zeeland et al., 1982; Yager and Wiencke, 1997; Vanni et al., 1998). Micronuclei may also arise as a secondary effect in response to cytotoxicity or regenerative hyperplasia. As indicated in the results, clear increases in cell proliferation (~40-fold) were seen at the high, presumably cytotoxic, dose of OPP administered to the rats in this study.
The observed induction of cell proliferation by OPP is consistent with previous reports in which OPP has been shown to produce superficial cytotoxicity of the urothelium with regenerative hyperplasia (Morimoto et al., 1987; Hasegawa et al., 1990; Smith et al., 1998). Cytotoxicity to the rat urothelium with subsequent cell proliferation has been proposed as a possible mechanism for OPP-induced carcinogenicity (Morimoto et al., 1987; Hasegawa et al., 1990). Somewhat surprisingly, labeling in the NaCl + OPP-treated rats was substantially lower than that observed in the OPP-treated rats. However, it should be noted that in this 2 week study, BrdU was administered only 24 h prior to killing of the rats. Cytotoxicity and other cellular alterations induced by NaCl + OPP may have occurred at a time point earlier in the study and thus may have eluded detection. Alternatively, the combination of NaCl + OPP might have had an unusual suppressive effect on cell proliferation in the bladder of the treated animals.
Labeling in the NaCl-treated rat bladder cells was also significantly higher than that of the controls. Previous studies of NaCl have shown weak promoting activity at a dose of 5–10%, but mixed results have been seen with respect to urothelial proliferation and tumor enhancement at a dose of 1% (Shibata et al., 1986, 1992; Fukushima et al., 1988; Cohen and Ellwein, 1991; Cohen, 1995; Lugli and Lutz, 1999). In vitro studies have shown that at high concentrations NaCl is capable of inducing structural chromosomal aberrations in cultured cells. These alterations are believed to be due to changes in osmolality of the treated cells (Scott et al., 1991). These results suggest that a similar clastogenic response can occur in vivo in animals given high doses of NaCl.
It has also been suggested that OPP may cause ploidy changes, which could possibly proceed to malignant aneuploidy (Sutou and Tokuyama, 1974; Tayama et al., 1989; Lambert and Eastmond, 1994). However, in the present study using FISH, no increase in the frequency of hyperdiploidy/polyploidy was seen in the treated rats over the controls. The inability to detect an increase in hyperdiploidy may be due to the high frequency of polyploid cells in the normal bladder epithelium of the controls. The frequency of cells with three or more hybridization signals in the controls was ~4%, which is higher than that typically seen in cells such as lymphocytes and granulocytes (Eastmond et al., 1995). The surface of the epithelium of the urinary bladder in rats has been shown to be covered by large, presumably polyploid, cells which are reported to increase in size and ploidy level from the basal to the superficial layer (Turusov and Mohr, 1990). The presence of polyploid cells combined with cytotoxicity and compensatory cell proliferation makes it difficult to determine if OPP is capable of inducing aneuploidy in the rat urothelium. It may be possible to overcome this problem in future studies by using BrdU labeling in conjunction with FISH and scoring only dividing cells.
In addition to cytotoxicity, cell proliferation and ploidy changes, several other possible mechanisms have also been proposed to explain the action of various rat bladder carcinogens. It has been hypothesized that an increase in urinary pH and Na+ concentration causes an increase in intracellular pH in the bladder epithelium which stimulates cell replication and can thus enhance tumor formation (Anderson, 1991). One of the most frequent causes of toxicity, regenerative hyperplasia and carcinogenesis in the rat bladder is the formation of urinary calculi (Cohen, 1995). Numerous chemicals, such as uracil, have been shown to produce urinary tract calculi and many of these chemicals are associated with the production of bladder cancer (Cohen, 1995). However, studies have indicated that the frequency of calculi is not increased in OPP-treated rats (Cohen, 1998). In our studies calculi were occasionally seen in both the control and treated rats, showing no association with treatment. The bladder carcinogenicity of sodium saccharin in rats has been attributed mainly to the occurrence of chronic regenerative hyperplasia in the bladder secondary to the presence of an amorphous urinary precipitate composed predominantly of calcium and potassium. As above, the occurrence of such a precipitate in OPP-treated rats has also been ruled out (Cohen, 1998;St John et al., 2001). The exact mechanism of OPP-induced carcinogenicity is still unclear, but our studies suggest that increased cell proliferation as well as chromosomal alterations in the rat bladder may play a significant role.
In summary, the experiments presented in this work have shown that OPP, in the absence of the sodium salt, can cause chromosomal alterations in urothelial cells following exposure of rats to OPP. The results also confirm the proliferative potential of OPP in the rat bladder. Furthermore, the results show that at high concentrations NaCl can cause chromosomal damage in rat bladder cells. Although in the present study we were unable to see an increase in hyperdiploidy in OPP-treated rat bladder cells, it is possible that by using BrdU labeling in conjunction with FISH and scoring only actively replicating cells it may be possible to detect chemically induced aneuploidy in the rat bladder.
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Acknowledgments |
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The DNA probe specific for rat chromosome 4 was a generous gift from Dr Barbara Hoebee (National Institute of Public Health and Environment, Bilthoven, The Netherlands). This work was supported in part by funds provided by the US Environmental Protection Agency (grant R826409-01-1).
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Notes |
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2 To whom correspondence should be addressed. Tel: +1 909 787 4497; Fax: +1 909 787 3087; Email: david.eastmond{at}ucr.edu
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Received on January 15, 2001; revised on August 27, 2001; accepted on September 10, 2001.
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