Mutagenesis, Vol. 14, No. 6, 621-632,
November 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Increased frequencies of diploid sperm detected by multicolour FISH after treatment of rats with carbendazim without micronucleus induction in peripheral blood erythrocytes
Laboratory of Health Effects Research, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands
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Abstract |
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The purpose of the present study was to determine the effect of a single oral dose of carbendazim (CARB) on the frequencies of numerical chromosome aberrations in sperm and on micronuclei in peripheral blood erythrocytes of rats. Dual colour FISH on epididymal sperm of rats treated 31 days before sacrifice (0, 50, 150, 450 and 800 mg/kg body wt CARB in corn oil), corresponding to exposure during late pachytene, revealed a clear induction of diploid sperm. Induction of aneuploid sperm was not observed. Although the absolute frequencies of diploidy were low, ranging from 0.03% in the control group to 0.22% in the highest dose group, the observed dose–response relationship was highly significant. In sperm of rats killed 50 days after treatment with CARB (corresponding to exposure of spermatogonial stem cells) the effect was no longer apparent. In a second experiment, in addition to more dose groups in the low dose range, the peripheral blood micronucleus assay was incorporated. Results of triple colour FISH on epididymal sperm of rats treated with CARB (0–800 mg/kg body wt) again showed induction of diploid, but not of aneuploid sperm. Induction was less prominent than in the first experiment, but the dose–response relationship for diploidy was again significant. In blood samples drawn from the tail vein 48 h after treatment with CARB induction of micronuclei in peripheral blood erythrocytes was not observed, whereas the micronucleus frequency was significantly increased after a single i.p. dose of mitomycin C (3 mg/kg body wt). In conclusion, the present results show that CARB induces diploidy in sperm, without an accompanying induction of micronuclei in erythrocytes. This finding suggests that in rats the peripheral blood micronucleus assay is a less sensitive indicator for the genotoxic potential of CARB than the epididymal sperm aneuploidy/diploidy assay.
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Introduction |
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Numerical chromosome abnormalities occur quite frequently in the human population and can give rise to pregnancy termination, congenital malformations and mental and physical retardation (Abruzzo and Hassold, 1995
). Fifty per cent of the spontaneous abortions harbour a chromosome aberration, the majority of which are of the numerical type. Trisomies are the most common (in 50% of these abortions), while 25% of these abortions are triploid or tetraploid. The incidence of numerical aberrations in live-born is 5/1000 births (Abruzzo and Hassold, 1995; Hassold et al., 1996; Griffin, 1996). In somatic cells numerical chromosome aberrations are consistently associated with some types of human cancer and may play a significant role in malignant transformation, genetic instability and tumour progression (Oshimura and Barret, 1986; Li et al., 1997).
Studies in germ cells show that up to 35% of human oocytes and 1.5% of human sperm may contain a numerical chromosome abnormality (Martin et al., 1991; Martin, 1993). For the detection of such aberrations in human sperm, the in situ hybridization technique with chromosome-specific probes is frequently used. In a large number of studies, aneuploidy and diploidy frequencies in sperm of normal healthy men have been determined using this technique (Wyrobek et al., 1990; Lähdetie et al., 1996; Rives et al., 1998; Baumgartner et al., 1999). In addition, the method has been applied to determine factors that may influence aneuploidy frequencies in human sperm, like age, smoking and drug exposure (Kinakin et al., 1997; Robbins et al., 1997b; Rubes et al., 1998) and to analyse sperm from infertile men (Guttenbach et al., 1997; Martin, 1998).
Several investigators observed an increase in diploid and/or aneuploid sperm in samples from infertile men or in fathers of recurrent abortions (Giorlandino et al., 1998; McInnes et al., 1998). Blanco et al. (1998) reported increased frequencies of disomy 21 in sperm of fathers of children with Down syndrome of paternal origin. Others have observed increased aneuploidy and diploidy frequencies in epididymal sperm after treatment with anti-cancer drugs (Brandriff et al., 1994; Robbins et al., 1997a) and Baumgartner et al. (1996) have shown that ingestion of high doses of diazepam increase aneuploidy frequencies in mature sperm.
To study the effect of external factors on the frequencies of aneuploid sperm in more detail, experiments in laboratory animals are needed (Wyrobek and Adler, 1996; Parry, 1996). Comparable with human studies, baseline aneuploidy frequencies in mature sperm of mice and rats have recently been determined by means of fluorescence in situ hybridization (FISH) (Adler et al., 1996; Wyrobek and Adler, 1996; Lowe et al., 1998). The results showed that multicolour FISH with chromosome-specific probes is a fast and sensitive technique to study aneuploidy in epididymal sperm and that the baseline frequencies of rat sperm with disomy Y were similar to those in mice and humans (Lowe et al., 1998).
The aim of the present study was to determine the effect of exposure to aneugenic compounds on the chromosome constitution of rat epididymal sperm, using FISH. The spindle poison carbendazim (CARB) was used as a model compound. The fungicide CARB, the active metabolite of benomyl (reviewed in World Health Organization, 1993), exerts its aneugenic action by binding to tubulin (Davidse and Flach, 1977) and consequently preventing the formation of microtubules (Albertini et al., 1988). It induces micronuclei in mouse bone marrow (Sarrif et al., 1994) and aneuploidy in human lymphocytes in vitro (Elhajouji et al., 1995, 1997; Marshall et al., 1996) and in hamster oocytes (Jeffay et al., 1996). From toxicological studies it is known that the testis is a target organ of CARB, indicating that the compound is capable of reaching the germ cells (Nakai et al., 1992; Lim and Miller, 1997). In the two experiments described in this paper, the effect of exposure of spermatogonial stem cells and late pachytene sperm to a single oral dose of CARB on aneuploidy/diploidy frequencies in epididymal sperm was studied. In addition, in order to compare the effects in germ cells with those in somatic cells, a peripheral blood micronucleus assay was incorporated in the second experiment.
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Materials and methods |
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Experimental design and chemicals
In the first experiment male rats of the outbred strain WU (Wistar Unilever, 13–14 weeks old, 5 rats/group) received a single oral dose of CARB (50, 150, 450 or 800 mg/kg body wt; Aldrich) in corn oil. The control group received corn oil only and one group was treated with CARB by i.p. injection (150 mg/kg body wt). The total volume of corn oil per rat applied orally was 1.85–2.5 ml and that i.p. 0.9–1.1 ml, depending on the weight of the rat 1 day before application.
In the pilot study for experiment II, the animals received a single oral dose of 2, 4 or 8 mg/kg body wt mitomycin C (MMC; Sigma) in phosphate-buffered saline i.p. (total volume 3.3–3.5 ml) or 400 or 800 mg/kg body wt CARB in corn oil by gavage (total volume 1.3–1.5 ml). One day before and 48 and 72 h after treatment peripheral blood samples were collected from the tail vein.
In experiment II groups of three rats received a single oral dose of CARB (2.5, 5, 10, 20, 30, 40, 50, 100, 150, 450, 800 mg/kg body wt) in corn oil. A group of four rats received MMC (3 mg/kg body wt i.p.) and the negative control group received corn oil only. The total volume applied (orally or i.p.) was 1.1–1.3 ml. One day before and 48 and 72 h after treatment peripheral blood samples were collected from the tail vein.
Body weight of the animals was regularly measured during the studies. Thirty-one or 50 days (only experiment I) after application of the test compound rats were killed by O2/CO2 asphyxiation, corresponding to exposure of the cells during the late pachytene stage or spermatogonial stem cell phase, respectively. At death, rats were briefly examined for gross pathological abnormalities before testes and epididymes were isolated and testes were weighed.
Sperm isolation and preparation and pretreatment of sperm smears
Epididymal sperm was isolated as described before (Lowe et al., 1998) and stored at –20°C in 2.2% sodium citrate. Sperm smears were prepared by smearing ~5 µl of sperm suspension on an ethanol-cleaned slide and allowed to air dry for at least one night. Sperm was decondensed before hybridization by incubating the slides in 10 mM dithiothreitol (DTT; Sigma) for 30 min on ice followed by incubation in 4 mM lithium 3,5-diiodosalicylic acid (LIS; Sigma) for 90 min at room temperature. Slides were again air dried completely before they were used for hybridization.
Fluorescence in situ hybridization on sperm smears
DNA probes specific for rat chromosomes 4 (25S5) and Y (9.1ES8) were used in the first experiment (for description of the probes see Essers et al., 1995; Hoebee and de Stoppelaar, 1996) and were labelled by nick translation with biotin and digoxigenin, respectively. For an additional FISH on a select group of slides from the first experiment (see Results), the chromosome 4 probe was labelled with biotin and the chromosome 19 probe (cos42-47) with digoxigenin.
For aneuploidy analysis in the second experiment an additional autosomal probe was included in a triple colour FISH, namely the chromosome 19 probe (cos42-47), which was labelled with biotin and digoxigenin (1:1 ratio).
Hybridizations were performed as previously described by Lowe et al. (1998) with some modifications. The probe mixture (final concentration of 20 ng probe in 55% formamide, 10% dextran sulphate, 2x SSC) was denatured at 80°C for 10 min. The sperm smears were denatured at 80°C for 10 min in 70% formamide, 2x SSC, pH 7.0, and dehydrated in an alcohol series. Following application of 10–15 µl of probe mixture, hybridization was performed at 37°C overnight in a humidified chamber. A total of five post-hybridization washings were carried out at 45°C in 50% formamide, 2x SSC, pH 7.0 (3x10 min) and PN buffer (0.1 M NaH2PO4, 0.1 M Na2HPO4, pH 8, 0.1% Nonidet P-40, 2x10 min). Visualization of the hybridized probes was achieved by a series of incubations, namely avidin–FITC, biotinylated anti-avidin/anti-digoxigenin–rhodamine and avidin–FITC/anti-sheep lissamine–rhodamine, all in 5% non-fat dry milk in PN buffer. Sperm nuclei were counterstained with 0.25 µg/ml 4,6-diamidino-2-phenylindole (DAPI) in antifade medium.
Scoring of the sperm slides
Fluorescent images were observed using a Zeiss Axioplan fluorescence microscope equipped with various fluorescent filters. A triple bandpass filter set was used to detect both red and green fluorescence along with blue dye (DAPI), the nuclear counterstain. The single bandpass emission filters, which identified red or green separately, were used to confirm the presence of the individual colour domains. Sperm were scored as having two fluorescent signals of the same colour (indicating the presence of two copies of the same chromosome) according to the following criteria: (i) the two fluorescent signals should be of similar size and intensity; (ii) the fluorescent signals should have similar sizes and intensities as the signals in the surrounding sperm; (iii) the two fluorescent signals should be separated by at least the diameter of one fluorescent signal. The two experiments were scored by different individuals, who were trained to use the same scoring criteria and did not have significantly different outcomes on the slides used for training.
Micronucleus test in peripheral blood
Approximately 200 µl of peripheral blood was collected from the tail vein in potassium/EDTA-coated vials and stored at 4°C until analysis (within 1–2 weeks after blood collection). For evaluation of the micronucleus frequency the acridine orange (AO) staining method was applied (Hayashi et al., 1990). Slides were coated with AO by smearing 10 µl of AO solution (1 mg/ml) on slides preheated to 70°C. One microlitre of peripheral blood, together with 4 µl of fetal calf serum was applied to the slide, covered with a coverslip and firmly pressed. Slides were scored within 24 h under an Axioplan fluorescence microscope. The fraction of polychromatic erythrocytes (PCE) was determined [no. PCE/2000 normochromatic erythrocytes (NCE) + PCE] and at least 2000 PCE types I and II were scored for the presence of micronuclei.
Statistical evaluation of results
The dose–response relationships were determined using the program PROAST (Possible Risk Obtained from Animal Studies), developed at RIVM. This program is used to fit a non-linear regression function to the data. There are two criteria for choosing a particular regression function. Firstly, the biological plausibility is considered, e.g. what is the dose–response relationship to be expected? Secondly, one may compare the fit of the various models to the data by comparing their associated log likelihood values and use the model with the best fit.
Statistical significance of the dose–response relationship can be determined using a likelihood ratio test, comparing the log likelihood of a fitted regression curve with the log likelihood of the so-called `null-model'. This `null-model' assumes no effect of the treatment, i.e. a straight horizontal line through the data (one parameter, Y = a). Twice the difference in log likelihoods is 
2 distributed. Thus, by comparing the value of twice the difference in log likelihoods with the 2 value at P = 0.05, significance can be determined. The number of degrees of freedom is given by the difference in the number of parameters between the dose–response model and the null model.
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Results |
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Experiment I
After killing the rats a brief pathological examination did not reveal any obvious clinical effect of treatment nor was there a significant effect on the average body weight or relative testes weight (data not shown). In the 450 mg/kg group (killed at day 31) only three rats remained for sperm analyses; one rat exhibited degenerate testes and epididymes and one rat died due to improper application of the test compound.
Aneuploidy and polyploidy in sperm.
For analysis of aneuploidy in sperm, dual colour FISH was performed in which chromosome 4 was stained in green (FITC) and chromosome Y in red (rhodamine, the X-chromosome was not labelled). Consequently, a normal sperm cell contained one green signal [4-(X) sperm] or one green and one red signal [4-Y sperm]. A hyperhaploid, or disomic, sperm cell contained two signals of a particular colour, for example a green-red-red fluorescent phenotype corresponds to a 4-Y-Y genotype (see Figure 1A). A diploid sperm cell contained two signals of each colour (see Figure 1B). Five thousand sperm per slide were scored, at least two slides per rat, amounting to a total of at least 10 000 sperm per rat. Exceptions were the rats treated i.p. and the rats killed at 50 days after treatment, for which only one slide was scored.
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Table I
shows the total frequencies of normal and aberrant sperm per group. The sex ratio (4/4-Y sperm) was not significantly different from the ratio 1.0, as expected. An increase in total hyperhaploid sperm was observed in the treated groups at 150 mg/kg and above (Table I) at 31 days after treatment. This effect was due to an increase in 4-4 and 4-4-Y sperm since the frequency of 4-Y-Y sperm was not increased in the treated groups. On the contrary, in the lowest (50 mg/kg body wt) and the highest (800 mg/kg body wt) dose group and the 150 mg i.p. treated group the frequency of Y-Y disomic sperm was decreased (Table I). A >10-fold increase in 4-4-Y-Y diploid sperm was observed in the three highest dose groups. Treatment with 150 mg CARB/kg i.p. did not result in an increase in total hyperhaploid or diploid sperm.
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In the rats killed 50 days after treatment no effect of CARB was observed in the 450 mg/kg group, the only group scored at this time point (Table I
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Distinction between diploidy and aneuploidy.
All sperm with the 4-4-Y-Y fluorescent phenotype, most likely diploid sperm, were about twice the size of the normal sperm (see Figure 1B). A more detailed analysis of the scoring results revealed that the majority of the sperm with a 4-4 or 4-4-Y fluorescent phenotype were also approximately twice the size of a normal sperm (see for example Figure 1C). It was assumed that these large sperm actually represented diploid sperm, namely 4-4-(XX) and 4-4-Y-(X) sperm. In order to confirm the assumption that large sperm represent diploid sperm, we performed a dual colour FISH with two autosomal probes, namely the chromosome 4 probe and a probe for chromosome 19 (cos42-47) (Hoebee and de Stoppelaar, 1996). Sperm from four rats were used for analysis, two controls and two rats that had the highest frequency of diploid cells in the previous analysis. Again, 5000 sperm per slide were scored and, as expected, all sperm with the 4-4-19-19 fluorescent phenotype (diploid sperm) had about twice the size of normal sperm (results not shown). Therefore, we conclude that 4-4 and 4-4-Y sperm of large size indeed represented diploid cells, while 4-4 and 4-4-Y sperm of normal size were attributed to actual disomy. Using this information, the frequencies of aneuploid and diploid sperm were recalculated. Results show that the observed CARB-induced increase in `hyperhaploid' sperm is entirely due to an increase in diploid sperm and not to aneuploidy (Table II). The absolute frequencies of diploidy were low, between 0.03 and 0.22% of sperm, but the observed induction was highly significant and dose related (see below). The increase in diploid sperm in the i.p. treated group (150 mg CARB/kg) was due to one rat with a high frequency of diploid sperm; the other four rats in this group had diploidy frequencies similar to the controls.
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Dose–response relationship for diploidy. To estimate the dose–response relationship, the data were analysed using the PROAST program. The frequencies of diploid sperm on all the slides scored (31 day death only, excluding the i.p. treated group) were plotted and a non-linear regression curve was fitted to the data. From various dose–response functions investigated, the Weibull function resulted in the best fit (Figure 2A
). The significance of a dose-related increase in diploid sperm is determined by comparing the log likelihood of the Weibull function with that of the null model. The log likelihood of the Weibull model (three parameters) is –2415.3 whereas that of the null model (one parameter) is –2465.0. Thus, the log likelihood increased by 50 compared with the null model, resulting in a 2 value of 100 with two degrees of freedom (i.e. the difference in the number of parameters in both models). The critical 2 value at P = 0.05 being 5.99, the effect of CARB is highly significant.
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= aa + (1 – aa) (1 – exp–bb.xcc), three parameters] describes the data with the best fit. (B) Experiment II: the quantal linear model [Meiosis I and II errors. By taking into account the distribution of the sex chromosomes among the sperm, errors in the first and second meiotic division can be distinguished from each other. Diploid sperm with the 4-4-Y fluorescent phenotype [4-4-Y-(X)] are assumed to originate in the first meiotic division (MI) whereas 4-4-Y-Y or 4-4-(XX) diploid sperm are assumed to represent an error in the second meiotic division (MII). Data on both MI and MII errors were plotted with the PROAST program and the resulting dose–response relationships were similar to that of total diploidy (data not shown). The dose-related increases in MI and MII errors were highly significant, indicating that CARB induces errors in both MI and MII.
Experiment II
The aim of the second experiment was three-fold: (i) confirmation of the finding that CARB induces diploidy and not aneuploidy in sperm; (ii) more accurate determination of the dose–response relationship by incorporating more dose groups, mainly in the low dose range; (iii) comparison of the effects in germ cells with the frequency of micronuclei in somatic cells by incorporating the peripheral blood erythrocyte micronucleus test in the experiment. In experiment II all the rats were killed 31 days after treatment, since 50 days after treatment the effect of CARB was no longer apparent (see experiment I).
Gross pathological examination revealed that in the higher dose groups some animals had soft testes and the epididymes contained less sperm. There was, however, no statistically significant effect on relative testes weight. No treatment-related effect on the average body weight gain of the different groups was observed, with the exception of the MMC group (data not shown). Rats lost weight after treatment with MMC but had recovered by the end of the study.
Aneuploidy and polyploidy in sperm. In addition to the probes for chromosomes 4 and Y (stained in green and red), a probe for chromosome 19 (cos42-47) was used in a triple colour FISH, labelled with both digoxigenin and biotin (stained in yellow). The use of an extra autosomal probe made it possible to score for diploidy more accurately. In this case, a normal sperm cell contains one green and one yellow signal [4-19-(X) sperm] or one green, one yellow and one red signal (4-19-Y sperm). A hyperhaploid sperm cell, or disomic sperm cell, contains two signals of a particular colour (for example, a green-green-yellow-red fluorescent phenotype corresponds to a 4-4-19-Y genotype; see Figure 1D). A diploid sperm cell harbours two signals of each autosome, in addition to no, one or two red signals, depending on the number of Y chromosomes present. An example of a 4-4-19-19-(XX) diploid sperm cell is shown in Figure 1E.
Five thousand sperm per slide were scored for the fluorescent pattern, two slides per rat, amounting to a total of 10 000 sperm per rat. The total frequencies per dose group of normal and aberrant sperm are shown in Table III. Again, the sex ratio (4-19 sperm/4-19-Y sperm) did not differ significantly from 1.0. Treatment with different doses of CARB did not influence the frequencies of disomic sperm for either of the chromosomes 4, 19 or Y. An induction of diploid sperm was again observed. Although this induction was much less prominent than in the first experiment, the dose–response relationship for diploidy induction was nevertheless significant (see below). In the MMC-treated rats there was no induction of disomy or diploidy, which is to be expected, since MMC is a clastogenic rather then an aneugenic compound. Large sized sperm that had the diploid fluorescent phenotype for only two of the three chromosomes were also observed (Table III). This occurred occasionally, but only in the CARB-treated groups. These sperm were probably `nearly diploid', meaning that they are diploid cells which have lost one or a few chromosomes during meiosis (chromosomes that were not included in the main nucleus when the nuclear membrane was formed). In the analysis of the dose–response relationship for diploidy induction these cells were included.
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Dose–response relationship for diploidy. Figure 2B
shows the dose–response relationship for diploidy induction in the second experiment. The frequencies of diploid sperm on all the slides scored (excluding the MMC group) were plotted and a regression curve fitted to the data. In order to achieve a better visual discrimination between the low dose groups, the dose was plotted on a log10 scale. The log likelihood of the quantal linear model (two parameters, the three parameter model did not improve the log likelihood of the fit) is –970.05 and of the null model (one parameter) –975.65. Thus, the log likelihood increased by ~5 compared with the null model, resulting in a 2 value of 10 with one degree of freedom (i.e. the difference in the number of parameters in both models). The critical 2 value at P = 0.05 is 3.84, again indicating that the effect of CARB is statistically significant.
Micronucleus assay in peripheral blood.
Using the AO staining method, micronuclei stain bright yellow and are easy to recognize (Figure 1F). In order to determine the appropriate MMC dose to be used as a positive control in the main assay, a pilot study was carried out in which the two highest CARB doses were also tested. Pretreatment blood samples of five rats were used to determine the micronucleus frequencies and the PCE/NCE ratios in untreated rats (Table IV). The frequency of micronuclei in PCE in pretreatment blood samples is consistently low, 0–0.5 per 1000 PCE.
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All doses of MMC induced micronuclei, but considering the low PCE/NCE ratios in the rats treated with 4 or 8 mg/kg body wt MMC (too low to score 2000 PCE on one slide; see Table IV
), we decided to use 3 mg/kg body wt as a positive control in the main experiment. In rats treated with CARB there was no induction of micronuclei in PCE at 48 or 78 h after treatment. In all treated rats the PCE/NCE ratios were significantly decreased compared with the ratios in pretreatment blood samples (95% confidence intervals did not overlap) (Table IV).
In the main experiment, the frequency of micronucleated PCE (MNPCE) in the CARB-treated groups was unchanged compared with the corn oil group and with the pretreatment blood samples (Table V). In contrast, the micronucleus frequency in erythrocytes of rats treated with 3 mg/kg body wt MMC was increased to an average of 6.2 MNPCE/1000 PCE. PCE/NCE ratios were decreased in the groups treated with the higher doses of CARB (150–800 mg/kg body wt) and with MMC (Table V). Although this decrease was strictly speaking not statistically significant compared with the corn oil group (the 95% confidence intervals overlapped marginally), this indicates exposure of the bone marrow.
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Compared with the pretreatment blood samples there was a significant decrease in the PCE/NCE ratios in blood samples taken from the same rats 48 h after treatment (12 rats, paired t-test), which was also observed in the pilot study. This decrease in PCE/NCE ratios after treatment compared with pretreatment was observed in all treatment groups, including the corn oil group and the low dose groups.
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Discussion |
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Considerations on experimental design
The present paper describes the effect of a single oral dose of CARB on the frequencies of numerical aberrations in sperm and on micronuclei in peripheral blood erythrocytes. Epididymal sperm was sampled 31 and 50 days after application of CARB, which means that, on the basis of the estimated duration of spermatogenesis (Adler, 1996
), the cells were exposed during the late pachytene stage or spermatogonial stem cell phase, respectively.
It has been shown that CARB in corn oil is well absorbed (80–85%) after a single oral dose and is excreted in the urine and faeces within 72 h after dosing (World Health Organization, 1993). Nevertheless, it is present in the body for a few days. Adding to this the possibility of a delay in spermatogenesis due to CARB treatment, it can be expected that the timing of the stage of exposure is not as precise as theoretically calculated.
CARB does not dissolve in aqueous solutions or corn oil and is therefore applied as a suspension in corn oil. Although homogenization of the suspension was attempted before every application (by shaking the suspension thoroughly), variations in effective dose cannot be excluded. The rats used in the present study are from an outbred strain, so variations in uptake, metabolism and excretion of CARB may be expected to a certain extent. These issues of dosing may reflect on the ultimate internal dose in the testes and this may contribute to the variation in diploidy frequencies observed (see Figure 2A and B).
In view of the above considerations, use of a repeated dosing regimen is recommended instead of administration of a single dose. In this way one can ensure exposure of the cells in the desired phase of spermatogenesis and possibly reach higher internal doses in the testes. In addition, we would recommend the use of multiple dose groups, followed by dose–response analysis. This reduces the risk of choosing an inadequate dose level, while systematic differences between dose groups due to other (unknown) experimental factors have less impact. It should be noted that this does not imply the use of more experimental animals: in a dose–response analysis the statistical power to detect a significant response is determined by the total number of animals used (see also Figure 2B). Therefore, a multiple dose study will generally be more sensitive in detecting effects. In addition, by analysing the data as presented here, the use of more dose groups with less animals per group provides more information on the whole dose range, instead of only on the particular doses used.
Aneuploidy and diploidy in sperm
The initial results at 31 days after treatment pointed in the direction of a pronounced increase in diploid sperm (4-4-Y-Y sperm), in addition to a 2-fold increase in hyperhaploid sperm (4-4 and 4-4-Y sperm). However, the observation that a majority of these 4-4 and 4-4-Y sperm had approximately twice the size of normal sperm prompted us to take a closer look at the results. The fact that all the 4-4-Y-Y (diploid) sperm were also twice the size of normal sperm let us to the assumption that the majority of these `hyperhaploid' sperm were actually diploid, namely 4-4-(XX) and 4-4-Y-(X) sperm. This assumption was confirmed by the results from slides hybridized with two autosomal probes for chromosomes 4 and 19, which showed that all the 4-4-19-19 (diploid) sperm were approximately twice the size of normal sperm. Thus, exposure of late pachytene sperm to CARB results in increased frequencies of diploid sperm but not of aneuploid sperm.
In the second experiment this result was confirmed but the induction of diploid sperm was less prominent than in the first experiment (mainly apparent in the highest dose group), although the dose–response relationship was significant. The weaker response in the second experiment was unexpected, in view of the almost identical protocol for the two experiments. The only difference in the experimental design was that the CARB was applied in a more concentrated suspension in the second experiment. The mean volume applied by gavage in the first experiment was 2 ml whereas in the second experiment it was 1 ml. Perhaps a more concentrated suspension in oil hampers absorption, resulting in a lower internal dose in the testes. Furthermore, if the rate of absorption of CARB differs between the two experiments, the stage of spermatogenesis exposed to CARB may also differ in the two experiments.
At 50 days after application of CARB initially only the control group and the 450 mg CARB/kg body wt group were scored. Since no effect of CARB was observed at 450 mg/kg, a dose that induced a clear increase in diploid sperm at 31 days after treatment, it was decided not to pursue scoring of the other groups. This finding was not unexpected since aneuploid spermatogonia are not likely to survive through the whole process of spermatogenesis, up to maturation of sperm in the epididymus. Transient aneuploidy/diploidy effects have also been observed in studies in humans. Robbins et al. (1997a) observed increased aneuploidy and diploidy frequencies in patients undergoing chemotherapy. However, ~100 days after the end of the therapy the frequencies of aneuploid and diploid sperm had declined to pretreatment levels. Other studies that measured aneuploidy in sperm several months to years after the patients underwent chemotherapy also yielded negative results (Genescà et al., 1990; Jenderny et al., 1992; Martin et al., 1997; Martin, 1998).
In both our experiments, sperm with fewer than the expected hybridization signals (hypohaploidy) were also encountered. Apart from actual loss of a chromosome, hypohaploidy can be due to inefficient hybridization of the probe. In addition, since the rat chromosome-specific probes are located on the arms of the chromosome and not in the centromere, loss of the signal could also be due to a break in the chromosome followed by subsequent loss of the part of the chromosome harbouring the hybridization signal. Sperm with a hypohaploid fluorescent phenotype were, therefore, not considered in the evaluation of the results.
Diploidy and not aneuploidy: possible mechanisms
The finding that CARB induced diploidy in sperm and not aneuploidy was rather unexpected. CARB is known to induce non-disjunction and chromosome loss in somatic cells in vivo and in vitro (Elhajouji et al., 1995, 1997; Marshall et al., 1996; de Stoppelaar et al., in preparation) and micronuclei in mouse bone marrow in vivo (see below). In addition, CARB and benomyl have been shown to induce aneuploidy in oocytes of hamsters and mice (Mailhes and Aardema, 1992; Jeffay et al., 1996). This is in contrast to our finding of an absence of aneuploidy induction in mature sperm. Perhaps the different stages at exposure, oogenesis (meiotic divisions) and spermatogenesis (late pachytene), could play a role here. On the other hand, the process of oogenesis is quite different from spermatogenesis with respect to duration, molecular regulators and checkpoints (LeMarie-Adkins et al., 1997; Sassone-Corsi, 1997).
In mitosis as well as in meiosis several cell cycle checkpoints operate to ensure proper replication and cell division, the metaphase–anaphase transition checkpoint being the most relevant in aneuploidy studies (reviewed in Gorbsky, 1997; Kirsch-Volders et al., 1998). The checkpoint in meiotic cells has not been as extensively studied as in somatic cells, but is believed to monitor synapsis of chromosomes and is probably triggered by the absence of tension on the kinetochores (Li and Nicklas, 1995; Nicklas, 1997; Nicklas et al., 1998). The presence of achiasmate chromosomes, or univalents, results in metaphase arrest in various species (Li and Nicklas, 1995; Odorisio et al., 1998). This arrest is not likely to continue for ever; eventually the arrested cells will enter one of the following pathways. (i) The arrested cell can enter apoptosis (Sassone-Corsi, 1997; Braun, 1998; Odorisio et al., 1998), which was not investigated in the present study. (ii) After a prolonged metaphase arrest the checkpoint may be relieved and the cell will enter anaphase and cytokinesis, resulting in aneuploid gametes. This has indeed been observed after exposure of mouse or rat spermatocytes to spindle poisons (Miller and Adler, 1992; Kallio et al., 1995). However, in the present study diploidy induction was observed and not aneuploidy induction, indicating that the cells may use an alternative pathway. (iii) A prolonged metaphase arrest in MI may disturb the normal timing of events in such a way that the second meiotic division will be skipped, resulting in diploid sperm. If this is the case, one would expect to find only sperm with an XX or YY configuration, indicative of MII suppression. However, XY diploid sperm were also observed. Possibly, the resulting XY configuration depends on the `pairing status' of the chromosomes, e.g. whether or not the chiasmata between the chromosomes have been resolved. In the normal sequence of events in MI the chiasmata resolve at anaphase onset and the homologous chromosomes will separate. However, in the case of prolonged metaphase arrest the chiasmata may perhaps eventually resolve, even though anaphase has not yet been initiated. In this case, when anaphase finally occurs the chromatids of both chromosomes X and Y will separate from each other, resulting in XY sperm.
On the other hand, a process may occur which is related to that in mitotic cells referred to as `mitotic slippage' (Andreassen and Margolis, 1994). Somatic cells arrested in metaphase by exposure to spindle poisons may eventually override the cell cycle checkpoint for anaphase onset. They may exit mitosis without chromosome segregation or cytokinesis and become diploid (Andreassen et al., 1996; Elhajouji et al., 1998). A similar observation was made in in vitro maturing mouse oocytes arrested in MI by chloral hydrate, due to the formation of asymmetric spindles and misaligned chromosomes (Eichenlaub-Rieter and Betzendahl, 1995; Eichenlaub-Rieter et al., 1996). Eventually, many oocytes that escaped the block became diploid, whereas no hyperhaploidy induction was observed. A related process may occur in male meiosis after exposure to CARB, resulting in diploid sperm.
Other studies in sperm
In contrast to our results, Tates et al. (1979) did not observe induction of diploidy by CARB or benomyl in sperm of the northern vole Microtus oeconomus. However, CARB did induce non-disjunction in early pachytene/zygotene spermatocytes in M.oeconomus. To our knowledge no other studies have specifically investigated polyploidy induction in sperm after CARB administration but there have been several other observations that may confirm our results. Evenson et al. (1987) used flow cytometry to investigate the effects of CARB on mouse germ cells and observed a significant correlation of CARB dose with the occurrence of 4n cell types. Nakai and Hess (1997) analysed in detail the effects of CARB on meiotic spermatocytes and spermiogenesis in the rat testis and observed that exposure of meiotic spermatocytes resulted in the formation of binucleated cells and large round spermatids (megaspermatids). The authors assumed that these large round spermatids were diploid cells, which would confirm our findings.
Using a similar technique as in the present study, Schmid et al. (1999) investigated the effect of exposure of mouse spermatocytes to the aneugenic compounds colchicine, thiabendazole and diazepam on the frequencies of aneuploid and diploid epididymal sperm. They observed induction of aneuploidy by a single dose of colchicine (1.5 and 3 mg/kg body wt) and diazepam (300 mg/kg) but the induction was only 2-fold. In contrast, the induction of diploidy by diazepam was 10-fold, as was observed in the first experiment with CARB described here.
Micronucleus assay in peripheral blood
A major advantage of the rodent peripheral blood micronucleus assay is that it can be incorporated into other (genetic) toxicology studies, in this case a study of germ cell aneuploidy, since the animals do not have to be killed when blood is drawn from the tail vein (Krishna et al., 1998; Wakata et al., 1998). The reports that the rat spleen eliminates circulating micronucleated erythrocytes led to the idea that the rat peripheral blood micronucleus assay might not be feasible (Schlegel and McGregor, 1984). However, recent publications conclude that the peripheral blood micronucleus assay can be used as an alternative to the bone marrow micronucleus assay (Hayashi and Sofuni, 1994; Holden et al., 1997). Following suggestions by Hayashi and colleagues (Hayashi and Sofuni, 1994; Wakata et al., 1998), the risk of elimination of micronucleated erythrocytes interfering with the results was reduced by scoring only young erythrocytes, namely type I and II reticulocytes. With the AO staining method the distinction between young and mature erythrocytes is clear and the micronuclei are unequivocally distinguished from artefacts (Tinwell and Ashby, 1989; Hayashi et al., 1990). In addition, since the background frequency was low (0–1 MNPCE per 1000 PCEs), the sensitivity of the assay was increased by scoring 2000 erythrocytes instead of 1000 (Wakata et al., 1998).
In the present study, a single oral administration of CARB did not induce micronuclei in erythrocytes at any dose tested, which is in contrast to the positive results reported for CARB and benomyl in the mouse bone marrow micronucleus test (Pandita, 1988; Barale et al., 1993; Sarrif et al., 1994). The doses used in the present study were lower than the 1650 and the 2x1000 mg/kg body wt CARB which scored positive in the bone marrow (Sarrif et al., 1994; Pandita, 1988). However, Barale et al. (1993) observed a 3-fold increase in micronucleated erythrocytes in mouse bone marrow after a single oral dose of 500 mg/kg body wt, which lies in the dose range tested in the present experiment. Since there have been no comparable experiments with CARB in rats, a species difference cannot be ruled out.
On the other hand, the sampling time of 48 h after treatment may not be the optimum time for detecting micronucleus induction by CARB. In the pilot study, with a 72 h sampling time, CARB also scored negative (Table IV), leaving the possibility that 24 h would have been an optimum time point. This does not, however, seem a likely explanation. In mice, Barale et al. (1993) observed the highest induction of micronuclei in mouse bone marrow PCE at 42 h after a single oral dose of CARB. Taking into account the fact that the PCE have to migrate to the blood, 48 h after dosing seems a reasonable sampling time for detection of CARB-induced micronuclei. In addition, with MMC clear micronucleus induction was observed 48 h after dosing.
Another possible explanation for the negative results obtained in the present study is that CARB did not reach the bone marrow. The PCE/NCE ratio was decreased in the high dose groups compared with the corn oil group, which was considered an indication of exposure of the bone marrow (although this decrease was not statistically significant). However, even if the decrease in PCE/NCE ratio was not considered relevant, this would not exclude exposure of the bone marrow to CARB, since CARB could also be non-toxic to erythropoiesis. In mouse bone marrow erythrocytes induction of micronuclei by CARB has been reported at doses that did not (yet) show an accompanying effect on PCE/NCE ratio (Pandita, 1988; Barale et al., 1993). In addition, analogous to CARB, MMC did not significantly decrease the PCE/NCE ratio but did induce micronuclei.
A remarkable finding was that the PCE/NCE ratios in blood samples taken after treatment were significantly lower than in pretreatment blood samples from the same rats, even in the control group and in the low dose groups. This shows that oral dosing of corn oil (with or without low doses of the test compound) is sufficient to induce a decrease in the PCE/NCE ratio. Most likely the stress of handling and dosing is enough to suppress erythropoiesis, unless corn oil is in itself toxic to the bone marrow. This makes the relevance of the PCE/NCE ratio as a measure of toxicity to the bone marrow questionable.
Hazard/risk assessment: comparison of sperm assay and micronucleus assay
The finding that CARB induced diploidy at doses that were below the lowest dose reported positive in the mouse bone marrow micronucleus test prompted us to investigate this further. In a second experiment we incorporated both end-points, aneuploidy/polyploidy in sperm and micronuclei in peripheral blood. The outcome of this experiment confirmed the induction of diploidy in sperm by CARB, although it was much less prominent than in the first assay, whereas induction of micronuclei in peripheral blood erythrocytes was not observed. This observation suggests that diploidy in sperm is induced at a lower dose than micronuclei in blood erythrocytes. It must of course be noted that the assays measure different end-points; polyploidy cannot be measured in the erythrocyte micronucleus assay and chromosome loss is not detected in the epididymal sperm assay. However, since the outcome of the (bone marrow) micronucleus test is generally considered an important indicator of the genotoxic potential of a compound in vivo, this is an important finding for hazard and risk assessment purposes. In conclusion, the results described here suggest that the erythrocyte micronucleus test is a less sensitive assay for the genotoxic potential of CARB in vivo in rats than the epididymal sperm aneuploidy/polyploidy assay. This implies that for a compound like CARB (a spindle poison with the testes as one of the target organs) a negative micronucleus assay does not automatically rule out possible effects in germ cells.
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Acknowledgments |
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We thank Marjo Poelen and Paul Reulen for biotechnical assistance, A.T. Natarajan for discussions and critically reading this manuscript and Jack Bishop and Ursula Eichenlaub-Rieter for helpful discussions.
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* To whom correspondence should be addressed. Tel: +31 30 274 2021; Fax: +31 30 274 4446; Email: J.van.Benthem{at}rivm.nl
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Received on June 14, 1999; accepted on August 9, 1999.
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