Simultaneous micronucleus and chromosome aberration assessment in the rat

Mutation Research, 264 (1991) 29-35 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$03.50 ADONIS 0165799291000798

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MUTLET 0526

Simultaneous micronucleus and c h r o m o s o m e aberration assessment in the rat G. Krishna, M.L. Kropko, V. Ciaravino and J.C. Theiss Molecular Toxicology Section, Department of Pathology and Experimental Toxicology, Parke-Davis PharmaceuticalResearch Division, Warner-Lambert Company, Ann Arbor. M148105 (U.S.A.) (Received 19 April 1991) (Revision received 30 April 1991) (Accepted 6 May 1991)

Keywords: Micronucleus assay; Chromosome aberration test; Cyclophosphamide; Sex comparison; (Rat)

Summary Using a cellulose column fractionation procedure to eliminate nucleated cells for micronucleus assessment, micronucleus and chromosome aberration endpoints in the same animal were compared in male and female rats following i.p. injection with cyclophosphamide (CP). Groups of 5 Wistar rats per sex were given single doses of CP at 0, 20, or 40 mg/kg. Two hours prior to sacrifice, animals were given colchicine (4 mg/kg) to arrest cells in metaphase. One femur from each animal was used for micronucleus assessment and the other for chromosome aberration assessment. In the micronucleus assessment, 2000 polychromatic erythrocytes (PCEs) per animal and in the chromosome aberration assessment, 50 metaphase cells per animal were scored. This experiment was repeated once. In both experiments, significant increases in micronucleated PCEs and chromosome aberrations were noted at both doses of CP in both sexes. In general, the clastogenic effects of CP were more pronounced in males than females. Both doses of CP caused a decrease in the proportion of PCEs and in mitotic index in both experiments, indicating toxicity of CP to the bone marrow. These results show the usefulness of this rat model for simultaneous evaluation of two cytogenetic endpoints in the same animal and indicate that assessment of MNPCE frequency in the bone marrow of male rats may be an appropriate model for screening test substances for in vivo clastogenic activity in this species.

Cytogenetic assays have been widely used in the genotoxicity assessment of test compounds under in vitro and in vivo conditions. Formation of

Correspondence: Dr. G. Krishna, Molecular Toxicology Section, ParkeoDavis Pharmaceutical Research Division, WarnerLambert Company, 2800 Plymouth Road, Ann Arbor, M1 48105 (U.S.A.).

micronuclei and chromosome aberrations are 2 important cytogenetic endpoints that are routinely used in genotoxicity evaluation (Heddle et al., 1983; Mavournin et al., 1990; MacGregor et al., 1987; Preston et al., 1981, 1987). Micronuclei are thought to arise from both clastogenic (chromosome breakage) and aneugenic (chromosome lagging and effects on spindle) el'-

30 fects, while chromosome aberrations are thought to arise from chromosome breakage and exchange. In the in vivo genotoxicity evaluation of test compounds, the mouse model has been routinely used in the micronucleus assay and the rat model has been primarily used in the chromosome aberration assay. O f these 2 rodent species, the rat plays a dominant role as an animal model in toxicology testing. Also, using rat, an enormous amount of data concerning pharmacokinetics, drug metabolism, distribution, and excretion is obtained during toxicological evaluation of a new compound under pharmaceutical development. However, the rat has been avoided for the most part in the micronucleus assay, mainly because granules from ruptured leukocytes resemble micronuclei and contaminate bone marrow smears. Recently, Romagna (1988) and Romagna and Staniforth (1989) described a cellulose column fractionation procedure to remove nucleated cells from the whole bone marrow and thus overcome the problem of mast granules in the micronucleus assay for the rat model. In the literature, there are reports describing procedures to simultaneously evaluate both micronucleus and chromosome aberrations in the same animal using the mouse model (Tates and Natarajan, 1976; Natarajan et al., 1983; SanSebastian et al., 1990). This approach of evaluating 2 endpoints in the same animal has several advantages such as reducing overall animal usage, clarifying marginal clastogenic responses of test agents and correlating genotoxicity results from multiple endpoints. However, while this multiple-endpoint approach has been described in the rat (Albanese, 1987), it has not been extensively used because of the mast cell granule contamination problem in bone marrow smears. Using the cellulose column fractionation method for micronucleus assessment in the present study, micronucleus and chromosome aberration endpoints in the same rat were compared. Cyclophosphamide was used as the test compound and the study was performed in both male and female rats in order to assess differences between sexes.

Materials and methods

Chemical Cyclophosphamide (CP, CAS No. 50-18-0) was obtained commercially from Sigma Chemical Company, St. Louis, MO. Animals 6-8-week-old Wistar male and female rats obtained from Charles River Breeding Laboratories (Kingston, NY) were housed individually in stainless steel wire mesh cages and acclimatized for 1 week before dosing. The animals were observed before initiation of the study to ensure that they were healthy. Only animals found to be in a clinically acceptable condition were assigned to the study. During the study, food (Purina Certified Rodent Chow ® 5002) was offered ad libitum in stainless steel food containers. Water was supplied ad libitum by an automatic watering system. An automatic timer provided the animal room with an alternating 12-h cycle of light and darkness. Each experimental group consisted of 5 animals per sex. Chemical treatment The drug, CP, was dissolved in sterile distilled water. Groups of 5 Wistar rats per sex were given single doses of CP at 0, 20, or 40 mg/kg intraperitoneally at a dosing volume of 10 ml/kg. These doses were selected based on published literature (Krishna et al., 1987, 1988, 1990; Romagna and Staniforth, 1989; Heddle et al., 1983; Mavournin et al., 1990; Preston et al., 1987) and our preliminary results with CP. The experiment was repeated once to confirm the results. A n i m a l sacrifice, bone marrow cell isolation, and slide preparation 2 h prior to sacrifice the animals were injected i.p. with 4 mg/kg of colchicine. At 24 h post dosing, both femurs were removed from each rat; one was used for micronucleus assessment and the other for chromosome aberration assessment. Bone marrow cells for micronucleus assessment were isolated and slides were prepared as described by Schmid (1975, 1976), Ashby and Mohammed

31 (1986), Romagna and Staniforth (1989) and Krishna et al. (1990). Briefly, rats were killed by CO2 asphyxiation and the femurs excised and trimmed immediately. The femur was severed with bone snips and the bone marrow was flushed from the channel into a tube using fetal bovine serum (approximately 3 ml/femur). Cell clumps were broken up by pipetting, and debris was removed by gravity sedimentation for approximately 5 min. The cell suspension was placed on a cellulose column and washed using Hanks' balanced salt solution (HBSS, without phenol red). The eluant was spun at 8 0 0 x g for 10 min and the supernatant discarded. The pellet was resuspended in an appropriate amount of serum and slides were prepared using a cytocentrifugation method. All slides were fixed in methanol and stained with Wright's Giemsa. The bone marrow for chromosome aberration assessment was prepared as described by Krishna et al. (1985, 1987) for rodents. Briefly, the bone marrow was flushed with HBSS, debris removed, and cells were centrifuged. The pellet was resuspended, then a hypotonic solution was added and incubated in a water bath at 37°C for 20 min. Following incubation, a few drops of cold fixative were added and centrifuged. The pellet was resuspended in cold fixative and tubes were refrigerated overnight. Cells were washed in fresh fixative 3 times before preparing the slides. To prepare slides, the final cell pellet was suspended and a few drops of fresh fixative were added to each tube. The cells were then dropped on labeled slides, flamed, air-dried, and stained in 10070 Giemsa.

Data collection In the micronucleus assessment, 4 replicate slides per animal were coded blind. The ratio of polychromatic erythrocytes (PCE) to 400 erythrocytes (Es) was determined to assess the toxicity of the test compound on erythrocytes. 2000 PCEs per animal were scored for micronucleus frequency. The criteria for scoring and identification of micronuclei were similar to earlier studies (Schmid, 1976; Heddle et al., 1983; Ashby and Mohammed, 1986; MacGregor et al., 1987).

In the chromosome aberration assessment, 50 suitable metaphase figures with chromosome number 2n = 42 + 2 were examined for each animal, whenever possible. Cells not scorable included those with poor morphology. A mitotic index (MI) based on 500 bone marrow cells per animal was also recorded to evaluate potential toxicity of the test compound in this assay.

Statistical analysis o f data For both the micronucleus and the chromosome aberration assessments, separate statistical analyses were conducted for each sex, taken separately and combined. In the micronucleus assessment, the primary measures were the frequency of PCEs in the erythrocyte population and the frequency of MNPCEs in the PCE population. In the chromosome aberration assessment, analyses were carried out for aberrations excluding chromatid and chromosome gaps as well as pulverized cells. Variables of interest were the MI, the percent ceils with at least one aberration, and the mean number of aberrations per cell. Student's t-test and Fisher exact test were used to analyze the data. Also, Pearson correlation coefficients (r) were calculated for dose and response to evaluate relative degree of association between these 2 parameters in each assay. Results

The micronucleus and chromosome aberration data from both experiments are shown in Table 1. Cyclophosphamide, at both doses and in both experiments, induced a significant increase in both cytogenetic endpoints compared to controls. In the first experiment, the controls had 0.4%, 1.2%, and 0.8°7o ceils with aberrations and 2.7, 1.0, and 1.9 MNPCEs per 1000 PCEs in males, females, and sexes combined, respectively. The cyclophosphamide-induced micronucleus frequency was significantly elevated in both sexes compared to controls; however, it was more pronounced in males than in females. This response was statistically significant between sexes. Based on the combined sex data, an about 6-fold increase in

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MNPCE frequency was noted in the cyclophosphamide-treated animals compared to controls at both 20 and 40 mg/kg (r=0.76). In the same animals, the chromosome aberration frequency was significantly elevated. The increased chromosome aberration frequency was more pronounced in males than in females, as was the case with the micronucleus frequency. The chromosome aberration response was clearly dose-dependent in both sexes (r=0.99). Based on the combined sex data, about a 9- and 22-fold increase in percent cells with aberrations at 20 and 40 mg/kg, respectively, was noted in cyclophosphamide-treated animals compared to controls. Cyclophosphamide primarily induced chromatid breaks and exchanges. Also, at doses tested, it caused gaps and pulverization of chromosomes in a dose-dependent manner; these anomalies were recorded but not included in the computation of total aberrations. In addition, it caused some toxicity to the bone marrow as evidenced by the decrease in MI and in percent PCEs. These observations were applicable for both sexes. In the repeat experiment (Expt. 2), the data in general were comparable with the first experiment especially regarding micronucleus frequency. However, in the chromosome aberration assessment, the percent of ceils with aberrations was somewhat higher in females compared to males. A comparison of data related to percent cells with acentric fragments and MNPCE based on all animals used in the study is shown in Table 2. This comparison was done to verify any general relationship between the formation of acentric fragments and MNPCEs in the same animal. In general, it is interesting to note that the majority of the animals (53 animals; about 88%) had relative agreement on the number of cells with acentric fragments and MNPCE frequencies, i.e., animals which had a lower percent ceils with acentric fragments had relatively lower MNPCE frequencies and animals which had a higher percent cells with acentric fragments had relatively higher MNPCE frequencies. The MNPCE frequency appeared to plateau and even decreased somewhat at very high levels of cells with acentric fragments but

TABLE 2 CLASTOGENICITY ANALYSIS IN THE RAT: COMP A R I S O N OF CELLS W I T H ACENTRIC FRAGMENTS A N D M I C R O N U C L E A T E D P O L Y C H R O M A T I C ERYTHROCYTES (MNPCE) Percent cells with acentric fragments

Mean MNPCE frequency per 1000 PCEs + SE

Number of animals

0 1-10 11-20 21-30 31-40 41-50 51-60

2.85 11.62 12.30 14.15 11.38 6.00 5.50

20 13 10 10 4 2 1

_+ _+ + + + + _+

0.58 3.18 2.27 2.12 2.71 0.00 0.00

the small number of animals with high levels of acentric fragments precludes drawing a definitive conclusion. Discussion The procedure developed by Romagna and Staniforth (1989) for automation of the bone marrow micronucleus test provided excellent preparations for the manual assessment of rat bone marrow erythrocytes in this study. The nucleated cell population, including mast cells, was essentially eliminated as a result of column fractionation and the detection of micronuclei was facilitated due to the flattening of cells resulting from cytocentrifugation. In general, there was good correlation among the clastogenic and toxic endpoints evaluated in this study of cyclophosphamide. This drug increased both the frequency of micronucleated PCEs and the frequency of nucleated cells with chromosome aberrations in the bone marrow, which agrees with results reported previously by Trzos et al. (1978) and Albanese (1987), and the drug decreased both the mitotic index and the proportion of PCEs in the erythrocyte population, indicating a good correlation between these two measures of toxicity. An increased micronucleated PCE frequency would be expected in this study since a high number of chromatid and chromosome breaks resulted from

34

cyclophosphamide exposure. It has been proposed that the formation of acentric fragments plays a major role in the mechanism of micronucleus formation (Heddle et al., 1983; MacGregor et al., 1987; Mavournin et al., 1990). The comparative data on the percent cells with acentric fragments and M N P C E frequencies in the same animal in this study support this proposition. There was a clear sex difference in this study with respect to the micronucleus response to cyclophosphamide, the males being more sensitive than females in both experiments. Similar results have been reported for this drug in mice (Collaborative Study Group, 1986). However, such a sex difference was not obvious with respect to the induction of chromosome aberrations. Males were more sensitive in the first experiment but females were as sensitive or slightly more sensitive in the second experiment. Also, in general, on a quantitative basis, the proportion of chromosome aberration-bearing mitotic cells was far greater than that of micronucleated PCEs. Such differences have been attributed to several possibilities such as their mechanism of formation, cellcycle kinetics, sex differences in their ability to metabolize the test drug and the probability of inclusion of a chromosome aberration in the subsequent PCEs (Heddle et al., 1983; Preston et al., 1981, 1987; Hayashi et al., 1984; MacGregor et al., 1987, 1990a,b; Roberts et al., 1986; Mavournin et al., 1990; Madle et al., 1986a,b, 1987). This study demonstrates the utility of the column fractionation/cytocentrifugation procedure in conducting simultaneous assessments of both micronucleus- and chromosome aberrationinducing activity of chemicals in the same rat. This approach offers the advantage that a fuller understanding of clastogenic activity can be achieved since multiple endpoints for clastogenicity are assessed. Also, since the rat plays a dominant role in both toxicity testing and pharmacokinetic/metabolism studies, the clastogenic activity of a chemical can be more closely related to its toxicologic and toxicokinetic profile. It has recently been suggested that the micronucleus assay might better be performed using 1 sex

of 2 different species rather than 2 sexes of 1 species (Arlett et al., 1989). Recent results support this contention (Albanese et al., 1988). Oral doses of 1,2-dimethylhydrazine effectively induced micronuclei in mice but not rats while 1,2-dibromochloropropane induced micronuclei in rats but not mice. Male mice are considered as sensitive as or more sensitive than female mice with respect to micronucleus induction. In this study we have demonstrated that micronucleus induction in male rats would make a suitable screening tool for in vivo clastogenicity. Thus, one could envision a screening test for in vivo clastogenicity utilizing male mice and rats rather than 2 sexes of 1 species. This approach has the advantage that genotoxicity information could be obtained in both species which are utilized in carcinogen bioassays.

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