oocytes. We conclude that when ET is administered during the preovulatory phase of meiosis, it is both an aneugen and a clastogen in mouse one-cell zygotes.
Mutagenesis vol.11 no.4 pp.357-361, 1996
Numerical and structural chromosome aberrations induced by etoposide (VP16) during oocyte maturation of mice: transmission to one-cell zygotes and damage to dictyate oocytes
John B.Mailhes1"3, Francesco Marchetti2, Daniel Young1 and S.N.London1 'Department of Obstetrics and Gynecology, Louisiana State University Medical Center, PO Box 33932, Shreveport, LA 71130 and 2Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA, USA -'To whom correspondence should be addressed
Introduction Etoposide (ET) or VP-16 is a podophyllotoxin-derived antineoplastic drug (Liu, 1989; Smith, 1990) that inhibits topoisomerase II (topo II) activity by forming a ternary complex involving DNA-topo n-ET (Chen et ai, 1984; Ross et ai, 1984). This complex impairs the DNA-strand-rejoining function of topo II and results in DNA single- and double-strand breaks and in DNA-protein cross-links (Wozniak and Ross, 1983; Tominaga et ai, 1986; Bender et ai, 1990). Such ET-induced DNA damage can be transformed into chromosome structural aberrations, fragmentation of chromatin, sister chromatid exchanges © UK Environmental Mutagen Society/Oxford University Press 1996
Materials and methods Animals ICR (Harlan Sprague-Dawley, Inc., Indianapolis, IN) mice 8-12 weeks old (25-34 g) were used in all experiments. They were maintained under a 12 h light/12 h dark photoperiod, at an ambient temperature of 21-23°C and relative humidity of 50 ± 59t. Food and water were provided ad libitum. Hormones and chemicals Follicular maturation was augmented by giving an i p. injection of 7.5 IU of pregnant mare's serum (PMS; Folligon, Intervet Ltd, Cambridge.UK). Fortyeight hours later, 5 IU of human chorionic gonadotrophin (HCG; Ayrcst, Inc., Philadelphia, PA) was given to induce ovulation. Etoposide [4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-p>-D-glucopyranoside)] was obtained from Sigma Chemical Company. The working solutions were formulated with 6% dimethylsulphoxide (DMSO) in 0.9 r i NaCI solution and were administered by i.p. injection within 1 h following preparation. Females were given either 20, 40 or 60 mg/kg ET 2 h post-HCG. Controls received 6% DMSO in 0.9'A NaCI 2 h after HCG. Preliminary experiments, including toxicity evaluation, were done to ascertain an appropriate treatment time and ET dose range (Mailhes et ai, 1994). Cell harvest and processing For the 1CI zygote study, females were paired (1:1) with males immediately following ET or 6CZ DMSO treatment. Seventeen hours later the males were
357
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The antineoplastic drug etoposide (ET) inhibits topoisomerase II (topo II) activity by forming a ternary complex (DNA-ET-topo II). This complex prevents the DNA-strandrejoining activity of topo II and may result in structural chromosome aberrations. Inhibition of topo II activity may also predispose cells to aneuploidy because this enzyme is needed for removing regions of DNA catenation prior to chromosome segregation. Our objectives were to study the dose response for ET-induced numerical and structural chromosomal aberrations in mouse one-cell zygotes, to compare these data with those obtained from a contemporary metaphase II (MO) oocyte study and to evaluate the sensitivity of dictyate oocytes to ET-induced aneuploidy. ICR female mice were superovulated and injected i.p. with either 6% dimethylsulphoxide (controls) or 20, 40 or 60 mg/kg ET 2 h after human chorionic gonadotrophin (HCG). ICR males were paired (1:1) with females immediately after treatment After 17 h the males were removed, and after 24 h the females with a vaginal plug were given colchicine. One-cell zygotes were harvested for cytogenetic analysis 17 h after colchicine. The percentages of hyperploid zygotes were 1.1, 5.7, 13.8 and 20.7 and of zygotes with structural aberrations were 2.5, 163, 37.7 and 64.7, for control, 20, 40 and 60 mg/kg ET respectively. The differences between each succeeding dose for both structural and numerical aberrations were statistically significant (P < 0.01). When the ET dose response aneuploidy data from zygotes were compared with similar data from a contemporary study involving metaphase II oocytes, the frequencies of hyperploidy were greater in zygotes than in oocytes. We conclude that when ET is administered during the preovulatory phase of meiosis, it is both an aneugen and a clastogen in mouse one-cell zygotes.
and micronuclei (Sieber et ai, 1978; Tominaga et ai, 1986; Pommierera/., 1988; Darroudi and Natarajan, 1989;Maraschin et ai, 1990; Agarwal et ai, 1994; Sjoblom et ai, 1994). Besides its DNA-strand-rejoining activity, topo II is also needed for chromatin condensation, chromosome segregation and cellular proliferation (Wang, 1985; Adachi et ai, 1991; Taagepera et ai, 1993; Dang et ai, 1994). Catenation or interwining of DNA duplexes occurs during the replication of chromatids (Sundin and Varshavsky, 1980). Topo II is needed to remove this catenation so that die segregation of chromosomes and chromatids can occur during meioses I and H (Gellert, 1981; Liu, 1989). Since ET interferes with the role of topo II during this decatenation process, the possibility exists that ET can induce abnormal chromosome segregation during female meiosis, resulting in aneuploidy. In fact, ET has been reported to induce numerical abnormalities in cultured mammalian cells (Pommier et ai, 1988; Downes et ai, 1991) and in mouse embryonic cells from treated dams (Sieber et ai, 1978). The objectives of this study were to estimate the dose response of ET-induced structural and numerical chromosome aberrations in mouse one-cell (1C1) zygotes and to compare these data with those from a contemporary study involving metaphase II (Mil) oocytes (Mailhes et ai, 1994). Also, since ET-induced aberrations are thought to originate by a mechanism different to that for compounds which alter the meiotic spindle (Murray and Szostak, 1985), we also studied the persistence of the ET-induced ternary complex in dictyate chromatin throughout several oestrous cycles. If a compound is capable of inducing genetic damage in germ cells, it is important to have information about the duration that such abnormal cells persist following treatment. This is a practical concern when consulting certain patients who are candidates for chemotherapy.
J.B.Mallhes el al removed, and those females with a vagina] plug were given 2X1O"3 M colchicine 7 h later (24 h post-ET). The zygotes were collected 17 h after the colchicine injection. This time frame was used to increase the probability of sampling cells that may have undergone chemically induced maturational delay prior to reaching the first cleavage stage (Mailhes and Marchetti, 1994a). For the aberration persistence study involving dictyate oocytes, Mil oocytes were collected 17 h post-HCG. The gonadotrophins were given at either 811, 14-16 or 21-23 days after 40 mg/kg ET. These intervals represent ~2, 3.5 and 5 spontaneous oestrous cycles of the laboratory mouse respectively (Schwartz, 1973; Whittingham and Wood, 1983). Cells were flushed from the oviducts and processed for cytogenetic analysis according to the procedure of Mailhes and Yuan (1987). This procedure involves collecting oocytes or zygotes from 10-20 females and processing them en masse. The time from killing the animals to preparing air-dried slides was ~3 h. Similar to cytogenetic techniques for other cell types, the number of cells placed onto slides was less than the number actually collected due to cell lysis during hypotonic treatment and fixation. Also, the number of cells actually analysed was less than the number placed onto slides because each cell could not be objectively analysed. The experiments represented in the report by Mailhes et al. (1994) involving Mil oocytes and those of this report were performed under similar experimental conditions and during the same time period.
Results Overall, the data in Tables I and II show that the frequencies of structural and numerical aberrations in one-cell zygotes were greater in ET-exposed animals than in the controls. ET did not elevate the frequency of triploidy over controls. CtFs were the most prevalent type of structural aberration and the differences between increasing ET doses were highly significant (P < 0.01; Table I, Figure 4). Also, the frequencies of both ChTs and the total number of zygotes containing at least one structural aberration of any type increased significantly (P < 0.01) with ET dose (Table I and Figure 4). The frequencies of MCFs at the two higher ET doses were significantly greater (P < 0.05) than those observed at either of the other two doses (Table I). The data in Table II and Figure 4 show that ET resulted in significantly (P < 0.01) higher levels of hyperploidy at each dose relative to controls and that the frequency of hyperploid zygotes significantly (P < 0.05) increased at each successive dose. 358
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Fig. 1. Hyperploid (2N = 44) mouse one-cell zygote with a chromatid fragment (CtF, arrow).
Fig. 2. Euploid (2N = 40) mouse one-cell zygote with a chromosome fragment (ChF, arrow).
Fig. 3. Mouse one-cell zygote with multiple chromosomal fragments (MCF, arrow) and chromosome translocation (ChT, arrowhead).
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Cytogenetic analysis and statistical analysis of data The chromosomes were C-banded according to the procedure of Salamanca and Armendares (1974) to enable a quantitative assessment of the number of centromeres. In each zygote analysed for aneuploidy and triploidy, the number of chromosomes was counted at X1250 magnification. The frequencies of hypoploid (2N = 30-39), diploid (IN = 40), hyperploid (2N = 41-50) and triploid cells (2N = 51-60) were determined relative to the total number of one-cell zygotes analysed. The range in chromosome numbers for each classification encompasses the actual observations. Since an unknown proportion of the hypoploid cells results from chromosome loss during slide preparation, the frequencies of hyperploid cells (Figure 1) were used for statistical analyses. The number and type of structural chromosome aberrations found in the zygotes were also recorded. These included: chromatid fragments (CtF, Figure I), chromosome fragments (ChF, Figure 2), multiple chromosomal fragments (MCF, Figure 3) and chromosome translations (ChT, Figure 3). The CtFs contained both reputed centric and acentric fragments; from C-banding alone, it was not always possible to objectively discern each class. The criterion used for distinguishing between CtFs and ChFs was that the former compnsed a single piece of chromatin whereas the latter contained two paired pieces of chromalin. This criterion recognizes the possibility that an unknown proportion of ChFs may actually be chromosome interstitial deletions Zygotes with MCF contained structural aberrations to the extent that the actual number of each aberration could not always be objectively counted. The criteria used for eliminating a cell from analysis included inadequate C-banding such that centromere numbers could not be objectively counted and excessive chromosome clumping or overlapping. Chi-square analyses, Fisher's exact test and a two-way factorial design with interaction (analysis of variance) were used for analysing the data.
* "
Etoposide-induced chromosome aberrations during oocyte maturation
Table I. Etoposide-induced structural chromosomal aberrations in mouse one-cell zygotes Total no.b of cells analysed
Etoposide" (mg/kg)
Controld 20 40 60
357 283 247 275
No. of aberrations (%)c Chromatid fragments (centric and acentric)
Multiple chromatid fragments
Chromosome fragments
Chromosome translocations
Total no. of zygotes with structural aberrations (
