Mutagenesis vol.7 no.4 pp.303-309, 1992

Benomyl-induced aneuploidy in mouse oocytes

John B.Mailhes1 and Marilyn J.Aardema2 'Department of Obstetrics and Gynecology, Louisiana State University Medical Center, Shreveport. LA 71130, USA and 2The Procter and Gamble Company. Miami Valley Laboratories. PO Box 398707, Cincinnati, OH 45239, USA

Materials and methods Introduction Benomyl (methyl-l-[butylcarbarnoyl]-2-benzimidazole carbamate) (Figure la) is the active compound in several, widely-used benzimidazole derivatives for controlling pathogenic fungi in plants (Clemons and Sisler, 1971). In aqueous solution, benomyl hydrolyzes to carbendazim or MBC (methyl-2-benzimidazole carbamate) (Figure lb) which is the stable, toxic form (Kilgore and White, 1970). Both benomyl and MBC are effective fungicides due to their ability to disrupt mitosis by binding to tubulin (Davidse, 1973; Davidse and Flach, 1977) and preventing polymerization in vitro (Kilmartin, 1981) and in vivo (Jacobs etal., 1988). Historically, benomyl has played a key role in drawing the attention of researchers and regulatory agencies to the possible risks of chemically-induced aneuploidy. In 1979, when the Environmental Protection Agency (EPA) was reviewing benomyl (Voytek, 1985) there were no cancer data available and the genotoxicity data were conflicting. However, the ability of benomyl to bind to and inhibit tubulin polymerization along with the observation that benomyl induced genetic instability in the fungus Aspergillus nidulans (Hastie, 1970) prompted the EPA to recommend further testing since the risk from exposure to aneuploidy-inducing agents was unknown. The EPA's concern was realized since it has been shown that benomyl induces hepatocellular tumors in mice (Salminen and Kivela-Ikonen, © Oxford University Press

Animals Virgin female ICR (Harlan Sprague-Dawley, Inc.) mice between 8 and 12 weeks of age (25-32 g) were maintained under a 12-h light/12-h dark photoperiod at a room temperature of 21 —23°C and relative humidity of 50 ± 5%. Feed was provided ad libitum. The number of mature follicles was increased by an i.p. injection of 7.5 IU pregnant marc's serum (PMS, Folligon, Intervet Ltd, Cambridge, UK). Ovulalion was induced by an i.p. injection of 5.0 IU human chorionic gonadotrophin (HCG, Ayerst, NY) 48 h after PMS. Chemicals and dosing Benomyl (CAS No. 17804-35-2) was provided by the Radian Corporation (National Toxicology Program source), lot No. 5-167, inventory control No. NT-004575, - 9 6 % pure. A suspension of benomyl in olive oil (Sigma Chemical Co., No. CONHCH2 CH2 CH2 CHj NHCO2CH3

Fig. 1. a, chemical structural formula of benomyl. b, chemical structural formula of carbendazim.

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Benomyl (methyl-l-[butylcarbamoyl]-2-benzimidazole carbamate) is a plant fungicide which acts by inhibiting tubulin polynierization. It is also a carcinogen and a teratogen. A substantial number of reports consistently show that benomyl inhibits cellular division and induces numerical chromosome changes in somatic cells, while results from standard genotoxicity assays which measure mutations and structural chromosome aberrations are conflicting. To define further the genotoxic effects of benomyl and to demonstrate the utility of the in vivo mouse oocyte assay for detecting chemicals which induce numerical chromosome changes, we investigated the ability of benomyl to induce numerical and structural chromosome aberrations in mouse oocytes. Superovulated female ICR mice were administered benomyl by oral gavage and oocytes were collected 17 h later. The proportions (and percentages) of hyperploid oocytes were 1/309(0.3), 6/155(3.9), 38/229(16.6), 46/130(35.4), 60/215 (27.9), 42/143(29.4) for control, 500, 1000, 1500, 1750 and 2000 mg/kg respectively. No increase in structural aberrations was observed. These results demonstrate that benomyl specifically induces numerical chromosome changes in mouse oocytes.

1982) and was teratogenic in both rats and mice (Kavlock et al., 1982). The EPA's position on benomyl and their conclusions that benomyl is carcinogenic and teratogenic in rodents, and can induce nondisjunction is summarized in the Federal Register (1982). The results for benomyl and MBC from genotoxicity assays designed to detect point mutations and structural chromosome aberrations are conflicting. As summarized in Table I, both positive and negative results have been obtained for benomyl and MBC from a variety of assay systems involving plant and mammalian cells. In contrast to the conflicting results for benomyl and MBC for the induction of point mutations and structural chromosome aberrations, there are consistent results demonstrating that benomyl and MBC interfere with cell division and induce numerical chromosome changes in somatic cells. As indicated in Table I, benomyl and MBC inhibit cell division, induce aberrant mitoses and/or induce nondisjunction in plant and animal somatic cells in vitro and in vivo. There are no data in the literature regarding the ability of benomyl to induce aneuploidy or other genotoxic effects in mammalian female germ cells. Therefore, the purpose of the experiments reported here was to investigate benomyl induced aneuploidy and chromosome aberrations in mouse oocytes. This study also continues our efforts to validate the mouse oocyte assay as a method for detecting chemicals that induce numerical chromosome changes in germ cells (Mailhes etal., 1986, 1990).

J.B.Mallhes and MJ.Aardema

Table I. Summary of the genotoxicity of benomyl and/or carbendazim (MBQ Organism/cell type

Point mutations

Fusarium oxysporum Escherichia coli Aspergillus nidulans Aspergillus nidukms Cladosporium cucumennum Mus musculus Salmonella typhimurium Salmonella typhimurium Salmonella typhimurium Streptomyces coelicolor Aspergillus nidulans

Dassenoy and Meyer (1973) Kappas et al. (1976) Kappas et al. (1974) Speakman and Nirenberg (1981) Speakman and Nirenberg (1981) Fahrig and Seder (1979) Seiler (1972) Carere et al. (1978) Fiscor et al. (1978) Carere et al. (1978) Bignami et al. (1977)

Structural chromosome aberrations

Allium cepa rat fetuses Pennisetum americanum Pennisetum americanum Secale cereale Human lymphocytes in vivo Human lymphocytes in vitro Rat bone marrow cells Chinese hamster bone marrow cells

Cell division abnormalities and nondisjunction in somatic cells

Rat bone marrow cells Mammalian cells in vitro Mammalian cells in vitro Botrytis cinerea Allium cepa Horderum vulgarc Human lymphocytes in vitro Aspergillus nidulans Aspergillus nidulans Aspergillus nidulans Aspergillus nidulans Mouse bone marrow cells Mouse bone marrow cells Rat bone marrow cells Human lymphocytes Chinese hamster ovary cells Mouse L-cells Microtus oeconomus Hamster bone marrow cells Human —mouse hybrid cell line V79/AP4 Chinese hamster cells Human lymphocytes Human x hamster hybrid cell line Rat dominant lethal Rat dominant lethal

Boyle (1973) Ruzicska et al. (1975) Sandhu and Dehsi (1984) Dhesi and Sandhu (1984) Boyle (1973) Ruzicska et al. (1975) Banduhn and Obe (1985) Ruzicska et al. (1975) Seller (1976) Styles and Gamer (1974) Debrabander ex al. (1976a,b) Styles and Gamer (1974) Richmond and Phillips (1975) Richmond and Phillips (1975) Nicoloff and Kappas (1987) Banduhn and Obe (1985) Davidse (1973) Bignami et al. (1977) Kappas et al. (1974) Gualandi and Bellincampi (1981) Seiler (1976)

0-1500, lot No. 40H-O1231) was prepared by mixing on a magnetic stirrer for 45 min. Dosages were administered by oral gavage immediately after mixing. In a preliminary study, five female mice dosed with 3000 mg/kg benomyl showed no overt signs of toxicity. For the actual study, female mice were given 500, 1000, 1500, 1750 or 2000 mg/kg benomyl in a volume of 0.5-0.64 ml per animal by oral gavage immediately after HCG. Controls received 0.2 ml olive oil/10 g body weight by oral gavage immediately following HCG. The basic experimental procedure involved harvesting oocytes en masse from 10—15 similarly treated females during a 3.5 h period. Thus, oocytes are harvested from controls and each benomyl group during a 2-week period. Due to differences among groups in the proportions of ovulated MI oocytes and in the quality of the cytogenetic preparations, additional experiments were performed for certain groups to increase me number of Mil oocytes analyzed. Metaphase D (MH) oocytes were collected from the oviducts of control and treated mice 17 h post-HCG and processed for cytogenetic analyses according to the procedure described by Mailhes and Yuan (1987a). Chromosomes were C-banded according to the procedure of Salamanca and Armendares (1974) to ensure the unequivocal identification of whole or separated chromosomes.

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Result

Reference

Tsuchimoto and Matter (1977) Georgieva et al. (1990) Raimondi et al. (1989) Eastmond and Tucker (1989) Stemes and Vig (1989) Tates (1978) Sheu et al. (1990) Athwal and Sandhu (1985) Rainaldi et al. (1987) Tenchini et al. (1983) Zelesco et al. (1990) Sherman et al. (1975) Georgieva et al. (1990)

Cytogenetic analyses The number of centromeres in C-banded oocytes was counted. Also the number and type of structural chromosome aberrations were recorded. The numbers of euploid (n = 20), hypoploid (n = 1 0 - 19.5), hyperploid (n = 20.5-29.5) and polyploid (n = 30-40) oocytes were recorded. The criteria for eliminating a cell from the total number analyzed included: insufficient C-banding to discriminate between whole chromosomes (dyads) or those separated at the centromere resulting in two chromatids, overlapped or clumped chromosomes that precluded accurate counts, or excessive chromosome scatter. The proportions of hyperploid oocytes were used for data analysis because an unknown proportion of hypoploid cells may result from technical artefact (Mailhes et al., 1986: Pacchierotti. 1988). Statistical differences in the percentage of hyperploid cells were determined by using a Fisher's Exact Test.

Results and discussion The chromosome distribution in Mil oocytes from females given 0, 500, 1000, 1750 and 2000 mg/kg benomyl by oral gavage

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Assay

BenomyHnduced aneuploidy

Table U. Distribution of chromosome number in mouse metaphase II oocytes after oral dosage of benomyl Benomyl (mg/kg) Control 500 1000 1500 1750 2000

Haploid number 10-14 15

15.5* 16

16.5 17

17.5 18

1 6 5 4 4 1

2 1 1 1 2

1

2

2

2 3 1

5 1

4 2 1

1 1

4 9 4 8 5 4

18 .5 19 6 13 14 13 11 12

2 2

19.5 20

20.5 21

2

1 1 5 4

2 4 6

295 115 158 44 122 79

5 3

21.5 22

3 15 18 39 15

1 6 1 1

1 12 11

6 14

22.5 23

3 1 1

23.5 24

2 3 3 3

24.5 25 25.5-29

1 1 2

2 2 2

"15 dyads plus one unpaired chromatid.

Table HI. Aneuploidy in ICR mouse metaphase II oocytes after oral gavage administration of benomyl No. of mice

Control 500 1000 1500 1750 2000

39 47 59 20 29 27

Historical controls

No. oocytes analyzed (%)b Total 309 155 242 142 241 168 5432

MI 0 0 10(4.2) 12(8.5) 19(79) 17(10.1) 0

No. Mils (%) Mil 309 155 229 130 215 143 5432

Polyploid0 0 0 3(1.2) 0 7(2.9) 8(4.8) 0

Hypoploidd 17(5.5) 34(21.9) 33(14.4) 40(30.8) 33(15.3) 22(15.4) 208(3.8)

Hyperploid1 1(0.3) 6(3.9)* 38(16.6)* 46(35.4)* 60(27.9)* 42(29.4)* 14(0.26)

"Administered immediately after HCG. b Oocytes collected 17 h post-benomyl. MII oocytes containing 30-40 dyads. d MII oocytes containing 10-19.5 dyads. C MII oocytes containing 20-29.5 dyads. f Olive oil solvent. *P < 0.01 (Fisher's Exact Test), control versus benomyl groups. C

is presented in Table II. The proportions of cells with numerical chromosome changes are summarized in Table HI. No structural aberrations in MI or Mil oocytes were observed. Examples of hyperploid oocytes are shown in Figure 2a and b. As indicated in Table III, benomyl induced an increase in ovulated MI oocytes indicating meiotic delay. Results have shown that colchicine (Mailhes and Yuan, 1987b), vinblastine sulfate (Russo and Pacchierotti, 1988), griseofulvin (Pacchierotti et al., 1989) and benomyl (this report) induced both MI oocyte arrest and aneuploidy. Although additional chemicals have been reported to cause aneuploidy in mammalian oocytes (reviewed in Mailhes et al., 1986; Pacchierotti, 1988), information about meiotic delay was not always presented. However, based on those studies where meiotic delay was reported and other studies in our laboratory involving vinblastine sulfate and griseofulvin (J.B.Mailhes, unpublished data) there seems to be an association between the ability of a chemical to induce meiotic delay (as evidenced by ovulated MI oocytes) and the induction of aneuploidy. This correlation has also been found in male mice in a study with seven chemicals which induced aneuploidy in spermatocytes and also induced meiotic delay (Miller and Adler, 1992). Thus, chemical induction of meiotic delay in germ cells may be a useful screening assay for detecting chemicals which can damage the spindle apparatus. Assessing the ability of a chemical to induce mitotic arrest has also been suggested as a method for detecting aneuploidy-inducing chemicals in vitro and in somatic cells in vivo (Satya-Prakash et al., 1984; Miller and Adler, 1989). However, as pointed out by Miller and Adler (1989), variables such as the treatment and harvest times,

determining the absolute number of mitotic cells, and subjectivity in classifying mitotic cells according to the degree of chromosome spreading and contraction may be responsible for negative c-mitotic effects in association with positive aneuploidy in mitotic cells. Along with the increase in meiotic delay, there was a clear increase in the frequency of hyperploid oocytes in all groups receiving benomyl as compared to concurrent controls. As another basis of comparison, the frequency of aneuploid cells in the benomyl-treated groups was greater than the historical control range of hyperploid oocytes found in our laboratory of 0-1.4% (mean 0.26%). All doses were statistically significant (P < 0.01) when compared to concurrent controls using a Fisher's Exact Test. Also, the difference in hyperploidy between the 500 and 1000 mg/kg benomyl and 1000 and 1500 mg/kg benomyl groups were highly significant (P < 0.01, chi-square test). We also noted polyploid Mil oocytes (Figure 3a and b) in some of the benomyl treated groups, whereas polyploid oocytes were not observed in concurrent control animals and have not been observed in the 5432 Mil oocytes in the historical control database. These cells had between 30 and 40 dyads and most likely represent oocytes that were previously blocked in MI and subsequently progressed to Mil without going through the reduction, division and formation of a polar body. Vinblastine has also been shown to induce a low frequency of polyploid MO oocytes (Russo and Pacchierotti, 1988). Similarly, colchicine resulted in triploid 1-cell zygotes when administered to female mice immediately following HCG (Mailhes et al., 1990). This latter finding suggests that ovulated MI (delayed) oocytes are

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Benomyl1 (mg/kg)

J.B.Mailbes and MJ.Aardema

c J

Fig. 2. a, mouse metaphase II hyperploid oocyte with extensive centromere separation in one dyad (I ) (n = 22 oocyte. n = 18 polar body), b. mouse metaphase II hyperploid oocyte (n = 21 oocyte, n = 19 polar body).

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J> '

Benomyl-induced aneuploidy

a

fertilized and reach at least the 1-cell zygote stage. Other reports (McGaughey and Chang, 1969; Albanese, 1987) have previously shown that triploid mouse zygotes result when the dams receive colchicine. Some investigators (Rainaldi et al., 1987; Zelesco et al., 1990) have proposed that polyploid cells result from a complete disruption of the mitotic apparatus following high doses of a chemical, whereas lower doses result in more subtle effects on the mitotic apparatus resulting in aneuploidy. The data presented here support this conclusion based on the increasing frequency of polyploid cells at higher doses of benomyl, which is preceded by the induction of aneuploid cells at lower doses. In contrast to the increase in numerical chromosome changes observed in mice given benomyl, structural chromosome aberrations were not found in MI or Mil oocytes. This indicates that benomyl induces only numerical changes in oocytes, and that a compound with these properties might go undetected in assays designed for assessing structural chromosome damage in germ cells. Although benomyl induced a significant increase in hyperploidy at all doses tested in this study, it has been reported by other investigators (Zelesco et al., 1990; Tenchini et al., 1983) that benomyl, and other chemicals which induce numerical

chromosome changes by a similar mechanism involving tubulin inhibition (colcemid, Cox and Puck, 1969; vinblastine sulfate, Russo and Pacchierotti, 1988), have a threshold for the induction of numerical chromosome changes. This would be expected based on the mechanism of benomyl-induced numerical chromosome changes which involves the binding of benomyl to tubulin and subsequent inhibition of microtubule polymerization. Seiler (1977) reported that an in vivo dose of MBC ~ 8 /tg/ml blood represents a true threshold based on an in vitro no effect level for MBC inhibition of tubulin association at this concentration. This is supported by his data indicating no increase in micronuclei in bone marrow at doses of MBC that resulted in blood levels of ~ 8 /tg/ml, a slight increase in micronuclei when the blood level of MBC reached - 1 1 jig/ml, and clear increases in micronuclei at doses of MBC that resulted in blood levels >20 /tg/ml. The positive results in our studies which employed doses of benomyl of 500 mg/kg and greater are as expected based on this threshold concentration. In general, chemicals which are genotoxic and/or carcinogenic via a mechanism that involves the inhibition of tubulin may be expected to have a threshold. Based on this, non-linear dose responses may be appropriate for this class of chemicals. To our knowledge, these data demonstrate for the first time 307

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Fig. 3. a, mouse metaphase II polyploid oocyte with extensive centromere separation in two dyads (I ) (n = 40). b, mouse metaphase II polyploid oocyte (n = 40).

J.B.Mailhes and MJ.Aardema

Acknowledgement The authors would like to express their appreciation to Dr R.Julian Preston for his suggestion for studying benomyl.

References AIbanese,R. (1987) Induction and transmission of chemically induced chromosome aberrations in female germ cells. Environ. Mol. Mutagenesis, 10, 231—243. Athwal,R.S. and Sandhu.S.S. (1985) Use of a human x mouse hybrid cell line to detect aneuploidy induced by environmental chemicals. Mutat. Res., 149, 73-81. Banduhn,N. and Obe.G. (1985) Mutagenicity of methyl 2-benzirnklazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sisterchromatid exchanges, C-mitoses, polyploidies and micronuclei. Mutat. Res., 156, 199-218. Bignami.M., Aulicino.F., Velcich.A., Carere,A. and Morpurgo,G. (1977) Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. Mutat. Res., 46, 395-402. Boyle,W.S. (1973) Cytogenetic effects of benlate fungicide on Allium cepa and Secale cereale. J. Hered, 64, 4 9 - 5 0 . Carere,A., Ortali.V.A., Cardamone.G., Torracca.A.M. and Raschetti.R. (1978) Microbiological mutagenicity studies of pesticides in vitro. Mutat. Res., 57, 277-286. Clemons.G.P. and Sisler.H.D. (1971) Localization of the site of action of a fungitoxic benomyl derivative. Pestic. Biochem. Physiol., 1, 32-43. Cox.D.M. and Puck,T.T. (1969) Chromosomal nondisjunction: the action of colcemide on Chinese hamster cdls in vitro. Cytogenetics, 8, 158-169. Dassenoy.B. and MeyerJ.A. (1973) Mutagenic effect of benomyl on Fusariwn oxysporum. Mutat. Res., 21, 119-120. Davidse,L.C. (1973) Antimitotic activity of methyl benzimidazole-2-yl carbamate (MBC) in Aspergillus nidulans. Pestic. Biochem. Physiol., 3, 317-325. Davidse.L.C. and Flach.W. (1977) Differential binding of methyl benzimidazole-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J. Cell Biol., 72, 174-193. DeBrabander.M., Van de Veire.R., Aerts.R, Geuens.S. and HoebekeJ. (1976a) A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells. J. Nail. Cancer Inst., 56, 357—363. DeBrabander.M.J., Van de Veirc.R., Aerts.F., Borgers.M. and Janseen.P. (1976b) The effects of methyl [5-

Benomyl-induced aneuploidy in mouse oocytes.

Benomyl (methyl-1-[butylcarbamoyl]-2-benzimidazole carbamate) is a plant fungicide which acts by inhibiting tubulin polymerization. It is also a carci...
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