hyperploidy, even if the higher dose was highly efficient as far as mitotic arrest is concerned. Polyploid cells were induced after injection of 3 mg /kg ; similarly to VBL treatment, they were observed only in M2 sampled 24 h after treatment. Neither VBL nor COL had any effect on SCE frequency. Spermatogonia and spermatocytes sampled after VBL treatment did not show any induction of non-disjunction, as expected on the basis of limited testis accessibility of the chemical. Preliminary germinal data on COL treatment do not show any induction of hyperploid spermato- cytes up to 18 h after injection.

A cytogenetic approach to aneuploidy assess- ment in vivo is proposed, which allows the simul- taneous estimation of mitotic arrest and chro- mosome malsegregation, as a tool to understand- ing the mechanisms of the origin of aneuploidy.

Partially supported by EEC Contract EV4C- 0044-1 (A).

89 Wang Xu, B. Miller, U. Kliesch and I.-D. Adler, Institut fiir S~iugetiergenetik, GSF, D-8042 Neu- herberg (F.R.G.)

Spindle inhibition and chromosomal breakage in mouse bone marrow by econazole (EZ) and hydro- quinone (HQ)

Econazole (EZ) and hydroquinone (HQ) were chosen for these comparative studies because of evidence of aneuploidy induction in fungi (EZ) or spindle effects and micronucleus induction (HQ). Five male and 5 female (101/E1 × C 3 H / E 1 ) F 1 mice, aged 12-14 weeks, were used per treatment group in the micronucleus (MN) test and the chromosome aberration (CA) analysis. The per- animal sample sizes were 2000 polychromatic erythrocytes and 50 metaphase cells, respectively. To evaluate mitotic effects 5 treated and 5 control males were used per dose group. The criteria were: (1) mitotic index (MI) (mitotic cells/1000 cel ls / animal); (2) c-mitoses (metaphase cells with in- creasing degrees of chromatid spreading/100 metaphases /animal) ; and (3) anaphase frequen- cies (anaphases/100 mitotic cells/animal). Both chemicals were tested at 3 doses in the range of 30-150 mg/kg .

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For EZ no clastogenic effects were found in the MN test and the CA analysis. However, EZ showed a significant ( p = 0.01), dose-dependent increase in MI accompanied by a significant reduction of anaphase stages and, at the highest dose, a signifi- cant excess of c-mitotic cells. HQ showed a dose- dependent induction of MN. It also induced a dose-dependent increase in CA. At 6 h after treat- ment single chromatid breaks were predominant while after 24 h the aberrant cells carried multiple exchanges. In contrast, the MI decreased signifi- cantly to about 50% of the concurrent control at the highest dose and the anaphase frequency was reduced to 50%. c-Mitotic cells were significantly more frequent at the 2 higher doses than in the concurrent controls.

It is concluded that EZ caused mitotic arrest in mouse bone marrow similar to colchicine but showed no clastogenic properties and did not in- duce micronuclei which could have been attrib- uted to lagging chromosomes. HQ, on the other hand, was clastogenic and induced micronuclei, but the mitotic changes indicated cytotoxicity prior to mitosis rather than mitotic arrest.

Research supported by the EEC Contract EV4V-0064-D (B).

90 Bishop, J.B. 1, W.M. Generoso 2, M. Katoh 2 and J.C. Rutledge 3, 1 National Institute of Environ- mental Health Sciences, Research Triangle Park, NC (U.S.A.), 2 Oak Ridge National Laboratory, Oak Ridge, TN (U.S.A.) and 3 Children's Medical Center, Dallas, TX (U.S.A.)

Treatment of mouse zygotes with germ cell muta- gens results in developmental abnormalities and fetal death

The zygotic stage of embryogenesis has been considered refractory to the effects of noxious agents, and the period of organogenesis, which is between days 7 and 13 of gestation for mice, a sensitive period for induction of developmental defects. However, Generoso et al. (1986; 1988) have demonstrated induction of morphological abnormalities among both the dead and surviving fetuses of female mice exposed post mating to ethylene oxide (ETO) by inhalation or to ethyl-

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nitrosourea (ENU), triethylenemelamine (TEM) or ethyl methanesulfonate (EMS) by intraperitoneal injection. Congenital defects have now also been observed following intraperitoneal injection of females with nocodazole (NOC), mitomycin C (MC), and acrylamide (AC). Of living fetuses ex- amined at mid-gestation following treatment of their mothers 1 or 6 h after mating (about the time of sperm entry or during the early pronuclear stage of the zygote, respectively), as many as 30% exhibited developmental anomalies such as eye defects, reduced or absent limbs, omphalocele, ectopia cordis, hydropia and exencephaly. With ETO and EMS these effects were not observed when females were treated at 9 h post mating or later. With NOC abnormalities were observed only when females were treated 1 h after mating. The peaks in the response to induction of developmen- tal defects with ENU, MC, EMS, NOC, AC, and ETO generally appeared to be concomitant with their peaks for fetal mortality, which occurred at the early pronuclear stage. The stage-dependent sensitivity and the varied nature of congenital defects suggest a genetic basis for the response. Further, the increase in developmental anomalies produced by NOC have now been associated with a similar increase in chromosome aberrations ob- served at first-cleavage metaphase.

91 Bulanova, M., B. Ivanov, I. Georgieva, V. Gerasimenko 1 and N. Ryzhov ~, Institute of Nuclear Medicine, Radiobiology and Radiation Hygiene, Sofia (Bulgaria) and t Academy of Medical Sciences Institute of Biomedical Prob- lems, Moscow (U.S.S.R.)

Chromosomal lesions produced in human somatic ceils by densely ionizing radiations

Findings are reported concerning the damaging action of accelerated charged particles and neu- trons on heritable structures in human peripheral lymphocytes, as obtained by the metaphase method for recording aberrant chromosomes. Samples were irradiated with 50 MeV protons, 4.6 GeV accelerated helium nuclei, 4.2 GeV deuterons (at the JINR, Dubna, U.S.S.R.), and 0.4 eV to 10 MeV fission neutrons (at the Atomic Reactor,

Sofia, Bulgaria), at doses ranging from 0.1 to 5 Gy. The results of cytogenetic analyses were compared to corresponding data from exposure to 6°Co y-radiation. For the radiation types studied, RBE coefficients were calculated based on each of 3 endpoints (frequencies of aberrant cells, di- centrics, and chromosome fragments) using the method of Kellerer (1973). The following RBE values were obtained:

Aberrant Dicentrics Chromosome cells fragments

Protons 0.9-1.9 0.9-1.9 0.9-1.9 Helium nuclei 0.8-1.0 0.8-2.3 0.8-1.0 Deuterons 1.5-1.9 1.8-2.8 1.8-1.9 Neutrons 2.4-6.5 5.0-6.7 0.4-0.8

Our results confirmed RBE dependence on radiation type and the endpoint examined. A trend was noted for figures to be higher with exposure to low-level doses, and coefficient values proved higher when based on dicentrics as compared to aberrant cells and chromosome fragments.

92 Bishop, J.B. 1, M. Katoh 2, K.T. Cain 2 and W.M. Generoso 2, ~ Cellular and Genetic Toxicology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) and 2 Oak Ridge Nat ional Laboratory, Biology Division, Oak Ridge, TN 37831-2009 (U.S.A.)

Exposure of female mice to nocodazole 1 h after mating results in abnormal chromosome numbers and early death among conceptuses

In the female mouse, the egg is ovulated with its nucleus in the metaphase 2 stage of meiosis and remains in this condition until sperm entry (about 1.5 h after copulation) triggers completion of meiosis. Thus, damage to the spindle may cause malsegregation of chromosomes during the second meiotic division of the pronucleus. When female (SEC × C57B1) or (C3H × C57B1)F t hybrid mice were treated 1 h after mating with an i.p. injection of nocodazole (10-35 mg/kg) , which is known to bind to tubulin, high frequencies of deciduomata (70%) were observed among the conceptuses. Treatments 1 -4 days before mating (exposure of