Quantitative Genetic Analysis

In vivo cyclophosphamide (CP)-induced sister chromatid exchanges (SCEs) were evaluated in females from five genetic strains of mice (C57BL/6J, C3H/ S, 129/ReJ, BALB/c and DBA/2) and their FI hybrids. Baseline (noninduced) SCE values differ significantly among strains, 129/ReJ having the lowest and DBA/2 having the highest mean SCE per cell values. In general, the baseline SCE of a given FI is within the range of its corresponding parental strains or near the lower parental value. Furthermore, there is a genotype-dependent increase in mean SCEs per cell with CP dose. Strain differences in SCE induction are noted particularly at the two higher CP doses (4.50 and 45.0 mg/kg). In general, FI hybrids involving a strain with high induced SCEs and a strain with low induced SCEs exhibit mean SCE values that are closer to the value of the lower strain. Fls involving two strains with high SCEs or two strains with low SCEs yield SCEs not different from parental strains. The method of diallel cross analysis showed the order of dominance of these strains in SCE induction to be 129/ReJ BALB/c CSH/S DBA/2 C57BL/6J. These results support the involvement of predominantly nonadditive genetic factors as major gene(s) in SCE induction. In addition, involvement of random and independent events in SCE induction is suggested by the distribution of SCEs which follows a Poisson distribution. ISTER chromatid exchanges (SCEs) represent the interchange of DNA beS tween replication products at apparently homologous loci (LATT et al. 1979). They presumably require DNA breakage and reunion (LATT 198 1), and the exchanges take place between double DNA strands (KATO 1977) having the same polarity (LATT and SCHRECK 1980). However, the molecular mechanism of SCE formation and its biological significance remain obscure. In spite of these uncertainties, this cytological phenomenon is considered to be a rapid and sensitive method to detect the effect of mutagens with few false-positive results in vivo (STETKA and WOLFF 1976a; NAKANISHI and SCHNEIDER 1979) and in vitro (STETKA and WOLFF 1976b; MADLE and OBE 1977), particularly in mammals. It has been extremely useful in differentiating among chromosome fragility diseases (LATT et al. 1980), providing information about the structure of the eukaryotic chromosome (Hsu and PATHAK 1976; REIMER and SINGH 1982) and is helping to elucidate the phenomenon of aging at the Genetics 105: 169-179 September, 1983 170 D. L. REIMER AND S. M . SINGH cellular level (KRAM et al. 1978; SCHNEIDER et al. 1979; SCHNEIDER and GILMAN 1979; NAKANISHI, DEIN and SCHNEIDER 1980; REIMER and SINGH 1983). It is natural to think of a genetic basis for the observations on SCE formation. In fact, a number of features from reported results strongly suggest genetic contributors to the phenomenon of SCE formation. These include mutagen specificity (LATT et al. 1981), species specificity (WOLFF and RODIN 1978; BARNETT and WALLACE 1982), the effect of genetically determined chromosome fragility disorders (LATT et al. 1980), cell type differences (MITCHELL, MEHER-HOMJI and BAKER 1982), strain differences in vivo (BIEGEL, BOGGS and CONNER 1980; GALLOWAY et al. 1980; DRAGANI, ZUNINO and SOZZI 1981; REIMER and SINGH 1982) and strain-dependent aging response (REIMER and SINGH 1983). Most of these generalities have been observed in vitro and in uivo in response to a number of chemicals and mutagens, some of which [e.g., cyclophosphamide (CP)] require metabolic activation prior to becoming effective SCE inducers (HILL 1975). The genetic determinants for SCE formation may affect genetically mediated metabolic activation or deactivation of the inducer (MITCHELL, MEHER-HOMJI and BAKER 1982), genetically determined enzymes providing protection against breaks [e.g., superoxide dismutase, catalase, etc. (MORGAN, CONE and ELGERT 1976; SPEIT, VOGEL and WULF 1982)] and/or genetically influenced rate of DNA replication and repair (SHIRAISHI, MINOWADA and SANDBERG 1979). It is realized (LATT et al. 1981) that appropriate studies on strains of mice or other rodents will be desirable in evaluating the role and nature of possible genetic determinants of SCE formation. These 211 vivo studies, using 5-bromo-2’-deoxyuridine (BrdU) administration, permit analyses that are complementary to in uitro trials and provide information which is otherwise unobtainable. We evaluated differences among genetic strains of mice in SCE formation zn vivo in response to CP. This metabolically activated alkylating agent induces high levels of SCEs with minimal chromosomal aberrations (NAKANISHI and SCHNEIDER 1979). Here, we report and discuss our results on CP-induced SCEs in five strains of mice and their F I hybrids. The results are discussed in the light of genetic determinants involved in the mechanism and rate of SCE formation. MATERIALS AND METHODS Five genetic strains of mice: C57BL/6J, 129/ReJ (The Jackson Laboratory, Bar Harbor, Maine), CSH/S, BALB/c and DBA/2 (Canadian Breeding Earm, Charles River, Quebec) were used in this study. Mice were maintained in the animal care facilities of the University of Western Ontario, London, Canada, under standard conditions. They were paired in breeding cages to produce all possible Fls and genotypes representing each of the five strains. Female mice (11.28 f 0.95 wk old) representig ten hybrids and five parental strains were treated with 9-hourly intraperitoneal (ip) injections of BrdU (-214 mg/kg/hr) following the method of REIMER and SINCH (1 982). This treatment was found to be satisfactory to differentially label metaphase chromosonies of the bone marrow cells. A single ip injection of 0, 0.045, 0.45, 4.50 or 45.0 mg/kg of CP was administered 15 min after the last BrdU injection. Twelve hours later, a single ip injection of -4 mg/kg of colcemid was given to each animal. Mice were sacrificed 2 hr later by cervical dislocation. The femoral bone marrow cells were collected. Slide preparation and fluorescence plus Giemsa staining followed the procedure of PERRY and WOLFF (1974). BrdU (Sigma), CP (Sigma), colcemid (Gibco) and other chemicals used were of analytical grade. All solutions were stored in light tight containers at 5” and used within 2 weeks. GENETICS OF CP-INDUCED SCES IN MICE 171 Total SCEs per cell in 20 well-spread complete second-replication metaphase cells were scored for each animal. A number of CP treatment and genotype combinations were repeated on two/ three animals; others represent values on a single individual. Analysis of variance was used to evaluate the significance of differences in SCE frequencies between individuals within a CP dose and genotype combination, between genotypes and between CP doses. Further analysis followed statistical comparisons of means and variances of appropriate combinations. The method of diallel cross analysis (HAYMAN 1954) was used to determine genetic components of variation in the induction of SCEs.