Michael L. Petras

Breeding Structure of The House Mouse, Mus Musculus, in A Population Cage

House mice ( Mus musculus ), released in a population cage consisting of a series of nest boxes connected by narrow runways, formed small breeding units or demes in each of four runs. Generally, each deme was composed of a “dominant” male, several females and several “subordinate” males. The formation of these breeding units appears to be primarily the result of male territoriality. However, females were also found to contribute to the maintenance of such units. Once formed, demes remained stable over a considerable period of time in spite of factors such as high animal density in some territories. In older demes “dominant” males were replaced by their offspring. Interdemic migration, once demes were established, was rare and appeared to be possible only through females. Attempts to introduce mice into established demes of five to eight animals failed unless at least four mice were introduced simultaneously. The existence of stable demes may explain a number of enigmatic situations encountered in natural populations of Mus musculus .

Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (comet) assay.

Pesticides are broadly used for pest control in agriculture despite possible negative impacts they may pose to the environment. Thus, we examined the DNA damage caused by five herbicides commonly used in southern Ontario (Canada). Erythrocytes from Rana catesbeiana (bullfrog) tadpoles were evaluated for DNA damage following exposure to selected herbicides, using the alkaline single‐cell gel DNA electrophoresis (SCG) or “comet” assay [Singh et al. (1988): Exp Cell Res 175:184–191; Ralph et al. (1996): Eviron Mol Mutagen 28:112–120]. This approach involves detection, under alkaline conditions, of DNA fragments that upon electrophoresis migrate from the nuclear core, resulting in a comet formation. The herbicides tested, along with their active ingredients, were AAtrex Nine‐O (atrazine), Dual‐960E (metalochlor), Roundup(glyphosate), Sencor‐500F (metribuzin), and Amsol (2,4‐D amine). Tadpoles were exposed in the laboratory for a 24‐hr period to several concentrations of the herbicides dissolved in dechlorinated water. Methyl methanesulphonate was used as a positive control. The herbicides AAtrex Nine‐O‐, Dual‐960E‐,Roundup‐, and Sencor‐500F‐treated tadpolesshowed significant DNA damage when compared with unexposed control animals, whereas, Amsol‐treated tadpoles did not. Unlike the other responding herbicides, Sencor‐500F did not show a relationship between dosage and DNA damage. In summary, the results indicate that at least some of the herbicides currently used in southern Ontario are capable of inducing DNA damage in tadpoles. Environ. Mol. Mutagen. 29:277‐288, 1997

Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single‐cell gel (SCG) (“comet”) assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a “comet‐with‐tail” formation. Exposure of R. clamitans tadpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time‐dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans tadpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay

To monitor genotoxicity in small bodies of water (e.g., creeks, ponds, and drainage ditches) we examined tadpole erythrocytes of two species: Rana clamitans and Rana pipiens, using the alkaline single cell gel DNA electrophoresis (SCG) or “comet” assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. Fifty‐six samples, a total of 606 tadpoles, from 18 sites in southern Ontario, collected between 1993 and 1995, were examined. Samples of R. clamitans tadpoles collected in 1994 and 1995, from regions with heavy agricultural activity, gave significantly higher (P < 0.001) DNA length to width ratios than samples of R. clamitans tadpoles collected from sites in the Bruce Peninsula and near the French River, which have little or no agriculture. Samples of R. pipiens tadpoles collected in 1994 from sites on the outskirts of Windsor, Ontario, sites which receive genotoxic inputs from nearby industries, gave significantly higher (P < 0.001) DNA ratios than samples from agricultural areas and the Bruce Peninsula. R. clamitans tadpoles showed significant annual variation in DNA damage which was greater in samples of tadpoles collected from agricultural areas than from the Bruce Peninsula. The higher levels of DNA damage in tadpoles collected from agricultural areas may be due to the pesticides used, and the increased variation in DNA damage in the same areas is likely due to the impact of crop rotation, including leaving fields fallow, the timing of rainfall, and/or the application of pesticides. R. clamitans tadpoles, especially those collected from agricultural areas, also showed significant seasonal variation in DNA damage. There was no significant (P > 0.05) seasonal or annual variation in the levels of DNA damage in R. pipiens tadpoles collected from the Tallgrass Prairie. This study indicates that both species are suitable for use in the alkaline SCG assay and as in situ sentinel organisms for environmental biomonitoring. Environ. Mol. Mutagen. 29:418–430, 1997.

Improved electrophoretic resolution of some hemoglobin variants in Mus musculus

Electrophoretic separation of house mouse (Mus musculus) hemoglobins on starch gel in a 0.2 M tris maleate NaOH pH 7.0 buffer facilitates the recognition of the three phenotypes controlled by locus Hbb. Evidence that Hbb does control the electrophoretic patterns with this buffer system is discussed.

Linkage of the locus for serum albumin in the house mouse, Mus musculus

An electrophoretic variant for serum albumin in Mus musculus has been used to map the structural gene for this protein to chromosome 5.

Serological survey of T-lymphocyte differentiation antigens in wild mice

Allelic distributions of Thy-1, Ly-l, and Ly-2 antigens in wild mice are characteristic of each Mus musculus subspecies. Eastern mice (M.m.molossinus, M.mmusculus, M.m.castaneus, M.m.bactrianus) express the Thy-1.1 antigen, whereas Western mice (M.m. domesticus, M.m.brevirostris) express the Thy-1.2. All mice from wild populations examined in this survey express the Ly-1.2. The Ly-2.1 is distributed in Eastern mice and some Western mice, and the Ly-2.2 is found in the remaining Western mice. Allelic distributions of these antigens were also examined in two other species, Mus spretus and Mus spicilegus. Allelic constitutions of Thy-1 and Ly-1 in these species are similar to those of Eastern mice. Some M.spicilegus, however, express the Ly-1.1 antigen. This antigenic type is not found in M.musculus. Some Eastern mice related to M.m.castaneus react weakly to Ly-1.2-specific and Ly-2.1-specific monoclonal antibodies in both the complement-mediated cytotoxicity test and the absorption test. These results suggest that M.m.castaneus has unique alleles in the Ly-1 and Ly-2 loci.

An inherited albumin variant in the house mouse, Mus musculus

In spite of extensive electrophoretic studies of numerous samples of North American house mouse (Mus musculus) populations and of a variety of inbred strains of this species, no albumin variants have as yet been reported (Petras et aI., 1969; Selander et al., 1969; Selander and Yang, 1969). The present report is concerned with the inheritance of an albumin variant uncovered in a single sample of mice. The albumin variant appears to be under control of an allele located at an autosomal locus and rather restricted in geographic distribution.

Comparison of sensitivity to methyl methanesulphonate among tadpole developmental stages using the alkaline single-cell gel electrophoresis (comet) assay.

In a previous study, we demonstrated that tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) for genotoxicity using the alkaline single‐cell gel DNA electrophoresis (SCG) or “comet” assay [Ralph and Petras, 1997]. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. In this initial study, most of the tadpoles collected were in the early stages of larval development, but this is not always possible. The present study evaluated the sensitivity of tadpoles, at different stages of larval development, to a range of concentrations of the genotoxicant methyl methane‐sulphonate (MMS). Four specific phases of Rana clamitans (green frog) larval development were examined: first‐year limbless tadpoles (Stage I as defined by Taylor and Kollros [1946]), second‐year limbless tadpoles (Stages II–III), second‐year tadpoles with only hindlimbs (Stages X–XVIII), and second‐year tadpoles with all four limbs evident and a tail undergoing resorption (Stages XXII–XXIII). Twenty‐four hour exposures to MMS of tadpoles in the three earliest phases produced a significant (P < 0.01) added variance component among tadpoles for DNA damage and there were significant increases (P < 0.05) in the length:width ratios of the DNA patterns at concentrations as low as 1.56 mg/l. However, tadpoles in the last phase studied (both pairs of limbs present) showed no significant (P > 0.05) added variance component and no significant increases (P > 0.05) in DNA damage upon exposure to any of the MMS doses tested. A nested ANOVA indicated that, for each of the tested concentrations of MMS, but not the dechlorinated water control, there was significant heterogeneity (P < 0.05) in DNA damage when tadpoles of all four phases studied were compared. However, when tadpoles of the last phase of development were removed from the comparison, there was no significant heterogeneity (P > 0.05) among tadpoles of the remaining three phases. Possible reasons for this insensitivity to MMS as animals enter the metamorphic climax were considered. The results indicate that pooling of the early tadpole phases of R. clamitans for SCG environmental genotoxicity biomonitoring is acceptable. Environ. Mol. Mutagen. 31:374–382, 1998.

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.