F. M. Johnson

Analysis of National Toxicology Program rodent bioassay data for anticarcinogenic effects

We reanalyzed data from 218 two-year rodent carcinogenicity studies carried out by the National Toxicology Program (NTP). These data were originally collected for the purpose of identifying potential human carcinogens. However, the objective of our analysis was to investigate the frequency of possible anticarcinogenic effects in these data, since recurring cases of chemical-associated tumor reductions have been noted in the course of these studies over time. Our analysis reveals that most (>90%) NTP-tested chemicals show at least one statistically significant (p<0.05) decrease in site-specific tumor incidence. Because of the large number of statistical comparisons made in a long-term bioassay, random variability can account for many of these tumor decreases. However, we found that certain tumors (predominantly those with a high spontaneous incidence) show chemically related decreases far more frequently than chance expectation. Many of these decreases, particularly those for pituitary and mammary gland tumors, adrenal pheochromocytoma and uterine polyps in rats and liver and lung tumors in mice, are associated with the reduced body weights frequently observed in the dosed groups. The chemically related decreased incidences of leukemia in rats appear to be related to spleen damage, i.e., chemically related splenic toxicity is evident for most chemicals showing decreased incidences of leukemia. While random variability, associations with body weight and splenic toxicity can account for most of the decreased tumor incidences observed in NTP studies, there are other tumor decreases that could not be totally explained by these factors. Further investigations of possible mechanisms of action are underway. These data are relevant to the concept of chemoprevention as well as to the task of using long-term laboratory animal studies to predict enhanced human environmental-cancer risk for regulatory purposes.

Variation in the shape of the mouse mandible: 1. Effect of age and sex on the results obtained from the discriminant functions used for genetic monitoring

The shape of the mandible of the mouse can be described by a series of discriminant functions which have been used to discriminate between and investigate relationships among strains of mice. The effect of sex and variation in age of the animal on the results obtained from these functions has been investigated. Significant sex differences in mandible shape were detected, but these were considerably smaller than the differences found between two inbred strains. A simple correction for expressing each measure as a proportion of the sum of the measures on each bone removes the effects of overall size. Significant age effects were found, but these were only large in animals under seven weeks of age where considerable changes are taking place in the relative lengths of bone measurements. Routine testing for genetic authenticity using the shape of the mandible is possible over a wide age range and may be an efficient method for monitoring genotypes at the end of long-term experiments.

Mouse spermatogonia exposed to a high, multiply fractionated dose of a cancer chemotherapeutic drug: Mutation analysis by electrophoresis

Male mice of the DBA/2J strain were injected with procarbazine at a dose of 200 mg/kg body weight twice weekly until an accumulated dose of 2400 mg/kg was reached. A concurrent control group, injected only with the vehicle (saline) was also established. Most of the treated animals died as a result of exposure and all survivors became temporarily sterile. After regaining fertility the few survivors were repeatedly mated with C57BL/6J females over several weeks time to generate a population of F1 animals. The parental animals and the F1 were subsequently analyzed by electrophoresis for the occurrence of newly arisen mutations of spermatogonial origin. A mutation in the gene Pep-3 was found.

Mutation-rate determinations based on electrophoretic analysis of laboratory mice.

Rate calculations are described for germinal mutations detected by an electrophoretic system as applied to mice in a series of recent experiments. The utility of the electrophoretic system is examined from several comparative viewpoints. The electrophoretic approach has a number of features to recommend it for future applications concerned with genetic hazard assessment. Its advantages include sensitivity, versatility, cost effectiveness and relevance to humans.

A variation in mouse kidney pyruvate kinase activity determined by a mutant gene on chromosome 9.

Pyruvate kinase activity was studied in kidney extracts of mice from various sources. One C57BL/6J female among several hundred examined expressed activity approximately one half of the normal level. This animal transmitted the trait, and a single gene responsible for the activity difference was identified and mapped to chromosome 9. Linkage with Mod-1 and dilute was established. A spontaneous mutation in the C57BL/6J strain apparently was responsible for the variant pyruvate kinase.

Dominant and Recessive Effects of Electrophoretically Detected Specific Locus Mutations

The heterozygous and homozygous effects of mutations recovered from a biochemical screen of mouse samples are described. Of the total of 28 electrophoretically detected mutations only one, a Pgm-2 mobility mutation appears to have caused a change to a previously known allelic form. All but one electrophoretically detected allele have been shown to be homozygous viable. Two mutations identified by other means are homozygous lethal; the first is a mutant detected because of its reduced PK-3 activity in heterozygotes, and the second is a morphologically detected mutation at the d locus. Three dominant visible mutations are also described briefly.

A null mutation at the mouse Phosphoglucomutase-1 locus and a new locus, Pgm-3

A null mutation at the phosphoglucomutase locus (Pgm-1) was discovered by electrophoretic analysis of the inbred mouse strain C57 BL/6J. The null allele (Pgm-1n) was shown to segregate as a Mendelian unit alternative to the Pgm-1a and Pgm-1b alleles. Mice expressing the Pgm-1n allele, either in the heterozygous or homozygous state, are viable, healthy, and fertile. The occurrence of the Pgm-1n mutant revealed a previously unreported genetic locus (Pgm-3) that controls the expression of a third phosphoglucomutase. Two electrophoretically expressed alleles of Pgm-3 (inherited without dominance) are found in the inbred mouse strains C57 BL/6J and DBA/2J. Linkage observed between the Pgm-3 locus, the dilute locus (d) and the cytoplasmic malic enzyme locus (Mod-1) has allowed assignment of the Pgm-3 locus to chromosome 9. A striking tissue specific expression of Pgm-1 and Pgm-3 was observed. Products of the Pgm-3 locus were detected in kidney, testes, brain, and heart. In contrast, Pgm-1 controlled isozymes were present in kidney, spleen, ovaries, and erythrocytes.

High resolution metrical analysis applied to the assessment of damage associated with induced mutations in the mouse.

Morphometric methods were used to investigate variation in the skeletons of 1030 offspring produced from matings of male DBA/2J by female C57BL/6J mice. 751 offspring originated from males that had received a single intraperitoneal injection of ethyl nitrosourea (EtNU) at a dose of 250 mg/kg. The remainder of the mice served as controls. The male parents of the controls were injected only with the buffer used as vehicle for the EtNU. Offspring were obtained for 3 weeks following injection. The treated males were then sterile for about 8 weeks. Immediately after the sterile period another sample of progeny was obtained. In the treated group, litter sizes at birth and weaning were reduced and survival to adulthood was lower. However, none of the differences were statistically significant. The skeletons were evaluated by two independent approaches. The first relied upon gross observation for unusual phenotypic variation, the second on a series of metrical measurement and coordinate data. A considerable amount of variation was recorded by both approaches. Some of the variants were severe but others were mild and perhaps of little or no importance to the health of the mice. The gross observation method produced no evidence for increased mild or severe variants in any group of offspring from the treated mice. The metrical methods also showed no evidence for treatment-related effects in offspring produced during the first 3 weeks of mating. However, in offspring produced after the sterile period, a pronounced, very highly statistically significant increase in all levels of metrical variation was observed. This treatment group revealed both increased variant measures and increased numbers of mice with variant measures. Much of this variation was so slight that it would have escaped notice were it not for the exacting measurements used in the analysis. Our morphometric approach is an analytically powerful tool, suitable for detecting variation in virtually any biological structure that can be measured. If the increased variation reported here is due to induced mutations, the effects would be consistent with that expected from slightly harmful mutations distributed throughout the mouse genome. It is appropriate to consider such effect in connection with genetic risk estimation.

THE HUMAN GENETIC RISK OF AIRBORNE GENOTOXICS: AN APPROACH BASED ON ELECTROPHORETIC TECHNIQUES APPLIED TO MICE

Among the most serious long-term adverse health effects potentially resulting from human exposure to airborne genotoxic agents is heritable damage. Environmentally-caused mutations transmitted through the gametes might not only persist for multiple human generations but, depending on the circumstances and the nature of the genetic alteration, may accumulate over time to be expressed as a gradual degradation in quality of life.