Mutant frequency of lacI in transgenic mice following benzo[a]pyrene treatment and partial hepatectomy.

The Big Blue, transgenic mouse provides an in vivo mutation system that permits the study of pharmacodynamic parameters on mutant frequency (MF) following xenobiotic exposure. We have studied the effects of cellular proliferation on the frequency of mutations in the lacl transgene by evaluating the MF in the liver of male C57B1/6 Big Blue mice following treatment with benzo[a]pyrene (B[a]P) and a partial hepatectomy. Mice received either 40 mg/kg of B[a]P in corn oil or corn oil alone by i.p. injection on three consecutive days, followed by a partial hepatectomy on the fourth day. Three days later (i.e., 7 days following the initial B[a]P injection), the animals were sacrificed and the MF in the liver was compared to the MF observed in the liver of the same mouse at the time of hepatectomy. Induction of cytochrome P-450 1A (CYP1A) following B[a]P treatment was evident by Western blot analysis. The MF in untreated control animals was not significantly different at hepatectomy (4.7 +/- 0.8 x 10(-5)) and 3 days later, at sacrifice (3.0 +/- 0.4 x 10(-5)). Neither was the MF observed in the B[a]P-treated mice at the time of sacrifice (12.0 +/- 2.1 x 10(-5)) significantly different from the MF observed at the time of hepatectomy (10.6 +/- 5.3 x 10(-5)). However, B[a]P-treatment resulted in a 4.0-fold increase in MF at sacrifice which was significantly different (p < 0.05), when compared to the untreated controls. The B[a]P-treated mice at hepatectomy showed a modest 2.2-fold increase in MF which was not statistically significantly different from the untreated controls. In addition, both control and B[a]P-treated tissues gave sectored mutant plaques. The sectored plaque frequency (SPF) was significantly elevated (p < 0.05) in the B[a]P-treated mice at hepatectomy (4.2 +/- 1.0 x 10(-5)) and sacrifice (7.3 +/- 2.4 x 10(-5)) as compared to the respective frequency in the control mice at hepatectomy (1.9 +/- 0.7 x 10(-5)) and sacrifice (1.4 +/- 0.2 x 10(-5)). One explanation for this data is the persistence of the B[a]P adducts in the mouse genomic DNA that was packaged into the lambda phage, and ultimately fixed as mutations in Escherichia coli.

Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice.

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.

Sources of variability in data from a lacI transgenic mouse mutation assay.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

Correlations between chemically related site-specific carcinogenic effects in long-term studies in rats and mice.

We examined a database of 379 long-term carcinogenicity studies in rats and mice to evaluate sex and species correlations in site-specific carcinogenic responses. Within a species, most target sites showed a strong correlation between males and females. For example, chemicals producing forestomach or liver tumors in males were likely to produce these same types of tumors in females. There was also a significant correlation between species for certain site-specific carcinogenic effects, most notably tumors of the forestomach, liver, and thyroid gland. In contrast, adrenal pheochromocytoma, preputial/clitoral gland neoplasms, and lung tumors showed no significant interspecies correlation. Many chemicals produced a syndrome of carcinogenic effects involving tumors of the skin, Zymbal gland, preputial/clitoral gland, mammary gland, and/or oral cavity. Regarding different target sites, there appeared to be a correlation between thyroid and liver tumors both within and between species. Further, all chemicals producing mesotheliomas in male rats also produced mammary gland neoplasms in female rats. In contrast, kidney and urinary bladder tumors showed no significant association with any other tumor type in rats or mice. If a chemical produced a site-specific carcinogenic effect in female rats or mice, there was approximately a 65% probability that the chemical would also be carcinogenic at that same site in males. The interspecies correlation was somewhat lower: approximately 36% of the site-specific carcinogenic effects observed in one species (rats or mice) were also observed in the other species.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessing overdispersion and dose-response in the male dominant lethal assay.

In dominant lethal studies the primary variables of interest are typically expressed as discrete counts or proportions (e.g., live implants, resorptions, percent pregnant). Simple statistical sampling models for discrete data such as binomial or Poisson generally do not fit this type of data because of extra-binomial or extra-Poisson departures from variability predicted under these simple models. Extra-variability in the fetal response may originate from parental contributions. These can lead to over- or under-dispersion seen as, e.g., extra-binomial variability in the proportion response. Utilizing a large control database, we investigated the relative impact of extra-variability from male or female contributions on the endpoints of interest. Male-related effects did not seem to contribute to overdispersion in our database; female-related effects were, however, evidenced. Various statistical methods were considered to test for significant treatment differences under these forms of sampling variability. Computer simulations were used to evaluate these methods and to determine which are most appropriate for practical use in the evaluation of dominant lethal data. Our results suggest that distribution-free statistical methods such as a nonparametric permutation test or rank-based tests for trend can be recommended for use.

A computer-aided system for the diagnosis of hepato-biliary diseases. A comparison with the performance of physicians.

A computer-aided system has been developed for the diagnosis of disease of the liver and biliary system. The program is based on the use of 30 indicants, all of which are available within six hours after patient's admission: 18 of them are clinical signs, 12 are laboratory parameters including routine liver-function tests. To date, the program concerns 52 hepato-biliary diseases. From the analysis of the 30 items collected in any patient, the diagnoses are computed according to Bayes' theorem and printed by decreasing order of probability. The performance of the program was tested using records of patients with fully proven diagnosis. The first diagnosis given by the computer was correct in 57 per cent of the cases. In 80 per cent, the right diagnosis was among the first four proposed. When the performance of the model was compared to that of physicians, the number of correct answers was roughly the same for the computer and for the specialists in hepatology; in contrast, the computer's responses were far better than those of general practitioners. These results demonstrate the efficiency of the program for the diagnosis of hepato-bilitary diseases and its potential interest for helping clinicians in decision-making.

The thymol turbidity test and determination of serum cholesterol : two tests to abandon in the routine evaluation of liver function.

The thymol turbidity test (Macglan's test) and the determination of serum cholesterol are still performed routinely as liver function tests in many laboratories. In this report, we have employed quantitative methods in order to evaluate the real usefulness of these two parameters. In a first step, the value of the thymol test and of cholesterol determination for the discrimination of the 13 most frequent hepato-biliary diseases was studied by analysis of variance, and compared to that of the following tests : serum bilirubin, serum glutamic pyruvic transaminase, serum alkaline phosphatase serum protein electrophoresis and prothrombin time. It was found that of all these parameters, the thymol test and cholesterol measurement had the lowest discriminatory powers. In a second step, the consequences of the suppression of the two tests were examined by linear discrimination analysis, which was done for all the possible pairs of diseases. It appeared that in each case, the loss of information due to the elimination of the thymol test or of cholesterol determination was nil or negligible. We conclude therefore that the thymol turbidity test and determination of serum cholesterol should be abandoned as routine tests of liver function.