James Holcroft

Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice

The advent of transgenic technology has greatly facilitated the study of mutation in animals in vivo. The Big Blue® mouse system, transgenic for the lacI gene, permits not only the quantification of mutations in different tissues but also provides for the generation of in vivo‐derived mutational spectra. This report details the sequence alterations of 348 spontaneous mutations recovered from the liver of 6–8‐week‐old male Big Blue® mice. The spectra recovered from two strains of mice, C57Bl/6 and B6C3F1, were compared and found to be very similar. The predominant mutations are G:C → A:T transitions, with 75% of these occurring at 5′‐CpG‐3′ sequences. This mutational bias is consistent with deamination‐directed mutation at methylated cytosine bases. The second most common class of mutations is G:C → T:A transversions. A significant clonal expansion of mutants was found in several animals, and this was used to make an approximate correction of the mutant frequency such that the most conservative estimate of mutation frequency is presented. The establishment of this substantial database of spontaneous mutations in the liver of Big Blue® mice is intended to serve as a reference against which mutations recovered after treatment can be compared. Environ. Mol. Mutagen. 30:273–286, 1997

Mutational specificity: An efficient laboratory protocol for the sequencing of large numbers of lacl mutants recovered from Big Blue® transgenic animals

Mutational spectra provide a powerful approach to investigate both the mutagenic potential and the mechanism of action of suspected mutagens and carcinogens. Recently, transgenic techniques have made it possible to generate mutational spectra in animals. Such a spectrum may consist of 50 to 200 mutants depending on the nature of the mutations, and many spectra can be generated depending on the design of the experiment. This report describes a practical approach for the processing and sequencing of large numbers of lacl mutants recovered from Big Blue® animals.

Coamplification of hprt cDNA and γ T-cell receptor sequences from 6-thioguanine resitant human T-lymphocytes

Summary The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged γ T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged γ TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient uantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and γ TCR gene respectively.

Spontaneous mutants recovered from liver and germ cell tissue of low copy number lacI transgenic rats

The finding of a large discordance between animal species in their response to a carcinogenic challenge, has led to the realization that the useful extrapolation of animal test data to humans requires a better understanding of animal interspecies differences. With the development of transgenic shuttle vector based animal systems we are now able to study mutation of the same genetic target in both mice and rats. We have begun to analyze mutants recovered from rat lines carrying low copy numbers of the same lambda/lacI constructs carried by the Big Blue mouse. A large database on mutations in lacI transgenic mice is already available for comparison. The data indicate that the differences between the mutations recovered from rat liver and germ cell tissues are similar to those recovered from transgenic mice, but when compared with a large database of mutations available for mice, some site-to-site differences may exist. This study represents the first interspecies look into the molecular nature of mutations in the lacI transgenic rodents.

The Use of lacI Transgenic Mice in Genetic Toxicology

Exposure to genotoxic agents can have a significant impact on human health. In each country, regulatory agencies have been created to establish safeguards to protect the public by minimizing these risks, usually by reducing or eliminating exposures. When this is not possible, estimates are required to quantitate risks so that a risk-benefit analysis is possible. To estimate risk, the field of genetic toxicology has embraced a number of assay systems to evaluate the genetic toxicity. Most well known is the Ames/Salmonella test, which uses a series of tester strains, with and without addition of metabolic activating S9 liver microsome extracts. This assay is generally considered to be the backbone of what has become known as “Tier One” testing and is used to screen all new chemicals and pharmaceuticals that may be introduced into the environment. The Ames assay is usually the first attempt to determine mutagenic potential (Maron and Ames, 1983). Cytogenetic toxicity is also evaluated using mammalian systems, such as the mouse lymphoma in vitro assay (MLA) (Oberly et al., 1984; Hozier et al., 1981), the micronucleus test (MN) (Wild, 1978), and chromosomal aberrations (CA) and sister chromatid exchange assay (SCE) (Latt, 1974). Damage to the germ line can be assessed in rodents by the specific locus test (Russell and Russell, 1992; Russell et al., 1981). Carcinogenic potential can be determined in the National Toxicology Program assay (Chhabra et al., 1990), a 2-year-long exposure of mice or rats to the potential carcinogen, a costly and time-consuming exercise that requires large numbers of animals.

Tris(2,3-dibromopropyl)phosphate causes a gradient of mutations in the cortex and outer and inner medullas of the kidney of lacI transgenic rats.

Tris(2,3‐dibromopropyl)phosphate (TDBP) is a kidney carcinogen in rats in which exposure results in tumors specifically in the outer medulla. We have previously shown that TDBP induces mutation in the rat kidney. Here we demonstrate that TDBP induces mutation in the kidney of the F344 Big Blue lacI transgenic rat in a gradient with the highest induction (6.4‐fold) in the cortex and lowest induction (2.2‐fold) in the inner medulla, when given at 2000 ppm in the feed for 45 days. Similar results were obtained at 100 ppm, although the gradient effect was less pronounced. Because exposure to TDBP results in increased cell proliferation in the outer medulla, our results suggest that tissue‐specific targeting of TDBP‐induced kidney tumors reflects the combination of cell proliferation and mutation induction. This is also the first known case when transgenic animals have been used to study mutation at the suborgan level. Environ. Mol. Mutagen. 36:1–4, 2000.

The effect of dietary restriction on PhIP-induced mutation in the distal colon and B[a]P- and ENU-induced mutation in the liver of the rat.

Abstract: A reduction in dietary intake has been shown to significantly increase the lifespan of rodents, lower the incidence of tumors, and reduce DNA damage. The objective of this study was to determine whether dietary restriction (DR) reduced the frequency of mutation induced by two environmentally relevant metabolically activated mutagens and one direct-acting mutagen in the lacI transgene of male and female Big Blue® rats. Both male and female rats were maintained on either an ad libitum (AL) or a 40%-reduced diet for 22 wk. The mutagenicity of a 100-mg/kg intraperitoneal injection with 2-amino-1-methyl-6-pheny-imidazo[4,5-b] pyridine (PhIP), benzo[a]pyrene (B[a]P), and N-ethyl-N- nitrosourea (ENU) was determined in the colon or liver. The results indicated that DR did not significantly alter the PhIP-induced mutant frequency in male or female colons. DR completely prevented mutagenicity induced by B[a]P in the female liver (2.6 ± 0.6 × 10-5 vs 10.9 ± 5.8 × 10-5 in AL females), yet increased the induced frequency in male livers (16.3 ± 3.7 × 10-5 vs 10.6 ± 1.5 × 10-5 in AL male livers). Although there was no difference in mutation frequency in the liver between AL and DR females treated with ENU, there was approximately a 40% decrease in induced frequency in DR males compared with AL males. These results indicate that a reduction in dietary intake has no preventive effect against PhIP-induced mutation in the colon, but has sex-dependent protective effects against B[a]P- and ENU-induced mutation in the liver.