Grantee Research Project Results
Final Report: Carcinogen Induced Deletions in the Mouse
EPA Grant Number: R825359Title: Carcinogen Induced Deletions in the Mouse
Investigators: Schiestl, Robert H.
Institution: Harvard University
EPA Project Officer: Aja, Hayley
Project Period: December 2, 1996 through December 1, 1999 (Extended to December 1, 2000)
Project Amount: $583,191
RFA: Exploratory Research - Human Health (1996) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The objective of this project was to: (1) determine dose response relations for "mutagenic" and "nonmutagenic" carcinogens for induction of DNA deletions in a short-term in vivo mouse assay based on the detection of DNA deletion events in the mouse embryo; (2) identify and quantify the effects on DNA deletions of toxicological interactions (such as additivity, synergism, and antagonism) in binary mixtures of pollutants; and (3) determine whether p53 mutant transgenic mice that may show increased sensitivity to DNA damaging agents can be used to improve the sensitivity of the mouse DEL assay.Summary/Accomplishments (Outputs/Outcomes):
A short-term in vivo mouse assay that readily detects both mutagenic and "nonmutagenic" carcinogens was used. The assay detects DNA deletion events in melanocyte precursor cells during embryonal development of the mouse that gives rise to a coat color change in the offspring that can be easily counted. The frequencies of deletions are determined by counting the number of spots on the coat or the eyes. This mouse assay would be useful in developing dose-response relationships, and in testing the effects of various combinations of carcinogenic agents. The same deletion events are detectable in yeast and in human cells and have been shown to be inducible by the same classes of carcinogens. Thus, assays scoring for the same genetic endpoint are available in cell culture to allow in vivo -in vitro comparisons. The yeast assay can readily distinguish between mutagenic and "nonmutagenic" carcinogens by different dose responses. If this mouse assay can distinguish between these two classes on the basis of their dose response this would have important implications for risk assessment. More widespread use of this mouse assay may provide better information for risk assessment and may lead to a more widespread use of the yeast and the cell culture assays with the same genetic endpoint, which in turn, may result in a reduction in the number of animals used in toxicity testing.We have completed much of the first objective on the ability of nonmutagenic carcinogens to induce DNA deletions and the dose-response relations of benzo(a)pyrene, chloroform, carbon tetrachloride, and trichloroethylene. Trichloroethylene seemed to induce DEL recombination at the highest dose of 5 g/kg with a nonlinear dose response. Chloroform seemed to induce DEL recombination at the highest dose of 700 mg/kg, maybe with spurious inductions or just fluctuations at lower doses. Carbon tetrachloride seemed to induce at the highest dose of 6g/kg. Benzo(a)pyrene showed a linear dose response with a large plateau between 6 and 150 mg/kg. We also did experiments with chemical interactions. Trichloroethylene and chloroform did not show any synergistic or additive effects, whereas chloroform and carbon tetrachloride seemed to show synergistic effects. The combination trichloroethylene and carbon tetrachloride was too toxic to obtain any offspring. We also have completed the third objective with the p53 mutant mice (see below). However, we found that the number of mice needed to complete Objectives 1 and 2 would go beyond the scope of this project. Therefore, we successfully developed a novel method to detect DNA deletion events in the mouse retina, which is much more cost effective and requires fewer mice, making the remaining experiments on dose responses and toxicological interactions much simpler. This development has delayed the progress of the original objectives. The data collected are still being evaluated.
Summaries of Completed Studies
Polychlorinated Biphenyls (PCBs) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Induce Intrachromosomal Recombination In Vitro and In Vivo. Polychlorinated aromatic hydrocarbons (PAHs), such as PCBs and TCDD, are extremely stable and widely distributed environmental pollutants. These chemicals are animal carcinogens and probable human carcinogens, TCDD possibly being one of the most potent toxins ever evaluated by the EPA. PAHs score negative in most genotoxicity assays including the Ames (Salmonella) assay. Although their mechanism of toxicity is not well understood, they induce aryl hydrocarbon hydroxylases and bind to the AH receptor, which is believed to mediate toxicity. We determined effects of PAHs in genotoxicity assays, which score for DNA deletions by intrachromosomal recombination in vivo and in vitro. In this study, TCDD, Aroclor 1221, and Aroclor 1260 induced deletions in vivo in the mouse embryo; Aroclor 1221 and Aroclor 1260 induced deletions in yeast. We also showed that the induced deletion events did not correlate with induction of AH hydroxylase. None of the tested compounds induced CYP1A associated ethoxyresorufin-O-deethylase activity in mouse embryos or in vitro. These results clearly demonstrate in vitro and in vivo a genotoxic activity of polychlorinated aromatic hydrocarbons, which is independent of induction of cytochrome P450 activity. Because genetic instability and deletions may be mechanistically involved in carcinogenesis, these results may encourage further research to determine whether such genotoxic mechanisms may be useful for cancer risk assessment of polychlorinated aromatic hydrocarbons.
p53 Involved in X-ray Induced Intrachromosomal Recombination in Mice. The p53 tumor suppressor gene is the most widely mutated gene in human tumorigenesis. DNA damage causes induction of p53 levels that result in G1 cell cycle arrest. The arrested cells have time to repair the DNA damage or, in case of irreparable damage, the cells undergo apoptosis. Thus, p53 has been proposed to play a key role in maintaining the integrity of genetic information after exposure of mammalian cells to DNA damaging agents. We sought to evaluate effects of p53 deficiency on spontaneous and DNA damage induced frequencies of DNA deletions in the mouse embryo. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70 kb internal fragment of the p gene. Spontaneous as well as carcinogen induced reversion events in homozygous pun/pun mice occur through deletion of one copy of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo give rise to black spots on the gray fur of the offspring. Pregnant dams, 10 days after conception were treated with x-rays (1 Gy) or benzo(a)pyrene (B[a]P, 150 or 30 mg/kg). Frequencies of spontaneous pun reversions were slightly elevated in p53 -/- and p53 +/- animals compared to p53 +/+ animals. X-ray treatment, increased the recombination frequency in weight and p53 +/-, but surprisingly not in the p53 -/- offspring, a statistically highly significant difference. However, B[a]P treatment caused induction of pun reversion frequencies in all three genotypes in a dose dependent manner. Thus, the lack of induction of deletions in p53 -/- animals are specific for x-ray induced events and may indicate different processing of DNA double strand breaks in the p53 -/- animals.
In conclusion, the p53 -/- mice did not show any enhanced sensitivity to detect B(a)P induced deletion events and showed a lack of inducibility by x-rays. Thus, p53 mutants are not useful for an improved more sensitive version of the assay.
Eye Spot Assay. Pink eyed unstable (pun/pun) mice have a dilute coat color as well as pink eyes. These eyes contain black eye spots that are thought to occur due to recombination events. Eyes from sacrificed C57BL/6J p+/+, p-/- and pun/pun mice were removed, mounted and dissected to remove the retinal pigmentation epithelium (RPE). p+/+ mice had a uniformly black RPE whereas p-/- mice had a uniformly translucent and colorless RPE. In contrast, pun mice showed in each RPE of 44 eyes one to 12 eye spots with an average of 5 spots per eye. Because the majority of these events comprised only one cell of the approximately 60,000 cells in a fully developed RPE (10 days following birth) a frequency of approximately one reversion per 104 cells can be calculated.
Benzo(a)pyrene and X-rays Induce Reversions of the Pink-Eye Unstable Mutation in the Retinal Pigment Epithelium of Mice. Recently, we used the eye spot assay to determine the frequency of spontaneous and B(a)P induced spotting. Using the same dose (150 mg/kg) and mode of application (i.p. of pregnant mothers) as for the fur spot assay, we have measured the inducibility of RPE reversion events at different times of development. We also have used gavage to apply the same dose of B(a)P to newborn mice. We assumed that adjacent pigmented cells, or no greater than one cell separated pigmented cells, form patches that resulted from one reversion event. B(a)P exposure results in a significant increase in spot frequency from 6.4 events in the control to 17.7 events at 10 days p.c. In comparison, x-ray exposure on day 10 p.c., resulted in an increased number of eye spots that was, however, not significant.
Position of Induced Reversion Events in the Retinal Epithelium. Dr. Sidman's laboratory previously described the development of the postnatal eye. The eye appears to have a front of dividing cells at the periphery of the RPE that moves away from the optic nerve as the epithelium grows. It is expected that only mitotically growing cells will exhibit an induced frequency of reversions, producing "patches" of revertant daughter cell. The number of spots, the size (number of cells) of the spots and the distance from the optic nerve were recorded. With just a few eyes we were able to determine a significant difference in the position of eye spots following either B(a)P or x-ray induction compared with the spontaneous eye spots. The eye spot assay in comparison with the fur spot assay requires fewer mice and less time for breeding and conducting the experiments; thus, it is less expensive. Both the frequency of offspring with fur spots and the frequency of eye spots are a true measure of deletion events and can be used to determine genetic and environmental effects.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 4 publications | 4 publications in selected types | All 4 journal articles |
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Aubrecht J, Secretan MB, Bishop AJR, Schiestl RH. Involvement of p53 in x-ray induced intrachromosomal recombination in mice. Carcinogenesis 1999;20(12):2229-2236. |
R825359 (Final) |
not available |
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Bishop AJR, Kosaras B, Carls N, Sidman RL, Schiestl RH. Susceptibility of proliferating cells to benzo(a)pyrene induced homologous recombination in mice. Carcinogenesis 2001;22(4):641-649. |
R825359 (Final) |
Exit |
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Bishop AJR, Kosaras B, Sidman RL, Schiestl RH. Benzo(a)pyrene and x-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice. Mutation Research 2000;457(1-2):31-40. |
R825359 (Final) |
not available |
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Schiestl RH, Aubrecht J, Yap WY, Kandikonda S, Sidhom S. Polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce intrachromosomal recombination in vitro and in vivo . Cancer Research 1997;57(19):4378-4383. |
R825359 (Final) |
not available |
Supplemental Keywords:
media, environmental, pollutants, risk assessment, exposure, effects, sensitive populations, dose response, carcinogen, mutagen, animal, mammalian, mice, in vivo assay, genetic predisposition, susceptibility, p53, chemicals, toxics, toxic substances, PAHs, PCBs, dioxin, solvents, organics, scientific disciplines, genetics, genomic instability, DNA deletions., RFA, Health, Scientific Discipline, Toxicology, Health Risk Assessment, Molecular Biology/Genetics, cancer risk, additivity, complex mixtures, hazard indices, p53 gene, interindividual variations, mutagenic, animal model, dose response, carcinogens, environmental toxicants, susceptability, cancer risk assessment, toxicological interactions, sensitivityProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.