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Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors
S Manam1, G A Shinder, D J Joslyn
1Department of Safety Assessment, Merck Research Laboratories, West Point, PA 19486, USA.
Abstract:
We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p. injections of 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine (DEN) were compared to those previously obtained for tumors induced by a single but higher dose of each carcinogen. The principal assay used was a direct tumor analysis involving sequencing of polymerase chain reaction (PCR)-amplified tumor DNA; additional mutations that were present in only a small fraction of tumor cells were detected using a transfection assay or a PCR-engineered restriction fragment length polymorphism method. Spontaneous liver tumors had a relatively low frequency of ras mutations, all found in Ha-ras codon 61, and most of these mutations were present in only a small fraction of tumor cells. With the exception of multiple-dose DEN, each group of single- and multiple-dose carcinogen-induced tumors exhibited a higher frequency of ras mutations compared with spontaneous tumors. For AAB, N-OH-AAF and DEN, the dosing regimen was found to affect significantly the profile of ras mutations. For each of these carcinogens, the multiple-dose tumor group (versus single-dose group) had fewer Ki-ras and N-ras mutations and more tumors in which the Ha-ras codon 61 (C-->A) mutation was present in a large fraction of cells. Our results demonstrate that the dosing procedure can materially affect the pattern of ras gene mutation in mouse liver tumors.
Insights
The dosing regimen significantly impacts ras gene mutations in mouse liver tumors. Multiple doses of carcinogens, compared to single doses, altered the mutation profile, favoring specific Ha-ras mutations.
Area of Science:
- Chemical carcinogenesis
- Molecular toxicology
- Cancer genetics
Background:
- Ras gene mutations are critical in chemical carcinogenesis.
- Understanding how carcinogen exposure affects mutation spectra is crucial for risk assessment.
Purpose of the Study:
- To investigate the influence of carcinogen dosing regimens on ras gene mutation patterns in chemically induced mouse liver tumors.
- To compare mutation spectra from single versus multiple doses of various carcinogens.
Main Methods:
- Chemically induced liver tumors in CD-1 mice using 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and N-nitrosodiethylamine (DEN).
- Analysis of ras gene mutations using polymerase chain reaction (PCR)-amplified tumor DNA sequencing.
- Detection of low-frequency mutations via transfection assays and PCR-engineered restriction fragment length polymorphism.
Main Results:
- Carcinogen-induced tumors showed higher ras mutation frequencies than spontaneous tumors, except for multiple-dose DEN.
- Dosing regimen significantly affected ras mutation profiles for AAB, N-OH-AAF, and DEN.
- Multiple-dose regimens, compared to single-dose, resulted in fewer Ki-ras and N-ras mutations and more Ha-ras codon 61 (C-->A) mutations present in a larger cell fraction.
Conclusions:
- The dosing procedure is a material factor influencing the pattern of ras gene mutations in mouse liver tumors.
- Different dosing strategies can lead to distinct molecular profiles in carcinogen-induced tumors.