7,12-dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk+/− mice: Automated scoring of lymphocyte clones using a fluorescent viability indicator

• Published in: Environmental and molecular mutagenesis 2000, Vol.36 (4), p.283-291
• Main Authors: Dobrovolsky, Vasily N., Shaddock, Joseph G., Heflich, Robert H.
• Format: Article
• Language: English
• Subjects: 5-bromodeoxyuridine; 9,10-Dimethyl-1,2-benzanthracene - toxicity; alamarBlue; Animals; Biological and medical sciences; Cell Survival; Chemical mutagenesis; Clone Cells; Female; Fluorescent Dyes; Hypoxanthine Phosphoribosyltransferase - genetics; loss of heterozygosity; Lymphocytes - drug effects; Male; Medical sciences; Mice; Mutagens - toxicity; Mutation; spleen lymphocytes; Thymidine Kinase - genetics; Toxicology
• ISSN: 0893-6692; 1098-2280
• DOI: 10.1002/1098-2280(2000)36:4<283::AID-EM4>3.0.CO;2-8

7,12‐Dimethylbenz[a]anthracene (DMBA) is a rodent carcinogen and a potent in vivo mutagen for the X‐linked hypoxanthine guanine phosphoribosyl transferase (hprt) gene of rats and for the lacI transgene of Big Blue mice and rats. Although DMBA is also a powerful clastogen, molecular analysis of these DMBA‐induced hprt and lacI mutations indicates that most are single base‐pair (bp) substitutions and 1‐ to 3‐bp frameshifts. In the present study, we evaluated the types of mutations induced by DMBA in the autosomal thymidine kinase (Tk) gene of Tk+/− mice. Male and female 5‐ to 6‐week‐old animals were injected i.p. with DMBA at a dose of 30 mg/kg. Five weeks after the treatment, hprt and Tk mutant frequencies were determined using a limiting dilution clonal assay in 96‐well plates. We established conditions for the automated identification of wells containing expanded lymphocyte clones using the fluorescent indicator alamarBlue. This procedure allowed the unbiased identification of viable clones and calculation of mutant frequencies. In male mice, DMBA treatment increased the frequency of hprt mutants from 1.8 ± 1.1 to 34 ± 9 × 10−6, and Tk mutants from 33 ± 12 to 78 ± 26 × 10−6; treated female mice had a significant but lower increase in hprt mutant frequency than did males. Molecular analysis of DMBA‐induced Tk mutants revealed that at least 75% had the entire wild‐type Tk allele missing. The results indicate that the predominant types of DMBA‐induced mutation detected by the autosomal Tk gene are different from those detected by the X‐linked hprt gene. The Tk gene mainly detects loss of heterozygosity mutation, whereas the majority of mutations previously found in the hprt gene were point mutations. Environ. Mol. Mutagen. 36:283–291, 2000. Published 2000 Wiley‐Liss, Inc.