Determination of Mutational Spectra Induced by Environmental Toxicants in Complex Human Cell Populations

  • Robert Gealy
  • Phouthone KeohavongEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)


Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and have potent mutagenic and carcinogenic activities. Studies of mutations induced by these compounds in human cells can help acquire an understanding of their mutagenic pathways. In this chapter, independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with benzo(a)pyrene (BaP) or dibenzo(a,l)pyrene (DBP), and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine resistance (6TGR). The kinds and positions of the mutations occurring in the third exon of the HPRT gene were analyzed in the mixed HPRTR mutant cell populations using a combination of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Mutant bands were excised from the gel, amplified using PCR, and sequenced to determine the kinds and positions, or spectrum of mutations.

Key words

Mutations Hotspot HPRT Human cells Benzo(a)pyrene Dibenzo(a,l)pyrene DGGE 


  1. 1.
    Benzer S, Freese E (1958) Induction of specific mutations with 5-bromouracil. Proc Natl Acad Sci U S A 44:112–119CrossRefGoogle Scholar
  2. 2.
    Coulondre C, Miller JH (1977) Genetic study of the lac repressor. IV. Mutagenic specificity in the lacl gene of Escherichia coli. J Mol Biol 117:577–606CrossRefGoogle Scholar
  3. 3.
    Fischer SG, Lerman LS (1983) DNA fragments different by single base-pair substitutions are separated in denaturing gradient gel: correspondence with melting theory. Proc Natl Acad Sci U S A 80:1579–1583CrossRefGoogle Scholar
  4. 4.
    Keohavong P, Thilly WG (1989) Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci U S A 86:9253–9257CrossRefGoogle Scholar
  5. 5.
    Crespi CL, Gonzalez FJ, Steimel JJT, Tumer TR, Gelboin HV, Penman B, Langelbach R (1991) A metabolically competent human cell line expressing five cDNA encoding procarcinogen-activating enzymes: application to mutagenicity testing. Chem Res Toxicol 4:566–572CrossRefGoogle Scholar
  6. 6.
    Leong PM, Thilly WG, Morgenthaler S (1985) Variance estimation in single-cell assays: comparison to experimental observations in human lymphoblasts at 4 gene loci. Mutat Res 150:403–410CrossRefGoogle Scholar
  7. 7.
    Furth E, Thilly WG, Penman B, Liber H, Rand W (1981) Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates. Anal Biochem 110:1–8CrossRefGoogle Scholar
  8. 8.
    Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G+ C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction resulted in improved detection of single base changes. Proc Natl Acad Sci U S A 86:232–236CrossRefGoogle Scholar
  9. 9.
    Cariello NF, Keohavong P, Kat AG, Thilly WG (1990) Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191. Mutat Res 231:165–176CrossRefGoogle Scholar
  10. 10.
    Cariello NF, Scott JK, Kat AG, Thilly WG, Keohavong P (1988) Resolution of missense mutation in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT-Munich. Am J Hum Genet 42:726–734PubMedPubMedCentralGoogle Scholar
  11. 11.
    Keohavong P, Thilly WG (1992) Mutational spectrometry: a general approach for hot-spot mutations in selectable genes. Proc Natl Acad Sci U S A 89:4623–4627CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Merck Research LaboratoriesWest PointUSA
  2. 2.Department of Environmental and Occupational Health, Graduate School of Public HealthUniversity of PittsburghPittsburghUSA

Personalised recommendations