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

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

Abstract

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 

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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

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