title: Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays.
abstract: Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of "misleading" positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of "misleading" positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by "misleading positive" genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK. Electronic address: James.Whitwell@covance.com.
authors.forename: James
authors.initials: J
authors.lastname: Whitwell
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Smith
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Karen
authors.initials: K
authors.lastname: Jenner
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Heather
authors.initials: H
authors.lastname: Lyon
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Deborah
authors.initials: D
authors.lastname: Wood
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Clements
authors.affiliation: Covance Laboratories Ltd., Harrogate, North Yorkshire, UK.
authors.forename: Kelly
authors.initials: K
authors.lastname: Aschcroft-Hawley
authors.affiliation: Exponent Inc., USA.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Gollapudi
authors.affiliation: Kirkland Consulting, Tadcaster, North Yorkshire, UK.
authors.forename: David
authors.initials: D
authors.lastname: Kirkland
authors.affiliation: Servier Group, Drug Safety Assessment, 45520 Gidy, France.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lorge
authors.affiliation: Procter & Gamble, Global Product Stewardship, Cincinnati, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pfuhler
authors.affiliation: ILSI Health and Environmental Sciences Institute, Washington DC, USA.
authors.forename: Jennifer Y
authors.initials: JY
authors.lastname: Tanir
authors.affiliation: Sanofi, Vitry sur Seine, France.
authors.forename: Veronique
authors.initials: V
authors.lastname: Thybaud
mesh_terms: D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002733:Chlorophenols / Q000494:pharmacology; D003474:Curcumin / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005054:Eugenol / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D015162:Micronucleus Tests / Q000379:methods*; D009154:Mutation; D009596:Nitrophenols / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D010796:Phthalic Anhydrides / Q000494:pharmacology; D011435:Propyl Gallate / Q000494:pharmacology; D015203:Reproducibility of Results; D012118:Resorcinols / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
chemical_list: D000179:Acrylates; D002733:Chlorophenols; D009596:Nitrophenols; D009930:Organic Chemicals; D010796:Phthalic Anhydrides; D012118:Resorcinols; D016159:Tumor Suppressor Protein p53; D062367:ortho-Aminobenzoates; D003571:Cytochalasin B; D005054:Eugenol; C040833:ethyl acrylate; D011435:Propyl Gallate; D003474:Curcumin; C004762:2,4-dichlorophenol; C043103:phthalic anhydride; C024836:4-nitrophenol; C031389:resorcinol