title: Technical modification of the Balb/c 3T3 cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol.
abstract: The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2microg/ml insulin, rather than the more-complex insulin-transferrin-ethanolamine-sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24-25 days to 31-32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl(2) and phenacetin, combining a post-treatment of 100ng/ml 12-O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl(2) and phenobarbital, with pre-treatment of 0.2microg/ml MCA at the initiation stage. In the present study, only phenobarbital was negative when tested by using the modified protocol.
authors.affiliation: Laboratory of Cell Carcinogenicity, Division of Genetic Toxicology, Hatano Research Institute, Food and Drug Safety Center, Hadano, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Hayashi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Sasaki
authors.forename: Shin
authors.initials: S
authors.lastname: Asada
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.forename: Isao
authors.initials: I
authors.lastname: Yoshimura
authors.forename: Noriho
authors.initials: N
authors.lastname: Tanaka
authors.forename: Makoto
authors.initials: M
authors.lastname: Umeda
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D041702:BALB 3T3 Cells / Q000187:drug effects*; D015197:Carcinogenicity Tests / Q000191:economics / Q000379:methods*; D002273:Carcinogens / Q000145:classification / Q000633:toxicity*; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced*; D003043:Cocarcinogenesis; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D008748:Methylcholanthrene / Q000145:classification / Q000633:toxicity; D051379:Mice; D008807:Mice, Inbred BALB C; D044967:Serum; D013997:Time Factors
chemical_list: D002273:Carcinogens; D003470:Culture Media; D004338:Drug Combinations; D008748:Methylcholanthrene