title: Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.
abstract: Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.
authors.affiliation: Food and Drug Administration, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Manjanatha
authors.forename: S D
authors.initials: SD
authors.lastname: Shelton
authors.forename: M
authors.initials: M
authors.lastname: Bishop
authors.forename: J G
authors.initials: JG
authors.lastname: Shaddock
authors.forename: V N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: R H
authors.initials: RH
authors.lastname: Heflich
authors.forename: P J
authors.initials: PJ
authors.lastname: Webb
authors.forename: L R
authors.initials: LR
authors.lastname: Blankenship
authors.forename: F A
authors.initials: FA
authors.lastname: Beland
authors.forename: K J
authors.initials: KJ
authors.lastname: Greenlees
authors.forename: S J
authors.initials: SJ
authors.lastname: Culp
mesh_terms: D000814:Aniline Compounds / Q000008:administration & dosage / Q000633:toxicity*; D000818:Animals; D030801:Animals, Genetically Modified; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002999:Clone Cells; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D005260:Female; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007763:Lac Operon; D008099:Liver / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects*; D015162:Micronucleus Tests; D015394:Molecular Structure; D009153:Mutagens / Q000008:administration & dosage / Q000633:toxicity*; D009154:Mutation; D051381:Rats; D012394:Rosaniline Dyes; D036401:Toxicity Tests, Chronic
chemical_list: D000814:Aniline Compounds; D009153:Mutagens; D012394:Rosaniline Dyes; C015320:leucomalachite green; D007041:Hypoxanthine Phosphoribosyltransferase