Trenbolone growth promotant: covalent DNA binding in rat liver and inSalmonella typhimurium, and mutagenicity in the Ames test

Abstract

DNA binding in vivo: [6,7-³H]β-trenbolone (β-TBOH) was administered p.o. and i.p. to rats. After 8 or 16 h, DNA was isolated from the livers and purified to constant specific radioactivity. Enzymatic digestion to deoxyribonucleotides and separation by HPLC revealed about 90% of the DNA radioactivity eluting in the form of possible TBOH-nucleotide adducts. The extent of this genotoxicity, expressed in units of the Covalent Binding Index, CBI = (μmol TBOH bound per mol nucleotide)/(mmol TBOH administered per kg body weight) spanned from 8 to 17, i.e. was in the range found with weak genotoxic carcinogens.Ames test: low doses of β-TBOH increased the number of revertants inSalmonella strain TA100 reproducibly and in a dose-dependent manner. The mutagenic potency was 0.2 revertants per nmol after preincubation of the bacteria (20 min at 37° C) with doses between 30 and 60 μg per plate (47 and 94 μg/ml preincubation mixture). Above this dose, the number of revertants decreased to control values, accompanied by a reduction in survival. The addition of rat liver S9 inhibited the mutagenicity.DNA binding in vitro: calf thymus DNA was incubated with tritiated β-TBOH with and without rat liver S9. Highest DNA radioactivities were determined in theabsence of the “activation” system. Addition of inactive S9 (without cofactors) reduced the DNA binding by a factor of up to 20. Intermediate results were found with active S9.DNA binding in Salmonella: β-TBOH was irreversibly bound to DNA isolated fromS. typhimurium TA100 after incubation of bacteria with [³H]β-TBOH.Conclusions: Covalent DNA binding appears to be the mechanism of an activation-independent (“direct”) mutagenicity of TBOH which is not easily detected because of the bactericidal activity. The genotoxicity risk arising from exposure of humans to trenbolone residues in meat was estimated using the in vivo data and compared to that from the exposure to unavoidable genotoxins aflatoxin B₁ and dimethylnitrosamine. It is concluded that trenbolone residues represent only a low genotoxic risk.