Oxazepam is mutagenic in vivo in Big Blue (R) transgenic mice

Although oxazepam (Serax(R)), a widely used benzodiazepine anxiolytic, does not induce gene mutations iii vitro or chromosomal aberrations in vivo, it was found to be a hepatocarcinogen in a 2 year bioassay in B6C3F1 mice. Th us, it was of interest to determine whether this carcinogen is mutagenic in vivo. Male B6C3F1 Big Blue(R) transgenic mice were fed 2500 p.p.m. oxazepa m or control diet alone for 180 days and killed on the nest day. The mutant frequency (MF) of lacI in control mice was 5.02 +/- 2.4 x 10(5), whereas t he R;IF in the oxazepam-treated mice was 9.17 +/- 4.82 x 10(-5), a signific ant increase (P < 0.05). Correction of the mutant frequency of lacI from th e oxazepam-treated mice for clonality resulted in a decrease in the mean mu tant frequency to 8.15 +/- 2.54 x 10-5. Although the mutant frequency diffe rence was small, sequencing of a random collection of the mutants from each oxazepam-exposed mouse showed a significant difference (P < 0.015) in the mutation spectrum compared with that from control mice, In the oxazepam-exp osed mice, an increase in G:C-->T:A and G:C-->C:G transversions and a conco mitant decrease in G:C-->A:T transitions were observed, Clonal expansion of mutations at guanines in 5'-CpG-3' sequencing contexts at three sites was noted. It is postulated that some of the mutations found in the oxazepam-de rived spectrum were due to oxidative damage elicited by induction of CYP2B isozymes as the result of chronic oxazepam administration. This study demon strates that the in vivo Big Blue(R) transgenic rodent mutation assay can d etect mutations derived from a carcinogen that did not induce gene mutation s in vitro or micronuclei in mouse bone marrow. Moreover, the sequencing of the recovered mutants can distinguish between the mutation spectrum from t reated mice compared with that from control mice, thereby confirming the ge notoxic consequences.