Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, aristolochic acids

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.75, 5.5, and 11mg/kg AAs for 28 days or with 0, 11, 22, and 30mg/kg AAs for 3 days. Day 1 being the first day of treatment, Pig-a mutant frequencies (MFs) were assayed in peripheral blood erythrocytes up to Day 56 for the 28-day treatment or Day 42 for the 3-day treatment; micronuclei were assayed in peripheral blood reticulocytes on Day 4 (both treatment protocols) and on Day 29 of the 28-day treatment protocol; and at the final sampling times (Day 59 or Day 42), the animals were sacrificed and Hprt mutant lymphocytes were measured. In a separate study, the Comet assay was performed on liver, kidney, and bone marrow of animals gavaged with 0, 11, 22, and 30mg/kg AAs for 4 days and sacrificed 3h after the last treatment. While only weak increases in micronucleated reticulocyte frequency were observed in treated animals, Pig-a MFs increased in a dose- and time-dependent manner with both treatment schedules. Lymphocyte Hprt mutant frequencies also increased dose dependently in treated animals, and the Comet assay detected elevated levels of DNA damage in all the tissues evaluated. These findings indicate that the DNA damage produced by AAs in rat bone marrow is a weak inducer of micronuclei but a relatively strong inducer of gene mutation.