Evidence of erythropoiesis rather than direct genotoxicity causing micronucleus induction by a new protoporphyrinogen-oxidase inhibitor.

Protoporphyrinogen-oxidase inhibitors (PPOi) are a class of herbicides that target chlorophyll synthesis in plants. Similarly, they affect heme synthesis in mammals, resulting in anemia. A new PPOi development candidate showed micronucleus formation in the bone marrow of rodents in vivo. Increased erythropoiesis has previously been described to increase micronucleus formation in the bone marrow. Hence, the micronuclei (MN) observed after administration of the new PPOi candidate may be caused by increased erythropoiesis rather than direct genotoxicity. To investigate this, the mutagenicity in vitro was examined and found to be clearly negative. In addition, micronucleus formation in vivo was investigated after administration of the new PPOi in doses that caused anemia. Both, erythropoietic and non-erythropoietic tissues, bone marrow and liver, were investigated. Moreover, genotoxicity was investigated by the Comet-assay in erythroid and non-erythroid cells of the bone marrow. The new PPOi did not show mutagenic effects in vitro. The new PPOi induced an increase in the micronucleus counts in the bone marrow of rats, whereas the hepatocytes were not affected. In another study in mice, micronucleus formation was observed in the new PPOi-exposed bone marrow. In parallel, bone marrow cells of the same animals were separated into subpopulations isolating erythroid (selected via their Ter119 surface marker) and non-erythroid (selected by their CD45 surface marker) cells, which were then assessed in a comet assay. Results showed a dose dependent increase in micronucleus frequencies while, increases in % DNA tail intensities in the comet assay were only observed in the erythroid Ter119+ subpopulation, but not in non-erythroid bone marrow cells. This provides evidence that the new PPOi induces MN only upon induction of anemia, and that the DNA-damage is only occurring in erythroid cells. The increased micronucleus-formation of the new PPOi in vivo is hence most likely caused by erythropoiesis and not by direct genotoxic actions.