Application of in vitro erythropoiesis from bone marrow derived progenitors to detect and study genotoxicity

Abstract

Assays that predict toxicity are an essential part of drug development and there is a demand for efficient models to better predict human responses. The in vivo micronucleus (MN) assay is a robust toxicity test that assesses the genotoxic effect of drugs on adult bone marrow (BM) using the metric of genotoxic damage to the reticulocyte population in mice. An in vitro correlate to this assay might facilitate extension to human cells and thus provide a highly predictive genotoxicity assay. As first steps in developing a toxicity assay, this thesis work (a) adapted a fetal liver-based in vitro erythropoietic culture system to induce optimized erythropoietic growth from the lineage-marker-negative (Lin) population in adult BM, as adult hematopoietic tissue is ultimately a feasible source of cells; and (b) demonstrated that exposure to alkylating agents induces physiological MN-formation in erythroid populations derived in vitro. The potential for increased efficiency in this in vitro model depends on the ability to stimulate terminal erythroid differentiation at an optimal level from adult BM.

(cont.) With this goal in mind, this thesis work employed experimental design strategies, erythroid-specific growth measurements, and multi-linear regression to model erythropoietic growth in this system and thus estimate the relative sensitivity of Lin7 BM to erythropoietic growth parameters, including Erythropoietin, Stem Cell Factor, p02, and Fibronectin, among others. From these erythroid-specific growth measurements, it is estimated that >1500 MN assays can be conducted using the BM of a single mouse. This throughput represents a significant improvement over the current in vivo test, which assays a single condition per mouse. This thesis work then quantified the genotoxic response to three alkylating agents (1,3-bis(2-chloroethyl)- 1-nitrosourea [BCNU], N-methyl-N -nitro-N-nitrosoguanidine [MNNG], and methylmethane sulfonate [MMS]) in this culture system and detected a significant cytotoxic response and concomitant increase in MN incidence in reticulocytes.

(cont.) This increase in MN frequency provides a clear signal of the genotoxic events that likely lead to global toxicity, and thus mimics the physiological hematopoietic response to alkylating chemotherapeutics. In addition, this thesis work determined that DNA repair-deficient (MGMT -/-) BM displayed sensitivity to genotoxic exposure in vivo compared with wild-type (WT) BM, and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, facilitating the study of genotoxic effects at specific developmental stages.