CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis.

BACKGROUND: A micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991. RESULTS: Rats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control. These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts. CONCLUSIONS: It was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects.

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society.

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society.

The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs-the PIGRET assay-has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.

The rat Pig-a assay using an erythroid HIS49 antibody in a single dose study of isopropyl p-toluenesulfonate.

As part of a collaborative study in the Mammalian Mutagenicity Study group of the Japanese Environmental Mutagen Society, we evaluated the in vivo mutagenicity of isopropyl p-toluenesulfonate (IPTS) using a peripheral blood Pig-a assay in rats. Pig-a mutant frequency (MF) data was obtained for both red blood cells (RBCs) and reticulocytes (RETs) at 1, 2 and 4 weeks after a single oral administration of IPTS at doses of 125, 250, or 500mg/kg. The results of the RBC Pig-a assay demonstrated that both the 250 and 500mg/kg treatment groups showed significant increases in Pig-a MF only at 4 weeks after IPTS treatment. In comparison, the PIGRET assay showed a clear and dose-related increase in Pig-a MF at 1 week after treatment, with a continuous increase until 4 weeks after treatment observed in the highest dose group. These results indicate that the both the RBC Pig-a assay and PIGRET assay can detect in vivo IPTS mutagenicity under a single dosing protocol. In particular, the PIGRET assay, which uses magnetic enrichment to analyze greater numbers of RETs in a high-throughput manner, showed an increase in Pig-a MF earlier than the RBC Pig-a assay. The PIGRET assay is also considered to be more sensitive than the RBC Pig-a assay because it exhibits a low spontaneous Pig-a MF. For this reason, the PIGRET assay clearly identified small increases in Pig-a MF as significant at the lower doses than in the RBC Pig-a assay under the conditions in this study.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

Report on the rat Pig-a assay using an anti-rat erythroid marker HIS49 antibody in a single dose study of 1,2-dimethylhydrazine.

As part of a collaborative study in the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society (JEMS), we investigated the in vivo genotoxicity profile of 1,2-dimethylhydrazine (DMH) using a Pig-a assay in total red blood cells (RBC Pig-a assay) or a reticulocyte Pig-a assay (PIGRET assay). We also assessed the genotoxic potential of DMH using both a bone marrow micronucleus test and a liver comet assay as follow-up studies. Single administration of 25, 50, 100mg/kg DMH to male rats did not show time- or dose-related increases in Pig-a mutant frequency (MF) in either the RBC Pig-a or PIGRET assays up to 4 weeks after treatment. The bone marrow micronucleus test under the same dose levels was judged positive, while the liver comet assay was judged inconclusive due to the high number of hedgehogs. Re-evaluation of the rat liver comet assay at lower dose levels (4, 10, and 25mg/kg DMH) showed a dose-related increase in%DNA in tail. Taken together, DMH showed a positive response in both the bone marrow micronucleus test and liver comet assay, while the increases in Pig-a MF in both the RBC Pig-a and PIGRET assays could hardly be detected after single dosing. These results suggest that DMH provides different genotoxicity outcomes depending on the endpoint following acute in vivo dosing.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Effective use of the Pig-a gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals.

The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time- and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10(-6) and 5.5 ×10(-6), respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.

Induction of cardiac fibrosis by ß-blocker in G protein-independent and G protein-coupled receptor kinase 5/ß-arrestin2-dependent Signaling pathways.

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and ß-arrestin-dependent signaling through GPCRs (biased agonism). Several ß-blockers are known as biased ligands. All ß-blockers inhibit the binding of agonists to the ß-adrenergic receptors. In addition to ß-blocking action, some ß-blockers are reported to induce cellular responses through G protein-independent and ß-arrestin-dependent signaling pathways. However, the physiological significance induced by the ß-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a ß(1)-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and ß-arrestin2-dependent pathway. Metoprolol, a ß-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between ß(1)-adrenergic receptor and ß-arrestin2, but not ß-arrestin1. The interaction between ß(1)-adrenergic receptor and ß-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in ß-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/ß-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and ß-arrestin-dependent signaling is a reason for the diversity of the effectiveness of ß-blockers.

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes.

Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.