Assessment of systemic genetic damage in pediatric inflammatory bowel disease.

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new-onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19-24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± SD = 3.1 ± 2.3 × 10-6 vs. 3.6 ± 5.6 x 10-6 , respectively). In contrast, 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new-onset (0/17) (p = .049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤0.42% MN-RET (p = .040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA-reactive hematopoietic carcinogen with potent and well-characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig-a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose-related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig-a assays even at the lowest dose tested. PCH-induced lacZ and Pig-a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505-512, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Empirical analysis of BMD metrics in genetic toxicology part II: in vivo potency comparisons to promote reductions in the use of experimental animals for genetic toxicity assessment.

Genotoxicity tests have traditionally been used only for hazard identification, with qualitative dichotomous groupings being used to identify compounds that have the capacity to induce mutations and/or cytogenetic alterations. However, there is an increasing interest in employing quantitative analysis of in vivo dose-response data to derive point of departure (PoD) metrics that can be used to establish human exposure limits or margins of exposure (MOEs), thereby supporting human health risk assessments and regulatory decisions. This work is an extension of our companion article on in vitro dose-response analyses and outlines how the combined benchmark dose (BMD) approach across included covariates can be used to improve the analyses and interpretation of in vivo genetic toxicity dose-response data. Using the BMD-covariate approach, we show that empirical comparisons of micronucleus frequency dose-response data across multiple studies justifies dataset merging, with subsequent analyses improving the precision of BMD estimates and permitting attendant potency ranking of seven clastogens. Similarly, empirical comparisons of Pig-a mutant phenotype frequency data collected in males and females justified dataset merging across sex. This permitted more effective scrutiny regarding the effect of post-exposure sampling time on the mutagenicity of N-ethyl-N-nitrosourea observed in reticulocytes and erythrocytes in the Pig-a assay. The BMD-covariate approach revealed tissue-specific differences in the induction of lacZ transgene mutations in Muta™Mouse specimens exposed to benzo[a]pyrene (BaP), with the results permitting the formulation of mechanistic hypotheses regarding the observed potency ranking. Lastly, we illustrate how historical dose-response data for assessments that examined numerous doses (i.e. induced lacZ mutant frequency (MF) across 10 doses of BaP) can be used to improve the precision of BMDs derived from datasets with far fewer doses (i.e. lacZ MF for 3 doses of dibenz[a,h]anthracene). Collectively, the presented examples illustrate how innovative use of the BMD approach can permit refinement of the use of in vivo data; improving the efficacy of experimental animal use in genetic toxicology without sacrificing PoD precision.

Part 2. Assessment of micronucleus formation in rats after chronic exposure to new-technology diesel exhaust in the ACES bioassay.

The formation of micronuclei (MN*) is a well-established endpoint in genetic toxicology; studies designed to examine MN formation in vivo have been conducted for decades. Conditions that cause double-strand breaks or disrupt the proper segregation of chromosomes during division result in increases in MN formation frequency. This endpoint is therefore commonly used in preclinical studies designed to assess the potential risks to humans of exposure to a myriad of chemical and physical agents, including inhaled diesel exhaust (DE). As part of the Advanced Collaborative Emissions Study (ACES) Phase 3B, which examined numerous additional toxicity endpoints associated with lifetime exposure to DE in a rodent model, this ancillary 24-month investigation examined the potential of inhaled DE to induce chromosome damage in chronically exposed rodents. The ACES design included exposure of both mice and rats to DE derived from heavy-duty engines that met U.S. Environmental Protection Agency (EPA) 2007 standards for diesel-exhaust emissions (new-technology diesel exhaust). The exposure conditions consisted of air (the control) and three dilutions of DE, resulting in four levels of exposure. At specific times, blood samples were collected, fixed, and shipped by the bioassay staff at Lovelace Respiratory Research Institute (LRRI) to Litron Laboratories (Rochester, NY) for further processing and analysis. In recent years, significant improvements have been made to MN scoring by using objective, automated methods such as flow cytometry, which allows the detection of micronucleated reticulocytes (MN-RET), micronucleated normochromatic erythrocytes (MN-NCE), and reticulocytes (RET) in peripheral blood samples from mice and rats. By using a simple staining procedure coupled with rapid and efficient analysis, many more cells can be examined in less time than was possible using traditional, microscopy-based MN assays. Thus, for each sample in the current study, 20,000 RET were scored for the presence of MN. In the chronic-exposure (12 and 24 months) bioassay, blood samples were obtained from separate groups of exposed animals at specific time points throughout the course of the study. The automated method using flow cytometry has found widespread use in safety assessment and is supported by regulatory guidelines, including International Conference on Harmonisation (ICH) S2(R1) (2011). Statistical analyses included the use of analysis of variance (ANOVA) to compare the effects of sex, exposure condition, and duration, as well asthe interactions between them. Analyses of blood samples from rats combined data from our earlier 1- and 3-month exposure studies (Bemis et al. 2012) with data from our current 12- and 24-month exposure studies. Consistent with findings from the preliminary studies, no sex-based differences in MN frequency were observed in the rats. An initial examination of mean frequencies across the treatment groups and durations of exposure showed no evidence of treatment-related increases in MN at any of the time points studied. Further statistical analyses did not reveal any significant exposure-related effects. An examination of the potential genotoxic effects of DE is clearly valuable as part of a large-scale chronic exposure bioassay. The results described in this report provide a comprehensive examination of chronic exposure to DE in a rodent model. Our investigation of chromosomal damage also plays an important role in the context of ACES, which was designed to assess the safety of emissions from 2007-compliant diesel engines.

Assessment of 5-fluorouracil and 4-nitroquinoline-1-oxide in vivo genotoxicity with Pig-a mutation and micronucleus endpoints.

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5-fluorouracil (5-FU) and 4-nitroquinoline-1-oxide (4NQO) as part of an international validation trial of the Pig-a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5-FU for three consecutive days (Days 1-3); blood was sampled on Days -1, 4, 15, 29, and 45. Pig-a mutant phenotype reticulocyte (RET(CD59-)) and mutant phenotype erythrocyte (RBC(CD59-)) frequencies were determined on Days -1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN-RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC(CD59-) and RET(CD59-) frequencies were determined on Days -1, 15, and 29, and MN-RET were quantified on Day 29. Whereas 5-FU was found to increase %MN-RET, no significant increases were observed for RBC(CD59-) or RET(CD59-) at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC(CD59-) and RET(CD59-) frequencies, and this same dose level caused a weak but significantly elevated increase in MN-RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig-a mutation and MN-RET into acute and 28-day repeat-dose studies.

Assessment of the genotoxic potential of azidothymidine in the comet, micronucleus, and Pig-a assay.

The genotoxic potential of azidothymidine (Zidovudine, AZT), chosen as a model compound for nucleotide analogs, was comprehensively assessed in vivo for gene mutation, clastogenicity, and DNA breakage endpoints. Male Wistar rats were treated by oral gavage over 7 days with AZT at dose levels of 2×0 (control), 2×250, 2×500, and 2×1000mg/kg/day with a final single dose given on day 8. DNA damage was then evaluated with the comet assay in liver, stomach, and peripheral blood and with the micronucleus test in bone marrow and peripheral blood (by flow cytometry) in the same animals. After a treatment-free period of upto 42 days, the Pig-a gene mutation assay was performed in peripheral blood of the high-dose animals. In the comet assay as well as the micronucleus test, AZT caused a considerable dose-dependent increase in DNA damage in all tissues evaluated and was highly cytotoxic to bone marrow and peripheral blood cells. These data are well in line with published results. Surprisingly, AZT did not significantly increase the number of Pig-a mutant cells. We speculate that two factors likely contributed to this negative result: a predominance of large deletions caused by AZT, and the relatively low statistical power of the first-generation scoring method used for this study.

Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity.

Part 2. Assessment of genotoxicity after exposure to diesel exhaust from U.S. 2007-compliant diesel engines: report on 1- and 3-month exposures in the ACES bioassay.

Micronucleus (MN*) formation is a well-established endpoint in genetic toxicology; studies designed to examine MN formation in vivo have been conducted for decades. Conditions that cause double-strand breaks or disrupt the proper segregation of chromosomes during division result in an increase in MN frequency. Thus this endpoint is commonly employed in preclinical studies designed to assess the potential risks of human exposure to a myriad of chemical and physical agents, including inhaled diesel exhaust (DE). As part of the Advanced Collaborative Emissions Study (ACES) this investigation examined the potential of inhaled DE to induce chromosome damage in chronically exposed rodents. The ACES design included exposure of both rats and mice to DE derived from 2007-compliant heavy-duty engines. The exposure conditions consisted of air control and dilutions of DE resulting in three levels of exposure. At specified times, blood samples were collected, fixed, and shipped by the bioassay staff to Litron Laboratories for further processing and analysis. Significant improvements have been made to MN scoring by using objective, automated methods such as flow cytometry, which allows for the detection of micronucleated reticulocytes (MN-RET), micronucleated normochromatic erythrocytes (MN-NCE), and reticulocytes (RETs) in peripheral blood samples from mice and rats. By using a simple staining procedure coupled with rapid and efficient analysis, many more cells were examined in less time than was possible in traditional, microscopy-based MN assays. Thus, for each sample, 20,000 RETs were scored for the presence of MN. In the chronic-exposure bioassay, blood samples were obtained from independent groups of exposed animals at specific time points throughout the course of the entire study. This automated method is supported by numerous regulatory guidelines and meets the requirements for an Organization of Economic Cooperation and Development (OECD)-compliant assay for genotoxicity. Statistical approaches employed analysis of variance (ANOVA) to compare effects of sex, exposure condition, and duration, as well as their interactions. This initial assessment of MN was performed on both mouse and rat blood samples from the 1-month and 3-month exposures. The data from mice demonstrate the well established, sex-based difference in MN-RET and MN-NCE frequencies regularly observed in this species, with females exhibiting slightly lower frequencies. There were no sex-based differences observed in rats. An examination of the mean frequencies across the exposure groups and durations of exposure did not show an appreciable induction of MN at the 1- or 3-month exposures in either species. Further statistical analyses did not reveal any significant exposure-related effects. An examination of the potential genotoxic effects of DE is clearly valuable as part of a large-scale chronic-exposure bioassay. The data and observations from the 1-and 3-month exposure studies will eventually be combined with the results from the 1- and 2-year exposure studies to provide a comprehensive examination of chronic exposure to DE in a rodent model. This examination of chromosome damage serves an important role in the context of the entire ACES bioassay, which was designed to assess the safety of diesel combustion engines.

The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment.

The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.

Assessment of genotoxicity associated with hydroxyurea therapy in children with sickle cell anemia.

Hydroxyurea induces fetal hemoglobin, improves laboratory parameters, and ameliorates clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety in this patient population remain incompletely defined. Although generally considered non-DNA reactive, an important safety concern is that hydroxyurea may indirectly cause genotoxic damage. To better address this safety issue of hydroxyurea in patients with SCA, we measured the production of micronuclei (MN) in red blood cells (RBCs) as a marker of genotoxicity. Blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (ClinicalTrials.gov NCT00305175). Flow cytometry quantified circulating MN-containing erythrocyte sub-populations before and during hydroxyurea exposure. The frequency of micronucleated reticulocytes (MN-CD71(+)) and micronucleated mature erythrocytes (MN-RBC) was then tested for associations with laboratory and clinical data. In cross-sectional analysis of 293 blood samples from 105 children with SCA and a median of 2 years of hydroxyurea therapy, exposure to hydroxyurea was associated with significantly increased frequencies of MN-CD71(+) and MN-RBC compared to baseline. The increases were evident by 3 months of therapy, and did not escalate further with up to 12 years of continuous drug exposure. In prospective longitudinal analysis, substantial inter-individual variation in the effect of hydroxyurea on %MN-CD71(+) was observed that was associated with the expected laboratory effects of hydroxyurea. In conclusion, clinically relevant exposure to hydroxyurea is associated with increased MN production consistent with erythroblast genotoxicity but with substantial inter-patient variability. Associations between increased %MN-CD71(+) and laboratory benefits suggest that hydroxyurea effects on MN production may be related to individual patient sensitivity to hydroxyurea within the bone marrow.

Cross-species genetic toxicity assessment accomplished by flow cytometric analysis of blood.

The formation of micronuclei in blood cells has been an established indicator of genotoxicity for decades. Standard microscopy methods are time-consuming and lack the objectivity that fully automated methods can provide. The ability of flow cytometric technology to rapidly and objectively survey thousands of cells for micronuclei can significantly improve the value of this endpoint. In addition, since many more cells can be scored, and specific populations can be targeted, species that historically have been difficult to obtain micronucleus data from, such as humans, can now be readily evaluated using this methodology. This unit describes a procedure for fixation, staining, and analysis of blood samples using materials supplied in MicroFlow kits (Litron Laboratories) and a single-laser flow cytometer. This methodology provides a reliable, robust assessment of chromosome damage that is used in basic science research as well as drug-safety screening programs at large pharmaceutical and chemical companies.

Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage.

This study evaluated the utility of human blood micronucleated reticulocyte (MNCD71+) frequency measurement as a cytogenetic damage biomarker. The analytical methodology was flow cytometry in conjunction with a previously described three color fluorescence labeling technique that includes anti-CD71 to focus analyses on the most immature fraction of reticulocytes [S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.K. Torous, S. Avlasevich, et al., Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ. Mol. Mutagen. 44 (2004) 427-435]. Blood specimens from 50 self-reported healthy adult volunteers were studied. In addition to MNCD71+ measurements, blood plasma folate and B12 levels were assessed, since these variables tend to influence other indices of cytogenetic damage. Time-course data are also provided for 10 cancer patients undergoing treatment. For these subjects, frequency of MNCD71+ was measured immediately before therapy, and daily during the first week of chemotherapy and/or fractionated radiotherapy. For the group of healthy volunteers, the variables of age, and folate and B12 levels demonstrated no significant effect on MNCD71+ frequency. In addition, no difference was observed between pre-treatment MNCD71+ values for cancer patients compared with healthy volunteers. Regarding chemotherapy and/or partial body radiotherapy, elevated frequencies were observed upon initiation of treatment for 9 of the 10 patients studied. Maximal effects were observed 3-5 days following initiation of therapy. The largest increases in frequency of MNCD71+ (up to 25.9-fold) were observed in those patients exposed to anti-neoplastic drugs, presumably due to the systemic red marrow exposure provided by these agents. Taken together, these data support the hypothesis that the MNCD71+ endpoint represents a valuable biomarker of cytogenetic damage that does not require cell culture or microscopy-based scoring.