In vitro human cell-based aneugen molecular mechanism assay.

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases (Bernacki et al., Toxicol. Sci. 170 [2019] 382-393). The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4 h treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxol-associated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol- and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Erythrocyte-Based Pig-a Gene Mutation Assay.

In addition to chromosomal damage, assessment of gene mutation is an important part of genotoxicity testing employed during preclinical safety testing. The Pig-a gene mutation assay is based on the loss of function of the Pig-a gene, which results in a lack of cell surface expression of specific proteins that are targeted to the surface by GPI anchors. This cell surface phenotype is readily assessed by flow cytometric analysis of red blood cells. This chapter describes a procedure for the collection, processing, and analysis of peripheral blood samples using materials supplied in MutaFlow® kits and a common benchtop flow cytometer.

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy.

The in vitro MultiFlow® DNA Damage Assay multiplexes γH2AX, p53, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. The current report describes a tiered sequential data analysis strategy based on data generated from exposure of human TK6 cells to a previously described 85 chemical training set and a new pharmaceutical-centric test set (n = 40). In each case, exposure was continuous over a range of closely spaced concentrations, and cell aliquots were removed for analysis following 4 and 24 hr of treatment. The first data analysis step focused on chemicals' genotoxic potential, and for this purpose, we evaluated the performance of a machine learning (ML) ensemble, a rubric that considered fold increases in biomarkers against global evaluation factors (GEFs), and a hybrid strategy that considered ML and GEFs. This first tier further used ML output and/or GEFs to classify genotoxic activity as clastogenic and/or aneugenic. Test set results demonstrated the generalizability of the first tier, with particularly good performance from the ML ensemble: 35/40 (88%) concordance with a priori genotoxicity expectations and 21/24 (88%) agreement with expected mode of action (MoA). A second tier applied unsupervised hierarchical clustering to the biomarker response data, and these analyses were found to group certain chemicals, especially aneugens, according to their molecular targets. Finally, a third tier utilized benchmark dose analyses and MultiFlow biomarker responses to rank genotoxic potency. The relevance of these rankings is supported by the strong agreement found between benchmark dose values derived from MultiFlow biomarkers compared to those generated from parallel in vitro micronucleus analyses. Collectively, the results suggest that a tiered MultiFlow data analysis pipeline is capable of rapidly and effectively identifying genotoxic hazards while providing additional information that is useful for modern risk assessments-MoA, molecular targets, and potency. Environ. Mol. Mutagen. 60:513-533, 2019. © 2019 Wiley Periodicals, Inc.

The incidence of acute angle closure in Scotland: a prospective surveillance study.

PURPOSE: To estimate the incidence, and describe the clinical features and short-term clinical outcomes of acute angle closure (AAC). METHODS: Patients with newly diagnosed AAC were identified prospectively over a 12-month period (November 2011 to October 2012) by active surveillance through the Scottish Ophthalmic Surveillance Unit reporting system. Data were collected at case identification and at 6 months follow-up. RESULTS: There were 114 cases (108 patients) reported, giving an annual incidence of 2.2 cases (95% CI 1.8 to 2.6) or 2 patients (95% CI 1.7 to 2.4) per 1 00 000 in the whole population in Scotland. Precipitating factors were identified in 40% of cases. Almost one in five cases was associated with topical dilating drops. Best-corrected visual acuity (BCVA) at presentation ranged from 6/6 to perception of light. The mean presenting intraocular pressure (IOP) was 52 mm Hg (SD 11). Almost 30% cases had a delayed presentation of 3 or more days. At 6 months follow-up, 75% had BCVA of 6/12 or better and 30% were found to have glaucoma at follow-up. Delayed presentation (≥3 days) was associated with higher rate of glaucoma at follow-up (22.6% vs 60.8%, p<0.001), worse VA (0.34 vs 0.74 LogMAR, p<0.0001) and need for more topical medication (0.52 vs 1.2, p=0.003) to control IOP. CONCLUSION: The incidence of AAC in Scotland is relatively low compared with the Far East countries, but in line with previous European data. Almost one in five cases were associated with pupil dilation for retinal examination.

A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.

The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration.

Erythrocyte-based Pig-a gene mutation assay.

In addition to chromosomal damage, assessment of gene mutation is an important part of genotoxicity testing employed during preclinical safety testing. The Pig-a gene mutation assay is based on the loss of function of the Pig-a gene, which results in a lack of cell surface expression of specific proteins that are targeted to the surface by GPI anchors. This cell surface phenotype is readily assessed by flow cytometric analysis of red blood cells. This unit describes a procedure for the collection, processing, and analysis of peripheral blood samples using materials supplied in MutaFlow® kits and a common benchtop flow cytometer.

Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood.

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.

Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans.

The frequency of micronuclei (also known as Howell-Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen's effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71-positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space.

Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy.

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.

Measurement of cytogenetic damage in rodent blood with a single-laser flow cytometer.

The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity. Double-strand chromosome breaks or dysfunction of the mitotic spindle apparatus can lead to micronuclei formation in dividing cells. Erythrocytes have become the target population of choice, as precursor cells are continuously dividing and micronuclei are readily observable after extrusion of nuclei. The traditional method has been to stain peripheral blood or bone marrow smears and microscopically determine the frequency of micronucleated erythrocytes. Because these events are rare, the process is tedious and time consuming. This unit describes a procedure for fixing and staining rodent peripheral blood for flow cytometric enumeration. The combination of reagents provides for differential labeling and enumeration of four subpopulations: mature erythrocytes, micronucleus-containing mature erythrocytes, young erythrocytes (reticulocytes), and micronucleus-containing young erythrocytes. Malaria-infected rodent erythrocytes, which closely mimic micronucleus-containing erythrocytes, serve as a biological standard to facilitate rational and consistent equipment calibration.

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer.

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.