Development of a novel flow cytometry‐based approach for reticulocytes micronucleus test in rat peripheral blood

Abstract

The micronucleus test (MNT) is the most widely applied short-term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71-fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague-Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCMbased method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM-based method correlated well with the micronucleus frequencies (r = 0.659-0.952), and the proportion of immature erythrocytes (r = 0.915-0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.

General toxicity and genotoxicity of altertoxin I: A novel 28‐day multiendpoint assessment in male Sprague–Dawley rats

“…The results of genotoxicity tests are shown in Figures 6 and 7 and Table 5. The results of the solvent control groups and the ENU group were all within the background levels reported previously (Chen et al, 2020; Huo et al, 2018; Liu et al, 2019; Zhu et al, 2021). For ENU group, the frequencies of RBC CD59− on Day 28; the frequencies of RET CD59− on Days 14 and 28; the incidence of MN‐RET in blood on Days 4, 14, and 28; the incidence of MN‐PCE in bone marrow on Day 29; and TI% of blood and liver cells on Days 28 and 29 were significantly different from those in PBS group ( P < 0.05).…”

“…474 and the China National Standards for Food Safety GB 15193.5‐2014 (China National Standards for Food Safety, 2014; OECD, 1997). Further details of this assay can be found in the references (Chen et al, 2020; Zeng et al, 2020). Briefly, blood samples (20 μl) were transferred to tubes containing 100‐μl antibody solution (2‐μg CD71‐FITC, 0.5‐μg CD45‐PE).…”

Determination of potential thresholds for N-ethyl-N-nitrosourea and ethyl methanesulfonate based on a multi-endpoint genotoxicity assessment platform in rats

“…474 and the China National Standards for Food Safety (GB 15,193.5–2014). More details of this method can be found in the references that we established (Liu et al 2019 ; Zeng et al 2020 ; Chen et al 2021 ). The induction of MN in peripheral blood RET was determined on days − 1, 4, 14, and 28.…”

“…We used the Pig-a mutation assay and micronucleus test that we previously established (Huo et al 2017 , 2019 ; Liu et al 2019 ; Zeng et al 2020 ; Chen et al 2021 ). On this basis, an in vivo multi-endpoint genotoxicity assessment platform was adopted by integrating the comet test and the above assays into 28-day repeat dose toxicology studies .…”

Genotoxicity of bisphenol AF in rats: Detrimental to male reproductive system and probable stronger micronucleus induction potency than BPA

“…474 (OECD, 1997) and partially modified. More details are available in the references (Chen et al, 2020).…”