Establishment and validation of liver micronucleus assay in rats using 4%neutral formaldehyde-fixed tissues
10.3867/j.issn.1000-3002.2024.06.005
- VernacularTitle:基于4%中性甲醛固定肝组织的大鼠肝微核实验方法的建立与验证
- Author:
Tiantian ZHAO
1
;
Weiwei HE
;
Changhui ZHOU
;
Zehao ZHAO
;
Zixuan YANG
;
Yan CHANG
Author Information
1. 中国医药工业研究总院上海益诺思生物技术股份有限公司,上海 201203
- Keywords:
liver micronucleus test;
formaldehyde fixation method;
collagenase digestion method
- From:
Chinese Journal of Pharmacology and Toxicology
2024;38(6):436-444
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To establish and validate a rat liver micronucleus test(LMNT)method based on fixation of liver tissue with 4%neutral formaldehyde(HCHO fixation)for preparation of hepa-tocytes(HEPs).METHODS ①The LMNT based on neutral HCHO fixation(HCHO fixation-LMNT)was established using the liver micronucleus positive compound N-nitrosodiethylamine(DEN).SD rats were divided into female and male groups,and each group was randomly subdivided into the vehicle control group and DEN 12.5 mg·kg-1 group,with five rats in each.The rats were ig administered with normal saline and DEN once a day for 14 consecutive days,after which liver tissues were collected.Some of the tissue was digested with collagenase to prepare HEP suspension,and the remaining tissue was used to prepare HEP suspension with HCHO fixation.After staining with SYBR Gold,the number of micronucleated hepatocytes(MN-HEP)and the number of HEPs in the mitotic phase were counted under a microscope.The micronucleus rate of HEP(MN-HEP rate)and the mitotic index were calculated,and an MN-HEP rate>0.07%was considered positive.②Male SD rats were divided into the quinoline(30,60,120 mg·kg-1)group,N-nitrosoopyrrolidine(NPYR,25,50,100 mg·kg-1)group,vehicle control group(deionized water for NPYR,and corn oil for quinoline),and positive control DEN(12.5 mg·kg-1)group,with 5-6 rats per group,and were ig administrated for 15 consecutive days.Body mass was recorded daily,and at the end of the experiment,the liver was removed to record the total liver weight,and calculate the liver coefficient.Liver function-related serum biochemical indicators including glutamic-pyruvic transaminase(GPT),glutamic-oxaloacetic transaminase(GOT)activities,and levels of total bili-rubin(T-BIL)were measured and direct bilirubin(D-BIL)using an automatic biochemical analyzer.The MN-HEP rate was determined using the collagenase digestion and HCHO fixation methods,and the peripheral blood MN assay and hepatocellular carcinoma comet assay were conducted to evaluate the genotoxicity of quinoline and NPYR.RESULTS ① Compared with the corresponding vehicle control groups(0.069%and 0.030%),the MN-HEP rate of male rats treated with DEN by formalin-LMNT was 1.10%,and the MN-HEP rate of female ones was 0.82%,both significantly increased(P<0.05).Com-pared with corresponding vehicle control groups(0.060%and 0.030%),the MN-HEP rate of male rats treated with DEN by collagenase digestion-LMNT was 1.45%,and that of female rats was 0.46%,both significantly increased(P<0.05),which were considered positive.The MN-HEP rate of male rats was significantly higher than that of females with both methods(P<0.05).There was no significant differ-ence in mitotic indexes between the DEN groups by collagenase digestion-LMNT and HCHO fixation-LMNT in male and female rats compared to corresponding vehicle control groups.② Compared to the vehicle control group,the body mass of rats in the NPYR 50 and 100 mg·kg-1 groups was significantly reduced 7 to 14 days into the ig administration(P<0.01),and the DEN group showed a significant reduction at days 8 to 14(P<0.01).The body mass of rats in the quinoline 120 mg·kg-1 group was signifi-cantly reduced 4 to 14 days into the ig administration(P<0.01),and the DEN group showed a signifi-cant reduction at days 10 to 14(P<0.05).Compared to the vehicle control group,both the liver weight and liver coefficient were significantly reduced in the NPYR 100 mg·kg-1 group(P<0.01)and the DEN group(P<0.05).The liver weight(P<0.01)and liver coefficient(P<0.05)were significantly increased in the quinoline 60 and 120 mg·kg-1 groups.Compared to the vehicle control group,the serum T-BIL level was significantly increased in the DEN group(P<0.01),and the activities of GPT and GOT,as well as the levels of D-BIL and T-BIL,were significantly increased in the NPYR 100 mg·kg-1 group(P<0.01).There were no significant changes in the NPYR 25,50 mg·kg-1 groups or any of the dose groups of quinoline.The MN-HEP rate by HCHO fixation-LMNT for NPYR was slightly higher than that by collage-nase digestion-LMNT,both considered positive.Compared with corresponding control group,the MN-HEP rate by formalin-LMNT for NPYR and the MN-HEP rate by collagenase digestion for NPYR were both significantly increased(P<0.05).The MN-HEP rate by HCHO fixation-LMNT for quinoline was comparable to that by collagenase digestion-LMNT,both considered positive.Compared with corresponding vehicle control group,the MN-HEP rate by HCHO fixation-LMNT for quinoline and the MN-HEP rate by collagenase digestion-LMNT for quinoline were both significantly increased(P<0.05).The correlation between the MN-HEP rates based on HCHO fixation and collagenase digestion for NPYR and quinoline was good(R2=0.8614 and 0.9279,respectively).In the NPYR groups,the periph-eral blood micronucleus assay were negative,while the comet assay results were positive.In the quino-line group,both the peripheral blood micronucleus assay and the comet assay results were negative.CON-CLUSION The HCHO fixation-LMNT has been established and validated,and the sensitivity of the LMNT method based on HCHO fixation-LMNT for detection of hepatocarcinogens is higher than that of collagenase digestion-LMNT.
