Leukemia risk assessment of exposure to low-levels of benzene based on the linearized multistage model.

Objectives: To assess leukemia risk in occupational populations exposed to low levels of benzene. Methods: Leukemia incidence data from the Chinese Benzene Cohort Study were fitted using the Linearized multistage (LMS) model. Individual benzene exposure levels, urinary S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t, t-MA) were measured among 98 benzene-exposed workers from factories in China. Subjects were categorized into four groups by rounding the quartiles of cumulative benzene concentrations (< 3, 3-5, 5-12, ≥12 mg/m3·year, respectively). The risk of benzene-induced leukemia was assessed using the LMS model, and the results were validated using the EPA model and the Singapore semi-quantitative risk assessment model. Results: The leukemia risks showed a positive correlation with increasing cumulative concentration in the four exposure groups (excess leukemia risks were 4.34, 4.37, 4.44 and 5.52 × 10-4, respectively; Ptrend < 0.0001) indicated by the LMS model. We also found that the estimated leukemia risk using urinary t, t-MA in the LMS model was more similar to those estimated by airborne benzene compared to S-PMA. The leukemia risk estimated by the LMS model was consistent with both the Singapore semi-quantitative risk assessment model at all concentrations and the EPA model at high concentrations (5-12, ≥12 mg/m3·year), while exceeding the EPA model at low concentrations (< 3 and 3-5 mg/m3·year). However, in all four benzene-exposed groups, the leukemia risks estimated by these three models exceeded the lowest acceptable limit for carcinogenic risk set by the EPA at 1 × 10-6. Conclusion: This study demonstrates the utility of the LMS model derived from the Chinese benzene cohort in assessing leukemia risk associated with low-level benzene exposure, and suggests that leukemia risk may occur at cumulative concentrations below 3 mg/m3·year.

Benzene metabolite hydroquinone enhances self-renewal and proliferation of preleukemic cells through the Ppar-γ pathway.

Benzene is a known hematotoxic and leukemogenic chemical. Exposure to benzene cause inhibition of hematopoietic cells. However, the mechanism of how the hematopoietic cells inhibited by benzene undergo malignant proliferation is unknown. The cells carrying leukemia-associated fusion genes are present in healthy individuals and predispose the carriers to the development of leukemia. To identify the effects of benzene on hematopoietic cells, preleukemic bone marrow (PBM) cells derived from transgenic mice carrying the Mll-Af9 fusion gene were treated with benzene metabolite hydroquinone in serial replating of colony-forming unit (CFU) assay. RNA sequencing was further employed to identify the potential key genes that contributed to benzene-initiated self-renewal and proliferation. We found that hydroquinone induced a significant increase in colony formation in PBM cells. Peroxisome proliferator-activated receptor gamma (Ppar-γ) pathway, which plays a critical role in carcinogenesis in multiple tumors, was significantly activated after hydroquinone treatment. Notably, the increased numbers of the CFUs and total PBM cells induced by hydroquinone were significantly reduced by a specific Ppar-γ inhibitor (GW9662). These findings indicated that hydroquinone can enhance self-renewal and proliferation of preleukemic cells by activating the Ppar-γ pathway. Our results provide insight into the missing link between premalignant status and development of benzene-induced leukemia, which can be intervened and prevented.

Pericarp starch metabolism is associated with caryopsis development and endosperm starch accumulation in common wheat.

The wheat pericarp is the main component of the caryopsis at the early development stage and ultimately converts into a tissue that covers the mature caryopsis. A large number of starch granules are accumulated in the pericarp, but the production of and the role of starch granules in caryopsis development remain- elusive. In the present study, the relationship between accumulated starch granules and starch metabolism-related genes in wheat pericarp was investigated using paraffin section observations, expression analysis, and mutant analysis. Starch synthesis is initiated before anthesis and is dependent on a sucrose uptake and conversion system similar to that in the endosperm. TaPTST2 is required to initiate the production of pericarp starch granules. Pericarp starch granules gradually disappeared at the filling stage with high expression levels of genes encoding ß-amylase, sucrose-phosphate synthase, and sucrose-phosphate phosphatase. As a maternal tissue adjacent to the endosperm and embryo, the pericarp plays a temporary reservoir for excess nutrients delivered into the caryopsis during the early development stage and exported at the filling stage. The pericarp contributes to the development of the endosperm and embryo as well as the accumulation of endosperm starch. The metabolism of pericarp starch may affect the weight of the wheat caryopsis.

Hydroquinone triggers pyroptosis and endoplasmic reticulum stress via AhR-regulated oxidative stress in human lymphocytes.

Benzene is a frequent component of environmental pollution and is abundant in petrochemicals, decorative materials, motor vehicle exhaust and cigarette smoke. Benzene is a well-known carcinogen in humans and animals, but the molecular mechanism has not yet been elucidated. Our earlier research indicated that hydroquinone (HQ), one of the main reactive metabolites of benzene, could activate aryl hydrocarbon receptor (AhR), which is essential for HQ-induced toxicity, including apoptosis and DNA damage. Since AhR is an important regulator of the immune system that integrates the environmental stimulus and immune response, we examined whether and how HQ-induced AhR activity could lead to NLRP3 inflammasome-dependent pyroptosis in JHP cells. Our results showed that HQ could cause inflammation process and resultant pyroptosis. In JHP cells, HQ also induced endoplasmic reticulum stress (ERS) by releasing excessive reactive oxygen species (ROS). The activation of pyroptosis induced by HQ treatment was reversed by an antioxidant (NAC) and an ERS inhibitor (4-PBA). Interestingly, the treatment of CH223191, an AhR inhibitor, reversed HQ-induced oxidative stress, ERS and pyroptosis. These data suggested that AhR-mediated HQ-induced ERS, ROS and inflammasome activation may play vital roles in the toxic effects of benzene. This work provides insights and prospective strategies into potential mechanisms for reducing benzene-induced hematotoxicity.

Using Urinary Biomarkers to Estimate the Benzene Exposure Levels in Individuals Exposed to Benzene.

Urinary benzene metabolites trans, trans-muconic acid (t, t-MA), and S-phenyl mercapturic acid (S-PMA) are often used as biomarkers of internal exposure to benzene. However, there are few reports on using urinary benzene metabolites to estimate airborne benzene concentrations in individuals exposed to benzene. In this study, t, t-MA, and S-PMA were analyzed by UPLC-MS/MS, and a simple pharmacokinetic model was used to calculate the daily intake (DI) of benzene based on the levels of urinary t, t-MA, and S-PMA in occupational individuals. The back-calculated airborne benzene levels (BCABL) were obtained from the DI of benzene. Among the exposed subjects (n = 84), the median BCABL (3.67 mg/m3) based on t, t-MA was very close to the median level of measured airborne benzene (3.27 mg/m3, p = 0.171), and there was no effect of smoking or dietary habits on t, t-MA-based BCABL. In the control subjects (n = 49), the levels of measured airborne benzene were all below the quantitation limit (0.024 mg/m3), and the BCABL (0.002-0.25 mg/m3) calculated by S-PMA was close to this background level. Our study suggests that the t, t-MA-based BCABL can reflect the actual airborne benzene level in a range of 1.10-86.91 mg/m3 and that the S-PMA-based BCABL is more reliable for non-professional benzene exposure.

New insight into the sucrose biosynthesis pathway from genome-wide identification, gene expression analysis, and subcellular localization in hexaploid wheat (Triticum aestivum L.).

Sucrose, the main synthesized product and transported form of photoassimilates, moves from leaves to support plant growth and storage substance biosynthesis occurring in the heterotrophic sink organs. Enhancing sucrose biosynthesis efficiency is a top priority for crop yield breeding programs. However, the molecular mechanism of sucrose biosynthesis is still elusive, especially in wheat. We performed transcriptome sequencing, subcellular localization, and bioinformatics analysis to identify key isoforms and metabolic branches associated with sucrose biosynthesis in wheat. Our analysis identified 45 genes from 13 families that exhibited high expression in young leaves with an evident diurnal change. The carbon flux from photoassimilates to sucrose was divided into two pathways. In the cytoplasm, assimilates initiating at phosphotrioses (TPs) exported by TaTPT1 from chloroplasts flowed along the TP-Sucrose branch formed by TaALD6, TaFBP5, TacPGI, TacPGM, TaUGP1, TaSPS5, and TaSPP1. Intermediates either from the Calvin cycle or TP-Sucrose branch were converted into ADPGlc to synthesize the simple starch, which was transiently degraded by a series of enzymes, including TaBAM4 and TaSEX4 in the chloroplast. Similar to the starch-biosynthesis branch in endosperms, the TP-Sucrose branch is the most prominent in leaves because each reaction can be catalyzed by at least one highly expressed isoform with expected cytosolic localization. The key isoforms and major branches highlighted in the wheat sucrose biosynthesis pathway expand our molecular understanding of crop sucrose biosynthesis and provide clues to increase wheat yield by enhancing the sucrose synthesis efficiency of leaves.

Industry Distribution Characteristics of Benzene-Induced Leukemia - 7 PLADs, China, 2005-2019.

What is already known about this topic?: In the 1980s. benzene-induced leukemia (BIL) mainly occurred in shoemaking and painting industries. Now the industry distribution of benzene-induced leukemia may have changed over time. What is added by this report?: BlL cases mainly occurred in the manufacturing industry from 2005-2019, especially in private enterprises and small/medium-sized enterprises. The industry with the largest number of new cases of BIL was the general and special equipment manufacturing. The number of leukemia cases in emerging industries such as computer/electronic product manufacturing was found to be increasing. What are the implications for public health practice?: Strengthening supervision and regulation of manufacturing, especially of small/medium-sized enterprises and emerging manufacturing industry, may be effective in reducing BIL.

Pressure-Induced Amorphization and Crystallization of Heterophase Pd Nanostructures.

Control of structural ordering in noble metals is very important for the exploration of their properties and applications, and thus it is highly desired to have an in-depth understanding of their structural transitions. Herein, through high-pressure treatment, the mutual transformations between crystalline and amorphous phases are achieved in Pd nanosheets (NSs) and nanoparticles (NPs). The amorphous domains in the amorphous/crystalline Pd NSs exhibit pressure-induced crystallization (PIC) phenomenon, which is considered as the preferred structural response of amorphous Pd under high pressure. On the contrary, in the spherical crystalline@amorphous core-shell Pd NPs, pressure-induced amorphization (PIA) is observed in the crystalline core, in which the amorphous-crystalline phase boundary acts as the initiation site for the collapse of crystalline structure. The distinct PIC and PIA phenomena in two different heterophase Pd nanostructures might originate from the different characteristics of Pd NSs and NPs, including morphology, amorphous-crystalline interface, and lattice parameter. This work not only provides insights into the phase transition mechanisms of amorphous/crystalline heterophase noble metal nanostructures, but also offers an alternative route for engineering noble metals with different phases.

Ligand-independent activation of AhR by hydroquinone mediates benzene-induced hematopoietic toxicity.

Although it has been well recognized that benzene exposure can cause hematopoietic disorders such as aplastic anemia and leukemia, the underlying molecular mechanism remains to be fully understood. Emerging evidence indicated that aryl hydrocarbon receptor (AhR) plays important roles in hematopoietic and immune systems. This study investigated the activation of aryl hydrocarbon receptor (AhR) by hydroquinone (HQ) and its role in HQ-induced DNA damage and apoptosis in cultured human lymphocytes (JHP cells). We also investigated the effect of ROS on AhR activation and functions in JHP cells exposed to HQ with and without regulator including N-acetyl-l-cysteine (NAC), a potent antioxidant, and tert-butylhydroquinone (TBHQ), a Nrf2 activator. Results showed that HQ can cause oxidative stress, DNA damage and apoptosis. Pretreatment of an AhR antagonist (CH223191) can significantly increase the cell survival and mitigate HQ-induced toxicities such as DNA damage and apoptosis. We found that HQ can obviously increase expressions of total protein of AhR and prompt nuclear translocation compared to the control group. Interestingly, NAC can block HQ-induced AhR activation and DNA damage and apoptosis. Conclusively, our results indicated that HQ toxicity is mediated by AhR which is in turn regulated by ROS generated by HQ. The interaction between AhR and ROS drive and amplify the hematopoietic toxicity of HQ. This study provided new insights of mechanism and potential targets for the prevention and treatment to benzene-induced hematopoietic toxicity.

Industry Distribution Characteristics of Benzene-Exposed Workers with Cytopenia - Four Provinces, China, 2020.

What is already known about this topic?: Benzene is harmful to the hematopoietic system and can cause leukemia. However, benzene is still being used in various industries including furniture, rubber, plastic products, and metal product manufacturing. What is added by this report?: The white blood cell count of workers in general equipment, special equipment, chemical raw materials, and chemical products manufacturing decreased significantly. The enterprises in which benzene concentration exceeded the occupational exposure limit were small enterprises and private enterprises. What are the implications for public health practice?: Regular health examinations are necessary for benzene-exposed workers. In addition, the monitoring of benzene concentration in small enterprises and private enterprises should be strengthened.

The Distribution and Concentration Monitoring of Benzene Industries - Six PLADs, China, 2020.

WHAT IS ALREADY KNOWN ABOUT THIS TOPIC?: Benzene is classified as a Class I human carcinogen by the International Agency for Research on Cancer. Long-term exposure to benzene increases the risk of chronic benzene poisoning and leukemia. However, benzene is still widely used in the manufacturing industry. WHAT IS ADDED BY THIS REPORT?: The scale of enterprises most exposed to benzene was small enterprises, and joint-equity enterprises had the highest number that exceeded the permissible concentration-time weighted average. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: It is still necessary to strengthen the monitoring of benzene concentrations in the manufacturing industry, especially in small enterprises. The occupational exposure limit of benzene should be appropriately reduced.

Changes in miR-222 expression, DNA repair capacity, and MDM2-p53 axis in association with low-dose benzene genotoxicity and hematotoxicity.

Mechanisms for hematotoxicity and health effects from exposure to low doses of benzene (BZ) remain to be identified. To address the information gap, our investigation was focused onto using appropriate populations and cell cultures to investigate novel BZ-induced effects such as disruption of DNA repair capacity (DRC). From our study, abnormal miRNAs were identified and validated using lymphocytes from 56 BZ-poisoned workers and 53 controls. In addition, 173 current BZ-exposed workers and 58 controls were investigated for key miRNA expression using RT-PCR and for cellular DRC using a challenge assay. Subsequently, the observed activities in lymphocytes were verified using human HL-60 (p53 null) and TK6 (p53 wild-type) cells via 1,4-benzoquinone (1,4-BQ) treatment and miR-222 interferences. The targeting of MDM2 by miR-222 was validated using a luciferase reporter. Our results indicate induction of genotoxicity in lymphocytes from workers with low exposure doses to BZ. In addition, miR-222 expression was up-regulated among both BZ-poisoned and BZ-exposed workers together with inverse association with DRC. Our in vitro validation studies using both cell lines indicate that 1,4-BQ exposure increased expression of miR-222 and Comet tail length but decreased DRC. Loss of miR-222 reduced DNA damage, but induced S-phase arrest and apoptosis. However, silencing of MDM2 failed to activate p53 in TK6 cells. In conclusion, our in vivo observations were confirmed by in vitro studies showing that BZ/1,4-BQ exposures caused genotoxicity and high expression of miR-222 which obstructed expression of the MDM2-p53 axis that led to failed activation of p53, abnormal DRC and serious biological consequences.

Performance of a novel in vitro assay for skin sensitization based on activation of T lymphocytes.

There are currently no skin sensitization assays based on T cell activation. We built a novel in vitro model to assess T cell activation and test its performance to discriminate skin sensitizers from non-sensitizers using 52 reference chemicals. Jurkat Clone E6-1 human T lymphocytes were exposed to a series of concentrations of test substances for 24 hours and CD69 expression was measured as a marker of early T cell activation with flow cytometry. Most tested sensitizers induced increased relative fluorescence intensity (RFI) of CD69 on the T cells, which was linearly correlated with the concentrations tested, indicating a statistically significant causal link between sensitizer concentration and increase of CD69 expression. CD69 RFI ≥ 1.5 was determined as the positive criterion for skin sensitizer classification. The sensitivity (79.4%), specificity (88.9%) and accuracy (82.7%) of the model for the 52 tested reference chemicals showed a good predictivity for skin sensitizers. The results were reproduced in at least two repeats; and the concurrent positive control, 2 mg/mL 2, 4-dinitrochlorobenzene, was found positive in all 25 independent runs conducted, indicating in-house reproducibility. The EC1.5 value (i.e., the concentration at which a test chemical induces a CD69 RFI of 1.5) may be used to assess skin sensitization potency of a chemical. This work may contribute to the development of an in vitro assay for skin sensitization based on the activation of T cells.

Characteristics in the Distribution of Chronic Benzene Poisoning Associated Industries - 6 PLADs, China, 2005-2019.

What is already known on this topic? Starting in the early 1950s, the main industries in China associated with chronic benzene poisoning (CBP) included painting, pharmaceuticals, and shoemaking. However, because of rapid socioeconomic development, the distribution of industries associated with CBP likely changed. What is added by this report? From 2005 to 2019, CBP has become an increasingly important type of chronic occupational poisoning (COP) in China. CBP was mainly found to have occurred in manufacturing industries, especially private enterprises and small and medium-sized enterprises. The sub-industry with the highest proportion of CBP cases was general and special equipment manufacturing, followed by chemical raw materials and chemical manufacturing. What are the implications for public health practice? CBP was found to be the main component of COP in China, so the supervision and management in manufacturing, especially in the medium-sized and small enterprises, need to be strengthened. Occupational benzene exposure limits should also be adjusted accordingly.

Benzene metabolite hydroquinone promotes DNA homologous recombination repair via the NF-κB pathway.

Benzene, a widespread environmental pollutant, induces DNA double-strand breaks (DSBs) and DNA repair, which may further lead to oncogenic mutations, chromosomal rearrangements and leukemogenesis. However, the molecular mechanisms underlying benzene-induced DNA repair and carcinogenesis remain unclear. The human osteosarcoma cell line (U2OS/DR-GFP), which carries a GFP-based homologous recombination (HR) repair reporter, was treated with hydroquinone, one of the major benzene metabolites, to identify the potential effects of benzene on DSB HR repair. RNA-sequencing was further employed to identify the potential key pathway that contributed to benzene-initiated HR repair. We found that treatment with hydroquinone induced a significant increase in HR. NF-κB pathway, which plays a critical role in carcinogenesis in multiple tumors, was significantly activated in cells recovered from hydroquinone treatment. Furthermore, the upregulation of NF-κB by hydroquinone was also found in human hematopoietic stem and progenitor cells. Notably, the inhibition of NF-κB activity by small molecule inhibitors (QNZ and JSH-23) significantly reduced the frequency of hydroquinone-initiated HR (-1.36- and -1.77-fold, respectively, P < 0.01). Our results demonstrate an important role of NF-κB activity in promoting HR repair induced by hydroquinone. This finding sheds light on the underlying mechanisms involved in benzene-induced genomic instability and leukemogenesis and may contribute to the larger exploration of the influence of other environmental pollutants on carcinogenesis.

Response to Comments on Zheng et al. "Association between Promoter Methylation of Gene ERCC3 and Benzene Hematotoxicity" Int. J. Environ. Res. Public Health 2017, 14, 1393.

We would like to thank Moshammer and Poteser for their comments [...].

Association between Promoter Methylation of Gene ERCC3 and Benzene Hematotoxicity.

Benzene is a primary industrial chemical and a ubiquitous environmental pollutant. ERCC3 is a key player in nucleotide excision repair. Recent studies suggested that site-specific methylation is a possible mechanism of the transcriptional dysregulation by blocking transcription factors binding. We previously found that the average promoter methylation level of ERCC3 was increased in benzene-exposed workers. In order to test whether specific CpG sites of ERCC3 play an important role in benzene-induced epigenetic changes and whether the specific methylation patterns are associated with benzene hematotoxicity, we analyzed the promoter methylation levels of individual CpG sites, transcription factor binding motif and the correlation between aberrant CpG methylation and hematotoxicity in 76 benzene-exposed workers and 24 unexposed controls in China. Out of all the CpGs analyzed, two CpG units located 43 bp upstream and 99 bp downstream of the transcription start site of ERCC3 (CpG 2-4 and CpG 17-18, respectively), showed the most pronounced increase in methylation levels in benzene-exposed workers, compared with unexposed controls (Mean ± SD: 5.86 ± 2.77% vs. 4.92 ± 1.53%, p = 0.032; 8.45 ± 4.09% vs. 6.79 ± 2.50%, p = 0.024, respectively). Using the JASPAR CORE Database, we found that CpG 2-4 and CpG 17-18 were bound by three putative transcription factors (TFAP2A, E2F4 and MZF1). Furthermore, the methylation levels for CpG 2-4 were correlated negatively with the percentage of neutrophils (ß = -0.676, p = 0.005) in benzene-exposed workers. This study demonstrates that CpG-specific DNA methylation in the ERCC3 promoter region may be involved in benzene-induced epigenetic modification and it may contribute to benzene-induced hematotoxicity.

Benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15.

LncRNA has been considered to play a crucial role in the progression of several diseases by affecting cell proliferation. However, its role in benzene toxicity remains unclear. Our study showed that the expression of lncRNA-OBFC2A increased accompanied with the change of cell proliferation related-genes in benzene-exposed workers. In vitro experiments, 1,4-Benzoquinone dose-dependently inhibited cell proliferation and simultaneously caused the decrease of NOTCH1 expression and the increase of KLF15 in AHH-1 cell lines. Meanwhile, 1, 4-Benzoquinone obviously increased the expression of lncRNA-OBFC2A, which was consistent with our previous population results. Therefore, we propose that lncRNA-OBFC2A is involved in benzene toxicity by regulating cell proliferation. Further, we successfully constructed a lentivirus model of interfering the expression of lncRNA-OBFC2A. After interfering lncRNA-OBFC2A, the cell proliferation inhibition and the expression of NOTCH1 and KLF15 induced by 1, 4-Benzoquinone were reversed. Subsequently, RNA fluorescence in situ Hybridization assay showed that lncRNA-OBFC2A was located in cell nuclei. These results suggest that benzene and its metabolite decreases cell proliferation via LncRNA-OBFC2A-mediated anti-proliferation effect involving NOTCH1 and KLF15. LncRNA-OBFC2A can be a potential biomarker for benzene toxicity.

Intra-laboratory study to determine the reproducibility of LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.

INTRODUCTION: The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for identification the skin sensitization potential of new chemicals. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. An intra-laboratory reproducibility study for the LLNA:BrdU-ELISA was conducted to demonstrate its adequate performance in our laboratory. METHODS: Ten independent LLNA:BrdU-ELISAs with the preferred positive controls (PCs), i.e., 25% hexyl cinnamic aldehyde (HCA) and 25% eugenol, were conducted within a period of less than one year. In addition, different concentrations of 2,4-dinitrochlorobenzene (DNCB, an extreme sensitizer) (0.01, 0.1 and 0.3%), HCA (10, 25 and 50%) and eugenol (10, 25 and 50%), were tested to determine the EC1.6 values. Special Pathogen Free female CBA/J mice of 8-10weeks old were randomly allocated to the groups, each group having 4 mice. 25µl of AOO (vehicle, acetone: olive oil=4:1, v/v) or HCA, eugenol, DNCB at the needed concentration was applied to the dorsum of each ear of the mice, daily for 3 consecutive days. A single intraperitoneal injection of 0.5ml of BrdU solution (10mg/ml) was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, and BrdU ELISA analysis was conducted. RESULTS: The result for each group is expressed as the mean Stimulation Index (SI). The mean of the 10 mean SIs for 25% HCA (2.58±0.95) and 25% eugenol (3.51±1.25) was not significantly different to that from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (i.e., the data on the formal validation study for the LLNA:BrdU-ELISA by the ICCVAM) (3.03±2.00 for 25% HCA, 6.13±6.06 for 25% eugenol) (P>0.05), with even smaller Coefficient of Variations (CV) (36.8% for 25% HCA, 35.6% for 25% eugenol) than that from the ICCVAM (66.0% for 25% HCA, 98.8% for 25% eugenol). In addition, the EC1.6 values for HCA, eugenol and DNCB (15.2, 12.5 and 0.25%, respectively) were consistent with that from the ICCVAM (12.92, 8.85 and 0.34%, respectively). DISCUSSION: The results indicate that the reliability for our laboratory to conduct the LLNA:BrdU-ELISA is successfully determined.