Empirical analysis of lead neurotoxicity mode of action and its application in health risk assessment.

The mode of action (MOA) framework is proposed to inform a biological link between chemical exposures and adverse health effects. Despite a significant increase in knowledge and awareness, the application of MOA in human health risk assessment (RA) remains limited. This study aims to discuss the adoption of MOA for health RA within a regulatory context, taking our previously proposed but not yet validated MOA for lead neurotoxicity as an example. We first conducted a quantitative weight of evidence (qWOE) assessment, which revealed that the MOA has a moderate confidence. Then, targeted bioassays were performed within an in vitro blood-brain barrier (BBB) model to quantitatively validate the scientific validity of key events (KEs) in terms of essentiality and concordance of empirical support (dose/temporal concordance), which increases confidence in utilizing the MOA for RA. Building upon the quantitative validation data, we further conducted benchmark dose (BMD) analysis to map dose-response relationships for the critical toxicity pathways, and the lower limit of BMD at a 5% response (BMDL5) was identified as the point of departure (POD) value for adverse health effects. Notably, perturbation of the Aryl Hydrocarbon Receptor (AHR) signaling pathway exhibited the lowest POD value, measured at 0.0062 µM. Considering bioavailability, we further calculated a provisional health-based guidance value (HBGV) for children's lead intake, determining it to be 2.56 µg/day. Finally, the health risk associated with the HBGV was assessed using the hazard quotient (HQ) approach, which indicated that the HBGV established in this study is a relative safe reference value for lead intake. In summary, our study described the procedure for utilizing MOA in health RA and set an example for MOA-based human health risk regulation.

Protein phosphatase 2A regulates cytotoxicity and drug resistance by dephosphorylating AHR and MDR1.

Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and metabolism. However, its specific actions in many of these critical pathways are unclear. To explore mechanisms underlying metabolic enzyme regulation in the liver, we investigated the key pathways involved in regulation of xenobiotic-metabolizing enzymes in a mouse model with hepatocyte-specific deletion of Ppp2r1a, encoding the Aα subunit of PP2A. We performed transcriptome and phosphoproteome analysis in mouse livers at the age of 3 months and identified 2695 differentially expressed genes and 549 upregulated phosphoproteins in homozygous knockout mouse livers compared with WT littermates. In particular, the expression of metabolic enzymes Cyp2e1, Cyp1a1, Cyp1a2, Mdr1a, and Abcg2 was dramatically altered in homozygous knockout mouse livers. We also demonstrated that activation of PP2A reversed the decline of metabolic enzyme expression in primary mouse hepatocytes. We found that specific PP2A holoenzymes were involved in metabolic enzyme induction through dephosphorylation of transcription factors, nuclear receptors, or the target enzymes themselves, leading to dysregulation of xenobiotic metabolism or drug-induced hepatotoxicity. Notably, we confirmed that a regulatory axis, PP2A B56α-aryl hydrocarbon receptor-Cyp1a1, was involved in benzo(a)pyrene-induced cytotoxicity through dephosphorylation of the metabolic nuclear receptor, aryl hydrocarbon receptor, at serine 36. In addition, we showed that PP2A B56δ complexes directly dephosphorylated the multidrug efflux pump MDR1 (encoded by multi-drug resistance gene 1), contributing to drug resistance against the chemotherapeutic 5-fluorouracil. Taken together, these novel findings demonstrate the involvement of PP2A in the regulation of liver metabolism.

Morphological alterations in C57BL/6 mouse intestinal organoids as a tool for predicting chemical-induced toxicity.

Intestinal organoid may serve as an alternative model for toxicity testing. However, the linkage between specific morphological alterations in organoids and chemical-induced toxicity has yet to be defined. Here, we generated C57BL/6 mouse intestinal organoids and conducted a morphology-based analysis on chemical-induced toxicity. Alterations in morphology were characterized by large spheroids, hyperplastic organoids, small spheroids, and protrusion-loss organoids, which responded in a concentration-dependent manner to the treatment of four metal(loid)s including cadmium (Cd), lead (Pb), hexavalent chromium (Cr-VI), and inorganic trivalent arsenic (iAs-III). Notably, alterations in organoid morphology characterized by abnormal morphology rate were correlated with specific intestinal toxic effects, including reduction in cell viability and differentiation, induction of apoptosis, dysfunction of mucus production, and damage to epithelial barrier upon repeated administration. The benchmark dose (BMDL10) values of morphological alterations (0.007-0.195 µM) were lower than those of conventional bioassays (0.010-0.907 µM). We also established that the morphologic features of organoids upon Cd, Pb, Cr-VI, or iAs-III treatment were metal specific, and mediated by Wnt, bone morphogenetic protein, apoptosis induction, and Notch signaling pathways, respectively. Collectively, these findings provide novel insights into the relevance of morphological alterations in organoids to specific toxic endpoints and identify specific morphological alterations as potential indicators of enterotoxicity.

CRISPR-based DNA methylation editing of NNT rescues the cisplatin resistance of lung cancer cells by reducing autophagy.

Cisplatin is recommended as a first-line chemotherapeutic agent against advanced non-small cell lung cancer (NSCLC), but acquired resistance substantially limits its clinical efficacy. Recently, DNA methylation has been identified as an essential contributor to chemoresistance. However, the precise DNA methylation regulatory mechanism of cisplatin resistance remains unclear. Here, we found that nicotinamide nucleotide transhydrogenase (NNT) was silenced by DNA hypermethylation in cisplatin resistance A549 (A549/DDP) cells. Also, the DNA hypermethylation of NNT was positively correlated to poor prognosis in NSCLC patients. Overexpression of NNT in A549/DDP cells could reduce their cisplatin resistance, and also suppressed their tumor malignancy such as cell proliferation and clone formation. However, NNT enhanced sensitivity of A549/DDP cells to cisplatin had little to do with its function in mediating NADPH and ROS level, but was mainly because NNT could inhibit protective autophagy in A549/DDP cells. Further investigation revealed that NNT could decrease NAD+ level, thereby inactivate SIRT1 and block the autophagy pathway, while re-activation of SIRT1 through NAD+ precursor supplementation could antagonize this effect. In addition, targeted demethylation of NNT CpG island via CRISPR/dCas9-Tet1 system significantly reduced its DNA methylation level and inhibited the autophagy and cisplatin resistance in A549/DDP cells. Thus, our study found a novel chemoresistance target gene NNT, which played important roles in cisplatin resistance of lung cancer cells. Our findings also suggested that CRISPR-based DNA methylation editing of NNT could be a potential therapeutics method in cisplatin resistance of lung cancer.

Effects of Florfenicol on Intestinal Histology, Apoptosis and Gut Microbiota of Chinese Mitten Crab (Eriocheir sinensis).

Excessive use of antibiotics in aquaculture causes residues in aquatic animal products and harms human health. However, knowledge of florfenicol (FF) toxicology on gut health and microbiota and their resulting relationships in economic freshwater crustaceans is scarce. Here, we first investigated the influence of FF on the intestinal health of Chinese mitten crabs, and then explored the role of bacterial community in FF-induced intestinal antioxidation system and intestinal homeostasis dysbiosis. A total of 120 male crabs (48.5 ± 4.5 g) were experimentally treated in four different concentrations of FF (0, 0.5, 5 and 50 µg/L) for 14 days. Responses of antioxidant defenses and changes of gut microbiota were assessed in the intestine. Results revealed that FF exposure induced significant histological morphology variation. FF exposure also enhanced immune and apoptosis characteristics in the intestine after 7 days. Moreover, antioxidant enzyme catalase activities showed a similar pattern. The intestinal microbiota community was analyzed based on full-length 16S rRNA sequencing. Only the high concentration group showed a marked decrease in microbial diversity and change in its composition after 14 days of exposure. Relative abundance of beneficial genera increased on day 14. These findings illustrate that exposure to FF could cause intestinal dysfunction and gut microbiota dysbiosis in Chinese mitten crabs, which provides new insights into the relationship between gut health and gut microbiota in invertebrates following exposure to persistent antibiotics pollutants.

Perturbation of Specific Signaling Pathways Is Involved in Initiation of Mouse Liver Fibrosis.

BACKGROUND AND AIMS: To identify the regulatory role of protein phosphatase 2A (PP2A) in the development of liver disease, we generated a mouse model with hepatocyte-specific deletion of Ppp2r1a gene (encoding PP2A Aα subunit). APPROACH AND RESULTS: Homozygote (HO) mice and matched wild-type littermates were investigated at 3, 6, 9, 12, 15, and 18 months of age. Pathological examination showed that PP2A Aα deficiency in hepatocytes resulted in progressive liver fibrosis phenotype from 9 months of age. No hepatocyte death was observed in HO mice. However, perturbation of pathways including epidermal growth factor receptor 1 (EGFR1), amino acid metabolism, and translation factors as well as leptin and adiponectin led to pronounced hepatic fibrosis. In vitro studies demonstrated the involvement of specific B subunit complexes in the regulation of EGFR1 signaling pathway and cross talk between defected hepatocytes and stimulation of interstitial hyperplasia. It is noteworthy that HO mice failed to develop hepatocellular carcinoma for as long as 22 months of age. We further demonstrate that PP2A Aß-containing holoenzymes played a critical role in preventing hepatocyte apoptosis and antagonizing tumorigenesis through specific pathways on Aα loss. Furthermore, PP2A Aα and Aß were functionally distinct, and the Aß isoform failed to substitute for Aα in the development of inflammation and liver fibrosis. CONCLUSIONS: These observations identify pathways that contribute to the pathogenesis of liver fibrosis and provide putative therapeutic targets for its treatment.

Assessing the neurotoxicity of airborne nano-scale particulate matter in human iPSC-derived neurons using a transcriptomics benchmark dose model.

Airborne nano-scale particulate matter (nPM) exposure is a risk factor for neurological diseases. However, to date, there has been no comprehensive evaluation of ambient nPM's neurotoxicity. We examined the toxic effects of nPM on human neurons derived from induced pluripotent stem cells (iPSCs) at doses ranging from 0 to 200 µg/mL, and employed whole-genome RNA-sequencing in different dose groups to gain further insight into the neurotoxicity of ambient nPM. Our findings showed that nPM was absorbed by neurons, and induced a variety of toxic effects. The apical benchmark dose lower confidence bound (aBMDL) values of each effect endpoint were ranked as follows, in ascending order: mitochondrial membrane potential, neurite length, early apoptosis, cell viability. BMD analysis based on transcriptomic data revealed that the point of departure (PoD) of the 20 pathways with the lowest p-values (0.75 µg/mL), the top 20 upstream regulators (0.79 µg/mL) and the neurological diseases (0.77 µg/mL) could be appropriate for nPM neurotoxicity evaluation. The transcriptomic PoDs (tPoDs) were similar to apical PoDs (aPoDs) since their absolute fold differences were within 10-fold. Further analysis of the transcriptomic data revealed that nPM exposure could disturb the pathways related to ferroptosis, neurotransmitters, xenobiotic metabolism, etc., which might be critical in regulating nPM neurotoxicity. We also found that low-dose nPM induced cytokine signaling pathways, while high doses of nPM activated cell-cycle regulation and DNA repair pathways. Our results indicate that BMD modeling based on transcriptomic data could be useful in illustrating the neurotoxic mechanism, and also could be a promising method for evaluating the potential health risks of nPM.

Association between exposure to a mixture of benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) and small airways function: A cross-sectional study.

BACKGROUND: Lung is one of the primary target organs of benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS). Small airways dysfunction (SAD) might be a sensitive indicator of early chronic respiratory disease. Here, we explored the relationships between exposure to BTEXS and small airways function, and identified the priority control pollutants in BTEXS mixtures. METHODS: 635 petrochemical workers were recruited. Standard spirometry testing was conducted by physicians. The cumulative exposure dose (CED) of BTEXS for each worker was estimated. The peak expiratory flow (PEF), forced expiratory flow between 25 and 75% of forced vital capacity (FEF25∼75%), and the expiratory flow rate found at 25%, 50%, and 75% of the remaining exhaled vital capacity (MEF25%, MEF50%, and MEF75%) were measured. SAD was also evaluated based on measured parameters. The associations between exposure to BTEXS individuals or mixtures and small airways function were evaluated using generalized linear regression models (GLMs) and quantile g-computation models (qgcomp). Meanwhile, the weights of each homolog in the association were estimated. RESULTS: The median CED of BTEXS are 9.624, 19.306, 24.479, 28.210, and 46.781 mg/m3·years, respectively. A unit increase in ln-transformed styrene CED was associated with a decrease in FEF25∼75% and MEF50% based on GLMs. One quartile increased in BTEXS mixtures (ln-transformed) was significantly associated with a 0.325-standard deviation (SD) [95% confidence interval (CI): -0.464, -0.185] decline in FEF25∼75%, a 0.529-SD (95%CI: -0.691, -0.366) decline in MEF25%, a 0.176-SD (95%CI: -0.335, -0.017) decline in MEF75%, and increase in the risk of abnormal of SAD [risk ratios (95%CI): 1.520 (95%CI: 1.143, 2.020)]. Benzene and styrene were the major chemicals in BTEXS for predicting the overall risk of SAD. CONCLUSION: Our novel findings demonstrate the significant association between exposure to BTEXS mixture and small airways function decline and the potential roles of key homologs (benzene and styrene) in SAD.

A toxicity pathway-based approach for modeling the mode of action framework of lead-induced neurotoxicity.

BACKGROUND: The underlying mechanisms of lead (Pb) toxicity are not fully understood, which makes challenges to the traditional risk assessment. There is growing use of the mode of action (MOA) for risk assessment by integration of experimental data and system biology. The current study aims to develop a new pathway-based MOA for assessing Pb-induced neurotoxicity. METHODS: The available Comparative Toxicogenomic Database (CTD) was used to search genes associated with Pb-induced neurotoxicity followed by developing toxicity pathways using Ingenuity Pathway Analysis (IPA). The spatiotemporal sequence of disturbing toxicity pathways and key events (KEs) were identified by upstream regulator analysis. The MOA framework was constructed by KEs in biological and chronological order. RESULTS: There were a total of 71 references showing the relationship between lead exposure and neurotoxicity, which contained 2331 genes. IPA analysis showed that the neuroinflammation signaling pathway was the core toxicity pathway in the enriched pathways relevant to Pb-induced neurotoxicity. The upstream regulator analysis demonstrated that the aryl hydrocarbon receptor (AHR) signaling pathway was the upstream regulator of the neuroinflammation signaling pathway (11.76% overlap with upstream regulators, |Z-score|=1.451). Therefore, AHR activation was recognized as the first key event (KE1) in the MOA framework. The following downstream molecular and cellular key events were also identified. The pathway-based MOA framework of Pb-induced neurotoxicity was built starting with AHR activation, followed by an inflammatory response and neuron apoptosis. CONCLUSION: Our toxicity pathway-based approach not only advances the development of risk assessment for Pb-induced neurotoxicity but also brings new insights into constructing MOA frameworks of risk assessment for new chemicals.

Influence of benzene exposure, fat content, and their interactions on erythroid-related hematologic parameters in petrochemical workers: a cross-sectional study.

BACKGROUND: Ubiquitously distributed benzene is a known hematotoxin. Increasing evidence has suggested that erythroid-related hematologic parameters may be sensitive to benzene exposure. Fat content, which is also closely associated with erythroid-related hematologic parameters, may affect the distribution and/or metabolism of benzene, and eventually benzene-induced toxicity. METHODS: To explore the influence of benzene exposure, fat content, and their interactions on erythroid-related hematologic parameters, we recruited 1669 petrochemical workers and measured their urinary S-phenylmercapturic acid (SPMA) concentration and erythroid-related hematological parameters. Indices for fat content included body fat percentage (BF%), plasma total cholesterol (TC) and triglycerides (TG), and occurrence of fatty liver. RESULTS: The dose-response curve revealed U-shaped nonlinear relationships of SPMA with hematocrit (HCT) and mean corpuscular hemoglobin concentration (MCHC) (P-overall < 0.001, and P-nonlinear < 0.015), as well as positive linear associations and r-shaped nonlinear relationships of continuous fat content indices with erythroid-related hematological parameters (P-overall ≤0.005). We also observed modification effects of fat content on the associations between benzene exposure and erythroid-related hematological parameters, with workers of lower or higher BF% and TG more sensitive to benzene-induced elevation of MCHC (Pinteraction = 0.021) and benzene-induced decrease of HCT (Pinteraction = 0.050), respectively. We also found that some erythroid-related hematologic parameters differed between subgroups of workers with different SPMA levels and fat content combination. CONCLUSIONS: Our study suggested that benzene exposure, fat content, and their interactions may affect erythroid-related hematological parameters in petrochemical workers in a complex manner that are worthy of further investigation.

Genome-wide study on genetic diversity and phylogeny of five species in the genus Cervus.

BACKGROUND: Previous investigations of phylogeny in Cervus recovered many clades without whole genomic support. METHODS: In this study, the genetic diversity and phylogeny of 5 species (21 subspecies/populations from C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii) in the genus Cervus were analyzed using reduced-representation genome sequencing. RESULTS: A total of 197,543 SNPs were identified with an average sequencing depth of 16 x. A total of 21 SNP matrices for each subspecies/population and 1 matrix for individual analysis were constructed, respectively. Nucleotide diversity and heterozygosity analysis showed that all 21 subspecies/populations had different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high expected and observed heterozygosity, while observed heterozygosity in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations. Phylogenetic ML tree of all Cervus based on the 21 SNP matrices showed 5 robustly supported clades that clearly separate C. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon. Within C. elaphus clade, 4 subclades were well differentiated and statistically highly supported: C. elaphus (New Zealand), C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. Phylogenetic ML tree of all samples based on individual SNP matrices, together with geographic distribution, have shown that there were 3 major subclades of C. elaphus and C. canadensis in China, namely C. e. yarkandensis (distributed in Tarim Basin), C. c. macneilli/C. c. kansuensis/C. c. alashanicus (distributed in middle west of China), and C. c. songaricus/C. c. sibiricus (distributed in northwest of China). Among them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8-2.2 Myr ago. D statistical analysis showed that there was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus. CONCLUSIONS: Our results provided new insight to the genetic diversity and phylogeny of Cervus deer. In view of the current status of these populations, their conservation category will need to be reassessed.

Full-length transcriptome and microRNA sequencing reveal the specific gene-regulation network of velvet antler in sika deer with extremely different velvet antler weight.

Velvet antler displays the fastest and most robust tissue proliferation in the animal world, it is a model for a complete organ development/regeneration, and alternative medicine, tonic made from velvet antler, was beneficial for human. The weight of velvet antler had high biomedical and economic value, but the related regulation mechanisms controlling velvet antler weight remain unclear. In this study, extremely heavy and light velvet antler groups were selected from a sika deer population of 100 individuals with extreme velvet antler weight. A combination of full-length transcriptome sequencing and microRNA sequencing to the proliferation zone in the tip of velvet antler was applied. A total of 55306 transcripts and 1082 microRNAs were identified. Some highly expressed genes (COL1A1, COL1A2, COL3A1, FN1, and ATP6) and microRNAs (miR-21, let-7i, and miR-27b) were highly correlated with the physiological and growth characteristics of velvet antlers. Among the 334 differentially expressed genes, we found that most of the genes were located in the developmental process, especially animal organ development process. It is exciting to see that more blood vessels were found in the growing tip of heavy velvet antler through histological observation, and GO term of blood vessel development was also significant different between two groups. The combination analysis with mRNA and microRNA data in velvet antler showed a specific regulation network involved in the development of bone, mesenchyme, cartilage, and blood vessel, and helped us clearly find out the candidate 14 genes and 6 microRNAs, which could be used for selecting significant DNA markers of velvet antler weight.

Transcriptome Changes in the Mink Uterus during Blastocyst Dormancy and Reactivation.

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

The effects of Nrf2 knockout on regulation of benzene-induced mouse hematotoxicity.

Previous studies have demonstrated that oxidative stress is implicated in benzene-induced hematotoxicity. However, the low dose-response effects and the mechanism underlying perturbation of hematopoiesis remain to be defined. This study aims to address the role of Nrf2 pathway in mediating benzene-induced hematotoxicity. Nrf2+/+ (wildtype, Nrf2-WT) and Nrf2-/- (knockout, Nrf2-KO) mice were administrated with benzene at doses of 0.1, 1.0, 10.0, 100.0 mg/kg by oral gavage for a consecutive 4 weeks (6 times/week). As a result, benzene exposure caused a decline of WBC and lymphocyte counts in a dose-dependent manner at a dose range from 1.0 to 100.0 mg/kg, while low dose benzene induced hormesis effects. Interestingly, Nrf2 deficiency seemed to relieve the decline of peripheral blood cell counts upon benzene exposure, indicating the involvement of Nrf2 in regulation of benzene-induced hematotoxicity. The suppression of phase II enzyme expression in Nrf2-KO mice resulted in considerable reduction in detoxification indicated by the decrease of urinary S-phenylmercapturic acid (SPMA), a metabolite of benzene. Ex vivo assay revealed enhanced cytotoxicity and oxidative stress were induced by benzene in Nrf2-KO mice. Notably, the depletion of Nrf2 triggered the proliferation and differentiation of hematopoietic cells, but induced aberrant morphological changes in periphery erythrocytes and bone marrow cells, implicating the compensatory effects carried on at the expense of induction of dysfunctional blood cells. Our findings provide a new insight into a low dose-response towards benzene-induced hematotoxicity and uncover the critical role of Nrf2 pathway in mediating abnormal hematopoiesis in response to oxidative stress.

Persistent phosphorylation at specific H3 serine residues involved in chemical carcinogen-induced cell transformation.

Identification of aberrant histone H3 phosphorylation during chemical carcinogenesis will lead to a better understanding of the substantial roles of histone modifications in cancer development. To explore whether aberrant H3 phosphorylation contributes to chemical carcinogenesis, we examined the dynamic changes of H3 phosphorylation at various residues in chemical carcinogen-induced transformed human cells and human cancers. We found that histone H3 phosphorylation at Ser10 (p-H3S10) and Ser28 (p-H3S28) was upregulated by 1.5-4.8 folds and 2.1-4.3 folds, respectively in aflatoxin B1 -transformed hepatocytes L02 cells (L02RT-AFB1 ), benzo(a)pyrene-transformed HBE cells (HBERT-BaP), and coke oven emissions-transformed HBE cells (HBERT-COE). The ectopic expression of histone H3 mutant (H3S10A or H3S28A) in L02 cells led to the suppression of an anchorage-independent cell growth as well as tumor formation in immunodeficient mice. In addition, an enhanced p-H3S10 was found in 70.6% (24/34) of hepatocellular carcinoma (HCC), and 70.0% (21/30) of primary lung cancer, respectively. Notably, we found that expression of H3 carrying a mutant H3S10A or H3S28A conferred to cells the ability to maintain a denser chromatin and resistance to induction of DNA damage and carcinogen-induced cell transformation. Particularly, we showed that introduction of a mutant H3S10A abolished the bindings of p-H3S10 to the promoter of DNA repair genes, PARP1 and MLH1 upon AFB1 treatment. Furthermore, we revealed that PP2A was responsible for dephosphorylation of p-H3S10. Taken together, these results reveal a key role of persistent H3S10 or H3S28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response (DDR) genes.

Transcriptome analysis of sika deer in China.

Sika deer is of great commercial value because their antlers are used in tonics and alternative medicine and their meat is healthy and delicious. The goal of this study was to generate transcript sequences from sika deer for functional genomic analyses and to identify the transcripts that demonstrate tissue-specific, age-dependent differential expression patterns. These sequences could enhance our understanding of the molecular mechanisms underlying sika deer growth and development. In the present study, we performed de novo transcriptome assembly and profiling analysis across ten tissue types and four developmental stages (juvenile, adolescent, adult, and aged) of sika deer, using Illumina paired-end tag (PET) sequencing technology. A total of 1,752,253 contigs with an average length of 799 bp were generated, from which 1,348,618 unigenes with an average length of 590 bp were defined. Approximately 33.2 % of these (447,931 unigenes) were then annotated in public protein databases. Many sika deer tissue-specific, age-dependent unigenes were identified. The testes have the largest number of tissue-enriched unigenes, and some of them were prone to develop new functions for other tissues. Additionally, our transcriptome revealed that the juvenile-adolescent transition was the most complex and important stage of the sika deer life cycle. The present work represents the first multiple tissue transcriptome analysis of sika deer across four developmental stages. The generated data not only provide a functional genomics resource for future biological research on sika deer but also guide the selection and manipulation of genes controlling growth and development.

[Effect of CCM3 gene defect on lead-induced cell genotoxicity in mouse embryonic fibroblasts].

OBJECTIVE: To investigate the effect of CCM3 gene defection on lead induced cell genotoxicity in mouse embryonic fibroblasts. METHODS: C57 female mice were mated with CCM3 gene heterozygous male mice. E13.5 embryos were taken to isolate primary mouse embryonic fibroblasts. After genotyping, wild type and heterozygous cells were treated with different doses of lead acetate. Cell viability, genotoxicity and protein expression were detected by MTS assay, CB micronucleus method and Western blot, respectively. RESULTS: Mouse embryonic fibroblasts with lead acetate treatment for 24 h, wild-type cells 100.00 µmol/L lead acetate-treated group (69.16±1.36) and the control group (100.00±2.33) compared to cells decreased by 30%, CCM3 heterozygous type cell 100.00 µmol/L lead acetate-treated group (87.16±5.50) and the control group (100.00±2.06) compared to cells decreased by 13%, the difference was statistically significant (F values were 98.59, 82.63, P<0.001). Lead acetate treatment after 48 h, wild-type cells 100.00 µmol/L lead acetate-treated group (51.99±5.62) and the control group (100.00±3.11) compared to cells decreased by 50%, heterozygous type cells 100.00 µmol/L lead acetate treatment group (66.33±4.06) and the control group (100.00±5.72) compared to cells decreased by 35%, the differences were statistically significant (F values were 82.63, 36.86, P < 0.001). The results of CBMN test showed that with increased dose, micronucleus cell rate of two genotypes showed an increasing trend, in the wild-type cells, the micronucleus cell rate (/1 000) for the control group, 29.6±2.2, 6.25 µmol/L dose group 47.3±6.6, 25 µmol/L dose group 55.5±9.1, 100.00 µmol/L dose group 66.8±3.5; heterozygous cells micronucleus cell rate (/1 000) for the control group, 35.3±5.6, 6.25 µmol/L dose of 50.0±8.3, 25.00 µmol/L dose group 57.0±8.5, 100.00 µmol/L dose group 58.8±2.1. Micronucleus cell rates (/1 000) were significant differences, in 100.00 µmol/L dose groups of two genotypes. Western blot results showed that wild-type cells CCM3 expression 100.00 µmol/L lead acetate-treated group (0.70±0.03) was 1.32 times higher than the control group (0.53±0.07), heterozygous cells CCM3 expression 100.00 µmol/L lead acetate-treated group (0.48±0.02) was 1.77 times higher than control group that of 0.27±0.04, there was statistically significant difference (F values were 14.77, 25.74, P < 0.001); wild-type cells γ-H2AX expression 100.00 µmol/L lead acetate-treated group (0.69±0.03) was 1.06 times higher than the control group (0.65±0.07), heterozygous cells γ-H2AX expression 100.00 µmol/L lead acetate-treated group (0.99±0.04) was 1.55 times higher than the control group CCM3 expression levels (0.64±0.06), there was statistically significant difference (wild-type cells: F = 7.08, P = 0.012, heterozygous type cell: F = 13.49, P = 0.002). CONCLUSION: CCM3 gene may play a role in lead-induced genetic toxicity of mouse embryonic fibroblasts, CCM3 gene-lead interactions effects on mouse embryonic fibroblasts cell toxicity.

Comparative Genomics Reveal Phylogenetic Relationship and Chromosomal Evolutionary Events of Eight Cervidae Species.

Cervidae represents a family that is not only rich in species diversity but also exhibits a wide range of karyotypes. The controversies regarding the phylogeny and classification of Cervidae still persist. The flourishing development of the genomic era has made it possible to address these issues at the genomic level. Here, the genomes of nine species were used to explore the phylogeny and chromosomal evolutionary events of Cervidae. By conducting whole-genome comparisons, we identified single-copy orthologous genes across the nine species and constructed a phylogenetic tree based on the single-copy orthologous genes sequences, providing new insights into the phylogeny of Cervidae, particularly the phylogenetic relationship among sika deer, red deer, wapiti and Tarim red deer. Gene family analysis revealed contractions in the olfactory receptor gene family and expansions in the histone gene family across eight Cervidae species. Furthermore, synteny analysis was used to explore the chromosomal evolutionary events of Cervidae species, revealing six chromosomal fissions during the evolutionary process from Bovidae to Cervidae. Notably, specific chromosomal fusion events were found in four species of Cervus, and a unique chromosomal fusion event was identified in Muntiacus reevesi. Our study further completed the phylogenetic relationship within the Cervidae and demonstrated the feasibility of inferring species phylogeny at the whole-genome level. Additionally, our findings on gene family evolution and the chromosomal evolutionary events in eight Cervidae species lay a foundation for comprehensive research of the evolution of Cervidae.

Muscle Transcriptome Analysis of Mink at Different Growth Stages Using RNA-Seq.

Mink is a kind of small and precious fur animal resource. In this study, we employed transcriptomics technology to analyze the gene expression profile of mink pectoral muscle tissue, thereby elucidating the regulatory mechanisms underlying mink growth and development. Consequently, a total of 25,954 gene expression profiles were acquired throughout the growth and development stages of mink at 45, 90, and 120 days. Among these profiles, 2607 genes exhibited significant differential expression (|log2(fold change)| ≥ 2 and p_adj < 0.05). GO and KEGG enrichment analyses revealed that the differentially expressed genes were primarily associated with the mitotic cell cycle process, response to growth factors, muscle organ development, and insulin resistance. Furthermore, GSEA enrichment analysis demonstrated a significant enrichment of differentially expressed genes in the p53 signaling pathway at 45 days of age. Subsequent analysis revealed that genes associated with embryonic development (e.g., PEG10, IGF2, NRK), cell cycle regulation (e.g., CDK6, CDC6, CDC27, CCNA2), and the FGF family (e.g., FGF2, FGF6, FGFR2) were all found to be upregulated at 45 days of age in mink, which suggested a potential role for these genes in governing early growth and developmental processes. Conversely, genes associated with skeletal muscle development (PRVA, TNNI1, TNNI2, MYL3, MUSTN1), a negative regulator of the cell cycle gene (CDKN2C), and IGFBP6 were found to be up-regulated at 90 days of age, suggesting their potential involvement in the rapid growth of mink. In summary, our experimental data provide robust support for elucidating the regulatory mechanisms underlying the growth and development of mink.