Intestinal carcinogenicity screening of environmental pollutants using organoid-based cell transformation assay.

The high incidence of colorectal cancer (CRC) is closely associated with environmental pollutant exposure. To identify potential intestinal carcinogens, we developed a cell transformation assay (CTA) using mouse adult stem cell-derived intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals, including Cd, iPb, Cr-VI, iAs-III, Zn, Cu, PFOS, BPA, MEHP, AOM, DMH, MNNG, aspirin, and metformin. We optimized the experimental protocol based on cytotoxicity, amplification, and colony formation of chemical-treated mASC-IOs. In addition, we assessed the accuracy of in vitro study and the human tumor relevance through characterizing interdependence between cell-cell and cell-matrix adhesions, tumorigenicity, pathological feature of subcutaneous tumors, and CRC-related molecular signatures. Remarkably, the results of cell transformation in 14 chemicals showed a strong concordance with epidemiological findings (8/10) and in vivo mouse studies (12/14). In addition, we found that the increase in anchorage-independent growth was positively correlated with the tumorigenicity of tested chemicals. Through analyzing the dose-response relationship of anchorage-independent growth by benchmark dose (BMD) modeling, the potent intestinal carcinogens were identified, with their carcinogenic potency ranked from high to low as AOM, Cd, MEHP, Cr-VI, iAs-III, and DMH. Importantly, the activity of chemical-transformed mASC-IOs was associated with the degree of cellular differentiation of subcutaneous tumors, altered transcription of oncogenic genes, and activated pathways related to CRC development, including Apc, Trp53, Kras, Pik3ca, Smad4 genes, as well as WNT and BMP signaling pathways. Taken together, we successfully developed a mASC-IO-based CTA, which might serve as a potential alternative for intestinal carcinogenicity screening of chemicals.

Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling.

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

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.

Genomic Characterization Revealed PM2.5-Associated Mutational Signatures in Lung Cancer Including Activation of APOBEC3B.

Fine particulate matter (PM2.5) exposure causes DNA mutations and abnormal gene expression leading to lung cancer, but the detailed mechanisms remain unknown. Here, analysis of genomic and transcriptomic changes upon a PM2.5 exposure-induced human bronchial epithelial cell-based malignant transformed cell model in vitro showed that PM2.5 exposure led to APOBEC mutational signatures and transcriptional activation of APOBEC3B along with other potential oncogenes. Moreover, by analyzing mutational profiles of 1117 non-small cell lung cancers (NSCLCs) from patients across four different geographic regions, we observed a significantly higher prevalence of APOBEC mutational signatures in non-smoking NSCLCs than smoking in the Chinese cohorts, but this difference was not observed in TCGA or Singapore cohorts. We further validated this association by showing that the PM2.5 exposure-induced transcriptional pattern was significantly enriched in Chinese NSCLC patients compared with other geographic regions. Finally, our results showed that PM2.5 exposure activated the DNA damage repair pathway. Overall, here we report a previously uncharacterized association between PM2.5 and APOBEC activation, revealing a potential molecular mechanism of PM2.5 exposure and lung cancer.

Type 1 diabetes and diet-induced obesity predispose C57BL/6J mice to PM2.5-induced lung injury: a comparative study.

BACKGROUND: Pre-existing metabolic diseases may predispose individuals to particulate matter (PM)-induced adverse health effects. However, the differences in susceptibility of various metabolic diseases to PM-induced lung injury and their underlying mechanisms have yet to be fully elucidated. RESULTS: Type 1 diabetes (T1D) murine models were constructed by streptozotocin injection, while diet-induced obesity (DIO) models were generated by feeding 45% high-fat diet 6 weeks prior to and throughout the experiment. Mice were subjected to real-ambient PM exposure in Shijiazhuang City, China for 4 weeks at a mean PM2.5 concentration of 95.77 µg/m3. Lung and systemic injury were assessed, and the underlying mechanisms were explored through transcriptomics analysis. Compared with normal diet (ND)-fed mice, T1D mice exhibited severe hyperglycemia with a blood glucose of 350 mg/dL, while DIO mice displayed moderate obesity and marked dyslipidemia with a slightly elevated blood glucose of 180 mg/dL. T1D and DIO mice were susceptible to PM-induced lung injury, manifested by inflammatory changes such as interstitial neutrophil infiltration and alveolar septal thickening. Notably, the acute lung injury scores of T1D and DIO mice were higher by 79.57% and 48.47%, respectively, than that of ND-fed mice. Lung transcriptome analysis revealed that increased susceptibility to PM exposure was associated with perturbations in multiple pathways including glucose and lipid metabolism, inflammatory responses, oxidative stress, cellular senescence, and tissue remodeling. Functional experiments confirmed that changes in biomarkers of macrophage (F4/80), lipid peroxidation (4-HNE), cellular senescence (SA-ß-gal), and airway repair (CCSP) were most pronounced in the lungs of PM-exposed T1D mice. Furthermore, pathways associated with xenobiotic metabolism showed metabolic state- and tissue-specific perturbation patterns. Upon PM exposure, activation of nuclear receptor (NR) pathways and inhibition of the glutathione (GSH)-mediated detoxification pathway were evident in the lungs of T1D mice, and a significant upregulation of NR pathways was present in the livers of T1D mice. CONCLUSIONS: These differences might contribute to differential susceptibility to PM exposure between T1D and DIO mice. These findings provide new insights into the health risk assessment of PM exposure in populations with metabolic diseases.

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.

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.

Assessment of intestinal injury of hexavalent chromium using a modified in vitro gastrointestinal digestion model.

Intestinal injury assessment of hexavalent chromium (Cr-VI) in humans is crucial for quantifying assessment of adverse health risk posed by the intake of Cr (VI)-contaminated water. To overcome the deficiency in simulating human gastric reduction and intestinal absorption, we modified the constituents of simulated gastric fluid in in vitro digestion method by adding reductants glutathione (18 µM) and ascorbic acid (180 µM), which incorporated with human intestinal epithelial model to construct an in vitro gastrointestinal digestion (IVGD) model for intestinal injury assessment. Cr-VI bioaccessibility results from IVGD model showed that weak gastric acidity significantly increased the intestinal accessible Cr-VI dose by 22.41-38.43 folds. The time-course intestinal absorption indicated prolongation of intestinal exposure destroyed the intestinal epithelium, and 24 h after Cr-VI treatment was a good time point to perform intestinal absorption and toxicity assessment. A series of cell-based bioassays provided initial warning of adverse effect, suggesting that epithelial integrity exhibited greatest sensitivity to Cr-VI exposure and might be used as a sensitive marker for the toxicity assessment of oral exposure to Cr-VI. Notably, this study provides a feasible strategy for delineation of Cr-VI biotransformation and intestinal injury following ingestion exposure, which contributes to address the toxicity data gap of low-dose exposure in humans and puts forward a reference for intestinal toxicity assessment of other chemicals.

Single-cell transcriptomics reveals immune dysregulation mediated by IL-17A in initiation of chronic lung injuries upon real-ambient particulate matter exposure.

BACKGROUND: Long-term exposure to fine particulate matter (PM2.5) increases susceptibility to chronic respiratory diseases, including inflammation and interstitial fibrosis. However, the regulatory mechanisms by which the immune response mediates the initiation of pulmonary fibrosis has yet to be fully characterized. This study aimed to illustrate the interplay between different cell clusters and key pathways in triggering chronic lung injuries in mice following PM exposure. RESULTS: Six-week-old C57BL/6J male mice were exposed to PM or filtered air for 16 weeks in a real-ambient PM exposure system in Shijiazhuang, China. The transcriptional profiles of whole lung cells following sub-chronic PM exposure were characterized by analysis of single-cell transcriptomics. The IL-17A knockout (IL-17A-/-) mouse model was utilized to determine whether the IL-17 signaling pathway mediated immune dysregulation in PM-induced chronic lung injuries. After 16-week PM exposure, chronic lung injuries with excessive collagen deposition and increased fibroblasts, neutrophils, and monocytes were noted concurrent with a decreased number of major classes of immune cells. Single-cell analysis showed that activation of the IL-17 signaling pathway was involved in the progression of pulmonary fibrosis upon sub-chronic PM exposure. Depletion of IL-17A led to significant decline in chronic lung injuries, which was mainly triggered by reduced recruitment of myeloid-derived suppressor cells (MDSCs) and downregulation of TGF-ß. CONCLUSION: These novel findings demonstrate that immunosuppression via the IL-17A pathway plays a critical role in the initiation of chronic lung injuries upon sub-chronic PM exposure.

GLIS3 mediated by the Rap1/PI3K/AKT signal pathway facilitates real-ambient PM2.5 exposure disturbed thyroid hormone homeostasis regulation.

Exposure to fine particulate matter (PM2.5) could damage multiple organs and systems. Recent epidemiological studies have shown that PM2.5 can disrupt dynamic balance of thyroid hormone (TH). However, the underlying mechanism by which PM2.5 interferes with TH remains unclear. This study evaluated the role of Gli-similar3 (GLIS3) in the effect of PM2.5 on TH synthesis in mice using a real-ambient exposure system, in Shijiazhuang City, Hebei Province. The PM2.5exposure group (PM) and filtered air group (FA) were placed in the exposure device for four and eight weeks. The results showed that the PM2.5 exposure altered the structure of the thyroid gland. Moreover, after PM2.5 exposure for eight weeks, the exposure level of free thyroxine (FT4) increased and the expression level of thyroid stimulating hormone (TSH) decreased in serum of mice. In addition, PM2.5 exposure significantly increased the expression of proteins related to thyroid hormone synthesis, such as sodium iodide transporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG). Next, we found that GLIS3 and thyroid transcription factor Paired box 8 (PAX8) also increased after PM2.5 exposure. In order to further explore the potential molecular mechanism, we carried out transcriptome sequencing. KEGG analysis of the top 10 pathways revealed that the Ras-associated protein 1 (Rap1) signaling pathway could activate transcription factors and is related to thyroid cell survival. Additionally, PM2.5 exposure significantly increased the protein levels of Rap1 and its active form (Rap1 +GTP). We speculate that the active state of Rap1 is believed to be involved in activating the expression of transcription factor GLIS3. In conclusion, PM2.5 exposure induces histological changes in the thyroid gland and thyroid dysfunction in mice. The exposure activates GLIS3 through the Rap1/PI3K/AKT pathway to promote the expression of proteins related to thyroid hormone synthesis, leading to increased dysregulating TH homeostasis.

Real ambient particulate matter-induced lipid metabolism disorder: Roles of peroxisome proliferators-activated receptor alpha.

A growing body of evidence associated particulate matter (PM) exposure with lipid metabolism disorders, yet, the underlying mechanism remains to be elucidated. Among the major lipid metabolism modulators, peroxisome proliferator-activated receptor (PPAR) alpha plays an important role. In the current study, an individually ventilated cage (IVC) system was used to expose C57/B6 mice to real-ambient PM for six weeks, with or without co-treatment of PPAR alpha agonist WY14,643. The general parameters, liver and adipose tissue pathology, serum lipids, metal deposition and lipid profile of liver were assessed. The results indicated that six weeks of real-ambient PM exposure induced dyslipidemia, including increased serum triglycerides (TG) and decreased high density lipoprotein cholesterol (HDL-C) level, along with steatosis in liver, increased size of adipocytes in white adipose tissue (WAT) and whitening of brown adipose tissue (BAT). ICP-MS results indicated increased Cr and As deposition in liver. Lipidomics analysis revealed that glycerophospholipids and cytochrome P450 pathway were most significantly affected by PM exposure. Several lipid metabolism-related genes, including CYP4A14 in liver and UCP1 in BAT were downregulated following PM exposure. WY14,643 treatment alleviated PM-induced dyslipidemia, liver steatosis and whitening of BAT, while enhancing CD36, SLC27A1, CYP4A14 and UCP1 expression. In conclusion, PPAR alpha pathway participates in PM-induced lipid metabolism disorder, PPAR alpha agonist WY14,643 treatment exerted protective effects on PM-induced dyslipidemia, liver steatosis and whitening of BAT, but not on increased adipocyte size of WAT.

DNA Methylation Biomarkers of IQ Reduction are Associated with Long-term Lead Exposure in School Aged Children in Southern China.

Although lead associated with intelligence decline in children has long been reported, studies combining intelligence determination, molecular mechanisms exploration, and biomarker screen are quite rare. In this study, based on 333 children aged 9-11, we determined the role of DNA methylation (DNAm) in the relationship of lead exposure with children's intelligence. DNAm was measured from children's blood DNA specimens, and mediation analysis was performed to identify DNAm biomarkers mediating the lead-intelligence relationship. We identified forty-three differentially methylated regions (DMRs), and two fragments (FAM50B1 and PTCHD3) significantly mediated the lead-intelligence relationship, with contribution rates of 30.36% (p = 0.010) and 60.36% (p < 0.001), respectively. In addition, blood lead levels (BLLs) lower than 100 µg/L still adversely affected children's IQs and DNAm of the two fragments. Our data suggests that DNAm mediates lead-associated cognitive delay in children and blood lead reference value for school-aged children (100 µg/L) should be revised, and the candidate biomarkers can be used in related neurological diseases in future.

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.

Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1.

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent ß-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of ß-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, ß-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.

Caloric restriction attenuates C57BL/6 J mouse lung injury and extra-pulmonary toxicity induced by real ambient particulate matter exposure.

BACKGROUND: Caloric restriction (CR) is known to improve health and extend lifespan in human beings. The effects of CR on adverse health outcomes in response to particulate matter (PM) exposure and the underlying mechanisms have yet to be defined. RESULTS: Male C57BL/6 J mice were fed with a CR diet or ad libitum (AL) and exposed to PM for 4 weeks in a real-ambient PM exposure system located at Shijiazhuang, China, with a daily mean concentration (95.77 µg/m3) of PM2.5. Compared to AL-fed mice, CR-fed mice showed attenuated PM-induced pulmonary injury and extra-pulmonary toxicity characterized by reduction in oxidative stress, DNA damage and inflammation. RNA sequence analysis revealed that several pulmonary pathways that were involved in production of reactive oxygen species (ROS), cytokine production, and inflammatory cell activation were inactivated, while those mediating antioxidant generation and DNA repair were activated in CR-fed mice upon PM exposure. In addition, transcriptome analysis of murine livers revealed that CR led to induction of xenobiotic metabolism and detoxification pathways, corroborated by increased levels of urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) and decreased cytotoxicity measured in an ex vivo assay. CONCLUSION: These novel results demonstrate, for the first time, that CR in mice confers resistance against pulmonary injuries and extra-pulmonary toxicity induced by PM exposure. CR led to activation of xenobiotic metabolism and enhanced detoxification of PM-bound chemicals. These findings provide evidence that dietary intervention may afford therapeutic means to reduce the health risk associated with PM exposure.

Silencing of solute carrier family 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells.

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet-induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma.

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.