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.

Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers.

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2'-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose-response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

Assessing the Effects of Nicotinamide Mononucleotide Supplementation on Pulmonary Inflammation in Male Mice Subchronically Exposed to Ambient Particulate Matter.

BACKGROUND: Chronic lung injury and dysregulated cellular homeostasis in response to particulate matter (PM) exposure are closely associated with adverse health effects. However, an effective intervention for preventing the adverse health effects has not been developed. OBJECTIVES: This study aimed to evaluate the protective effects of nicotinamide mononucleotide (NMN) supplementation on lung injury and elucidate the mechanism by which NMN improved immune function following subchronic PM exposure. METHODS: Six-week-old male C57BL/6J mice were placed in a real-ambient PM exposure system or filtered air-equipped chambers (control) for 16 wk with or without NMN supplementation in drinking water (regarded as Con-H2O, Exp-H2O, Con-NMN and Exp-NMN groups, respectively) in Shijiazhuang City, China (n=20/group). The effects of NMN supplementation (500mg/kg) on PM-induced chronic pulmonary inflammation were assessed, and its mechanism was characterized using single-cell transcriptomic sequencing (scRNA-seq) analysis of whole lung cells. RESULTS: The NMN-treated mice exhibited higher NAD+ levels in multiple tissues. Following 16-wk PM exposure, slightly less pulmonary inflammation and less collagen deposition were noted in mice with NMN supplementation in response to real-ambient PM exposure (Exp-NMN group) compared with the Exp-H2O group (all p<0.05). Mouse lung tissue isolated from the Exp-NMN group was characterized by fewer neutrophils, monocyte-derived cells, fibroblasts, and myeloid-derived suppressor cells induced by subchronic PM exposure as detected by scRNA-seq transcriptomic analysis. The improved immune functions were further characterized by interleukin-17 signaling pathway inhibition and lower secretion of profibrotic cytokines in the Exp-NMN group compared with the Exp-H2O group. In addition, reduced proportions of differentiated myofibroblasts and profibrotic interstitial macrophages were identified in the NMN-supplemented mice in response to PM exposure. Furthermore, less immune function suppression and altered differentiation of pathological cell phenotypes NMN was related to intracellular lipid metabolism activation. DISCUSSION: Our novel findings suggest that NMN supplementation mitigated PM-induced lung injury by regulating immune functions and improving lipid metabolism in male mice, providing a putative intervention method for prevention of human health effects associated with PM exposure. https://doi.org/10.1289/EHP12259.

Moderate body lipid accumulation in mice attenuated benzene-induced hematotoxicity via acceleration of benzene metabolism and clearance.

Recent population and animal studies have revealed a correlation between fat content and the severity of benzene-induced hematologic toxicity. However, the precise impact of lipid deposition on benzene-induced hematotoxicity and the underlying mechanisms remain unclear. In this study, we established a mouse model with moderate lipid accumulation by subjecting the mice to an 8-week high-fat diet (45% kcal from fat, HFD), followed by 28-day inhalation of benzene at doses of 0, 1, 10, and 100 ppm. The results showed that benzene exposure caused a dose-dependent reduction of peripheral white blood cell (WBC) counts in both diet groups. Notably, this reduction was less pronounced in the HFD-fed mice, suggesting that moderate lipid accumulation mitigates benzene-related hematotoxicity. To investigate the molecular basis for this effect, we performed bioinformatics analysis of high-throughput transcriptome sequencing data, which revealed that moderate lipid deposition alters mouse metabolism and stress tolerance towards xenobiotics. Consistently, the expression of key metabolic enzymes, such as Cyp2e1 and Gsta1, were upregulated in the HFD-fed mice upon benzene exposure. Furthermore, we utilized a real-time exhaled breath detection technique to monitor exhaled benzene metabolites, and the results indicated that moderate lipid deposition enhanced metabolic activation and increased the elimination of benzene metabolites. Collectively, these findings demonstrate that moderate lipid deposition confers reduced susceptibility to benzene-induced hematotoxicity in mice, at least in part, by accelerating benzene metabolism and clearance.

An integrative analysis of lipidomics and transcriptomics in various mouse brain regions in response to real-ambient PM2.5 exposure.

Exposure to Fine particulate matter (PM2.5) has been associated with various neurological disorders. However, the underlying mechanisms of PM2.5-induced adverse effects on the brain are still not fully defined. Multi-omics analyses could offer novel insights into the mechanisms of PM2.5-induced brain dysfunction. In this study, a real-ambient PM2.5 exposure system was applied to male C57BL/6 mice for 16 weeks, and lipidomics and transcriptomics analysis were performed in four brain regions. The findings revealed that PM2.5 exposure led to 548, 283, 304, and 174 differentially expressed genes (DEGs), as well as 184, 89, 228, and 49 distinctive lipids in the hippocampus, striatum, cerebellum, and olfactory bulb, respectively. Additionally, in most brain regions, PM2.5-induced DEGs were mainly involved in neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, and calcium signaling pathway, while PM2.5-altered lipidomic profile were primarily enriched in retrograde endocannabinoid signaling and biosynthesis of unsaturated fatty acids. Importantly, mRNA-lipid correlation networks revealed that PM2.5-altered lipids and DEGs were obviously enriched in pathways involving in bile acid biosynthesis, De novo fatty acid biosynthesis, and saturated fatty acids beta-oxidation in brain regions. Furthermore, multi-omics analyses revealed that the hippocampus was the most sensitive part to PM2.5 exposure. Specifically, dysregulation of Pla2g1b, Pla2g, Alox12, Alox15, and Gpx4 induced by PM2.5 were closely correlated to the disruption of alpha-linolenic acid, arachidonic acid and linoleic acid metabolism in the hippocampus. In summary, our findings highlight differential lipidomic and transcriptional signatures of various brain regions by real-ambient PM2.5 exposure, which will advance our understanding of potential mechanisms of PM2.5-induecd neurotoxicity.

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.

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.

Explored Normalized Cut With Random Walk Refining Term for Image Segmentation.

The Normalized Cut (NCut) model is a popular graph-based model for image segmentation. But it suffers from the excessive normalization problem and weakens the small object and twig segmentation. In this paper, we propose an Explored Normalized Cut (ENCut) model that establishes a balance graph model by adopting a meaningful-loop and a k-step random walk, which reduces the energy of small salient region, so as to enhance the small object segmentation. To improve the twig segmentation, our ENCut model is further enhanced by a new Random Walk Refining Term (RWRT) that adds local attention to our model with the help of an un-supervising random walk. Finally, a move-making based strategy is developed to efficiently solve the ENCut model with RWRT. Experiments on three standard datasets indicate that our model can achieve state-of-the-art results among the NCut-based segmentation models.

CpG site-specific methylation as epi-biomarkers for the prediction of health risk in PAHs-exposed populations.

Environmental insults can lead to alteration in DNA methylation of specific genes. To address the role of altered DNA methylation in prediction of polycyclic aromatic hydrocarbons (PAHs) exposure-induced genetic damage, we recruited two populations, including diesel engine exhausts (low-level) and coke oven emissions (high-level) exposed subjects. The positive correlation was observed between the internal exposure marker (1-hydroxypyrene) and the extents of DNA damage (P < 0.05). The methylation of representative genes, including TRIM36, RASSF1a, and MGMT in peripheral blood lymphocytes was quantitatively examined by bisulfite-pyrosequencing assay. The DNA methylation of these three genes in response to PAHs exposure were changed in a CpG-site-specific manner. The identified hot CpG site-specific methylation of three genes exhibited higher predictive power for DNA damage than the respective single genes in both populations. Furthermore, the dose-response relationship analysis revealed a nonlinear U-shape curve of TRIM36 or RASSF1a methylation in combined population, which led to determination of the threshold of health risk. Furthermore, we established a prediction model for genetic damage based on the unidirectional-alteration MGMT methylation levels. In conclusion, this study provides new insight into the application of multiple epi-biomarkers for health risk assessment upon PAHs exposure.

PP2A-mTOR-p70S6K/4E-BP1 axis regulates M1 polarization of pulmonary macrophages and promotes ambient particulate matter induced mouse lung injury.

To identify key signaling pathways involved in ambient particulate matter (PM)-induced pulmonary injury, we generated a mouse model with myeloid-specific deletion of Ppp2r1a gene (encoding protein phosphatase 2 A (PP2A) A subunit), and conducted experiments in a real-ambient PM exposure system. PP2A Aα-/- homozygote (Aα HO) mice and matched wild-type (WT) littermates were exposed to PM over 3-week and 6-week. The effects of PM exposure on pulmonary inflammation, oxidative stress, and apoptosis were significantly enhanced in Aα HO compared to WT mice. The number of pulmonary macrophages increased by 74.8~88.0% and enhanced M1 polarization appeared in Aα HO mice upon PM exposure. Secretion of M1 macrophage-related inflammatory cytokines was significantly increased in Aα HO vs. WT mice following PM exposure. Moreover, we demonstrated that PP2A-B56α holoenzyme regulated M1 polarization and that the mTOR signaling pathway mediated the persistent M1 polarization upon PM2.5 exposure. Importantly, PP2A-B56α holoenzyme was shown to complex with mTOR/p70S6K/4E-BP1, and suppression of B56α led to enhanced phosphorylation of mTOR, p70S6K, and 4E-BP1. These observations demonstrate that the PP2A-mTOR-p70S6K/4E-BP1 signaling is a critical pathway in mediating macrophage M1 polarization, which contributes to PM-induced pulmonary injury.

Determination of tipping point in course of PM2.5 organic extracts-induced malignant transformation by dynamic network biomarkers.

The dynamic network biomarkers (DNBs) are designed to identify the tipping point and specific molecules in initiation of PM2.5-induced lung cancers. To discover early-warning signals, we analyzed time-series gene expression datasets over a course of PM2.5 organic extraction-induced human bronchial epithelial (HBE) cell transformation (0th~16th week). A composition index of DNB (CIDNB) was calculated to determine correlations and fluctuations in molecule clusters at each timepoint. We identified a group of genes with the highest CIDNB at the 10th week, implicating a tipping point and corresponding DNBs. Functional experiments revealed that manipulating respective DNB genes at the tipping point led to remarkable changes in malignant phenotypes, including four promoters (GAB2, NCF1, MMP25, LAPTM5) and three suppressors (BATF2, DOK3, DAP3). Notably, co-altered expression of seven core DNB genes resulted in an enhanced activity of malignant transformation compared to effects of single-gene manipulation. Perturbation of pathways (EMT, HMGB1, STAT3, NF-κB, PTEN) appeared in HBE cells at the tipping point. The core DNB genes were involved in regulating lung cancer cell growth and associated with poor survival, indicating their synergistic effects in initiation and development of lung cancers. These findings provided novel insights into the mechanism of dynamic networks attributable to PM2.5-induced cell transformation.

Association between H3K36me3 modification and methylation of LINE-1 and MGMT in peripheral blood lymphocytes of PAH-exposed workers.

To explore the epigenetic alterations in response to DNA damage following polycyclic aromatic hydrocarbons (PAHs) exposure and the crosstalk between different epigenetic regulations, we examined trimethylated Lys 36 of histone H3 (H3K36me3) and methylation of 'long interspersed element-1 (LINE-1)' and 'O 6-methylguanine-DNA methyltransferase (MGMT)' in peripheral blood lymphocytes (PBLCs) of 173 coke oven workers (PAH-exposed group) and 94 non-exposed workers (control group). The PAH-exposed group showed higher internal PAH exposure level, enhanced DNA damage and increased MGMT expression (all P < 0.001). Notably, the methylation of LINE-1 and MGMT decreased by 3.9 and 40.8%, respectively, while H3K36me3 level was 1.7 times higher in PBLCs of PAH-exposed group compared to control group (all P < 0.001). These three epigenetic marks were significantly associated with DNA damage degree (all P < 0.001) and PAH exposure level in a dose-response manner (all P < 0.001). LINE-1 hypomethylation is correlated with enhanced H3K36me3 modification (ß = -0.198, P = 0.002), indicating a synergistic effect between histone modification and DNA methylation at the whole genome level. In addition, MGMT expression was positively correlated with H3K36me3 modification (r = 0.253, P < 0.001), but not negatively correlated with MGMT methylation (r = 0.202, P < 0.05). The in vitro study using human bronchial epithelial cells treated with the organic extract of coke oven emissions confirmed that H3K36me3 is important for MGMT expression following PAH exposure. In summary, our study indicates that histone modification and DNA methylation might have synergistic effects on DNA damage induced by PAH exposure at the whole genome level and H3K36me3 is more essential for MGMT expression during the course.

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.

FLT1 hypermethylation is involved in polycyclic aromatic hydrocarbons-induced cell transformation.

Coke oven emissions (COEs) are common particle pollutants in occupational environment and the major constituents of COEs are polycyclic aromatic hydrocarbons (PAHs). Previously, we identified aberrant methylation of the fms related tyrosine kinase 1 (FLT1) gene over the course of benzo(a)pyrene (BaP)-induced cell transformation via genome-wide methylation array. To quantify FLT1 methylation, we established a bisulfite pyrosequencing assay and examined the FLT1 hypermethylation in several human cancers. The results revealed that 70.0% (21/30 pairs) of lung cancers harbored hypermethylated FLT1 and concomitant suppression of gene expression compared to the adjacent tissues. This implies that FLT1 hypermethylation might play a role in malignant cell transformation. In addition, FLT1 hypermethylation and gene suppression appeared in primary human lymphocytes in a dose-response manner following COEs treatment. To explore whether FLT1 methylation is correlated with COEs exposure and DNA damage, we recruited 144 male subjects who had been exposed to high levels of COEs and 84 male control subjects. Notably, the FLT1 methylation in peripheral blood lymphocytes (PBLCs) of the COEs-exposed group (19.8 ±â€¯3.2%) was enhanced by 17.9% compared to that of the control group (16.8 ±â€¯2.8%) (P < 0.001). The FLT1 methylation status was positively correlated with urinary 1-hydroxypyrene (1-OHP) levels, an internal exposure marker of PAHs (ß = 0.029, 95% CI = 0.010-0.048, P = 0.003) and positively correlated with DNA damage (ßOTM = 0.024, 95% CI = 0.007-0.040, P = 0.005; ßTail DNA = 0.035, 95% CI = 0.0017-0.054, P < 0.001) indicated by comet assay. Taken together, these findings indicate that FLT1 might be a tumor suppressor, and its hypermethylation might contribute to PAHs-induced carcinogenicity.

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.