Benzene-induced Hematotoxicity Research Articles

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells

ObjectiveHydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. MethodsIn this study, we treated K562 cells with 40 μmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. ResultsOne hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. ConclusionOur work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

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.

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.

Analysis of self-renewing and differentiation-related microRNAs and transcription factors in multilineage mouse hematopoietic stem/progenitor cells induced by 1,4-benzoquinone.

HSPCs are targets for benzene-induced hematotoxicity and leukemogenesis. However, benzene toxicity targeting microRNAs (miRNAs) and transcription factors (TF) that are involve in regulating self-renewing and differentiation of HSPCs comprising of different hematopoietic lineages remains poorly understood. In this study, the effect of a benzene metabolite, 1,4-benzoquinone (1,4-BQ) exposure, in HSPCs focusing on the self-renewing (miRNAs: miR-196b and miR-29a; TF: HoxB4, Bmi-1) and differentiation (miRNAs: miR-181a, TF: GATA3) pathways were investigated. Freshly isolated mouse BM cells were initially exposed to 1,4-BQ at 1.25 to 5 µM for 24 h, followed by miRNAs and TF studies in BM cells. Then, the miRNAs expression was further evaluated in HSPCs of different lineages comprised of myeloid, erythroid and pre-B lymphoid progenitors following 7-14 days of colony forming unit (CFU) assay. Exposure to 1,4-BQ in BM cells significantly (p<0.05) reduced the miR-196b (2.5 and 5 µM), miR-181a (1.25, 2.5 and 5 µM) and miR-29a (1.25 µM) along with upregulation of miR-29a at 2.5 µM. Meanwhile, 1,4-BQ exposure in HSPCs significantly increased the miR-196b expression level (p<0.05) only in myeloid and pre-B lymphoid progenitors at 2.5 and 5 µM. Significant (p<0.05) reduction in expression of miR-181a in myeloid (1.25 µM), erythroid (5 µM) progenitors along with miR-29a in myeloid (1.25 µM) and pre-B lymphoid (5 µM) progenitors were noted following exposure to 1,4-BQ. Meanwhile, increased expression of miR-181a was observed in pre-B lymphoid progenitor upon exposure to 1,4-BQ, but only at 5 µM. As for TF studies, expression of HoxB4 protein was significantly increased (p<0.05) at all 1,4-BQ concentrations as compared to Bmi-1 and GATA3, which were significantly (p<0.05) elevated starting at 2.5 µM of 1,4-BQ. 1,4-BQ induces aberration of miRNAs and transcription factors protein expression that are involved in regulating self-renewing and differentiation pathways of HSPCs. Moreover, epigenetic toxicity as evidenced from the miRNAs expression was found to be mediated by a lineage-driven mechanism. The role of cell lineage in governing the toxicity of 1,4-BQ in HSPCs lineages deserves further investigation.

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

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

ROS-directed activation of Toll/NF-κB in the hematopoietic niche triggers benzene-induced emergency hematopoiesis.

Hematopoietic stem cells/progenitor cells (HSC/HPCs) orchestrate the hematopoietic process, effectively regulated by the hematopoietic niche under normal and stressed conditions. The hematopoietic niche provides various soluble factors which influence the differentiation and self-renewal of HSC/HSPs. Unceasing differentiation/proliferation/high metabolic activity of HSC/HPCs makes them susceptible to damage by environmental toxicants like benzene. Oxidative stress, epigenetic modifications, and DNA damage in the HSC/HPCs are the key factors of benzene-induced hematopoietic injury. However, the role of the hematopoietic niche in benzene-induced hematopoietic injury/response is still void. Therefore, the current study aims to unravel the role of the hematopoietic niche in benzene-induced hematotoxicity using a genetically tractable model, Drosophila melanogaster. The lymph gland is a dedicated hematopoietic organ in Drosophila larvae. A group of 30-45cells called the posterior signaling center (PSC) in the lymph gland acts as a niche that regulates Drosophila HSC/HPCs maintenance. Benzene exposure to Drosophila larvae (48h) resulted in aberrant hemocyte production, especially hyper-differentiation of lamellocytes followed by premature lymph gland dispersal and reduced adult emergence upon developmental exposure. Subsequent genetic experiments revealed that benzene-induced lamellocyte production and premature lymph gland dispersal were PSC mediated. The genetic experiments further showed that benzene generates Dual oxidase (Duox)-dependent Reactive Oxygen Species (ROS) in the PSC, activating Toll/NF-κB signaling, which is essential for the aberrant hemocyte production, lymph gland dispersal, and larval survival. Together, the study establishes a functional perspective of the hematopoietic niche in a benzene-induced hematopoietic emergency in a genetic model, Drosophila, which might be relevant to higher organisms.

Lnc-TC/miR-142-5p/CUL4B signaling axis promoted cell ferroptosis to participate in benzene hematotoxicity

Exposure to benzene causes damage to the hematopoietic system, but the mechanisms are still unclear. Competitive endogenous RNAs (ceRNAs) are the epigenetic regulatory axis of long non-coding RNA (lncRNA)-microRNA-mRNA, which are shown to play roles in benzene-induced hematotoxicity. Ferroptosis, a lipid peroxidation-dependent cell death, has been reported to be regulated by ceRNAs. We hypothesized that ceRNAs regulated ferroptosis to participate in benzene hematotoxicity. In this study, we observed that the expression of lncRNA TC (Lnc-TC) and CUL4B were increased, but miR-142-5p was decreased in benzene-exposed workers. Correlation analysis suggested that the ceRNAs had co-expression relationships, and were associated with blood cell counts. We further explored the role of ceRNA in vitro, and discovered that 1,4- benzoquinone (1,4-BQ) stimulated ferroptosis in AHH-1 cells by inhibiting the expression of GPX4 and SLC7A11, which was partially relieved by knockdown of Lnc-TC and CUL4B. Finally, by interfering with Lnc-TC and miR-142-5p expression, we confirmed that Lnc-TC acted as a microRNA sponge to reduce the accessibility and inhibition of miR-142-5p to CUL4B, thus increasing the expression of CUL4B. In summary, Lnc-TC/miR-142-5p/CUL4B signaling axis promoted cell ferroptosis to participate in benzene hematotoxicity, and was a potential biomarker for risk screening and health surveillance of benzene-exposed workers.

Let-7e-5p, a promising novel biomarker for benzene toxicity, is involved in benzene-induced hematopoietic toxicity through targeting caspase-3 and p21

Benzene is a common industrial chemical and environmental pollutant. However, the mechanism of hematotoxicity caused by exposure to low doses of benzene is unknown. Let-7e-5p pathway regulatory networks were constructed by bioinformatics analysis using a benzene-induced aplastic anemia (BIAA) mouse model. The MTT assay, EdU staining, flow cytometric analysis, dual luciferase reporter gene assay, and RIP assay were utilized to evaluate the effects of benzoquinone (1,4-BQ) on let-7e-5p pathway. This study consisted of 159 workers with a history of low-level benzene exposure and 159 workers with no history of benzene exposure. After the confounding factors were identified, the associations between let-7e-5p expression and hematotoxicity were assessed by multiple linear regression. Furthermore, we used four machine learning algorithms (decision trees, neural network, Bayesian network, and support vector machines) to construct a predictive model for detecting benzene-causing hematotoxicity in workers. In this study, compared with respective controls, let-7e-5p expression was decreased in BIAA mice and benzene-exposed workers. After 1,4-BQ exposure, let-7e-5p overexpression negatively regulated caspase-3 and p21 expression, protected cells from apoptosis, and facilitated cell proliferation. RIP assays, and dual luciferase reporter gene assays confirmed that let-7e-5p could target p21 and caspase-3 and regulate the cell cycle and apoptosis. The support vector machines classifier achieved the best prediction of benzene-induced hematotoxicity (prediction accuracy = 88.27, AUC = 0.83) by statistically characterizing the internal dose of benzene exposure and the oxidative stress index, as well as the expression levels of let-7e-5p pathway-related genes in benzene-exposed workers. Let-7e-5p may be a potential therapeutic target of benzene-induced hematotoxicity, provide a basis for evaluating the health hazards of long-term and low-dose benzene exposure in workers, and supply a reference for revising occupational health standards.

Early hematopoietic injury triggered by benzene characterized with inhibition of erythrocyte differentiation involving the mollicutes_RF39-derived citrulline.

Benzene poisoning is a common adverse blood outcome in occupational workers, manifested by hematopoietic dysfunction. However, the specific phenotype and its mechanisms of early hematopoietic toxicity caused by benzene remain unclear. After 15 days of exposure, the WBC levels were not significantly altered in benzene-exposed mice. However, the level of red blood cells (RBC) showed a significant decrease, and it was significantly and negatively correlated with urinary S-phenylmercapturic acid (SPMA). Notably, 5mg/kg benzene exposure significantly inhibited the renewal capacity and the number of colony formation of hematopoietic stem progenitor cells in mice, especially erythrocyte differentiation. These results suggested that the early hematopoietic toxicity phenotype caused by benzene was dominated by inhibition of erythroid differentiation rather than WBC-related inflammation. To further understand the underlying mechanisms of benzene-induced early hematopoietic toxicity, 16S rRNA sequencing and plasma metabolites analysis were conducted to investigate the impact of benzene exposure for 15 days on microbial composition and metabolic profile of mice. We found that short-term benzene exposure induced disturbances in gut microbiota and metabolism. The relative abundance of Mollicutes_RF39 at order levels was significantly reduced in benzene-exposed mice and was strongly correlated with hematopoietic indicators and urinary benzene markers. Interestingly, Mollicutes_RF39 might disturb the levels of eight metabolites, whereas Citrulline was highly linked to Mollicutes_RF39 (r=0.862, P=0.000). Consequently, Mollicutes_RF39-derived Citrulline might be the key regulator of early hematopoietic injury induced by benzene exposure. These findings promote the understanding of early hematotoxicity phenotypes and provide new perspectives on the underlying mechanisms of benzene-induced hematotoxicity.

Benzene Exposure Leads to Lipodystrophy and Alters Endocrine Activity In Vivo and In Vitro.

Benzene is a ubiquitous pollutant and mainly accumulates in adipose tissue which has important roles in metabolic diseases. The latest studies reported that benzene exposure was associated with many metabolic disorders, while the effect of benzene exposure on adipose tissue remains unclear. We sought to investigate the effect using in vivo and in vitro experiments. Male adult C57BL/6J mice were exposed to benzene at 0, 1, 10 and 100 mg/kg body weight by intragastric gavage for 4 weeks. Mature adipocytes from 3T3-L1 cells were exposed to hydroquinone (HQ) at 0, 1, 5 and 25 μM for 24 hours. Besides the routine hematotoxicity, animal experiments also displayed significant body fat content decrease from 1 mg/kg. Interestingly, the circulating non-esterified fatty acid (NEFA) level increased from the lowest dose (p trend < 0.05). Subsequent analysis indicated that body fat content decrease may be due to atrophy of white adipose tissue (WAT) upon benzene exposure. The average adipocyte area of WAT decreased significantly even from 1 mg/kg with no significant changes in total number of adipocytes. The percentages of small and large adipocytes in WAT began to significantly increase or decrease from 1 mg/kg (all p < 0.05), respectively. Critical genes involved in lipogenesis and lipolysis were dysregulated, which may account for the disruption of lipid homeostasis. The endocrine function of WAT was also disordered, manifested as significant decrease in adipokine levels, especially the leptin. In vitro cell experiments displayed similar findings in decreased fat content, dysregulated critical lipid metabolism genes, and disturbed endocrine function of adipocytes after HQ treatment. Pearson correlation analysis showed positive correlations between white blood cell (WBC) count with WAT fat content and plasma leptin level (r = 0.330, 0.344, both p < 0.05). This study shed light on the novel aspect that benzene exposure could induce lipodystrophy and disturb endocrine function of WAT, and the altered physiology of WAT might in turn affect benzene-induced hematotoxicity and metabolic disorders. The study provided new insight into understanding benzene-induced toxicity and the relationship between benzene and adipose tissue.

Ameliorative Effects of Bovine Lactoferrin on Benzene-Induced Hematotoxicity in Albino Rats.

Benzene (Bz) is one of the major products of the petrochemical industry globally, which induces aplastic anemia and leukemia in humans and animals. This study aimed to investigate the modulatory effects of bovine lactoferrin (bLf) on Bz-induced hematotoxicity in albino rats. Eighty male rats were randomly divided into eight groups: corn oil group [2 mL/kg body weight (BW)], bLf groups (100, 200, and 300 mg/kg BW), Bz group (Bz 2 mL/kg BW; corn oil 2 mL/kg BW), and Bz + bLf groups (Bz 2 mL/kg BW; corn oil 2 mL/kg BW; bLf 100, 200, and 300 mg/kg BW). Hematobiochemical results exhibited marked pancytopenia, a significant decrease in total protein, albumin, α2- and γ-globulin, ferritin, serum iron, and total iron-binding capacity (TIBC), and an increase in serum bioactivities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and erythropoietin hormone levels in Bz-treated rats. Histopathological examination revealed a marked reduction in all hematopoietic cell lines in the bone marrow (BM), necrosis in the white pulp of the spleen and cytosolic hydrops, and apoptosis of hepatocytes in the Bz-treated group. Rats treated with bLf (300 mg/kg BW) revealed marked increases in total protein, albumin, α2- and γ-globulin, ferritin, serum iron, and TIBC levels and decreases both in ALP and LDH bioactivities and erythropoietin hormone levels compared with the Bz-treated group. Histopathological results were concomitant with hematobiochemical parameters in rats treated with bLf (300 mg/kg BW), almost showing restoration of the normal cellularity of BM, the architecture of red and white pulps of the spleen, and even the normal hypertrophy of hepatocytes compared with the control groups. To conclude, bLf (300 mg/kg BW) can be recommended to treat Bz-induced hematotoxicity.

Ferroptosis is involved in the benzene-induced hematotoxicity in mice via iron metabolism, oxidative stress and NRF2 signaling pathway

Benzene is a pollutant that widely exists in the environment and in occupational workplaces. Its exposure is closely associated with hematological disorders and even leukemia, which poses a significant threat to public health. Thus, the underlying mechanisms should be explored. In the current study, it was investigated whether ferroptosis plays a role in benzene hematopoietic toxicity and related mechanisms. Mice were subcutaneously injected with benzene at 150 mg/kg b.w. to establish a hematotoxicity model. Four weeks later, the mice exposed to benzene exhibited a decrease in white blood cells, red blood cells, and hemoglobin level, as well as reduction in frequency of hematopoietic stem and progenitor cells (HS/PCs) and the colony forming abilities of CFU-G, CFU-M, CFU-GM, and CFU-GEMM. Simultaneously, apart from ferroptosis features in the mitochondrial morphology, decreased ATP and mitochondrial membrane potential, alterations in biochemical indices and gene expression were also observed, such as increased intracellular iron and lipid peroxidation, glutathione (GSH) depletion, and reduced glutathione peroxidase (GSH-Px) level, and upregulated PTGS2. Meanwhile, markedly altered expression of SLC7A11, GPX4, GCLC, NOX1, TFRC, FTH1, and FTL hinted that redox imbalance and dysfunction of iron uptake and storage are vital to induce ferroptosis. Additionally, decreased cytoplasmic NRF2 and increased nuclear NRF2 were also found, suggesting the activation of the NRF2 pathway. More importantly, inhibition of ferroptosis with ferrostatin-1 (Fer-1) or deferoxamine (DFO) partially relieved the hematopoietic injuries. Our findings imply that dysregulation in the system Xc−/GPX4 axis, iron metabolism, and activation of the NRF2 pathway play a crucial role in benzene-induced ferroptosis, and reveals that taking ferroptosis as a target may be a potential intervention strategy for benzene-induced hematotoxicity.

Plasma metabolomics study reveals the critical metabolic signatures for benzene-induced hematotoxicity

Metabolomics has been used to explore the molecular mechanism and screen biomarkers. However, the critical metabolic signatures associated with benzene-induced hematotoxicity remain elusive. Here, we performed a plasma metabolomics study in 86 benzene-exposed workers and 76 healthy controls, followed by a validation analysis in mice, to investigate the dynamical change of the metabolic profile. We found that 8 fatty acids were significantly altered in both benzene-exposed worker and benzene-exposed animal models. These metabolites were significantly associated with S-phenylmercapturic acid and WBC, and they mediated the benzene-induced WBC decline. Furthermore, in vivo results confirm that fatty acid levels were dynamically altered, characterized by a decrease at 15 days and then sharp increases at 30 and 45 days. Following these identified fatty acids, the potential metabolic pathways were investigated. Fatty acids, as precursors for fatty acid oxidation, may disturb the balance of fatty acid biosynthesis and degradation. Our results reveal that fatty acid metabolism was strongly reprogrammed after benzene exposure. This abnormal change of fatty acids might be the key metabolic signature associated with benzene-induced hematotoxicity.

LincRNA-p21 promotes p21-mediated cell cycle arrest in benzene-induced hematotoxicity by sponging miRNA-17-5p

Benzene is widely employed in manufacturing and causes hematotoxic effects and leukemia in humans. A long intergenic noncoding RNA (lincRNA)-microRNA (miRNA)-mRNA coexpression and competing endogenous RNA (ceRNA) regulatory network was constructed by bioinformatics analysis based on a benzene-induced aplastic anemia (BIAA) mouse model. In this population-based study, we observed a trend consistent with that in the BIAA mice: lincRNA-p21 and p21 were upregulated, while miRNA-17-5p expression was downregulated in benzene-exposed workers. Moreover, multiple linear regressions indicated that lincRNA-p21 was negatively associated with white blood cell (WBC) counts. Predictive thresholds of hematotoxicity were identified by ROC curve analysis with S-phenylmercapturic acid (SPMA) and lincRNA-p21 showing a better predictive ability than the other parameters and the combination of SPMA and lincRNA-p21 exhibiting the highest predictive value for hematotoxicity. LincRNA-p21 was predominantly present in the cytoplasm of bone marrow cells (BMCs) and K562 cells as assessed by fluorescence in situ hybridization (FISH). Upon exploring the underlying mechanism by which lincRNA-p21 mediates benzene-induced hematotoxicity, we observed that the negative regulation of 1,4-benzoquinone (1,4-BQ) on cell cycle arrest and inhibition of K562 cell proliferation was partially relieved by lincRNA-p21 knockdown, which can inhibit the expression of P21 and thereby suppress the toxic effects of 1,4-BQ. Finally, dual-luciferase reporter gene and RIP assay showed that, by acting as a sponge, lincRNA-p21 reduced the activity of miRNA-17-5p and consequently increased the expression of p21. In conclusion, our research suggested that benzene induces hematotoxicity via the lincRNA-p21/miRNA-17-5p/p21 signaling which might contribute to the underlying mechanism of lincRNA-p21 in benzene-induced hematotoxicity. Therefore, lincRNA-p21 can serve as a potential biomarker for the early detection of hematopoiesis inhibition in individuals with long-term exposure to low-dose benzene.

GCN5-mediated PKM2 acetylation participates in benzene-induced hematotoxicity through regulating glycolysis and inflammation via p-Stat3/IL17A axis

Benzene is a common environmental carcinogen that induces leukemia. Studies suggest that metabolic disorder has a relationship with the toxicity of benzene. Pyruvate kinase M2 (PKM2) is a key rate-limiting enzyme in glycolysis. However, the upstream and downstream regulatory mechanisms of PKM2 in benzene-induced hematotoxicity and the therapeutic effects of targeting PKM2 in vivo are unclear. This study aims to provide insights into the new mechanism of benzene-induced hematotoxicity and reveal the therapeutic significance of targeting PKM2. Herein, we demonstrated that PKM2-dependent glycolysis contributes to benzene-induced hematotoxicity by regulating inflammation reaction. Mechanistically, acetylated proteomics revealed that 1,4-benzoquinone (1,4-BQ) induced acetylation of PKM2 at position K66, and this modification contributed to the increase of PKM2 expression and can be inhibited by inhibition of acetyltransferase GCN5. Meanwhile, the elevated PKM2 was shown to prompt the activation of nuclear phosphorylated Stat3 (p-Stat3) and IL17A. Clinically, pharmacological inhibition of PKM2 alleviated the blood toxicity induced by benzene, which was mainly characterized by an increase in routine blood parameters and improvement of hematopoietic imbalance. Besides, elevated PKM2 is a promising biomarker in people occupationally exposed to benzene. Overall, we identified PKM2/p-Stat3/IL-17A axis participates in the hematotoxicity of benzene, and targeting PKM2 has certain therapeutic implications in hematologic diseases.

Metabolome-wide association study of occupational exposure to benzene.

Benzene is a recognized hematotoxin and leukemogen; however, its mechanism of action in humans remain unclear. To provide insight into the processes underlying benzene hematotoxicity, we performed high-resolution metabolomic profiling of plasma collected from a cross-sectional study of 33 healthy workers exposed to benzene (median 8-h time-weighted average exposure; 20 ppma), and 25 unexposed controls in Shanghai, China. Metabolic features associated with benzene were identified using a metabolome-wide association study (MWAS) that tested for the relationship between feature intensity and benzene exposure. MWAS identified 478 mass spectral features associated with benzene exposure at false discovery rate < 20%. Comparison to a list of 13 known benzene metabolites and metabolites predicted using a multi-component biotransformation algorithm showed five metabolites were detected, which included the known metabolites phenol and benzene diolepoxide. Metabolic pathway enrichment identified 41 pathways associated with benzene exposure, with altered pathways including carnitine shuttle, fatty acid metabolism, sulfur amino acid metabolism, glycolysis, gluconeogenesis and branched chain amino acid metabolism. These results suggest disruption to fatty acid uptake, energy metabolism and increased oxidative stress, and point towards pathways related to mitochondrial dysfunction, which has previously been linked to benzene exposure in animal models and human studies. Taken together, these results suggest benzene exposure is associated with disruption of mitochondrial pathways, and provide promising, systems biology biomarkers for risk assessment of benzene-induced hematotoxicity in humans.

The effects of glucose-6-phosphate dehydrogenase deficiency on benzene-induced hematotoxicity in mice

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme deficiency. Our previous study revealed the level of G6PD changed in wild type (WT) mice after benzene exposure. In this study, the pentose phosphate pathway (PPP) in regulation of benzene-induced hematotoxicity was investigated and other potential pathways were discovered in a G6PD deficiency mouse model. WT and G6PD mutation (G6PDmut) mice were exposed to benzene (diluted in corn oil) at doses of 0 and 160 mg/kg by subcutaneous injection for 5 days/week, 4 weeks. Peripheral blood samples and bone marrow cells (BMCs) were obtained and measured. The levels of nicotinamide adenine dinucleotide phosphate (NADPH),reduced glutathione (GSH) and malondialdehyde (MDA) were detected and comet assay was analyzed for DNA damage in BMCs. Finally, RNA sequencing (RNA-seq) of BMCs was performed. The results showed that white blood cells decreased significantly in G6PDmut mice compared with WT mice after benzene treatment. The ratio of hematopoietic stem/progenitor cells significantly decreased in G6PDmut mice exposed to benzene. The reduction of NADPH and GSH revealed the effect on PPP with G6PD deficiency, which then caused the increase of MDA and DNA damage. Finally, RNA-seq results suggested potential genes including SHROOM4, CAMK2B and REN1 played potential roles of G6PD deficiency on benzene-induced hematotoxicity. Renin-angiotensin system and cAMP signaling pathway were potentially involved in the process. Our study provides a better understanding for the effects of G6PD deficiency on benzene-induced hematotoxicity.

Glycine/glycine N-methyltransferase/sarcosine axis mediates benzene-induced hematotoxicity

Benzene, an important and widely used industrial chemical, is the cause of different types of blood disorders. However, the mechanisms of benzene-induced hematotoxicity are still unclear. This study aimed to explore the effects of benzene on metabolism, especially in amino acid metabolism, in human peripheral blood B lymphocyte cells (AHH-1 cells) treated with 1,4-benzoquinone (1,4-BQ) and in benzene-exposed population based on the un-targeted and targeted metabolomics platforms. The results showed that 1,4-BQ disturbed the metabolic activity, such as arginine biosynthesis, citrate cycle, glycine, serine, and threonine metabolism pathways, and significantly upregulated the ratio of sarcosine/glycine in vitro. Meanwhile, the targeted metabolomics further showed that the ratio of sarcosine/glycine was also increased in the benzene exposure population. Notably, the expression of glycine N-methyltransferase (GNMT), an enzyme catalyzing the transformation of glycine to sarcosine, was upregulated both in 1,4-BQ treated AHH-1 cells and benzene-exposed workers. These results imply that the glycine/GNMT/sarcosine axis was involved in benzene-induced hematotoxicity. Such evidence will help to develop a better understanding of the underlying mechanism of benzene-induced hematotoxicity at the level of amino acid metabolism.

Kefir milk alleviates benzene-induced immunotoxicity and hematotoxicity in rats.

The adverse health effects of benzene occupational and circumstance pollution exposure are an increasing concern. It leads to damage to various human tissues including bone marrow and ovarian tissues and many vital physiological processes. Previous studies showed that kefir is a rich probiotic, having protective effect, thanks to its antioxidant, anti-inflammatory, and immunomodulatory capacity. The purpose of this study was to evaluate the potential efficacy of kefir to remediate benzene toxicity in rat. Thirty-two female rats were randomly allocated and administered orally with benzene and/or kefir during a period of 21 consecutive days. At the end of the experiment, hematological and bone marrow cell changes were estimated. The animals exposed to benzene exhibited anemia and a significant decrease in the levels of white blood cell. Moreover, benzene led to the activation of gene expression of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6), a myelotoxicity in bone marrow cells. Our data showed that kefir treatment alleviated benzene-associated weight loss and increased the number of whole blood cells in peripheral blood and nucleated cells in the bone marrow. Furthermore, these physiological results were observed with animals showing high concentrations of lactic acid bacteria (LAB) determined from fecal samples, which are considered an indicator of kefir-associated microorganisms. Our study suggests that kefir is a potential nutritional supplement target to attenuate hematotoxicity induced by benzene.

Pyridoxine deficiency modulates benzene inhalation-induced hematotoxicity associated with hepatic CYP2E1 activity in B6C3F1 mice.

Pyridoxine is a co-factor in many enzymatic reactions and impacts of deficiency have been observed in affected populations. A possible modifying effect of pyridoxine deficiency on benzene toxicity was assessed in male B6C3F1 mice fed either a pyridoxine-deficient diet or a control diet. This treatment was combined with benzene inhalation exposure (100 ppm) or no benzene treatment. Pyridoxine-deficient mice exposed to 100 ppm benzene had significantly lower body, thymus and spleen weights. While total white blood cell counts, percentage of lymphocytes, hematocrit and hemoglobin levels were lower, the percentage of neutrophils was significantly higher in deficient and benzene-exposed mice compared to non-exposed controls. Hepatic CYP2E1 protein expression and activity in the deficient and exposed mice were also significantly higher compared to the non-exposed controls. A significant correlation between CYP2E1 activity and several hematological parameters was observed. These results demonstrated that pyridoxine deficiency significantly impacted benzene-induced hematotoxicity. Moreover, the observed agonistic effect of pyridoxinedeficiency and benzene inhalation exposure on CYP2E1 would seem to indicate an involvement of metabolism, but this needs to be further assessed.