Characterization of gut microbiota, metabolism and cytokines in benzene-induced hematopoietic damage.

Benzene exposure leads to hematopoietic dysfunction and is characterized clinically by a decrease in blood cells, but the underlying mechanisms remain elusive. Disturbed gut microbiota may induce host metabolic, immune disorders and the onset of disease. However, the characterization of gut microbiota, metabolism, cytokines and their association with benzene-induced hematopoietic toxicity lacks systematic evidence. Here, the microbiomics, metabolomics and cytokine network were applied to find out the critical characteristics of gut microbiota, metabolism and cytokines in mice involved in the benzene-induced hematopoietic toxicity. We found that the decline in hematopoietic stem cells was earlier than the hematological changes in the 5 mg/kg and 25 mg/kg benzene exposure groups. While 125 mg/kg benzene exposure resulted in a significant decline in whole blood cells. High-throughput sequencing results showed that benzene exposure disrupted homeostasis of gut microbiota, metabolism and cytokine in mice. 6 bacteria, 12 plasma metabolites and 6 cytokines were associated with benzene-induced hematopoietic damage. Notably, IL-5 was significantly increased in benzene exposure group in a dose-dependent manner, and a significant negative correlation was found between IL-5 and hematopoietic damage. We further found that increased Family_XIII_AD3011_group at the genus level and decreased Anaerotruncus_sp at the species level in benzene-exposed group were strongly associated with hematopoietic toxicity and IL-5. Furthermore, the abundance of Family_XIII_AD3011_group and Anaerotruncus_sp were negatively correlated with Adipic acid and 4-Hydroxyproline, respectively. Our findings indicated that altered flora structure of gut microbiota affects the metabolic phenotype which acts as messengers for the gut microbes, affecting host inflammation. This preliminary study provides new insight into the potential mechanisms of benzene-induced hematopoietic toxicity, further exploration by functional studies is required in the future.

Targeting IGF2BP1 alleviated benzene hematotoxicity by reprogramming BCAA metabolism and fatty acid oxidation.

Benzene is the main environmental pollutant and risk factor of childhood leukemia and chronic benzene poisoning. Benzene exposure leads to hematopoietic stem and progenitor cell (HSPC) dysfunction and abnormal blood cell counts. However, the key regulatory targets and mechanisms of benzene hematotoxicity are unclear. In this study, we constructed a benzene-induced hematopoietic damage mouse model to explore the underlying mechanisms. We identified that Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) was significantly reduced in benzene-exposed mice. Moreover, targeting IGF2BP1 effectively mitigated damages to hematopoietic function and hematopoietic molecule expression caused by benzene in mice. On the mechanics, by metabolomics and transcriptomics, we discovered that branched-chain amino acid (BCAA) metabolism and fatty acid oxidation were key metabolic pathways, and Branched-chain amino acid transaminase 1 (BCAT1) and Carnitine palmitoyltransferase 1a (CPT1A) were critical metabolic enzymes involved in IGF2BP1-mediated hematopoietic injury process. The expression of the above molecules in the benzene exposure population was also examined and consistent with animal experiments. In conclusion, targeting IGF2BP1 alleviated hematopoietic injury caused by benzene exposure, possibly due to the reprogramming of BCAA metabolism and fatty acid oxidation via BCAT1 and CPT1A metabolic enzymes. IGF2BP1 is a potential regulatory and therapeutic target for benzene hematotoxicity.

Gut microbiota-palmitoleic acid-interleukin-5 axis orchestrates benzene-induced hematopoietic toxicity.

Due to the annual increase in its production and consumption in occupational environments, the adverse blood outcomes caused by benzene are of concern. However, the mechanism of benzene-induced hematopoietic damage remains elusive. Here, we report that benzene exposure causes hematopoietic damage in a dose-dependent manner and is associated with disturbances in gut microbiota-long chain fatty acids (LCFAs)-inflammation axis. C57BL/6J mice exposed to benzene for 45 days were found to have a significant reduction in whole blood cells and the suppression of hematopoiesis, an increase in Bacteroides acidifaciens and a decrease in Lactobacillus murinus. Recipient mice transplanted with fecal microbiota from benzene-exposed mice showed potential for hematopoietic disruption, LCFAs, and interleukin-5 (IL-5) elevation. Abnormally elevated plasma LCFAs, especially palmitoleic acid (POA) exacerbated benzene-induced immune-inflammation and hematopoietic damage via carnitine palmitoyltransferase 2 (CPT2)-mediated disorder of fatty acid oxidation. Notably, oral administration of probiotics protects the mice against benzene-induced hematopoietic toxicity. In summary, our data reveal that the gut microbiota-POA-IL-5 axis is engaged in benzene-induced hematopoietic damage. Probiotics might be a promising candidate to prevent hematopoietic abnormalities from benzene exposure.

Oxidative stress-affected ACSL1 hydroxymethylation triggered benzene hematopoietic toxicity by inflammation and senescence.

Long-term benzene exposure is harmful and causes hematopoietic dysfunction. However, the mechanism of benzene hematopoietic toxicity is still unclear. Acyl-CoA Synthetase Long-Chain Family Member 1 (ACSL1) has been found to participate in the progress of a variety of benign and malignant diseases, but there is no research about its effect on benzene-induced hematopoietic toxicity. Herein, We exposed C57BL/6J mice to benzene to construct an in vivo model. Human peripheral blood mononuclear cells (THP-1 cells) were treated with benzene metabolite 1, 4-BQ to construct an in vitro model. We observed that the ACSL1 expression was upregulated both in vivo and in vitro. Moreover, inhibition of ACSL1 relieved inflammation and senescence development in vitro, suggesting that ACSL1 mediates inflammation and senescence. As for the regulation mechanism of ACSL1 expression, it is closely related to hydroxymethylation modification. This was proved by hydroxymethylated DNA immunoprecipitation (hMeDIP) experiments. Furthermore, oxidative stress influenced the hydroxymethylation process. These results showed that benzene hematopoietic toxicity occurs through the induction of oxidative stress and thus the regulation of ACSL1 hydroxymethylation, which in turn mediates inflammation and senescence. Thus, this study might be of great significance in identifying and preventing benzene exposure in the early stage.

Probiotics ameliorate benzene-induced systemic inflammation and hematopoietic toxicity by inhibiting Bacteroidaceae-mediated ferroptosis.

The intestinal microbiota is associated with the development of benzene-induced hematopoietic toxicity. Modulation of intestinal homeostasis by probiotic supplementation has been considered an effective strategy to prevent adverse health effects. However, the role and mechanism of probiotics in benzene-induced hematopoietic toxicity are unclear. After 45 days of exposure, benzene caused bone marrow hematopoietic toxicity in mice. Furthermore, we found that benzene altered the intestinal barrier in mice, leading to an increase in the abundance of Bacteroidaceae and the activation of systemic inflammation. Interestingly, Fe2+ accumulation, lipid peroxidation, and differential expression of ferroptosis proteins were observed in the intestinal tissues of benzene-exposed mice. After fecal microbiota transplantation, stool microbes from benzene-exposed mice led to the development of intestinal ferroptosis in recipient mice. In particular, oral probiotics significantly reversed elevated Bacteroidaceae and intestinal ferroptosis, ultimately improving benzene-induced hematopoietic damage. We further used the benzene metabolite 1,4-BQ to treat human normal colonic epithelial cells (NCM460) and intervened with the ferroptosis inhibitor liproxstatin-1 (Lip-1) to validate the relationship between intestinal ferroptosis and inflammation. The results showed that 1,4-BQ treatment resulted in increased intracellular ROS levels and abnormal expression of ferroptosis proteins and the inflammatory factors IL-5 and IL-13. However, the use of Lip-1 significantly inhibited oxidative stress, ferroptosis, and inflammation in NCM460 cells. This result suggested that ferroptosis might be involved in benzene-induced hematopoietic toxicity by mediating Th2-type systemic inflammation. Overall, these findings revealed a role for Bacteroidaceae-intestinal ferroptosis-inflammation in benzene-induced hematopoietic toxicity and highlighted that probiotics could be a promising strategy to prevent adverse hematologic outcomes.

The role of N6-methyladenosine modification in benzene-induced testicular damage and the protective effect of melatonin.

Benzene is a universal ambient pollutant. Population-based studies have shown that benzene exposure affects male fertility. However, the mechanism of benzene-induced reproductive toxicity is unknown. Here, we established a dynamic inhalation model and exposed C57BL/6J mice to 0, 10, and 50 ppm benzene (6 h/day, 6 days/week, 7 weeks). Our study revealed that benzene exposure caused testicular injury, including structural damage to spermatogenic tubules, reduced semen quality, and decreased testosterone levels. In addition, the decrease in the global level of N6-Methyladenosine (m6A) and the change of m6A important regulatory enzymes in mice testes suggested that m6A was involved in the benzene-induced testicular injury. Further genome-wide m6A methylation analysis showed that 1469 differential m6A peaks were present in the testes of control and benzene groups, indicating that benzene exposure modulated m6A methylation in testes. Furthermore, the comprehensive analysis of m6A-sequencing and transcriptome revealed that hypermethylated Rara and its consequent reduced expression impaired the sperm production process. In particular, melatonin alleviated benzene-induced testicular injury by modulating m6A-related genes. Overall, our research provides a new idea and fundamental knowledge into the possible mechanisms of m6A modifications in benzene-induced testicular impairment, as well as a new experimental basis for benzene-induced male fertility therapy.

Genus unclassified_Muribaculaceae and microbiota-derived butyrate and indole-3-propionic acid are involved in benzene-induced hematopoietic injury in mice.

Benzene is a group I carcinogen determined by IARC. The prevalence of benzene in occupational and general environments increases the risk of acute myeloid leukemia (AML) among workers and childhood leukemia. However, the mechanism of hematotoxicity induced by benzene remains unclear. Recently, the gut microbiota has been regarded as a pivotal part of normal and malignant hematopoiesis. Therefore, in this study, we explored the function of gut microbiota in hematopoietic injury induced by benzene by 16S rRNA sequencing. We found that benzene exposure caused bone marrow damage, hematopoietic stem and progenitor cells (HSPCs) dysfunction, and peripheral blood cell reduction. Moreover, intestinal barrier damage and gut microbiota dysbiosis were also observed in benzene-exposed mice. Interestingly, two gut flora, Lachnospiraceae_NK4A136_group and unclassified_Muribaculaceae, were significantly up-regulated and associated with hematopoietic indicators, suggesting that gut-host crosstalk might mediate benzene hematotoxicity. Microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), bile acids, and tryptophan metabolites, are the primary mediators of the gut-host crosstalk. Therefore, we conducted absolute quantitative metabolomics to investigate the impact of benzene exposure on these metabolites in mice. The results showed that the concentration of SCFA butyrate, tryptophan metabolites kynurenine, and Indole-3-propionic acid (IPA) were significantly altered after benzene exposure. However, no difference was found in bile acids. Significant correlations were found between altered metabolites and hematopoietic indicators. We then investigated the flora that derived these metabolites. Lachnospiraceae_NK4A136_group and unclassified_Muribaculaceae were enriched in the butyrate metabolism and tryptophan metabolism pathways. Correlation analysis further suggested that unclassified_Muribaculaceae was positively associated with butyrate (r = 0.588, P < 0.05) and IPA (r = 0.59, P < 0.05). The above results demonstrated that unclassified_Muribaculaceae and microbiota-derived butyrate and IPA were involved in hematopoietic toxicity caused by benzene. This study provides insight into gut microbiota-derived metabolites-host crosstalk in benzene hematopoietic toxicity.

The gut-brain axis involved in polystyrene nanoplastics-induced neurotoxicity via reprogramming the circadian rhythm-related pathways.

The production of plastic is still increasing globally, which has led to an increasing number of plastic particles in the environment. Nanoplastics (NPs) can penetrate the blood-brain barrier and induce neurotoxicity, but in-depth mechanism and effective protection strategies are lacking. Here, C57BL/6 J mice were treated with 60 µg polystyrene NPs (PS-NPs, 80 nm) by intragastric administration for 42 days to establish NPs exposure model. We found that 80 nm PS-NPs could reach and cause neuronal damage in the hippocampus, and alter the expression of neuroplasticity-related molecules (5-HT, AChE, GABA, BDNF and CREB), and even affect the learning and memory ability of mice. Mechanistically, combined with the results of hippocampus transcriptome, gut microbiota 16 s ribosomal RNA and plasma metabolomics, we found that the gut-brain axis mediated circadian rhythm related pathways were involved in the neurotoxicity of NPs, especially Camk2g, Adcyap1 and Per1 may be the key genes. Both melatonin and probiotic can significantly reduce intestinal injury and restore the expression of circadian rhythm-related genes and neuroplasticity molecules, and the intervention effect of melatonin is more effective. Collectively, the results strongly suggest the gut-brain axis mediated hippocampal circadian rhythm changes involved in the neurotoxicity of PS-NPs. Melatonin or probiotics supplementation may have the application value in the prevention of neurotoxicity of PS-NPs.

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, 5 mg/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, 16 S 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.

Melatonin and probiotics ameliorate nanoplastics-induced hematopoietic injury by modulating the gut microbiota-metabolism.

Plastic pollution has become a non-negligible global pollution problem. Nanoplastics (NP) can reach the bone marrow with blood circulation and develop hematotoxicity, but potential mechanisms and prevention strategies are lacking. Here, we report the biological distribution of NP particles in the bone marrow of mice and hematopoietic toxicity after exposure to 60 µg of 80 nm NP for 42 days. NP exposure inhibited the capability of bone marrow hematopoietic stem cells to renew and differentiate. Notably, probiotics and melatonin supplementation significantly ameliorated NP-induced hematopoietic damage, and the former was superior to the latter. And interestingly, melatonin and probiotic interventions may involve different microbes and metabolites. After melatonin intervention, creatine showed a stronger correlation with NP-induced gut microbiota disorders. In contrast, probiotic intervention reversed the levels of more gut microbes and plasma metabolites. Of these, threonine, malonylcarnitine, and 3-hydroxybutyric acid might be potential performers in the regulation of hematopoietic toxicity by gut microbes, as they had a more significant relationship with the identified microbes. In conclusion, supplementation with melatonin or probiotics may be two candidates to prevent hematopoietic toxicity attributable to NP exposure. Also, the multi-omics results may lay the foundation for future investigations into in-depth mechanisms.

Iron-dependent ferroptosis participated in benzene-induced anemia of inflammation through IRP1-DHODH-ALOX12 axis.

Benzene, a widely existing environmental pollutant, gives huge harm to the hematopoietic system. Iron is one of the raw materials for the creation of blood cells, but the role of iron in the blood toxicity of benzene is still unknown. Here, we examined the role of iron homeostasis in benzene-induced toxicity both in vivo and in vitro. In this study, mice exposed to benzene at 50 ppm for 8 weeks demonstrated the anemia of inflammation, mainly manifested as the decreased serum Fe2+, increased serum ferritin and inflammation factors (TNF-α, IL6, IL1ß) in the plasma of mice. Furthermore, we found that iron maldistribution in the spleen and bone marrow is accompanied by inflammation reaction and ferroptosis. In the vitro study, benzene metabolite 1,4-BQ stimulated the obvious ROS production and ferroptosis activation in the normal B lymphocytes cells. Meanwhile, from the molecular perspective, the combined proteomics and transcriptome enriched the ferroptosis pathway, and we further confirmed the increased expression of iron regulator IRP1, ferroptosis-regulator DHODH, and fatty acids metabolism enzyme ALOX12 were the crucial participators in regulating benzene-mediated iron metabolism imbalance and ferroptosis. Particularly, the targeted and un-targeted metabolomics in the vivo and vitro study further emphasized the importance of DHODH in benzene-induced ferroptosis. In conclusion, this study revealed that iron-dependent ferroptosis participated in benzene-induced anemia of inflammation and provided a constructive perspective on targeting ferroptosis for the prevention and control of benzene toxicity.

Shikonin targets to m6A-modified oxidative damage pathway to alleviate benzene-induced testicular injury.

Benzene exposure causes reproductive toxicity through oxidative damage. However, the specific mechanisms of benzene-induced testicular damage and the potential therapeutic drugs remain poorly understood. In the present study, C57BL/6J mice have been exposed to 0 and 150 mg/kg benzene for four weeks. Then, we found that benzene exposure induced testicular damage in mice, mainly manifested by decreased testicular coefficients, abnormal semen parameters and HE-stained intraepithelial vacuolation. On the mechanism, benzene exposure activated Kelch Like ECH Associated Protein 1/Nuclear factor-erythroid 2 related factor 2 (Keap1/Nrf2) and NF-κB signaling pathway, then promoted apoptosis and inflammatory responses in testes. In vitro, massive reactive oxygen species (ROS) production and a large number of apoptotic cells were observed after 1,4-BQ treatment of GC-2 cells. Furthermore, benzene altered the expression of three important RNA methylation modulator genes, methyltransferase-like 3 (Mettl3), AlkB homolog 5 (Alkbh5) and YTH domain containing 2 (Ythdc2). Moreover, both m6A modification and mRNA levels of NF-κB increased with benzene exposure. Inspiringly, shikonin alleviated benzene-induced male reproductive damage by targeting m6A-modified NF-κB in mice testes. Our study provides new insights into molecular mechanisms of RNA m6A modification in benzene-induced reproductive injury and directions to find potential drugs for the treatment of male infertility.

Polystyrene micro-/nanoplastics induced hematopoietic damages via the crosstalk of gut microbiota, metabolites, and cytokines.

Micro-/nanoplastics (MNPLs), novel environmental pollutants, widely exist in the environment and life and bring health risks. Previous studies have shown that NMPLs can penetrate bone marrow, but whether they cause hematopoietic damage remains uncertain. In this study, C57BL/6J mice were treated with polystyrene MNPLs (PS-MNPLs, 10 µm, 5 µm and 80 nm) at 60 µg doses for 42 days by intragastric administration. We evaluated the hematopoietic toxicity induced by MNPLs and potential mechanisms via combining 16S rRNA, metabolomics, and cytokine chips. The results demonstrated that PS-MNPLs induced hematopoietic toxicity, which was manifested by the disorder of bone marrow cell arrangement, the reduction in colony-forming, self-renewal and differentiation capacity, and the increased proportion of lymphocytes. PS-MNPLs also disrupted the homeostasis of the gut microbiota, metabolism, and inflammation, all of which were correlated with hematotoxicity, suggesting that abnormal gut microbiota-metabolite-cytokine axes might be the crucial pathways in MNPLs-induced hematopoietic injury. In conclusion, our study systematically demonstrated that multi-scale PS-MNPLs induced hematopoietic toxicity via the crosstalk of gut microbiota, metabolites, and cytokines and provided valuable insights into MNPLs toxicity, which was conducive to health risk assessment and informed policy decisions regarding PS-MNPLs.