Benzene-induced Toxicity Research Articles

Extracellular vesicles-derived long noncoding RNAs participated in benzene hematotoxicity by mediating apoptosis and autophagy

Benzene is a common contaminant in the workplace and wider environment, which induces hematotoxicity. Our previous study has implicated that lncRNAs mediated apoptosis and autophagy induced by benzene. Nevertheless, the roles of extracellular vesicle(EVs)-derived lncRNAs in benzene toxicity are unknown. However, the role of EVs and EVs-derived lncRNAs in benzene-induced toxicity remains unclear. In this research, we explored the function of EVs and EVs-derived lncRNAs in cell-cell communication through benzene-induced apoptosis and autophagy. Our findings demonstrated that EVs derived from 1,4-BQ-treated cells treated cells and coculture with 1,4-BQ-treated cells enhanced apoptosis and autophagy via regulating the pathways of PI3K-AKT-mTOR and chaperone-mediated autophagy. Treating with GW4869 in 1,4-BQ-treated cells significantly inhibited EV secretion, which reduced apoptosis and autophagy. Furthermore, we identified a set of differentially expressed autophagy- and apoptosis-related lncRNAs using EVs-derived lncRNA sequencing. Among them, 8 candidate lncRNAs were upregulated in EVs derived from 1,4-BQ-treated cells, as determined by lncRNA sequencing and qRT–PCR. Importantly, these lncRNAs were also increased in the serum EVs of benzene-exposed workers. 1,4-BQ-treated cells released EVs that transfer differentially expressed lncRNAs, thereby inducing apoptosis and autophagy in the recipient cells. The above results support the hypothesis that EVs-derived lncRNAs participate in intercellular communication during benzene-induced hematotoxicity and function as potential biomarkers for risk assessment of benzene-exposed workers.

Ameliorative Potentials of Quercetin in Some Histo-Physiological and Biochemical Parameters Against Alterations Induced Benzene Inhalation in Albino Rats

Present study investigates the protective role of quercetin in reducing benzene-induced toxicity in rats. Sixteen adult rats, weighing 200-250 g, were selected. They were divided into four groups: 1. Control group, 2. Benzene inhaled group, 3. Quercetin group and 4. Group of benzene and quercetin in combination. Biochemical, spermatological parameters, and histopathological changes in lungs were recorded. Results of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea and creatinine levels in the serum of rats were higher in benzene exposed rats than in unexposed rats. Conversely, exposure to benzene led to a decrease in sperm quality compared to the unexposed rats. Histopathological examinations of the lung tissues revealed structural changes in exposed rats, including emphysema, thinning of the wall of the alveolar sac, congestion between the alveolar sacs and more elasticity in the wall of smooth muscles. The present study showed that quercetin treatment can reverse the negative effects of benzene in approximately all studied parameters and showed amelioration in the histological anomalies induced by benzene toxicity.

Effect of Spanish Black Radish Extract Against Benzyne Induced Toxicity in Rats

Benzene is one of the main products of the petrochemical industry worldwide, which causes aplastic anemia and leukemia in both people and animals. This study was designed to examine the modulatory effects of black radish on Benzene-induced toxicity in male rats. Thirty-five rats were divided into 7 groups (5 rats in each group), G1 negative control group; G2 positive control group (benzene treated group); G3 and G4 were treated after development of leukemia with Black radish aqueous extract (100mg/kg and 200 mg/kg respectively), while G5 and G6 were treated with Black radish ethanolic extract (100mg/kg and 200 mg/kg and G7 was treated with cyclophosphamide. Significant pathological variations in the liver, kidney and spleen tissues were detected in all experimental groups except for the control group. Our results indicate that benzyne induces irreversible adverse effects on the livers, kidneys, and spleen of rats.

Characterization of lymphocyte subsets and intestinal short-chain fatty acids in benzene-induced immunosuppressive mice.

Exposure to benzene causes immunosuppression, but the mechanism has not been clarified. In this study, mice were subcutaneously injected with different concentrations (0, 6, 30 and 150 mg/kg) of benzene for four weeks. The lymphocytes of bone marrow (BM), spleen and peripheral blood (PB) and the level of short-chain fatty acids (SCFAs) in mouse intestine were measured. The results showed that benzene exposure led to a reduction in CD3+ and CD8+ lymphocytes in mouse BM, spleen and PB, and CD4+ lymphocytes were increased in mouse spleen but decreased in mouse BM and PB after 150 mg/kg benzene exposure. In addition, Pro-B lymphocytes were reduced in mouse BM in the 6 mg/kg group. Besides, the levels of IgA, IgG, IgM, IL-2, IL-4, IL-6, IL-17a, TNF-α and IFN-γ in mouse serum were reduced after benzene exposure. Furthermore, the levels of acetic, propionic, butyric and hexanoic acid were reduced in mouse intestine, and the AKT-mTOR signaling pathway was activated in mouse BM cells after benzene exposure. Our results demonstrate that benzene induced immunosuppression in mice, and the B lymphocytes in BM are more sensible to benzene-induced toxicity. The reduction in mouse intestinal SCFAs as well as the activation of AKT-mTOR signaling may be related to the occurrence of benzene immunosuppression. Our study provides new insight for further mechanistic research on benzene-induced immunotoxicity.

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 50ppm for 8 weeks demonstrated the anemia of inflammation, mainly manifested as the decreased serum Fe2+, increased serum ferritin and inflammation factors (TNF-α, IL6, IL1β) 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.

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.

Linalool reverses benzene-induced cytotoxicity, oxidative stress and lysosomal/mitochondrial damages in human lymphocytes

Benzene exposure results in bone marrow suppression, leading to a decrease in the number of circulating white blood cells, an increased risk of chronic lymphocytic leukemia, acute myeloid leukemia and aplastic anemia. Since the mechanism of induction of benzene toxicity is due to active metabolites through cytochrome p450 enzymes and production of reactive oxygen species (ROS), we hypothesized that natural compound such linalool with anti-inflammatory/antioxidant properties could be effective in reducing its toxicity. Lymphocytes isolated from healthy individuals were simultaneously cotreated with different concentrations of LIN (10, 25 and 50 µM) and benzene (50 µM) for 4 h at 37 °C. After incubation, the toxicity parameters such cytotoxicity, ROS formation, lysosomal membrane integrity, mitochondria membrane potential (ΔΨm) collapse, oxidized/reduced glutathione (GSH/GSSG) and malondialdehyde (MDA) were analyzed using biochemical and flow cytometry evaluations. Our data showed that benzene (50 µM) induced a significant increase in cytotoxicity, ROS formation, mitochondrial membrane potential (MMP) collapse, lipid peroxidation and oxidative stress while LIN with antioxidant potential reversed the toxic effects of benzene on isolated human lymphocytes. Our results suggest that LIN reduces and reverses benzene-induced cytotoxicity, oxidative stress and lysosomal/mitochondrial damages in human lymphocyte. This study demonstrated that cotreatment of LIN with benzene can reduce several parameters indicative of oxidative stress. As such, LIN could represent a potential therapeutic agent in reducing certain aspects of benzene-induced toxicity.

Screening of biomarkers in exhaled breath of mice exposed to benzene

Objective: To screen the biomarkers in the exhaled breath of mice exposed to benzene by using exhaled breath online analysis system. Methods: Thirty 8-week-old male C57BL/6 mice were randomly divided into six groups (0, 3, 32, 324, 648, and 1 296 mg/m3) and treated with benzene vapour for 28 days. At the end of the exposure, the peripheral blood cell counts and blood glutathione (GSH) were detected. The content of malondialdehyde (MDA) in HL60 cells treated by mice plasma was examined. Exhaled breath data from mice were collected by Secondary electrospray ionization source high resolution mass spectrometry (SESI-HRMS). Targeted analysis underlying benzene metabolites and oxidative stress metabolites was performed to screen the biomarkers in exhaled breath. Results: After benzene exposure, the number of peripheral blood cells was decreased in different degrees, particularly in the white blood cells (WBC) number. The WBC in 32 and 324 mg/m3 groups was declined by 27.76% and 52.87%, respectively compared to that in control group (P<0.05). Meanwhile, compared with the control group, the GSH content of peripheral blood cells from 324 mg/m3 group decreased by 13.16% (P<0.05). In addition, MDA content was increased by 18.11% in HL60 cells treated with plasma from 324 mg/m3 group mice (P<0.05). The phenol, hydroquinone/catechol, benzenetriol and trans, trans-Muconic acid (t,t-MA) in the exhaled gas of mice could be used as biomarkers for benzene exposure (R2>0.8, P<0.001). The peak intensity of five small molecular metabolites related to oxidative stress (ω-carboxylic fatty acid C5H10O3, ω-carboxylic fatty acid C6H12O3, glutamate, cysteine and MDA) increased with the increase of benzene concentration (P<0.05), which was negatively correlated with WBC decline (P<0.001), suggesting that these molecules mignt be used as biomarkers of benzene-induced toxicity. Conclusions: Phenol, hydroquinone/catechol, benzenetriol and trans, trans-Muconic acid (t,t-MA) in exhaled breath of mice could be used as biomarkers for benzene exposure; ω-carboxylic fatty acid C5H10O3, ω-carboxylic fatty acid C6H12O3, glutamate, cysteine and MDA might be used as markers of benzene-induced toxicity.

Association between benzene exposure, serum levels of cytokines and hematological measures in Chinese workers: A cross-sectional study

BackgroundLow benzene exposure leads to hematotoxicity, but we still lack sensitive early monitoring and early warning markers. Benzene is associated with inflammation, which is mainly mediated by cytokines network. However, until now few studies have conducted high-throughput detection of multi-cytokines to get a global view of cytokine changes and screen for markers of benzene-induced toxicity. We hypothesized that cytokine profiles mediate benzene-induced hematotoxicity. Methods228 subjects consisting of 114 low benzene exposed workers and 114 healthy controls were recruited at Research Center of Occupational Medicine, Peking University Third Hospital, Beijing. The serum concentrations of 27 cytokines were detected by cytokinomics array, urinary benzene series metabolites were measured by UPLC-MS/MS, and peripheral blood cell counts were observed by basic blood test. ResultsAmong 27 cytokines, IL-9 and MIP1-α were significantly lower, but IL-4, IL-10, IL-15, MCP-1, TNF-α and VEGF were significantly higher in benzene exposure group than controls. Urinary benzene metabolite S-phenylmercapturic acid (S-PMA) was significantly higher in benzene exposure group and had a negative linear relationship with WBC count. S-PMA was only significantly associated with IL-9, meanwhile IL-9, IL-15 and VEGF had a positive linear relationship with WBC count. The bootstrapping mediation models showed that the effect of S-PMA on WBC count was partially explained by IL-9 for 10.11%. ConclusionThis study suggests that exposure to benzene was associated with alternation of blood cell count and cytokine profiles in workers exposed to low levels of benzene, especially decreases of WBC count and IL-9. We also found IL-9 partially mediated the effect of low benzene exposure on WBC count, which may be a potential and promising early monitoring and early warning marker of benzene hematotoxicity.

Toxicity in hematopoietic stem cells from bone marrow and peripheral blood in mice after benzene exposure: Single-cell transcriptome sequencing analysis

Benzene is a ubiquitous, occupational, and environmental hematotoxic and leukemogen. Damage to hematopoietic stem cells (HSCs) induced by benzene and its metabolites is a key event in bone marrow (BM) depression and leukemogenesis. There are no reports on transcriptome profiles of HSCs following benzene exposure. Here, Smart-seq2 single-cell transcriptome sequencing was used to detect transcriptomic alternations in BM HSCs and peripheral blood HSCs (PBSCs) in male C57B/6 mice exposed to benzene. We found that benzene caused hematotoxicity which was confirmed by routine blood test, pathological examination, and HSCs percentage analysis. A total of 1514 differentially expressed genes (DEGs) in BM HSCs and 1703 DEGs in PBSCs were screened after treatment with benzene. Weighted gene correlation network analysis revealed that pathways in cancer, transcriptional misregulation in cancer, and hematopoietic cell lineage are vital pathways involved in benzene-induced toxicity in BM HSCs, whereas hematopoietic cell lineage and leukocyte transendothelial migration are critical pathways in PBSCs. Of note, there were 164 common DEGs in both HSCs, out of which 53 genes were co-regulated in both types of HSCs. Subsequent pathway analysis of these 53 genes indicated that the most relevant pathways involved neutrophil degranulation and CD93 localized in the core of the network of the 53 genes, which are known to regulate leukemia stem cell self-renewal and quiescence. Our results could enhance our understanding of HSC responses to benzene, facilitate the identification of potential molecular biomarkers and future studies on its mechanism of toxicity toward HSCs.

Genetic, epigenetic, and lineage-directed mechanisms in benzene-induced malignancies and hematotoxicity targeting hematopoietic stem cells niche.

Benzene is a known hematotoxic and leukemogenic agent with hematopoietic stem cells (HSCs) niche being the potential target. Occupational and environmental exposure to benzene has been linked to the incidences of hematological disorders and malignancies. Previous studies have shown that benzene may act via multiple modes of action targeting HSCs niche, which include induction of chromosomal and micro RNA aberrations, leading to genetic and epigenetic modification of stem cells and probable carcinogenesis. However, understanding the mechanism linking benzene to the HSCs niche dysregulation is challenging due to complexity of its microenvironment. The niche is known to comprise of cell populations accounted for HSCs and their committed progenitors of lymphoid, erythroid, and myeloid lineages. Thus, it is fundamental to address novel approaches via lineage-directed strategy to elucidate precise mechanism involved in benzene-induced toxicity targeting HSCs and progenitors of different lineages. Here, we review the key genetic and epigenetic factors that mediate hematotoxicological effects by benzene and its metabolites in targeting HSCs niche. Overall, the use of combined genetic, epigenetic, and lineage-directed strategies targeting the HSCs niche is fundamental to uncover the key mechanisms in benzene-induced hematological disorders and malignancies.

The effects of grape seed extract against toxicity of benzene on liver and kidney tissues of albino mice: biochemical evaluation

Objective: The objective of the present study was to investigate the possible effects of grape seed extract (GSE) against benzene-induced toxicity in Swiss albino mice. Methods: The animals were randomly divided into six groups each containing six mice. Group I, treated with distilled water; Group II and III orally treated with 50 mg/kg and 150 mg/kg body weight GSE, respectively. Group IV, orally treated with 250 mg/kg body weight benzene by using feeding cannula; Group V, orally treated with 50 mg/kg body weight GSE + 250 mg/kg body weight benzene; Group VI, orally treated with 150 mg/kg body weight GSE + 250 mg/kg of body weight benzene for 50 consecutive days. At the end of experimental period all mice were sacrificed; blood, liver and kidney tissues were removed after post-mortem examination. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine levels were analyzed from serum. Malondialdehyde (MDA) and reduced glutathione (GSH) levels were analyzed from isolated tissues. And also histopathological examinations of liver and kidney tissues were investigated. Results: Serum AST, ALT, ALP, BUN and creatinine levels were slightly increased in Group IV compared with the other tested groups (p&lt;.05). Benzene-induced toxicity caused a significant decrease in GSH levels and a significant rise in MDA levels of liver and kidney tissues. Oral treatment with GSE significantly ameliorated the indices of hepatotoxicity, nephrotoxicity and lipid peroxidation induced by benzene. Both doses of GSE provided significant protection and the strongest effects were observed at the dose level of 150 mg/kg. Conclusion: Consequently, it was found that GSE has a significant positive effect in benzeneinduced toxicity, and its GSE effect is dose dependent.

The Cytotoxic Effect of the Benzene Metabolite Hydroquinone is Mediated by the Modulation of MDR1 Expression via the NF-κB Signaling Pathway

Background/Aims: Benzene is a toxic chemical whose leukemogenic effects have been studied for decades. The mechanisms of benzene-induced toxicity and leukemogenicity are not fully understood, although the involvement of several pathways has been suggested, including oxidative stress, DNA damage, cell cycle regulation and programmed cell death. In the present study, we investigated the effect of hydroquinone (HQ), a major benzene metabolite, on the viability of bone marrow derived mesenchymal stem cells (BMSCs) and explored the underlying mechanisms. Methods: First, we study the the effect of HQ on BMSCs cell viability, apoptosis and the expressions of MDR1 and NF-κB. Then we investigate the MDR1 on cell viability and cell apoptosis for BMSCs under HQ treatment. Finally, we studied the impact of nuclear factor κB (NF-κB) on the expression of MDR1. Results: Our results showed that HQ decreased cell viability and promoted cell apoptosis of BMSCs, as determined by the MTT assay and flow cytometry. Western blotting and quantitative PCR showed that HQ downregulated the expression of the MDR1 gene by inhibiting the activation and nuclear translocation of the transcription factor NF-κB. Overexpression of MDR1 attenuated the inhibitory effect of HQ on cell viability in BMSC. Conclusion: The results of the present study suggest the involvement of the multidrug resistance membrane transporter MDR1 and the NF-κB pathway in the cytotoxicity of benzene and its metabolites. Further studies are necessary to clarify the role of the pathways involved and the crosstalk between them in mediating the effects of HQ in bone marrow progenitor cells.

Influence of testosterone on phase-II enzymes in liver and kidney of benzene-treated rats

Testosterone manifests some protective effects against benzene-induced toxicity in rats. However, mechanism of protection remains to be established. Data showed that testosterone modulates conjugation of reactive metabolites of benzene by influencing phase-II enzymes viz. glutathione-S-transferase, glutathione peroxidase, and catalase in both liver and kidney. These observations are supported by the opposite results obtained in castrated rats. It is postulated that testosterone decreases the formation of reactive oxygen species resulting into an increase in phase-II enzymes. Enhanced activity of these antioxidant enzymes is responsible for DNA strand repair as demonstrated by short comet tail length in liver and kidney of benzene and testosterone treated rats. Castration alters benzene pharmacokinetics by influencing these enzymes, a response which may be abolished by testosterone supplementation.

The protective role of grape seed extract against chronic toxicity of benzene in swiss albino mice

Benzene is an aromatic hydrocarbon often used for industrial purposes. The objective of the present study was to investigate the possible in vivo protective effects of grape seed extract (GSE) against benzene-induced toxicity in Swiss albino mice. 36 mice were divided into six groups of six animals of each, having free access to food and water ad libitum. The control group received distilled water alone, whereas mice in the treatment groups received GSE alone (50 or 150 mg/kg of body weight), benzene alone (250 mg/kg of body weight), or GSE + benzene for 50 weeks. All mice (100%) survived until the end of experiment and were sacrificed at the end of 24 hours. Bone marrow tissue was analyzed for micronucleus (MN) frequency, chromosomal aberrations (CAs), and pathological damages. Benzene alone–treated mice presented higher frequencies (P<.05) of MNs, CAs, and abnormal metaphases compared with the controls; moreover, the mitotic index was lower than in controls (P<.05). Oral treatment with GSE significantly ameliorated the indices of hepatotoxicity, nephrotoxicity, lipid peroxidation, and genotoxicity induced by benzene. Both doses of GSE tested provided significant protection against benzene induced toxicity, and its strongest effect was observed at the dose level of 150 mg/kg of body weight. Consequently, it was found that GSE has a significant protective effect against benzene-induced toxicity, and its protective effect is dose dependent.

Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ

Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005–5μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater.

Metabonomics Biomarkers for Subacute Toxicity Screening for Benzene Exposure in Mice

Benzene is known to produce hematotoxicity in occupational exposure workers. This study examined the utility of metabonomic biomarkers to ascertain subacute toxicity produced by benzene in male C3H/He mice. A 30-d intermittent collection of urine was obtained from mice in this experiment. The relative organ weights, blood parameters, and bone marrow smears were examined to identify specific changes of benzene-induced toxicity. In addition, an integrated analytical approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to map metabolic responses in urine. Five endogenous metabolites, hypoxanthine, spermidine, 4-aminohippuric acid, indolelactic acid, and glutamylphenylalanine, were identified as potential biomarkers of benzene-induced toxicity, indicating that pathways of purine, spermidine, fatty acid, tryptophan, and peptides metabolism might be disturbed in benzene-exposed mice. Our findings showed that the use of urine metabonomics was a more sensitive tool to detect benzene-induced toxicity compared to body weight or blood parameter changes.

Myeloperoxidase-Derived Halogenative Stress, a Possible Cause of Benzene Leukemogenesis

Abstract 4404 Background:Benzene, widely used in the chemical industry, is known to be myelotoxic, and to cause acute myeloid leukemia in humans. Although the mechanism has not been elucidated, benzene toxicity is considered to occur only after metabolic activation. Myeloperoxidase (MPO) is synthesized and secreted by myeloid cells catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant derived from chloride ions and hydrogen peroxide (H2O2). HOCl plays key roles in host defense by oxidizing the cellular constituents of invading pathogens. At the same time, HOCl is also capable of damaging proteins and nucleic acids in host tissue. By damaging the DNA of host cells, MPO-induced DNA halogenation (halogenative stress) might contribute to the carcinogenesis. Objectives:Because the findings of in vivo and in vitro research so strongly implicate the involvement of reactive oxygen species (ROS) in benzene-induced toxicity, focusing on 1,2,4-benzenetriol (BT), a benzene metabolite that generates ROS by autoxidation, we designed a study to investigate the toxicity of benzene leading to leukemogenesis, specifically examining the cytotoxic effects of BT on a human myeloid cell line, a class of cells from the organ mainly affected by benzene. Methods:After exposing human myeloid cell line HL-60 to BT, we investigated the cellular effects, including apoptosis, ROS generation, DNA damage (halogenated-DNA and 8-oxo-deoxyguanosine (8-oxo-dG)), and protein damage (halogenated tyrosine). We also investigated how the cellular effects of BT were modified by H2O2 scavenger catalase, HOCl scavenger methionine, and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase-specific-inhibitor. Apoptosis was measured by Annexin V-fluorescein isothiocyanate and propidium iodide double-labeling kits. Intracellular ROS were determined flow-cytometrically using ROS-sensitive fluorescent probes. Halogenated-DNA was measured by flow cytometry after immunostaining, using a novel monoclonal antibody (mAb2D3) which recognizes the HOCl-modified 2'-deoxycytidine residue, 5-chloro-2'-deoxycytidine. 8-oxo-dG was measured by electrochemical detection after HPLC separation and halogenated tyrosines were detected immunocytochemically. Results:BT increased the levels of apoptosis and of ROS including superoxide (O2•−), H2O2, HOCl, and the hydroxyl radical (•OH). Catalase, ABAH, and methionine each inhibited the increased apoptosis caused by BT; and catalase and ABAH inhibited increases in HOCl and •OH. Indicating that HOCl was generated via the H2O2–MPO–HOCl system, this inhibition of HOCl generation by both catalase and ABAH demonstrates that, in HL-60 cell samples exposed to BT, H2O2 was certainly metabolized to HOCl by MPO. It also suggests that HOCl might trigger BT-induced apoptosis of HL-60 cells. We then investigated whether this cytotoxicity of BT was related to the induction of DNA damage. We evaluated the halogenation of DNA by HOCl, and 8-oxo-dG induction by •OH. While BT exposure increased halogenated DNA, this increase was inhibited by catalase, methionine, and ABAH. BT exposure did not, however, increase 8-oxo-dG. To confirm the induction of halogenative stress by BT, we investigated HOCl-induced protein damage in the form of halogenated tyrosines. BT exposure also increased the amount of halogenated tyrosines. Conclusions:In this study we have constructed a novel hypothesis (Figure 1): exposure to BT increases O2•− generation, possibly by autoxidation; this O2•− is chemically or enzymatically converted to H2O2; this H2O2 is metabolized to HOCl by MPO; whereupon this HOCl halogenates DNA and proteins, thus inducing myelotoxicity or leukemogenesis. The high expression of MPO from myeloid cells, along with the fact that halogenated DNA can cause gene mutation and epigenetic changes, may explain how benzene is involved in bone marrow disorders or myeloid leukemia. Previous studies of benzene toxicity have reported that MPO plays a role in the bioactivation of benzene's phenolic metabolites. Here, we have shown for the first time that a benzene metabolite, BT, is capable of generating HOCl and consequent halogenative damage via the H2O2–MPO–HOCl system. Our findings lend strong support to the conjecture that BT-induced DNA halogenation is a primary reaction in the leukemogenesis associated with benzene. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

United States voluntary children’s chemical evaluation program (VCCEP) risk assessment for children exposed to benzene

As part of the Voluntary Children’s Chemical Evaluation Program (VCCEP) program, a risk assessment was performed to evaluate the risks to children from environmental benzene exposures. This paper summarizes this risk assessment. Risk was characterized using two distinct methods: USEPA’s default type of risk assessment, which used the Reference Dose (RfD) and Cancer Slope Factor (CSF) to characterize non-cancer and cancer risks, as well as a Margin of Safety (MOS) approach that utilized a point of departure (POD). The exposures for most scenarios evaluated in this VCCEP risk assessment are lower than both the cancer and non-cancer PODs by several orders of magnitude, indicating a large MOS and corresponding low potential for toxicity at these exposures. The highest benzene exposures likely experienced by children, associated with the lowest MOS, are from cigarette smoke. In addition, the potential for age-related differences in the sensitivity towards benzene-induced toxicity was investigated. In general, this risk assessment does not indicate that children are likely to be at a elevated risk of AML or hematopoietic toxicity associated with environmental exposures to benzene.

Protective effects of zinc and selenium against benzene toxicity in rats

The presented study investigates the protective role of zinc (Zn) and selenium (Se) in attenuating benzene-induced toxicity in rats. Male Sprague-Dawley rats were injected with benzene (0.5 mL/kg body weight ip) and received a diet supplement containing Zn and Se. Several hematological and biochemical parameters (representing antioxidant status) were estimated. Histopathological examinations were performed. Results showed that food intake and body weight gain of benzene-injected rats were significantly lower than that of the control rats. Benzene-injected rats showed increased plasma malondialdehyde (MDA) and decreased activity of: glutathione peroxidase (GSH-Px), catalase, superoxide dismutase (SOD) enzymes, as well as reduced glutathione (GSH) when compared to the control group. Histopathological investigations revealed structural changes in benzene-injected rats' liver. Supplementation with Zn and Se resulted in a significant decrease in MDA, elevation in GSH, GSH-Px, SOD and catalase levels. This study shows that Zn and Se supplementation can improve the activity of antioxidant enzymes in rats and decrease the histological anomalies induced by benzene toxicity as well.