Benzene-induced Hematotoxicity Research Articles

Benzene Exposure Alters Expression of Enzymes Involved in Fatty Acid β-Oxidation in Male C3H/He Mice.

Benzene is a well-known hematotoxic carcinogen that can cause leukemia and a variety of blood disorders. Our previous study indicated that benzene disturbs levels of metabolites in the fatty acid β-oxidation (FAO) pathway, which is crucial for the maintenance and function of hematopoietic and leukemic cells. The present research aims to investigate the effects of benzene on changes in the expression of key enzymes in the FAO pathway in male C3H/He mice. Results showed that benzene exposure caused reduced peripheral white blood cell (WBC), red blood cell (RBC), platelet (Pit) counts, and hemoglobin (Hgb) concentration. Investigation of the effects of benzene on the expression of FA transport- and β-oxidation-related enzymes showed that expression of proteins Cpt1a, Crat, Acaa2, Aldh1l2, Acadvl, Crot, Echs1, and Hadha was significantly increased. The ATP levels and mitochondrial membrane potential decreased in mice exposed to benzene. Meanwhile, reactive oxygen species (ROS), hydrogen peroxide (H2O2), and malondialdehyde (MDA) levels were significantly increased in the benzene group. Our results indicate that benzene induces increased expression of FA transport and β-oxidation enzymes, mitochondrial dysfunction, and oxidative stress, which may play a role in benzene-induced hematotoxicity.

MiR-133a regarded as a potential biomarker for benzene toxicity through targeting Caspase-9 to inhibit apoptosis induced by benzene metabolite (1,4-Benzoquinone)

Benzene is an environmental and industrial chemical which is widely utilized in various applications. Our previous study showed that miR-133a expression was down-regulated in chronic benzene poisoning workers, but the mechanism of miR-133a in benzene-induced hematotoxicity remains unclear. In this population-based study, benzene-exposed group recruited workers whose concentration of air benzene was 3.50±1.60mg/m3, and control workers who were exposed to 0.06±0.01mg/m3 air benzene. By comparison, Caspase-9 and Caspase-3 was up-regulated while miR-133a expression decreased in benzene-exposed workers. Pearson correlation analysis showed that miR-133a was reversely correlated with pro-apoptotic gene Caspase-9 in population-based study. Moreover, multiple linear regressions indicated that miR-133a was positively associated with blood cells count. To explore the underlying mechanism of miR-133a in benzene-induced hematotoxicity, AO/EB staining and TEM ultrastructural analysis were conducted to verify the activation of apoptosis in Human Leukemic U937 Cells induced by benzene metabolites (1,4-Benzoquinone, 1,4-BQ), while the mechanism of miR-133a in 1,4-BQ-induced apoptosis was performed using lentivirus vectors transfection. The results demonstrated that 1,4-BQ evidently induced mitochondria-mediated apoptosis and increased pro-apoptotic genes (Caspase-9 and Caspase-3) expression in a dose-dependent manner. The mechanistic study showed 1,4-BQ decreased miR-133a expression and miR-133a over-expression attenuated 1, 4-BQ-caused upregulation of Caspase-9, Caspase-3 and apoptosis. In conclusion, our research suggested that benzene induced hematotoxicity by decreasing miR-133a and caspase-dependent apoptosis which might contribute to the underlying mechanism of miR-133a in benzene-induced hematotoxicity.

Plasma microRNA expression in workers occupationally exposed to benzene

Background: Benzene is a confirmed organic carcinogen, and one of the most common environmental pollutants. However, the mechanism underlying benzene-induced hematotoxicity and leukemogenicity has not been fully elucidated. Our study aimed to explore whether benzene exposure has an effect on the plasma microRNA (miRNA) expression. Methods: In the discovery stage, we used a microarray assay to detect the miRNA expression profiles of two pooled plasma samples from ten healthy benzene-exposed individuals and ten non-benzene exposed controls. Subsequently, we conducted an expanded validation of three candidate miRNAs in 118 healthy benzene-exposed individuals and 91 non-exposed controls. Results: In the discovery stage, 5 miRNAs demonstrated at least a 1‑fold higher expression, and 12 miRNAs showed at least a −1‑fold lower expression in the exposure group as compared with the expression levels in the control group. In the expanded validation stage, the plasma level of miR-638 was significantly up-regulated (2.68±0.12 vs. 1.38±0.10, P vs. 1.44±0.12 for miR-221-3p and 1.91±0.10 vs. 2.86±0.13 for miR-122-5p, P Conclusions: Our investigation suggested that benzene exposure might be related to elevated expression of miR-638 and decreased expression of miR-221-3p and miR-122-5p in human plasma.

Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα.

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.

MiR-146a affects the alteration in myeloid differentiation induced by hydroquinone in human CD34+ hematopoietic progenitor cells and HL-60 cells.

The MiR-146a/TRAF6/NF-κB axis is important for the regulation of hematopoiesis and the immune system. To identify the key axis that regulates benzene-induced hematotoxicity or even leukemia, we investigated miR-146a expression in human CD34+ hematopoietic progenitor cells (HPCs) and human acute promyelocytic leukemia cells (HL-60) during the differentiation process. By performing a colony formation assay and flow cytometry on cells in the differentiation process after hydroquinone treatment, we found that hydroquinone induced a marked reduction of differentiation toward myeloid cells and immune cells in CD34+ cells (5 days exposure) as well as in HL-60 cells (3 h exposure). Further study using real-time PCR and western blot showed that the impaired myeloid differentiation was accompanied by the up-regulation of miR-146a and the down-regulation of TRAF6 and NF-κB. Using the miR-146a-5p inhibitor to suppress miR-146a expression could relieve the inhibitory effect on myeloid differentiation induced by hydroquinone to a certain extent. At the same time, the level of TRAF6 protein, as well as the phosphorylated IκBα protein which indicates NF-κB transcriptional activity was restored to the same levels as the control group. These results suggested that hydroquinone induced a dysregulation of miR-146a and its downstream NF-κB transcriptional factor pathway, which might be an early event in the generation of benzene-induced differentiation disturbance and subsequent leukemogenesis.

Benzene Induces Hematotoxicity Via Acetyltransferase p300 Inhibition-Mediated Autophagy

Objective: Chronic exposure to benzene is known to be associated with hematotoxicity and is involved in the development of aplastic anemia and leukemia. However, the mechanism of benzene-induced hematotoxicity has not been well understood. Accumulating studies have reported that benzene metabolite hydroquinone (HQ) and analogous chemicals formaldehyde can induce autophagic cell death. So it is meaningful to investigate the role of autophagy and the underlying mechanisms in the benzene-induced hematotoxicity.Materials and Methods: Human mononuclear cells were isolated from the heparinized bone marrow of healthy donors or patients with chronic benzene exposure using Ficoll gradient centrifugation, mainly consisted of progenitor and stem cells, and were measured for the levels of autophagy and the expression of p300. Totally five patients, 2 female and 3 male, all were shoe workers and had experience of contact with benzene for at least 6 months, diagnosed as occupational benzene poisoning. Five age-matched healthy donors were enrolled as controls. Besides, bone marrow mononuclear cells isolated from healthy donors were incubated with HQ 20 μM for 12 hours to determine the levels of autophagy and the expression of p300. Furthermore, thirty mice weighting 23 - 25 g were randomized into benzene group (benzene 1 ml/kg; corn oil 1 ml/kg; n=15) and corn oil group (2 ml/kg; n=15). After subcutaneous injected 12 times, three times a week, the mice executed to study the hemototoxicity of benzene with the blood cell counts, bone marrow aspiration and the pathology of the femurs. Similarly, the bone marrow mononuclear cells isolated from mice were determined for the levels of autophagy and acetylation and the expression of p300.Results: All five enrolled patients with chronic benzene exposure had hematotoxicity. Increased autophagy, deacetylation of autophagic components and histone H3 occurred in patients with chronic benzene exposure. Furthermore, both the expression of P300 mRNA and protein were decreased significantly in patients with chronic benzene exposure compared with those in healthy donors, which arouses our interest to investigate whether in vitro exposure to benzene metabolite HQ promotes autophagy. Interesting, HQ induced cell autophagic cell death without apoptosis, which could be reversed by autophagy inhibitor 3-methyladenine and exaggerated by autophagy activator rapamycin. HQ caused deacetylation, especially decreased the acetylation of autophagy components including ATG7 and LC3. HQ also significantly decreased the acetyltransferase activity of p300 demonstrated by reduced acetylation of histone H3 and the expression of p300 mRNA and protein. HQ treatment decreased the physical interaction between p300 and autophagy components including ATG7, LC3, and ATG3. Both C646, a 'specific' antagonist of p300, and knockdown of p300 induced cell autophagy and deacetylation, similar with HQ, further identifying that p300 was a major endogenous repressor of autophagy. However, the deacetylation effect could be completely inhibited by the addition of acetyl-CoA at a concentration of 100 μM. Taken together, these data suggest that HQ promotes autophagy by inhibiting the activity of P300. We next investigate whether modulation of P300 expression regulates the autophagic flux induced by HQ. Intriguingly, Momordica Antiviral Protein 30 Kd (MAP30), a ribosome-inactivating protein isolated from dietary bitter melon, could increase p300 expression and attenuate autophagy and cell death induced by HQ. The mouse model of benzene-induced hematotoxicity was injected subcutaneously with benzene and corn oil. The levels of hemoglobin, and the numbers of white blood cells and platelets decreased significantly in benzene-treated mice compared with the vehicle mice, and the benzene-induced hematotoxicity was further verified by the bone marrow aspiration and the pathology of the femur. Similarly, the increased autophagic flux and p300 inhibition occurred in the benzene-treated mice.Conclusion: We have demonstrated that benzene and its metabolite HQ induced autophagy by inhibiting p300, which may be a main cause of benzene-induced hematotoxicity. MAP30 can restore decreased p300 expression induced by HQ and attenuate autophagic cell death. Therefore, MAP30 may be a therapeutic drug for patients with chronic benzene exposure. DisclosuresNo relevant conflicts of interest to declare.

Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice.

Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin− cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin−c-Kit+ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs.

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p<0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e+ cells but reduced the total counts of Sca-1+, CD11b+, Gr-1+, and CD45+ cells at 7 and 12μM (p<0.05). Furthermore, the CFU assay showed reduced (p<0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage.

Preventive effects of garlic oil against the benzene-induced hematotoxicity in mice

To investigate the preventive effects of garlic oil (GO) on benzene-induced hematotoxicity in mice. Specific pathogen-free male Kunming mice were randomly divided into 5 groups, i.e., control group, model group, and low-, middle-, and high-dose GO groups (n = 20 in each group). Mice in GO groups were orally given GO at 20, 40, or 80 mg/kg BW, while mice in the other two groups received an equal volume of corn oil. Two hours later, mice in model group and GO groups were orally given benzene (20%, v/v, dissolved in corn oil, 10 ml/kg BW) for 21 days consecutively. On the 22nd day, blood was collected from the orbital sinus, to determine the counts of red blood cells (RBC), white blood cells (WBC), and platelets (PLT) and hemoglobin level using an automatic blood cell counter. The mice were sacrificed thereafter. The spleen was excised and weighed for calculation of the spleen index (spleen weight/body weight×100%). The counts of WBC, RBC, and PLT and Hb level in the model group were reduced by 40%, 18%, 28%, and 23.6%, respectively, as compared with those in the control group (P < 0.01). Compared with those in the model group, WBC and PLT counts in the high-dose GO group increased by 95% and 66%, respectively (P < 0.01), wherein lymphocytes and monocytes increased by 142% and 100%, respectively (P < 0.01); the RBC count and Hb level in the low-dose GO group increased by 15% and 16%, respectively (P < 0.05). GO significantly suppressed benzene-induced decreases in spleen weight and spleen index. GO is capable of suppressing benzene-induced hematotoxicity in mice. One possible mechanism may be promotion of hematopoiesis in the spleen.

PTEN methylation involved in benzene-induced hematotoxicity

It is well known that benzene is a hematotoxic carcinogen. PTEN promoter methylation is a representative example of transcriptional silencing of tumor suppressor genes. However, the effect of PTEN methylation on benzene-induced hematotoxicity has not yet been elucidated. In this study, the animal model of benzene hematotoxicity was successfully established. WBC significantly decreased in experimental groups (P<0.01). Compared with the control group, the weight of rats increased slowly and even declined with increasing doses of benzene in the benzene-treated groups. An increase in the level of PTEN methylation was observed in the low dose group, and PTEN methylation level increased significantly in a dose-dependent manner. However, it was interesting that PTEN mRNA expression increased in the low dose group, but declined with increasing doses of benzene. The decrease of tumor suppressor function caused by PTEN methylation may be an important mechanism of benzene hematotoxicity. Furthermore, lymphoblast cell line F32 was incubated by benzene and then treated with 5-aza and TSA, alone or in combination. A dramatic decrease in the PTEN mRNA expression and a significant increase of PTEN methylation level in benzene-treated cells were also shown. PTEN mRNA expression was up regulated and PTEN methylation level was reduced by the epigenetic inhibitors, 5-aza and TSA. In conclusion, PTEN methylation is involved in benzene-induced hematotoxicity through suppressing PTEN mRNA expression.

Investigation into variation of endogenous metabolites in bone marrow cells and plasma in C3H/He mice exposed to benzene.

Benzene is identified as a carcinogen. Continued exposure of benzene may eventually lead to damage to the bone marrow, accompanied by pancytopenia, aplastic anemia or leukemia. This paper explores the variations of endogenous metabolites to provide possible clues for the molecular mechanism of benzene-induced hematotoxicity. Liquid chromatography coupled with time of flight-mass spectrometry (LC-TOF-MS) and principal component analysis (PCA) was applied to investigate the variation of endogenous metabolites in bone marrow cells and plasma of male C3H/He mice. The mice were injected subcutaneously with benzene (0, 300, 600 mg/day) once daily for seven days. The body weights, relative organ weights, blood parameters and bone marrow smears were also analyzed. The results indicated that benzene caused disturbances in the metabolism of oxidation of fatty acids and essential amino acids (lysine, phenylalanine and tyrosine) in bone marrow cells. Moreover, fatty acid oxidation was also disturbed in plasma and thus might be a common disturbed metabolic pathway induced by benzene in multiple organs. This study aims to investigate the underlying molecular mechanisms involved in benzene hematotoxicity, especially in bone marrow cells.

Aberrant Hypomethylated KRAS and RASGRF2 as a Candidate Biomarker of Low Level Benzene Hematotoxicity

Benzene is an important industrial chemical and an environmental contaminant. The mechanisms of low level benzene-induced hematotoxicity are unresolved. Aberrant DNA methylation, which may lead to genomic instability and the altered gene expression, is frequently observed in hematological cancers. The purpose of the present study was to conduct a genome-wide investigation to examine comprehensively whether low level benzene induces DNA methylation alteration in the benzene-exposed workers. Infinium 450K methylation array was used to compare methylation levels of the low level benzene-exposed individuals and health controls and the differentially expressed DNA methylation pattern critical for benzene hematotoxicity were screened. Signal net analysis showed that two key hypomethylated KRAS and RASGRF2 associated with low level benzene exposure were identified. Further, the hypomethylated RASGRF2 gene played central roles through regulation of Rho protein, MAPK, small GTPase mediated signal transduction. While the hypomethylated KRAS gene played important roles through small GTPase, Ras protein, MAPK cascade, Gap junction, Axon guidance, Tight junction, GnRH, T cell receptor signaling pathway, Acute myeloid leukemia, B cell receptor signaling pathway, Chronic myeloid leukemia, ErbB signaling pathway. Our preliminary study indicated that aberrant hypomethylated KRAS and RASGRF2 might be a potential methylated biomarker of low level benzene hematotoxicity.

Assessment of benzene-induced hematotoxicity using a human-like hematopoietic lineage in NOD/Shi-scid-IL2Rγnull mice

Assessment of benzene-induced hematotoxicity using a human-like hematopoietic lineage in NOD/Shi-scid-IL2Rγnull mice

Microsomal epoxide hydrolase (EPHX1) polymorphisms are associated with aberrant promoter methylation of ERCC3 and hematotoxicity in benzene‐exposed workers

Benzene is an important industrial chemical and widespread environmental pollutant known to induce leukemia and other blood disorders. To be carcinogenic, benzene must be metabolized to produce toxic metabolites. To investigate whether single nucleotide polymorphisms (SNPs) in the metabolic enzyme genes are associated with benzene-induced alterations in DNA methylation and hematotoxicity, we genotyped four commonly studied SNPs in three metabolic enzymes genes CYP1A1, EPHX1 and NQO1; and analyzed promoter DNA methylation status in 11 genes which have been reported to be associated with benzene-induced hematotoxicity (BLM, CYP1A1, EPHX1, ERCC3, NQO1, NUDT1, p15, p16, RAD51, TP53 and WRAP53) in 77 benzene-exposed workers and 25 unexposed controls in China. ERCC3, a DNA repair gene, showed a small but statistically significant increase of promoter DNA methylation in the exposed group compared with the unexposed group (mean ± SD: 4.73 ± 3.46% vs. 3.63 ± 1.96%, P = 0.048). We also observed that an increased number of C allele for rs1051740 in EPHX1 was associated with decreased ERCC3 methylation levels in benzene-exposed workers (P(trend) = 0.001), but not in unexposed controls (P(trend) = 0.379). Interestingly, another EPHX1 SNP (rs2234922) was associated with lower white blood cell (WBC) counts (P(trend) = 0.044) in benzene-exposed workers. These associations remained the same when ERCC3 promoter methylation and WBCs were dichotomized according to the 90th percentile (≥6%) of methylation levels in controls and a leucopenia cutoff (<4 × 10(9) /L), respectively. Our findings suggest that benzene exposure may be associated with hypermethylation in ERCC3, and that genetic variants in EPHX1 may play an important role in epigenetic changes and hematotoxicity among benzene-exposed workers.

Influence of acetylsalicylic acid on hematotoxicity of benzene

The aim of the study was to evaluate the influence of acetylsalicylic acid (ASA) on benzene hematotoxicity in rats. The study was carried out on rats exposed for 2, 4 and 8 weeks to benzene vapour at a concentration of 1.5 or 4.5 mmol/m(3) of air (5 days per week, 6 hours per day) alone or together with ASA at the doses of 5, 150 or 300 mg/kg body weight (per os). Benzene at a concentration of 4.5 mmol/m(3) caused a slight lymphopenia, granulocytosis and reticulocytosis in blood. In bone marrow traits of megaloblastic renewal, presence of undifferentiated cells and giant forms of granulocytes as well as an increase in myeloperoxidase and decrease in chloroacetate esterase activity and lipids content were noted. ASA (150 and 300 mg/kg b.w.) influenced some of hematological parameters, altered by benzene intoxication. ASA limited the solvent-induced alteration in blood reticulocyte count and in the case of bone marrow in the erythroblasts count. Traits of megaloblastic renewal in bone marrow were less pronounced. Besides, higher activity of myeloperoxidase and the decrease in the level of lipids in granulocytes were noted. Our results suggest that ASA limited the benzene-induced hematotoxicity.

Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2Rγnull Mice

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγnull (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.

Clinical study of correlation between the expression of ICBP90 and hematopoietic suppression in patients with chronic benzene poisoning

To observe the change of ICBP90 expression in patients with chronic benzene poisoning and explore the correlation between the expression of ICBP90 and benzene-induced hematotoxicity. The bone marrow samples were from 13 chronic benzene poisoning cases with hematopoietic suppression, 11 chronic benzene poisoning cases with hematopoietic regeneration and 10 controls. Western-blot was applied to detect the ICBP90 expression in bone marrow mononuclear cells (BMNCs). The correlation between ICBP90 expression and hematopoietic suppression in patients with chronic benzene poisoning was analyzed. The ICBP90 expression of BMNCs in 13 chronic benzene poisoning cases with hematopoietic suppression was significantly lower than that in controls (P < 0.01). The ICBP90 expression of BMNCs in 11 chronic benzene poisoning cases with hematopoietic regeneration was significantly higher than those in controls and 13 chronic benzene poisoning cases with hematopoietic suppression (P < 0.05 or P < 0.01), respectively. There were good correlations between the expression of ICBP90 and white blood cell and platelet counts in patients with chronic benzene poisoning (r(1) = 0.555,P = 0.006; r(2) = 0.854,P < 0.01). The ICBP90 expression of BMNCs in the chronic benzene poisoning cases with hematopoietic suppression decreased significantly, and the ICBP90 expression of BMNCs in the chronic benzene poisoning cases with hematopoietic regeneration increased significantly. There was good correlation between hematopoietic suppression and ICBP90 expression in patients with chronic benzene poisoning.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials.

Stem Cell and Benzene-Induced Malignancy and Hematotoxicity

The biological effect of benzene on the hematopoietic system has been known for over a century. The rapid advancement in understanding the biology of hematopoietic stem cells (HSCs) and cancer stem cells (CSCs) in recent years has renewed interest in investigating the role of stem cells in benzene-induced malignancy and bone marrow depression. The interplay between benzene and stem cells is complex involving the stem cell, progenitor, and HSC niche compartments of the bone marrow. In this prospect, benzene metabolites formed through metabolism in the liver and bone marrow cause damage in hematopoietic cells via multiple mechanisms that, in addition to traditionally recognized chromosomal aberration and covalent binding, incorporate oxidative stress, alteration of gene expression, apoptosis, error-prone DNA repair, epigenetic regulation, and disruption of tumor surveillance. However, benzene-exposed individuals exhibit variable susceptibility to benzene effect that arises, in part, from genetic variations in benzene metabolism, DNA repair, genomic stability, and immune function. These new studies of benzene leukemogenesis and hematotoxicity are expected to provide insights into how environmental and occupational chemicals affect stem cells to cause cancer and toxicity, which impact the risk assessment, permissible level, and therapy of benzene exposure.

Effect of Methanolic Leaf Extract ofOcimum basilicumL. on Benzene-Induced Hematotoxicity in Mice

The aim of the present study was to investigate the protective role of methanolic leaf extract of Ocimum basilicum L. against benzene-induced hematotoxicity in Swiss albino mice. GC analysis and subacute toxicity level of the extract were tested. Mice were randomly divided into three groups among which II and III were exposed to benzene vapour at a dose 300 ppm × 6 hr/day × 5 days/week for 2 weeks and group I was control. Group III of this experiment was treated with the leaf methanolic extract at a dose of 100 mg/kg body weight, a dose in nontoxic range. Hematological parameters (Hb%, RBC and WBC counts), cell cycle regulatory proteins expression and DNA fragmentation analysis of bone marrow cells was performed. There was an upregulation of p53 and p21 and downregulation of levels of CDK2, CDK4, CDK6, and cyclins D1 and E in leaf extract-treated group. DNA was less fragmented in group III compared to group II (P < 0.05). The present study indicates that the secondary metabolites of O. basilicum L. methanolic leaf extract, comprising essential oil monoterpene geraniol and its oxidized form citral as major constituents, have modulatory effect in cell cycle deregulation and hematological abnormalities induced by benzene in mice.