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

Abstract

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.

Keywords

Benzene hematopoietic stem cell niche genetic epigenetic lineage-directed strategy

Introduction

Benzene (C₆H₆) is an extensively used industrial solvent in the petrochemical industry.¹ The International Agency for Research of Cancer has categorized benzene as a group 1 carcinogen affecting both human and animals.² Beside occupational exposure, other major sources of benzene exposure are cigarette smoke, gasoline vapor, vehicle emissions, and water or soil contaminated with benzene.³ Benzene is known as a hematotoxic and leukemogenic agent, exposure to which can cause bone marrow failure, leading to such hematological disorders and malignancies such as aplastic anemia, myelodysplasia syndrome, and acute myeloid leukemia.⁴

Bone marrow is the primary site for hematopoietic stem cells (HSCs) niche that maintain hematopoiesis.⁶ The niche consists of HSCs and lineage-committed progenitors with the ability to self-renew and differentiate into mature and functional blood cells under the regulation of intrinsic (e.g. signaling pathway or transcription factors) and extrinsic (e.g. cytokines or growth factors) factors.⁷ Thus, balanced regulation of self-renewal and differentiation by the HSCs and progenitor cells is crucial, as any disruption of these activities can lead to altered hematopoiesis and probable malignancies.⁸ Previous studies have reported that benzene induced genotoxicity in exposed HSCs by causing chromosome aberration,^9
–11 sister chromatid exchange,¹² DNA damage,¹³ and epigenetic alterations.^14,15 However, despite numerous reports regarding how benzene toxicity affects targeting bone marrow and HSCs, understanding exact mechanism linking benzene to the HSCs niche dysregulation is challenging due to the complexity of its microenvironment. Thus, this review will focus on the genetic, epigenetic, and lineage-directed factors in governing the mechanism of benzene-induced malignancies and hematotoxicity targeting HSCs niche. In summary, this review is based on 155 references comprising of 110 original research and 45 review articles that focus on benzene toxicity, HSC, as well genetic and epigenetic studies. Five epidemiological studies reporting cohort studies of benzene toxicity among industrial workers exposed to benzene have also been included in this review.

Metabolic pathway for benzene metabolism

Benzene metabolism as summarized in Figure 1 is vital for understanding benzene toxicity effects.^5,16–
18 The first step of metabolic pathways in benzene biotransformation involves the epoxidation process mediated by the major cytochrome P450 liver enzyme, namely CYP 2E1, producing benzene oxide and oxepine (Figure 1). Most of the benzene oxide is then further oxidized into phenol and subsequently metabolized into hydroquinone or catechol mediated by CYP 2E1.¹⁹ These metabolites are then transported to bone marrow, where subsequent secondary metabolism occurs. The high level of myeloperoxidase (MPO) enzyme presence in bone marrow plays a vital role in the oxidization of hydroquinone to a more stable and toxic metabolite known as p-benzoquinone or 1,4-benzoquinone (1,4-BQ) which is believed to be involved in benzene-mediated carcinogenicity.²⁰ In the context of benzene toxicity targeting bone marrow, NAD(P)H:quinone oxidoreductase 1 (NQO1), which is produced by bone marrow stromal cell, protects against benzene toxicity by catalyzing the obligatory two-electron reduction of 1,4-BQ into HQ.^21
–23 Thus, NQO1 plays an important role in 1,4-BQ detoxification and cellular protection against oxidative stress following BQ exposure.²⁴ Overall, the risk of toxicity following benzene exposure targeting the hematopoietic system can be determined by the activity of metabolic enzymes involved in benzene activation and detoxification.

Figure 1.

Metabolic pathway of benzene metabolism in the liver and bone marrow.⁵ CYP: cytochrome P450; GST: glutathione S-transferase; MPO: myeloperoxidase; NQO1: NAD(P)H:quinone oxidoreductase 1.

An overview of hematopoiesis

Hematopoiesis is the process by which HCSs produce functional hematopoietic cells comprised of erythroid, myeloid, lymphoid, and megakaryocytes lineages.⁸ The bone marrow provides an optimum microenvironment for stem cell survival, self-renewal, and differentiation into lineage-committed progenitors and terminally differentiated matured blood cells.²⁵ It is composed of stromal cells and a microvascular network that supports HSCs growth by producing growth factors necessary for stem cell survival. Thus, HSCs play a fundamental role in the maintenance of hematopoiesis.

The fundamental role of HSC niche for the maintenance of hematopoiesis

A stem cell is one which has the ability to undergo self-renewal division to produce more stem cells, while maintaining an undifferentiated stem cell pool. Stem cells also have the remarkable potential to carry out differentiation to develop specialized progenitor cells and other different cell types.²⁶ A stem cell niche is the in vivo microenvironment where stem cells reside and receive stimuli that regulate their fate.²⁷ Thus, the niche is not merely a physical location but also as a place where numerous external signals interact and integrate to influence stem cell behavior. These stimuli include cell-to-cell and cell–matrix interactions, as well as signaling molecules that activate and/or repress genes expression and transcriptional activities. These interactions are essential in regulating the fate of stem cells to remain in a dormant undifferentiated state, undergo self-renewal, or to differentiate to lineages-committed and terminally differentiated matured cells.⁸

HSCs are a population of immature cells with self-renewal and pluripotency properties which distinguish them from other hematopoietic cells. The HSCs are rare and they occur at the ratio of about one in every 20 million nucleated cells in bone marrow. Although the exact phenotype of the HSC is unknown, immunological testing has shown that human HSCs express surface markers CD 34⁺, CD 38⁻, and negative for lineage markers (Lin⁻) with the noted appearance of a small or medium-sized lymphocyte. Cell differentiation occurs from the HSCs via committed hematopoietic progenitors that have restricted developmental potential as compared to the primitive HSCs.⁶ Hence, self-renewal and differentiation properties of HSCs and hematopoietic progenitors are vital for the maintenance of functional hematopoiesis.

The stem cell niche provides a microenvironment composed of cellular structures and extracellular matrix in which stem cells are maintained as undifferentiated.²⁶ Hematopoiesis is a complex process regulated by specific genetic program which begins from a nonsymmetrical division, producing new stem cells through self-renewal and multipotential progenitors to terminally differentiated matured blood cells through differentiation (Figure 2).^6,7,28 Thus, the balance between self-renewal and differentiation activities of HSCs is crucial to avoid accumulation of immature and undifferentiated stem cells and/or progenitor cells that could lead to clonal formation of leukemic stem cell (LSC).

Figure 2.

Hematopoiesis is the process by which immature stem cells undergo self-renewal forming new pool of stem cells and differentiate into lineage-committed progenitors that will undergo further differentiation into terminally differentiated mature blood cells.⁶ LT-HSC: long-term hematopoietic stem cell; ST-HSC: short-term hematopoietic stem cell; MPP: multipotent progenitors; CMP: common myeloid progenitor; CLP: common lymphoid progenitor; MEP: myeloid, erythroid progenitor; GMP: granulocytic myeloid progenitor; NK cell: natural killer cell.

Regulation of HSCs self-renewal and differentiation

Genetic regulation

HSCs undergo self-renewal, differentiation, and apoptosis to maintain optimal homeostasis of the hematopoietic system. The self-renewal and differentiation activities of HSCs are regulated by specific genetic programs.²⁹ To date, HOXB4, Bmi-1, GATA], and Wnt families are among the mostly studied transcription factors due to their critical role in the regulation of HSC self-renewal and differentiation.

HOXB4 is one of the HOX gene family members. It is an important transcription factor that regulates self-renewal and differentiation process at the early stage of hematopoiesis.^30
–32 Overexpression of HOXB4 gene enhances expression of downstream self-renewal-regulated genes such as Sox4, Meisl, and Runx2, as well as the cell cycle–regulated gene, namely cyclin D1, promoting self-renewal and proliferation activities in HSCs. HOXB4 overexpression has been shown to increase murine HSCs self-renewal activity and competitive repopulation capacity, without compromising their differentiation potential and homeostasis of HSCs pool size.³¹ However, a study using human HSCs has reported that forced expression of HOXB4 was able to dysregulate lymphoid and myeloid differentiation, whereby the engraftment levels were found to be disproportionately higher in bone marrow and thymus relative to spleen and peripheral blood, while multilineage analysis of bone marrow showed a predominance of myeloid lineages.³³ The results demonstrate the potential role of HOXB4-forced expression to alter the balance and distribution of hematopoiesis, highlighting the need for a careful evaluation of future clinical gene therapy application concerning the modification of HOXB4 expression.

In addition to HOXB4, Bmi-1 is another transcription factor that belongs to the polycomb group (PcG). It also regulates the self-renewal activity of HSCs by controlling the cell cycle.^34,35 Bmi-1 is expressed in the population of HCSs, and its expression level decreases following hematopoietic differentiation. The PcG gene Bmi-1 has been identified as a key epigenetic regulator of cell fates during different stages of development in multiple murine tissues.³⁶ Rizo et al. found that enforced expression of Bmi-1 in cord blood CD34⁺ cells resulted in a long-term maintenance and self-renewal of human hematopoietic stem and progenitor cells.³⁷ Long-term culture-initiating cell frequencies were increased upon stable expression of Bmi-1 and greater engraftment in non obese diabetic-mice severe combined immunodeficiency disease (NOD-SCID) mice was noted. Serial transplantation studies in NOD-SCID mice revealed that secondary engraftment was only achieved with cells overexpressing Bmi-1. Significantly, Bmi-1-transduced cells proliferated in stroma-free cytokine-dependent cultures for more than 20 weeks, as compared to control cells which lost their engraftment potential after 10 days of ex vivo culture in the absence of stroma cells. Thus, the finding indicates that self-renewal of human HSCs is enhanced by Bmi-1, which is classified as an intrinsic regulator of human stem cells/progenitor cells self-renewal.

Cell differentiation plays an essential role in hematopoiesis for the production of matured blood cells.^38
–40 The GATA group genes, which include GATA1, GATA2, and GATA3, are the critical transcription factors that regulate hematopoietic differentiation. GATA1 is mostly expressed in erythroid progenitor cells. It regulates erythropoiesis and is also crucially involved in the maturation of megakaryocyte, mast cell, and eosinophil.^41
–43 Meanwhile, GATA2 is expressed in megakaryocyte, erythroid, and myeloid progenitors and is involved in promoting maturation of monocyte and macrophage through interaction with other myeloid transcription factors, namely PU 1.⁴⁴ As for the function of GATA3, it plays an essential role in the maturation of T lymphocyte cell.^45,46 A previous study has reported that a deficiency of GATA transcription factors affects cellular differentiation process. For example, a decrease in GATA-1 depresses the erythropoiesis potency of myeloid progenitor and reduced formation granulocyte-macrophage (colony forming unit-granulocyte-macrophage [CFU-GM]) progenitors.⁴⁰ Thus, GATA transcription factors are fundamental to maintain sufficient pool of matured blood cells.

Wnt signaling plays a key role in the proliferation and differentiation of multiple cell types.⁴⁷ Wnt signaling is also involved in the differentiation regulation of different hematopoietic lineages.⁴⁸ An example is in T-cell development, where fetal and LSCs may require higher Wnt activity than normal adult HSCs.^49,50 Both early myeloid and B-cell progenitors display detectable levels of Wnt signaling activity, which is downregulated as these cells differentiate to more mature stages.⁵¹ A differential optimum of Wnt signaling activity was observed in HSCs, myeloid development, and early thymocytes, with HSCs having lower requirements and early thymocytes having the highest ones.⁵² Hence, very high Wnt signaling activities are important in contributing toward the impairment of both HSC self-renewal and differentiation along the hematopoiesis involving differential types of hematopoietic lineages.

Overall, a balanced regulation between self-renewal and differentiation activities within HSCs and hematopoietic progenitors niche is vital in maintaining homeostasis of the hematopoietic system. Therefore, any interruption to these biological activities may lead to interfered hematopoiesis and the possibility of subsequent development of hematological abnormalities and malignancies. With regards to benzene exposure, HSCs niche in the bone marrow has been reported as the potential target for benzene-induce hematotoxicity and leukemogenicity. A previous study has reported that the genes controlling self-renewing (HOXB4 and Bmi-1) and differentiation (GATA3) of HSCs and hematopoietic progenitors are significantly affected following 1,4-BQ exposure, leading to the fundamental discovery of lineage-directed mechanism.⁵³ Hence, further investigation of the genes regulating the self-renewal and differentiation properties of HSCs and hematopoietic progenitors may develop novel understandings of the mechanisms of benzene-induced hematotoxicity and leukemogenicity targeting HSCs niche.

Epigenetic regulation

The term “epigenetic” refers to inheritable changes in phenotype and function without alteration of DNA sequences.⁵⁴ In other words, epigenetic modulation regulates gene expression and subsequent protein expression and functions without changes to DNA sequences. The epigenetic regulation of gene expression can occur through multiple modes of epigenetic modifications that include DNA methylation, histone modifications, and non-coding RNAs expression. In the context of DNA methylation, methyl groups were added to DNA, typically at CpG dinucleotide region, causing conformational changes of DNA structure and subsequent alteration of downstream gene expression.⁵⁵ Histone modification also affects gene expression through posttranslational modifications of histone proteins involving acetylation, phosphorylation, methylation, and ubiquitination.⁵⁶ Other than DNA methylation and histone modification, another fundamental epigenetic modifier is micro RNAs (miRNAs) which reportedly play a major role in the posttranscriptional regulation of protein expression in physiological and pathological cellular processes.^57,58

Changes in both gene expression and epigenetic regulation, especially DNA methylation and demethylation, have been characterized during the development and differentiation of HSCs and their downstream hematopoietic lineages.^58,59 DNA methylation is carried out by a family of proteins known as DNA methyltransferases (DNMTs) with CpG islands being the major target for methylation by DNMTs. The four members of the DNMT family are DNMT1, DNMT3A, DNMT3B, and DNMT3L. DNMT3L, unlike the other DNMTs, does not possess any inherent enzymatic activity.⁶⁰ The other three family members are active on DNA. DNMT1 is the most abundant DNMT in adult cells. It binds to hemi-methylated DNA (DNA with only one strand methylated), at CpG sties. After DNA replication, while the parent strand remains methylated, the newly synthesized strand is not. DNMT1 binds to these hemi-methylated CpG sites and methylated the cytosine on the newly synthesized stand. This maintains established CpG methylation patterns through mitosis.⁶¹

DNMT3A and DNMT3B are examples of DNMTs that add new methyl groups to DNA which are important for cell fates determination.⁶² DNMT3A and DNMT3B have been shown to play essential roles in hematopoiesis. Loss of both DNMT3A and DNMT3B leads to more severe defects in HSC proliferation and differentiation. DNA methylation is reversible, and there are additional “Ten-eleven-translocation” (TET) enzymes vital for DNA demethylation by removing the DNMT-mediated methyl marks to unmethylated DNA.^63
–65 Mutations of TET enzymes have been reported in hematopoietic malignancies in which it leads to a significant decrease in myeloid, lymphoid, and erythroid populations.^66
–68 Thus, DNMTs and TET enzymes are both required for the maintenance of optimal balance between DNA methylation and demethylation that are critical for cellular activity.

The nucleosome, the basic unit of chromatin, consists of an octamer of histone proteins wrapped with DNA.⁶⁹ The histone octamer consists of four core histone proteins: H2A, H2B, H3, and H4. Histone proteins contain a globular C-terminal domain and an unstructured N-terminal tail that receive a variety of posttranslational modifications, including acetylation and methylation. Histone lysine acetylation occurs at several different locations in the histone tail (e.g. H3K9, H3K14, and H3K27) and are associated with active transcription by allowing access to the transcriptional factor through the open structure of chromatin.⁷⁰ Meanwhile, histone lysine methylation leads to transcriptional activation or repression, depending on which residue is modified and the degree of methylation. Histone H3 lysine 4 (H3K4), H3K36, and H3K79 methylation are associated with transcriptional activation, while dimethylation and trimethylation of H3K9 (H3K9me2, H3K9me3) and H3K27 (H3K27me2, H3K27me3) are related to transcriptional repression.^70,71 Histone methylation is regulated by histone lysine methyltransferases (KMT) and lysine demethylases (KDM) targeting specific lysine in histones.⁷¹ Several KDM have been shown to play a vital role in hematopoiesis and the alteration of KMT has been implicated in hematopoietic neoplasms.⁶⁸

To date, a number of miRNAs have been shown to be instrumental in hematopoiesis. miRNAs are the short RNA sequence with 19–24 nucleotides that play an important role in gene expression regulation via mRNA degradation or transcriptional inhibition at the transcriptional and posttranscriptional level.⁷² It is involved in all stage of hematopoiesis, including control between self-renewal and differentiation as shown in Figure 3.⁷³ The pathways of hematopoietic regulation involving miRNAs are often intertwined with transcription factor expression, both as targets and regulators. miRNAs also play an essential role in regulating hematopoiesis process where it is expressed with transcription factors such as HOX genes that regulate self-renewal and differentiation of HSC.⁷⁴ Thus, miRNAs are critical regulators of hematopoietic cell proliferation and differentiation, as alteration of their expression has been identified as playing a significant pathogenic role in hematological malignancies.

Figure 3.

Role of miRNAs in hematopoiesis. miRNAs that are related to transcriptional repression are indicated in gray, whereas miRNAs that are related to transcriptional activation are indicated in black.⁷² LT-HSC: long-term hematopoietic stem cell; ST-HSC; short-term hematopoietic stem cell; MPP: multipotent progenitors; CMP: common myeloid progenitor; CLP: common lymphoid progenitor; MEP: myeloid, erythroid progenitor; GMP: granulocyte megakaryocyte progenitor; RBC: red blood cell; miRNA: micro RNA.

LSC and benzene carcinogenesis

Leukemia and LSC

Leukemia is a blood cancer in which myeloid and lymphoid progenitor cells undergo uncontrolled cell division and unable to carry out the differentiation process to produce functional and matured blood cells.^75,76 It is a clonal hematological malignancy that arises as a result of chromosomal aberration or epigenetic changes affecting HSCs or multipotential hematopoietic progenitor cells.^77,78 LSC theory has been introduced to further understanding of mechanism of leukemogenesis and characteristics of leukemic cells. LSC is known as a rare cancer stem cell population in leukemia that possesses similar properties as stem cell.⁷⁷ To date, LSC is known to be produced from HSCs and/or hematopoietic progenitor cells that undergo mutation in which they have maintained or reacquired the capacity for indefinite and uncontrolled proliferation through accumulated mutations (Figure 4).^79,80 Despite their critical importance, the developmental origin of LSC and the mechanisms responsible for their emergence remain to be explored.

Figure 4.

Hypothesis on the evolution of LSC. HSCs and/or hematopoietic progenitor cells are believed to undergo mutation, giving rise to a clonal expansion of LSC. The mutated LSC has the capacity for indefinite and uncontrolled proliferation through accumulated mutations which eventually form a population of leukemic blast cells.⁷⁹ LSC: leukemic stem cell; HSC: hematopoietic stem cell.

Leukemia is a clonal disease as LSC maintains its clonal characteristics through self-renewing activity.^79,81 LSC is produced when mutation occurs in HSCs or progenitor cells during hematopoiesis. Genetic and epigenetic changes in LSC can increase the level of gene expression that regulates cell division or inactivates the apoptotic genes, which leads to leukemia.^77,82,83 Benzene exposure has been linked to the incidences of leukemia, particularly acute myelocytic leukemia (AML). Epidemiological studies have reported a higher risk of AML among factory workers exposed to benzene, compared to a control population without benzene exposure.⁸⁴ AML is a clonal hematopoietic malignancy that produces immature myeloid cells (blast) thereby inhibiting the cell differentiation processes.⁸⁵ Thus, uncontrolled accumulations of blast cells that are unable to undergo the maturation process in the hematopoietic system can lead to subsequent hematological disorders and malignancies, including leukemia.

Benzene-induced hematotoxicity and leukemogenicity: A mechanistic review

Chronic benzene exposure has been linked to impaired bone marrow function, leading to a number of hematological disorders and malignancies, including aplastic anemia, myelodysplasia syndrome, and acute myeloid leukemia.^4,86 Based on reported epidemiological studies, benzene exposure induces hematological disorders affecting myeloid and erythroid lineage such as aplastic anemia (erythroid progenitor), myelodysplasia syndrome (myeloid progenitor), and leukemia (myeloid progenitor).³ Moreover, industrial workers who had been exposed to benzene showed significantly lower white and red blood cell counts in the body compared to control groups.^85,87 These findings indicate that benzene toxicity toward the hematopoietic system may affect the survivability and functionality of multi-lineage blood cells. Furthermore, previous research reported that exposure to benzene also reduces the number of lymphocyte cells in blood circulation and induces genotoxicity such as chromosomal translocations on lymphoid progenitors.^88
–90 Hence, these findings indicate that benzene exposure has different toxicity effects caused by the different types of hematopoietic lineages. The hematotoxicological effects of specific benzene metabolites are further discussed in the following section.

Role of liver-derived benzene metabolites

Phenol, catechol, and hydroquinone are the three liver-derived metabolites of benzene which are known to demonstrate myelotoxicity (marrow-damaging) and hematotoxicity (blood-system damaging). The toxicity effects are mediated through the interactions of these metabolites with target cells in the bone marrow niche, including HSCs.^91
–93

Phenol and hydroquinone are further oxidized in the bone marrow to become reactive metabolites that have genotoxic, clastogenic, and cytotoxic effects. Phenol and phenol-derived metabolites (e.g. 4,4′-diphenoquinone) inhibit topoisomerase II and promote the genotoxic effects, which is a major concern as the inhibition of topoisomerase II has been linked to increased risk of leukemogenesis.^94,95 This indicates the potential ability of benzene to cause leukemia. The genotoxic effects of benzene metabolites have been reported in studies of the ability of phenol and hydroquinone to induce micronuclei and oxidative DNA damage in the bone marrow of treated mice,^92,95 promotion of the DNA adducts formation following the exposures to the combinations of the phenolic metabolites,^96,97 and induction of DNA damage in human peripheral blood lymphocytes, and in proliferating human T-lymphocytes by catechol.^98,99

Besides the genotoxic effects, benzene exposure has been shown to interfere with hematopoiesis by targeting the biological properties of HSCs. Hydroquinone has been shown to disrupts HSCs proliferation and differentiation in the myeloid compartment that includes the alteration of apoptosis pathways and clonal expansion of blood progenitor cells.¹⁰⁰ These events are mediated through multiple mechanisms, including alteration of inflammatory mediators, growth factors, and other cellular messengers activities.^101
–103 Moreover, when mouse embryonic yolk sac hematopoietic stem cells and adult bone marrow hematopoietic stem cells are exposed to hydroquinone, apoptosis, inhibited cell proliferation, differentiation, and clonogenic potential of both HSCs are promoted.¹⁰⁴ It is thus evident that liver-derived benzene metabolites play an important role in benzene-induced toxicity effects targeting bone marrow and HSCs niche.

Role of bone marrow-derived benzene metabolite

The benzene metabolites produced in liver are transported to bone marrow and further oxidized by MPO to 1,4-BQ or also known as 2,5-cyclohexadiene-1,4-dione. 1,4-BQ is categorized as group 3, that is, not carcinogenic to humans,² as there are insufficient epidemiological data indicating that 1,4-BQ is carcinogenic.^105,106

1,4-BQ has been reported to cause cytotoxicity to murine HSCs at a series of concentrations ranging from 10 µM to 40 µM.¹⁰⁷ Exposure to 1,4-BQ induced concentration-dependent cytotoxicity in human CD34⁺ cells.¹⁰⁶ Previous studies have reported that 1,4-BQ is more toxic than other benzene metabolites (HQ, benzenetriol catechol, and PH) on human mononuclear peripheral blood cells.^108
–110 Thus, it has been speculated that 1,4-BQ is a benzene metabolite that is capable of inducing genotoxicity in hematopoietic cells.

The in vitro studies investigating the genotoxic effects of 1,4-BQ include a report that 1,4-BQ altered the expression of genes involve in the regulation cell cycle, apoptosis, DNA damage, and DNA repair in HSCs^107
–109 as well as promoting the formation of micronucleus in human lymphocyte cells.^109,111,112 In addition, 1,4-BQ has been shown to possess genotoxic effects even at lower concentration of exposure following its ability to induce sister chromatid exchange (SCE) on human T-lymphocyte cell and DNA damage in peripheral blood mononuclear cell (5μM and 10 μM in comparison to other metabolites.)^109,111

Apart from known genotoxic and hematotoxic effects, recent studies have also come up with new findings with regards to the mechanism of 1,4-BQ toxicity, which effects are mediated through lineage-dependent toxicity. This was evidenced when 1,4-BQ exposure induces significant lineage-dependent cytotoxicity toward HSCs population (Sca-1⁺), B lymphocyte (CD45⁺), and myeloid (CD11b⁺ and Gr-1⁺) compared to T lymphocyte cell (CD3⁺).^53,113 Besides that, exposure to 1,4-BQ induces lethal cytotoxic effect on erythroid (CFU-E and BFU-E) and myeloid (CFU-G) progenitors, compared to other progenitors (CFU-GM and CFU-GEMM), implying that greater toxicity is observed in single-lineage committed progenitors than multi-lineages committed progenitors.¹¹⁴ In addition to lineage-dependent toxicity, the activity of genes regulating self-renewal and differentiation of HSCs was also affected following 1,4-BQ exposure. This was evidenced when exposure to 1,4-BQ causes a significant upregulation of HoxB4, GATA3, and Bmi-1 gene expressions. This demonstrates the ability of 1,4-BQ to modulate hematopoietic stem/progenitor cells (HSPCs) by altering the self-renewal and differentiation of related genes.

These findings indicate there is a link between hematopoietic lineages and the hematotoxic effects of benzene. Despite the reported results, the precise mechanism of 1,4-BQ, particularly via genetic and epigenetic pathways affecting HSCs niche, comprises of committed progenitors of different lineages remains to be explored. Thus, it is vital to elucidate lineage-directed mechanism of benzene toxicity in targeting HSCs niche that could lead to stem cells dysfunction and subsequent hematological malignancies and disorders.

Benzene-induced leukemia and other hematological malignancies

Benzene is a bone marrow carcinogen that causes acute myeloid leukemia and possibly other hematological malignancies. Some studies of workers who have been exposed to benzene have reported that benzene is a causal factor for acute lymphoblastic leukemia and chronic myeloid leukemia.² Association between benzene exposure and chronic lymphocytic leukemia has also been reported in numerous epidemiological studies.^115,116

Incidence of leukemia among workers who had been exposed to benzene between 1928 and 1938 was first recorded by Penati and Vigliani of the Institute for Industrial Medicine, University of Turin, in 1938.¹¹⁶ They reported 60 cases of aplastic anemia and 10 cases of leukemia. A later study by Vigliani and Forni of 66 cases of workers exposed to benzene during 1942–1975 identified the cause of death as aplastic anemia in 7 cases and leukemia in 11 cases. They also found 137 cases of hemopathy leading to death from aplastic anemia in 3 cases and leukemia in 13 cases in another study in Pavia.¹¹⁷ Besides, the studies by Akshoy et al. during the period 1955–1960 in Istanbul, Turkey, have demonstrated multiple cases of leukemia among shoemaking workers.¹¹⁸

Furthermore, in the mid-1970s, the US Occupational and Safety and Health Administration (OSHA) funded a new study of workers in the rubber industry who had been exposed to benzene. Infante et al. reported a significant fivefold increased risk of all leukemia and a 10-fold increase of deaths from myeloid and monocytic leukemias in benzene-exposed workers at a rubber plant in Akron, Ohio.¹¹⁹ As a result of Infante’s study, the US OSHA lowered the permissible exposure level to benzene from 10 ppm to 1 ppm (time weighted average). In 1987, in China, a major study of 74,497 workers who had been exposed to benzene and 35,805 control individuals was conducted. The data indicated that workers who had been exposed to benzene were much more likely to develop acute myelogenous leukemia, malignant lymphoma, myelodysplastic syndrome (MDS), and aplastic anemia.¹²⁰

These epidemiological studies have revealed a strong association between benzene exposure, even at low levels, and AML and MDS. In addition, chronic exposure of benzene has also been increasingly associated with other forms of malignancy, such as lymphoma and multiple myeloma.¹²¹ The study by Glass et al. associated benzene exposure with chronic lymphocytic leukemia, now classified as a form of non-Hodgkin’s lymphoma (NHL).¹²² Several studies have reported an increased risk of NHL among workers who had been exposed to benzene or a mixture of organic solvents, instead of benzene alone.^123

–126 For example, a report by a National Institute of Cancer found a threefold increase in risk for NHL among the Chinese workers exposed to benzene, with risk rising to fourfold for workers with 10 or more years of benzene exposure.¹²⁰

Benzene exposure also has been linked to multiple myeloma in few studies. Sailors exposed to cargo vapor from gasoline and other light petroleum products on tankers had an increased risk of multiple myeloma.¹²⁷ Further evidence of an association between benzene exposure and the risk of multiple myeloma includes an increased risk of multiple myeloma(AML) among petroleum workers.¹²⁸ Moreover, studies have reported that people living near oil or gas fields have increased risk of lymph hematopoietic cancers, including multiple myeloma.^129
–131 As all hematopoietic malignancies arise from damaged stem cells, thus researchers believed that all kinds of myeloid and lymphoid malignancies can be caused by occupational exposure to benzene.

Genetic and epigenetic mechanisms in benzene-induced malignancies and hematotoxicity

The precise mechanism of which benzene is mediating hematotoxicity and leukemogenicity remains unclear. It has been reported that benzene must be metabolized to produce metabolites with greater toxicity such as PH, catechol, HQ, and 1,4-BQ to become carcinogenic to hematopoietic system.^3,18 One of the mechanisms involves the binding of reactive benzene metabolites to DNA, causing oxidative DNA damage and topoisomerase-IIα inhibition.¹³² Benzene metabolites such as HQ, 1,4-BQ, and catechol can bind covalently to DNA and protein causing oxidative stress toward the cell.^132

–135 In addition, reactive oxygen species (ROS) produced from oxidative stress is an important factor causing DNA damage. Topoisomerase IIα, a nuclear enzyme involved in the replication process and chromosome recombination, is inhibited when exposed to benzene metabolites causing DNA strand breakage.¹³⁶ Finally, this DNA damage will lead to the occurrence of mitotic recombination, chromosome translocation, and chromosome aneuploidy.^3,137 Failure in cell repair mechanism upon the DNA damage eventually will result in carcinogenicity.

Thus, benzene is able to induce toxicity toward the hematopoietic system via multiple modes of actions targeting HSCs niche.³ It has been reported that benzene specifically suppresses hematopoietic functions and causes leukemia. This indicates the role of the bone marrow niche as a target for benzene-induced hematotoxicity and leukemogenicity. Benzene metabolism can promote oxidative stress causing genomic instability to the targeted HSCs which can be manifested through chromosome aberration, gene mutation, error-prone DNA repair, and epigenetic alteration. Both epigenomic and genomic instability cause the clonal formation of LSC with increased proliferation leading to the subsequent increase in a leukemic cell population as shown in Figure 5.¹³⁷

Figure 5.

Multiple key events and modifying factor involved in benzene-induced leukemia. Benzene metabolism in liver and bone marrow produces benzene metabolites that act via multiple mechanisms involving both genetic and epigenetic alteration that cause various damages to the hematopoietic cells.¹³⁷

Chromosomal aberration

Benzene exposure has been associated with increased incidence of leukemia, especially AML. According to the French American British classification, AML has been categorized into nine groups from M0 to M7 based on the types of cells involved.¹³⁸ Meanwhile, the World Health Organization (WHO) has categorized AML according to chromosomal translocation detected in AML patients. Examples of chromosomal translocation for AML group that has been classified by WHO are t(8;21)(q22;q22), t(16:16)(p13.1;q22), t(15;17)(q22;q12), t(9;11)(p22;q23), t(6;9)(p23;q34), t(3;3)(q21;q26.2), and t(1;22)(p13;q13). In addition, monosomy of chromosome 7 and trisomy 8 are also chromosomal changes observed in AML.^10,139 Numerous studies have reported that benzene exposure induced leukemia-related chromosomal changes that are commonly observed in AML can be detected in the peripheral blood lymphocytes of workers who have been heavily exposed to benzene.^{11,12,140,141} The presence of monosomy 5 and monosomy 7 has been observed in the human lymphocytes from healthy workers exposed to benzene.¹⁴⁰ Benzene metabolites also produce AML-related chromosomal changes in human CD34⁺ progenitor cells, providing evidence that chromosomal aberrations, such as aneuploidy and translocations, may be the genetic pathway for induction of AML by benzene.^141,142

Furthermore, a study using OctoChrome fluorescent in situ hybridization and the micronucleus–centromere assay reported that benzene exposure causes monosomy of chromosomes 5, 6, 7, and 10 and trisomy of chromosomes 8, 9, 17, 21, and 22.¹⁴³ Chromosomal aberrations such as translocation t(14;18) and dose-dependent induction of long-arm deletion of chromosome 6 [del(6q)] have been reported in workers who have been exposed to benzene.¹⁴⁴ Benzene can cause leukemia with chromosomal translocations and inversions known to be induced by topo II inhibitors through HQ and BQ.¹³⁶ Conversion of HQ to BQ by peroxidase increases topo II inhibition, thus making BQ, is more potent than HQ in the inhibition of topo II in vitro. Taken together, these observations suggest that benzene is a known inducer of topo II inhibition leading to chromosomal abnormalities which subsequently could lead to leukemia.

Error-prone DNA repair mechanism

DNA repair pathways are involved in the removal of benzene-induced DNA lesions, such as oxidative DNA lesions, DNA adducts, and apurinic sites. DNA repair is vital for cell survival and maintenance of tissue homeostatis.¹⁴⁵ Examples of major DNA repair pathway are base excision repair, nucleotide excision repair, and double-strand break (DSB) repair. DSB repair involves two types of mechanisms: (1) homologous recombination that uses templates which are sister chromatid exchange for proper repairing only in cycling cells and (2) error-prone nonhomologous end joining (NHEJ)^145,146 (Figure 6). NHEJ-mediated DNA repair can take place at any stage of the cell cycle and has a limited requirement for sequence homology which may contribute to genomic instability. Benzene metabolites such as HQ and BQ can cause double-stranded DNA breakage via inhibition of topoisomerase II enzyme and ROS formation.¹³⁶ This breakage of double-stranded DNA will eventually initiate phosphorylation process on H2AX histone which is a minor protein histone in nucleosome.¹⁴⁶ Phosphorylation of H2AX histone has been identified as one of the initial processes that take place after DNA strand breakage to maintain the integrity of the genome by participating in DSBs repair.¹⁴⁷ Therefore, γ-H2AX (phosphorylated H2AX histone protein) is used as a highly sensitive indicator in genotoxicity research.¹⁴⁸ Many in vitro studies have stated that benzene metabolites such as phenol, HQ, and BQ in low dose could lead to the formation of γ-H2AX foci, which is the sensitive indicator of double-stranded DNA breakage (Figure 7).^146,149 Thus, to study the mechanism mediating benzene leukemogenicity, the γ-H2AX can be used as a first indicator to look into the ability of benzene metabolites to induce double-strand DNA breakage.

Figure 6.

Promotion of DNA repair mechanisms by benzene. Benzene metabolites effectively inhibit Topo II and produce DNA strands with DNA adducts generating DSBs. The excessive and unrepaired DNA damage will lead to programed cell death (apoptosis). The two complementary mechanisms by which eukaryotic cells repair DSBs are HR and NHEJ. HR-mediated DNA repair uses a template for accurate repair, usually via sister chromatid exchange pathway producing error-free DNA repair. However, the NHEJ-mediated DNA repair is an error-prone pathway that often leads to misrepaired DSBs that may result in chromosomal deletions, insertions, or translocations, and subsequent genomic instability.^145,146 DSB: double-strand break; HR: homologous recombination; NHEJ: non-homologous end joining.

Figure 7.

Summary of pathways in benzene metabolites mediating γ-H2AX generation that includes mechanisms via ROS production, inhibition of Topo II, and replication of DNA strands containing DNA adducts.¹⁴⁹ ROS: reactive oxygen species.

Formation of γ-H2AX foci will activate Ku70 and Ku80, the regulatory subunits of DNA-dependent protein kinase (DNA-PK), which will eventually facilitate activation of DNA-PKcs. DSB will activate DNA-PK, which will then initiate NHEJ for DSB repair. In vitro studies have shown that benzene metabolites (phenol, HQ, and BQ) induce DSB and DNA-PKcs that cause NHEJ for DSB repair which in turn increases genomic instability that leads to leukemogenesis.^150,151 Furthermore, following DNA damage, human HSCs that are in quiescent state prefer to undergo DNA repair via NHEJ instead of initiating apoptosis, causing HSCs to be highly susceptible to benzene-induced leukemogenesis.¹⁴⁵ Overall, these findings provide mechanistic insights into benzene-induced DNA DSB and error-prone repair, which are vital in benzene-induced leukemogenesis.

Epigenetic regulation

In addition to genomic instability, reactive benzene metabolites may also induce epigenetic alterations toward HSCs.³ Epigenetic regulation has been found to be involved in the regulation of gene expression, proliferation, and differentiation of stem cell and stem cell tumor formation.⁵⁸ The previous study has reported that the epigenetic mechanism plays an important role in benzene-induced bone marrow toxicity by affecting the regulation of gene expression and stem cell proliferation and differentiation.³ Changes in the pattern of DNA methylation can be observed even at low dose of benzene exposure, as evidenced by the study conducted among petrol station workers and traffic police.¹⁵² Besides that, methylation on DNA promoter may cause a decrease in p15 and p16 gene expression, as shown by the study of patients who had been poisoned by benzene.¹⁵³ In addition to DNA methylation, exposure to benzene also caused an alteration in miRNA expression profile. Previous studies have reported that benzene induces hematotoxicity and leukemogenesis by altering miRNA expression in HSCs.^154,155 Taken together, further molecular studies to identify the deregulated miRNAs potentially involved in benzene-induced hematotoxicity and leukemogenicity targeting HSCs niche may open new paths for benzene poisoning prevention through pharmacological interventions.

Conclusion

Benzene toxicity is commonly linked to bone marrow depression and leukemogenesis. The events are believed to be mediated through interactions between benzene metabolites against the targeted HSCs and lineage-committed progenitors that represent fundamental population of cells in HSCs niche. Benzene metabolites may act via multiple mode of actions in targeting the HSCs and lineage-committed progenitors. These actions can lead to LSC clonal evolution and expansion, followed by subsequent development of hematological disorders and malignancies with varying phenotypes. Despite the critical role of benzene toxicity in causing hematological disorders is reported by numerous studies, uncovering the precise mechanism benzene toxicity uses to target HSCs niche is still a challenging exploration. This is due to the complexity of HSCs niche microenvironment which comprises of mixed population of HSCs and progenitor cells of different lineages. These cells may possess different biological properties and intrinsic mechanisms that may determine differential response toward benzene toxicity. Thus, application of novel lineage-directed strategy combined with toxicogenomic profiling to focus on genetic and epigenetics investigations is an emerging field in hematotoxicological studies targeting fundamental property of HSCs niche that are crucial for maintenance of hematopoiesis. In conclusion, elucidation of genetic and epigenetic mechanisms responsible for cellular transformation of HSCs and committed progenitors is fundamental to gaining greater understanding of the etiologies of hematological disorders and malignancies caused by benzene toxicity. The resulted findings will be useful in developing preventive methods and therapies for benzene-induced blood disorders, which will be informative for the research of other hematotoxic interactions targeting HSCs niche.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

R Dewi

SR Abdul Razak

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