Robert Snyder

Current Concepts of Chronic Benzene Toxicity

AbstractThe maladies that have beset mankind throughout history may in general be classified as parasitic (infections, infestations, etc.), nutritional/endocrine disease (hyper- or hypofunctional endocrine organs, etc.), or traumatic injury (accidents, war, etc.). With the development of an industrial society, new forms of disease arose in which people succumbed to illness induced by exposure to toxic materials in the course of their labor. Occupational diseases have been with us for many centuries,1, 2 but the incidence of industrial toxicity increased markedly with the advent of the industrial revolution. Although efforts to protect workers against job-related illness have been vigorous in recent years, economic necessity compels the continued use of hazardous chemicals, and benzene is an excellent case in point. It has been estimated by the National Institute for Occupational Safety and Health that about 2, 000, 000 persons in the national work force have potential exposure to benzene.

Effects of toluene on the metabolism, disposition and hemopoietic toxicity of [3H]benzene.

Abstract The administration of [3H]benzene to mice resulted in the decreased incorporation of 59Fe into red cells and the accumulation of benzene and its metabolites in bone marrow and other tissues. Toluene protected against the benzene-induced depression of red cell 59Fe uptake and reduced the levels of benzene metabolites in bone marrow without affecting the level of benzene in this tissue. The results of this study suggest that toluene exerted its protective effect by inhibiting benzene metabolism and that a metabolite of benzene probably mediates the observed hemopietic toxicity of benzene.

Partial hepatectomy reduces both metabolism and toxicity of benzene.

Removal of 70--80% of the liver reduced both the metabolism and the toxicity of benzene in rats. Metabolism was evaluated by measuring the levels of urinary metabolites in both sham-operated and partially hepatectomized rats given 2200 mg/kg [3H]benzene sc. Toxicity was evaluated by measuring the incorporation of 59Fe into circulating erythrocytes according to the method of Lee et al. The observation that partial hepatectomy decreases benzene metabolism and protects against benzene toxicity indicates that the liver may play a primary role in the development of benzene-induced bone marrow toxicity. The fact that benzene administration also reduces the ability of the liver to regenerate after partial hepatectomy suggests that the regenerating liver may serve as a model system in lieu of the bone marrow for studying the mechanism by which benzene inhibits cell proliferation.

OVERVIEW OF THE TOXICOLOGY OF BENZENE

Santesson (1897) and Selling (1916) reported that chronic exposure to benzene in the workplace resulted in marked decreases in circulating blood cells and subsequent fatality. An overview of benzene-induced bonemarrow depression can be derived from industrial studies where large groups of workers were exposed and various stages in the severity of the disease could be identified among the worker population (e.g., Helmer, 1944; Greenburg et al., 1939; Hamilton-Patterson & Browning, 1944; Savilahti, 1956; Aksoy et al., 1971). Chronic exposure to low concentrations of benzene may produce reversible decreases in blood cell numbers. However, higher chronic exposures lead to the onset of irreversible bonemarrow depression, which is characterized by anemia, leukocytopenia, with emphasis on lymphocytopenia, and/or thrombocytopenia. Decrease in all three cell types is defined as pancytopenia, which in the case of benzene toxicity results from severely damaged bone marrow, and the disease is termed aplastic anemia. The recognition that benzene was a leukemogen was established on the basis of studies among exposed worker populations by in Italy by Vigliani and Forni (1976), in Turkey by Aksoy, Erdem, and Dincol (1974), and Aksoy and Erdem (1978), in the United States by Infante et al. (1977), and in China by Yin et al. (1987). Decreases in circulating blood cells may also be observed in the presence of a dysplastic marrow. Myelodysplastic syndrome (MDS) is a preleukemic state characterized by abnormal marrow architecture, inadequate hematopoiesis, and many cells demonstrating chromosome damage. MDS is a clonal disease (Janssen et al., 1989), which may result in response to chemotherapy by alkylating agents (Kantarjian & Keating, 1987) or to chronic benzene exposure (Forni & Moreo, 1967, 1969; van den Berghe et al., 1979). Chronic benzene toxicity leading to MDS usually proceeds to acute myelocytic leukemia (AML). Chromosome damage has been associated with benzene-induced leukemia since the observations of Pollini and

A Perspective on Benzene Leukemogenesis

Although benzene is best known as a compound that causes bone marrow depression leading to aplastic anemia in animals and humans, it also induces acute myelogenous leukemia in humans. The epidemiological evidence for leukemogenesis in humans is contrasted with the results of animal bioassays. This review focuses on several of the problems that face those investigators attempting to unravel the mechanism of benzene-induced leukemogenesis. Benzene metabolism is reviewed with the aim of suggesting metabolites that may play a role in the etiology of the disease. The data relating to the formation of DNA adducts and their potential significance are analyzed. The clastogenic activity of benzene is discussed both in terms of biomarkers of exposure and as a potential indication of leukemogenesis. In addition to chromosome aberrations, sister chromatid exchange, and micronucleus formation, the significance of chromosomal translocations is discussed. The mutagenic activity of benzene metabolites is reviewed and benzene is placed in perspective as a leukemogen with other carcinogens and the lack of leukemogenic activity by compounds of related structure is noted. Finally, a pathway from exposure to benzene to eventual leukemia is discussed in terms of biochemical mechanisms, the role of cytokines and related factors, latency, and expression of leukemia.

Benzene metabolism in mouse liver microsomes

Abstract Mouse liver microsomes metabolized benzene more rapidly than microsomes prepared from rat and rabbit liver. Treatment of mice with benzene increased the metabolism of benzene in vitro without increasing cytochrome P-450 concentrations. Conversely, treatment of mice with phenobarbital increased cytochrome P-450 values but did not increase benzene metabolism. Benzene metabolism was inhibited by compounds known to interact with the mixed function oxidase system, e.g., aniline, metyrapone, aminopyrine, SKF-525A and cytochrome c , but not by KCN or 3-amino-1,2,4-triazole. CO also inhibited benzene metabolism and the Warburg partition coefficient was similar to that obtained for other drugs metabolized by cytochrome P-450. Addition of benzene to mouse liver microsomes yielded a type I binding spectrum. Induction with benzene increased the magnitude of the type I spectral change ( ΔE max ) by a factor approximately equal to the increase in benzene metabolism. The evidence suggests that benzene metabolism is mediated by the mixed function oxidase and binding of benzene to cytochrome P-450 is a significant factor in determining the rate of benzene metabolism.

Leukemia and Benzene

Excessive exposure to benzene has been known for more than a century to damage the bone marrow resulting in decreases in the numbers of circulating blood cells, and ultimately, aplastic anemia. Of more recent vintage has been the appreciation that an alternative outcome of benzene exposure has been the development of one or more types of leukemia. While many investigators agree that the array of toxic metabolites, generated in the liver or in the bone marrow, can lead to traumatic bone marrow injury, the more subtle mechanisms leading to leukemia have yet to be critically dissected. This problem appears to have more general interest because of the recognition that so-called “second cancer” that results from prior treatment with alkylating agents to yield tumor remissions, often results in a type of leukemia reminiscent of benzene-induced leukemia. Furthermore, there is a growing literature attempting to characterize the fine structure of the marrow and the identification of so called “niches” that house a variety of stem cells and other types of cells. Some of these “niches” may harbor cells capable of initiating leukemias. The control of stem cell differentiation and proliferation via both inter- and intra-cellular signaling will ultimately determine the fate of these transformed stem cells. The ability of these cells to avoid checkpoints that would prevent them from contributing to the leukemogenic response is an additional area for study. Much of the study of benzene-induced bone marrow damage has concentrated on determining which of the benzene metabolites lead to leukemogenesis. The emphasis now should be directed to understanding how benzene metabolites alter bone marrow cell biology.

Covalent binding of benzene and its metabolites to DNA in rabbit bone marrow mitochondria in vitro

Rabbit bone marrow mitochondria, stripped of their outer membrane (mitoplasts), have been shown to carry out the NADPH-dependent bioactivation of radiolabelled benzene in vitro to metabolites capable of covalently binding to mtDNA, thereby inhibiting transcription. The metabolites of benzene produced in bone marrow cells by the microsomal cytochrome P-450 are thought to be phenol, catechol, hydroquinone and p-benzoquinone (Andrews et al., Life Sci., 25 (1979) 567; Irons et al., Chem.-Biol. Interact., 30 (1980) 241). Incubation of mitoplasts from rabbit bone marrow cells in vitro with varying concentrations of the putative microsomal metabolites showed a concentration-dependent inhibition of RNA synthesis. The 50% inhibitory molar concentration (IC50) for each metabolite was determined to be: 1,2,4- benzenetriol , 6.3 X 10(-7); p-benzo-quinone, 2 X 10(-6); phenol, 2.5 X 10(-5); hydroquinone, 5 X 10(-5); catechol, 2 X 10(-3); benzene, 1.6 X 10(-2). DNA, isolated from rabbit bone marrow cell or rat liver mitoplasts prelabelled in DNA with [3H]dGTP and exposed to [14C]benzene in vitro, was enzymatically hydrolyzed to nucleosides which were chromatographed on a Sephadex LH-20 column to separate free nucleosides from nucleoside-adducts. The elution profiles indicated that rat liver mtDNA contained six guanine nucleoside-adducts and rabbit bone marrow cell mtDNA contained seven guanine nucleoside-adducts. Incubation of bone marrow mitoplasts in vitro in the presence of benzene and the hydroxyl radical scavenger, mannitol, resulted in the inhibition of formation of four of the guanosine-adducts. When [3H]dATP was substituted as the prelabelled precursor nucleotide, the LH-20 column profile indicated that two adenine nucleoside-adducts were also formed from benzene in vitro. Furthermore, a comparison of the Sephadex LH-20 column profiles of purine adducts derived from [14C]benzene- and [3H]dGMP-labelled mtDNA with profiles generated by individually incubating each of the putative unlabelled metabolites with bone marrow mitoplasts in vitro has indicated that p-benzoquinone, phenol, hydroquinone and 1,2,4- benzenetriol form adducts with guanine. One of the two adenosine-adducts may arise from hydroquinone; the compound forming the other adduct is unknown at the present time. Exposure of mitoplasts to catechol in vitro resulted in the formation of a guanine nucleoside-adduct that was present in rat liver mtDNA but absent from the DNA isolated from rabbit bone marrow cell mitoplasts exposed to [14C]benzene in vitro. This suggests that catechol is probably not a major metabolite of benzene formed in bone marrow cell mitochondria.

Bone marrow depressant and leukemogenic actions of benzene

Abstract Chronic benzene toxicity is expressed as bone marrow depression resulting in leucopenia, anemia or thrombocytopenia. With continued exposure the disease progresses to pancytopenia resulting from a bone marrow aplasia. In recent years evidence has accumulated implicating benzene in the etiology of leukemias in workers in industries where benzene was heavily used. It has been suggested that leukemia is as frequent a cause of death from chronic benzene exposure as is aplastic anemia. This review explores some current ideas on the mechanisms by which benzene may produce these diseases and emphasizes recent work suggesting that the causative agent is a metabolite of benzene.

The metabolism of benzene in vitro

Benzene-14C metabolism was studied in rabbit and rat liver preparations. Phenol and its conjugates, phenyl sulfate and phenyl glucuronide, were the major metabolites. At low benzene concentrations most of the phenol was conjugated. At higher benzene concentrations considerably more free phenol remained. Pretreatment of animals with phenobarbital or benzene resulted in a stimulation of benzene metabolism in vitro. The stimulation of benzene metabolism by benzene was accompanied by a stimulation of amino acid incorporation into microsomal protein, suggesting that the stimulation of benzene metabolism resulted from induction of more benzene-metabolizing enzymes.