Charlotte M. Witmer

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.

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.

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.

Metabolic Correlates of Benzene Toxicity

The basic concept that underlies the work presented in this and the preceding volume of this series (Jollow et al., 1977) is that the toxicity of many chemicals is seen only after metabolic activation of the original chemical to a more toxic form. Several lines of evidence indicate that benzene also must be metabolically activated in order to exert its characteristic toxicity on bone marrow. Most of the hydroxylated benzene metabolites known today had been described earlier by Parke and Williams (1953). These authors found phenol, catechol, hydroquinone, resorcinol and some trihydroxylated derivatives in the urine of rabbits given 14C-benzene; they suggested some of these may be the toxic metabolite(s). Dustin (1950) reported that di- and trihydroxylated benzenes probably act in their quinone forms to inhibit mitosis. Nomiyama (1965) treated rats with phenol, phenylsulfate, pyrocatechol, hydroquinone, hydroxyhydroquinone and other metabolites of benzene and reported that only catechol caused a reduction in circulating leukocytes. He reasoned that catechol was the toxic metabolite. Andrews et al. (1977) found that in mice given benzene by subcutaneous injection the concentrations of benzene metabolites in the bone marrow exceeded those in blood by a factor of ten. This was the highest organ to blood ratio found when comparing other organs with marrow. Similar data demonstrating sequestration of benzene metabolites in bone marrow were obtained by Rickert et al. (1979) who administered the benzene by inhalation. Andrews et al. (1977) also found that toluene inhibited benzene metabolism competitively, reducing benzene metabolite levels in urine and in the bone marrow and protecting the animals against benzene-induced bone marrow depression. It may be concluded on the basis of these and other related observations that benzene toxicity is due to the formation of a toxic metabolite of benzene.

Toxicological and biochemical effects of repeated administration of benzene in mice

Repeated dosing of mice with benzene led to a dose-related decrease in red cell production as measured by the incorporation of 59Fe into developing erythrocytes. Phenol, catechol, and hydroquinone were observed in the urine, largely conjugated with glucuronic acid and ethereal sulfate. During repeated dosing, toluene-soluble radioactivity derived from labeled benzene was found to accumulate in blood, liver fat, and, most significantly, bone marrow. Greater accumulation was observed when water-soluble metabolites of benzene were examined in these organs. Covalent binding of benzene metabolites was also observed in liver and marrow during repetitive treatment. Both covalently bound and soluble metabolites accumulated in bone marrow, liver, and kidney over a 24-h period after a single administration of benzene. The highest levels of covalent binding were seen in kidney and liver after 3 d of dosing at 880 mg/kg, two doses per day. Studies in vitro demonstrated the necessity for metabolic activation to produce covalent binding from benzene. These studies demonstrate that increasing benzene toxicity during repetitive treatment of mice is accompanied by increases in the levels of both water-soluble and covalently bound benzene metabolites.

An Overview of the Problem of Benzene Toxicity and Some Recent Data on the Relationship of Benzene Metabolism to Benzene Toxicity

Historically, both bone marrow depression and leukemogenesis have been associated with exposure to benzene in the industrial setting. As early as 1897, Santesson (1) described chronic benzene poisoning in Sweden and, around the time of World War I, Selling (2), at Johns Hopkins, described benzene-induced aplastic anemia in both man and animals. Impaired immunological mechanisms in benzene-treated animals were first reported before World War I (3–8). Prior to the development of modern industrial hygiene, the uncontrolled use of benzene in industry expanded despite the growing appreciation of the dangers of working in an atmosphere laden with this highly volatile solvent.

Urinary Metabolites of Benzene in the Mouse

Chronic benzene toxicity, which is characterized by decreases in circulating blood cell levels due to depression of the bone marrow, is thought to be caused by a metabolite of benzene. Our understanding of the metabolism of benzene derives largely from the reports of R.T. Williams and his co-workers (Porteus and Williams, 1949a, 1949b; Parke and Williams, 1952, 1953a, 1953b), who were the first to systematically identify benzene metabolites in rabbit urine after administering benzene to rabbits. Forty-five percent of the administered benzene was recovered. in the expired air, 43% of the administered dose being unchanged benzene and the remaining 2% was carbon dioxide. Urine contained 35% of the administered radioactivity, mostly in the form of conjugated phenolic metabolites of benzene. Upon acid hydrolysis of the conjugates, phenol was found to account for 24% of the dose while other metabolites included catechol (2.2%), hydroquinone (4.8%), and hydroxyhydroquinone (0.3%). 1-Phenylmercapturic acid (0.5%) and trans-trans-muconic (1.3%) acid were also recovered in the urine.

Benzene Metabolism and Toxicity

Chronic exposure to benzene leads to blood dyscrasias characterized by progressive depression in the levels of circulating leukocytes, thrombocytes, and/or erythrocytes, eventually leading to pancytopenia and aplastic anemia (1). The literature contains many descriptions of human benzene toxicity following chronic inhalation (2–4) and the process has been reproduced in several animal species either by exposing animals to atmospheric benzene (5,6) or by parenteral administration of benzene (7,8). The mechanism by which benzene produces bone marrow depression has been explored in a series of studies by Kissling and Speck (9,10) and by Boje et al. (11), who demonstrated that nucleic acid synthesis in bone marrow was inhibited in chronic benzene toxicity, but the molecular site of action is as yet unknown.

Mixed Function Oxidase Enzyme Responses to in Vivo and in Vitro Chromate Treatment

Chromium (Cr) is an essential nutrient in humans which aids in the metabolism of cholesterol, glucose and fats. The trivalent state (Cr(III)) is essential in trace doses, while Cr(VI) is toxic to mammals in acute and subchronic doses [Langard and Norseth, 1986] and long term exposure has been associated with respiratory cancer [Chiazze and Wolf, 1980; Langard and Norseth, 1975; Mancuso, 1975]. Unusually high rates of lung cancer have been reported in workers in chrome plating, leather tanning, and other Cr related industries [Chiazze and Wolf, 1980; Langard and Norseth, 1975; Mancuso, 1975]. The mechanisms for the toxic and genotoxic effects of Cr are only partially understood. The differences in toxicity of the two most common oxidation states, Cr(VI) and Cr(III), are due to the relative lack of ability of cationic Cr(III) compounds to cross cell membranes, while Cr(VI) as the chromate anion, crosses biological membranes freely [Aaseth et al., 1982; Wiegand et al., 1985]. The intracellular reduction of Cr(VI) has recently been shown to result in both Cr(V) and Cr(III) production, both of which are putative DNA damaging agents [Goodgame et al., 1982; Jennette, 1982]. Compounds such as glutathione (GSH) [Aaseth et al., 1982; Connett and Wetterhahn, 1983], ascorbate [Connett and Wetterhahn, 1983], and hydrogen peroxide [Cupo and Wetterhahn, 1985] participate in the intracellular reduction of Cr(VI). Reduction takes place both in mitochondria [Alexander et al., 1982], and the endoplasmic reticulum [Gruber and Jennette, 1978]. When GSH is the reductant a toxic glutathionyl radical (GS) may be formed [Wetterhahn, 1990, in press]. Cr(III) binds to nucleophiles including some sulfhydryl (SH) containing enzymes, with some resulting enzyme inhibition. Thus the metabolism of Cr(VI) is important for the interaction of Cr with DNA [Tsapakos and Wetterhahn, 1983], with GSH [Wiegand et al., 1985] and with SH groups on other cellular macromolecules. Previous studies with microsomes have indicated that cytochrome P-450 (P-450), an SH containing enzyme, acts as an electron donor in the microsomal reduction of Cr(VI) [Gruber and Jennette, 1978].