Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Partial hepatectomy reduces both metabolism and toxicity of benzene D. Sammett , E. W. Lee , J. J. Kocsis & R. Snyder To cite this article: D. Sammett , E. W. Lee , J. J. Kocsis & R. Snyder (1979) Partial hepatectomy reduces both metabolism and toxicity of benzene, Journal of Toxicology and Environmental Health, 5:5, 785-792, DOI: 10.1080/15287397909529789 To link to this article: http://dx.doi.org/10.1080/15287397909529789
Published online: 15 Oct 2009.
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Date: 26 September 2015, At: 23:34
PARTIAL HEPATECTOMY REDUCES BOTH METABOLISM AND TOXICITY OF BENZENE D. Sammett, E. W. Lee, J. J. Kocsis, R. Snyder
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Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania
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 59 Fe 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.
INTRODUCTION Chronic benzene exposure progressively depresses bone marrow function until eventually the marrow fails to produce normal circulating blood cells (Snyder and Kocsis, 1975). The mechanism by which benzene acts has not yet been completely established, but studies of red cell development indicate that benzene selectively damages the early proliferating cell forms—pronormoblasts and normoblasts—without affecting resting stem cells or reticulocytes (Lee et al., 1974; Snyder et al., 1977a, 1977b; Steinberg, 1949; Moeschlin and Speck, 1967; Rondanelli et al., 1970). Andrews et al. (1977) reported that the extent of benzene metabolism closely followed the degree of benzene-induced inhibition of erythrocyte 59 Fe uptake and suggested that a benzene metabolite mediates benzene toxicity. Although the liver is generally accepted as the major site of benzene metabolism, the observation that metabolite concentrations in marrow exceed those in blood and liver raises questions concerning the relative importance of liver and marrow in the production of toxic benzene metabolites. The studies described here were designed to further delineate the role of the liver in producing benzene-induced bone marrow depression. This work was supported by U.S. Public Health Service grant ES00322. Requests for reprints should be sent to Robert Snyder, Department of Pharmacology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania 19107. 785 Journal of Toxicology and Environmental Health, 5:785-792, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/040785-08$2.25
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MATERIALS AND METHODS Before surgery, male Sprague-Dawley rats (250-300 g) had free access to laboratory chow and tap water. After surgery, rats were maintained on 5% dextrose water, sugar cubes, and laboratory chow ad libitum. Partial hepatectomy was performed according to Higgens and Anderson (1931) and resulted in removal of 75 ± 8% (n = 16) of the liver. Sham operations were performed by making a midline incision reaching 1-1.5 cm posteriorly from the xiphoid process of the sternum, exposing the median and left lateral lobes of the liver, and then closing the area with no liver excision. All rats were sacrificed 72 h after surgery. Control rats received olive oil at a dose of 0.5% body weight and the benzene-treated animals received 2200 mg/kg (2.5 ml/kg) benzene sc as a 50% (v/v) benzene-olive oil solution. All injections were given 8 h after surgery. Generally labeled [ 3 H]benzene (approximately 80,000 dpm//imol, Amersham-Searle, Arlington Heights, III.) was administered to determine benzene metabolites in urine. After the injection, urine was collected at regular intervals from rats kept in metabolism cages, and urine samples were counted in a liquid scintillation counter. Total radioactivity in urine was reported as benzene equivalents in micromoles. To determine radioiron utilization, rats were given 59 Fe (1.0 nC\, 40-80 ng Fe) in the form of ferrous citrate (New England Nuclear, Boston, Mass.), ip 2 d after benzene administration. Blood (1.0 ml) was collected from the inferior vena cava 24 h after 59 Fe administration and analyzed in a gamma counter. The percentage of S9 Fe incorporated into erythrocytes was calculated, assuming a blood volume of 6% of the body weight. Protein was estimated by the biuret method (Gorrall et al., 1949), using bovine serum albumin as a standard. DNA was isolated from rat liver according to the Schmidt-Thannhauser method as modified by Munro and Fleck (1966) and estimated by a modified diphenylamine reaction, using calf thymus DNA as a standard. Liver cell nuclei were counted as a measure of cell number by using a modified orcinol reaction. A 10% (w/v) homogenate was prepared from fresh liver tissue in a 0.15 M KCI, 0.10 M phosphate buffer, pH 7.4. An aliquot of the homogenate (0.25 ml) was shaken with 5.0 ml orcinol reagent and then taken up to 10.0 ml with the reagent and shaken again. Nuclei were counted on a Neubauer-type hemocytometer and the results expressed as the number of nuclei per gram (wet weight) of liver. Tests of significance were performed by the Student f-test. RESULTS Effect of Partial Hepatectomy on S9 Fe Uptake into Erythrocytes To determine the importance of the liver in the production of benzene toxicity, the effect of partial hepatectomy on benzene-induced reduction
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TABLE 1. Effect of Partial Hepatectomy on Erythrocyte " F e Utilization in Control and Benzene-Treated Rats 59
Fe uptake in erythrocytes (%)°
Treatment
Sham-operated rats
Partially hepatectomized rats
Control Benzene (2200 mg/kg)
31.6 ± 13.3 (9)^ 15.4 ± 6.7 {\0)c>d
29.1 ± 6.5 (10) 36.4 ± 16.2 (iO) e
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"Based on administered dose. Mean ± SD; the number of animals is given in parentheses. c Significantly different from control sham-operated rats (p < 0.01). Significantly different from benzene-treated partially hepatectomized rats (p < 0.01). e Not significantly different from control partially hepatectomized rats.
of S9 Fe incorporation into erythrocytes was studied in rats given a single dose of benzene (Table 1). Although sham-operated and partially hepatectomized control rats showed no difference in 59 Fe uptake, benzene reduced 59 Fe uptake to about half of control values in sham-operated animals. No reduction in S9 Fe uptake, however, was observed in the benzene-treated partially hepatectomized rats, indicating that partial hepatectomy protected rats against benzene-induced bone marrow depression. Effect of Partial Hepatectomy on Benzene Metabolism Table 2 shows the results of two experiments in which partially hepatectomized and sham-operated rats were given a single dose of [ 3 H ] benzene (2200 mg/kg). For each animal the cumulative urinary excretion of labeled metabolites was monitored for a 36-h period. Partial hepatectomy markedly reduced benzene metabolism in both experiments, demonstrating the essential role of the liver in benzene metabolism. TABLE 2. Urinary Excretion of Labeled Metabolites after a Single Injection of [ 3 H] Benzene (2200 mg/kg, sc)° Benzene equivalents Treatment Sham operation Partial hepatectomy
132.2 ± 52.2 (4) 6 40.5 ± 19.8 (5) c
"Measured as 36-h cumulative urinary radioactivity. Mean ± SD; the number of animals is given in parentheses. c Significantly different from control sham-operated rats
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TABLE 3. Characteristics of Normal and Regenerated Liver in Control and Benzene-Treated Rats
Operation
Treatment0
Protein content (mg/g liver, wet weight)
DNA content (mg/g liver, wet weight)
Nucleated cell count (X 10*/g liver, wet weight)
Liver weight (g)
Sham Partial hepatectomy Sham Partial hepatectomy
Olive oil
189.9 ± 52.8 (3)b
1.5 ± 0.3 (3)
135.6 ± 17.7 (3)
13.2 ± 3.4 (3)
Olive oil Benzene
190.0 ± 57.6 (5) 186.0 ± 35.5 (4) c
3.1 ± 1.2 (5) 2.0 ± 0.3 (4) c
136.2 ± 10.6 (5) 143.5 ± 22.7 (4) c
9.8 ±1.3 (5) 12.6 ± 1.3 (4) c
Benzene
169.6 * 60.4 (4) c
2.9±1.4(4) c
134.6 ± 38.3 (4) c
6.8 ±1.1 (4)°
Injections were given approximately 8 h after surgery. Benzene was administered at a dose of 2200 mg/kg, sc; controls received olive oil at a dose of 0.5% body weight. Animals were sacrificed 72 h after surgery. "Mean ± SD; the number of animals is given in parentheses. c Not significantly different from respective controls. Significantly different from partially hepatectomized controls (p < 0.01).
Effect of Benzene on Liver Regeneration Benzene inhibits liver regeneration. Table 3 shows that the total amount of liver regenerated 72 h after hepatectomy was reduced in rats given a single dose of benzene compared with control rats. Protein and DNA concentrations and nucleated cells per gram of liver remained the same. After sham operation, no difference was found between the livers of control and benzene-treated rats with respect to protein content, DNA content, nucleated cell counts, or whole liver weights. The fact that there was no significant difference in nucleated cell counts between shamoperated and hepatectomized control and benzene-treated rats supports the concept that, during regeneration, hyperplasia is a more important factor than hypertrophy in restoring the liver (Bucher, 1963; Bresnick, 1971). DISCUSSION Previous studies have generally focused on the liver as the major site of benzene metabolism (Snyder et al., 1967; Gonasun et al., 1973; Drew and Fouts, 1974; Drew et al., 1975; Ikeda and Ohtsuji, 1971; Ikeda et al., 1972). The work of Andrews et al. (1977), however, suggested that the bone marrow itself may metabolize benzene and thus lead to hematotoxicity. Protection against bone marrow toxicity by partial hepatectomy supports the central role of the liver in the conversion of benzene to an ultimate toxic species. The covalent binding of benzene metabolites in the bone marrow indicates that benzene can be metabolically activated in marrow; however, a fully functional liver seems to be necessary for benzene to produce bone marrow depression. The covalent
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binding of benzene in liver suggests that reactive metabolites are formed there. It seems unlikely that reactive metabolites escape binding in the liver, survive transport in the circulation, and enter and covalently bind in the marrow. Therefore, a reasonable scheme for the production of toxic benzene metabolites (Fig. 1) is that a metabolite (I) of benzene is formed in the liver and transported to the marrow, where it is further metabolized to a reactive metabolite that causes benzene toxicity. Benzene is hydroxylated by the microsomal mixed function oxidase system to phenol and other hydroxylated benzene derivatives, probably through intermediate formation of benzene epoxide. The epoxide may rearrange nonenzymatically to phenol, conjugate with glutathione to produce a premercapturic acid, be hydrated by epoxide hydratase and oxidized to catechol, or react with various cellular constituents (Snyder and Kocsis, 1975). Phenol and its conjugates, as well as catechol and hydroquinone, are sufficiently stable to constitute, alone or in some combination, metabolite I. Autoxidation or superoxide-mediated oxidation of catechol and/or hydroquinone may lead to the formation of a reactive intermediate. Although this scheme is highly speculative, there is, by analogy to other compounds, theoretical support for the involvement of semiquinones or quinones. Covalent binding of estrogens appears to involve semiquinones or quinones as intermediates (Bolt and Kappus, 1974), and covalent binding of
Blood
Liver
Benzene other metabolites
Metabolite
reactive intermediate
covalent binding to nucleic acids (DNA.RNA.etc.) and protein
Bone Marrow (site of toxicity) Benzene
— Metabolite
Metabolite I
other metabolites
reactive intermediate
covalent binding to macromolecules
FIGURE 1. Postulated scheme for the production of toxic benzene metabolites and eventual toxicity.
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D. SAMMETTETAL.
methyldopa may proceed similarly (Dybing, 1976; Dybing et al., 1976). In addition, quinone intermediates such as 1,2- and 1,4-benzoquinone have been evaluated as cytotoxic through their reactivity as sulfhydryl reagents (Graham et al., 1978a, 1978b). Thus, although catechol and hydroquinone constitute a relatively small fraction of benzene metabolites in mice, they could still be the precursors (metabolite I) to the reactive intermediate that binds to macromolecules. So far, a number of metabolites have been proposed as mediators of benzene toxicity, including phenol (Tunek et al., 1978), catechol (Nomiyama, 1965), and benzene oxide (Jerina and Daly, 1974). However, experimental evidence to show that these compounds produce bone marrow toxicity is meager. The mechanism by which benzene metabolites produce bone marrow depression and possibly leukemia has been difficult to study because of the inaccessibility and relatively amorphous nature of the marrow and the small amounts available for study. The techniques of. bone marrow cell colony formation (McLeod et al., 1974) may provide a suitable method for monitoring the effect of toxic substances on cultured hematopoietic cells. It would also be of value to develop a model system in which the inhibition of cell proliferation in bone marrow could be mimicked with another organ. Although liver cells ordinarily undergo mitosis slowly, the regenerating liver cell shares with bone marrow the capacity for rapid cell division. The studies reported here indicate that benzene inhibits liver regeneration. Previous reports have shown that benzene covalently binds to DNA in liver (Lutz and Schlatter, 1977) and to solid residues of both liver and bone marrow (Snyder et al., 1978). Moeschlin and Speck (1967) and Boje et al. (1970) demonstrated inhibition of nucleic acid synthesis in bone marrow of benzene-treated animals. These observations suggest that benzene may interfere with nucleic acid synthesis, which is a critical factor in cell proliferation. It may now be possible to use the regenerating liver cell instead of the bone marrow as a model for studying the mechanism by which benzene inhibits cell proliferation.
REFERENCES Andrews, L. S., Lee, E. W., Witmer, C. M., Kocsis, J. J., and Snyder, R. 1977. Effects of toluene on the metabolism, disposition and hematopoietic toxicity of 3H-benzene. Biochem. Pharmacol. 26:293-300. Boje, V. H., Benkel, W., and Heiniger, H. J. 1970. Untersuchungen zur Leukopoese im Knochmark der Ratte nach Benzol-Inhalation. Blood 21:250-257. Bolt, H. M. and Kappus, H. 1974. Irreversible binding of ethynyl-estradiol metabolites to protein and nucleic acids as catalyzed by rat liver microsomes and mushroom tyrosinase. J. Steroid Biochem. 5:179-184. Bresnick, E. 1971. Regenerating liver: An experimental model for the study of growth. Methods Cancer Res. 6:347-397.
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Bucher, N. L. R. 1963. Regeneration of mammalian liver. Int. Rev. Cytol. 15:245-300. Drew, R. T. and Fouts, J. R. 1974. The lack of effects of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats. Toxicol. Appl. Pharmacol. 27:183-193. Drew, R. T., Fouts, J. R., and Harper, C. 1975. Species differences in benzene hydroxylation to phenol by pulmonary and hepatic microsomes. Drug. Metab. Dispos. 3:381-388. Dybing, E. 1976. Organ and species differences in microsomal activation of methyldopa. Drug. Metab. Dispos. 4:513-516. Dybing, E., Nelson, S. D., Mitchell, J. R., Sasame, H. A., and Gillette, J. R. 1976. Oxidation of α-methyl-dopa and other catechols by cytochrome P-450-generated superoxide anion: Possible mechanism of methyldopa hepatitis. Mol. Pharmacol. 12:911-920. Gonasun, L. M., Witmer, C. M., Kocsis, J. J., and Snyder, R. 1973. Benzene metabolism in mouse liver microsomes. Toxicol. Appl. Pharmacol. 26:398-406. Gorrall, A. G., Bardawill, G. J., and David, M. M. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-756. Graham, D. G., Tiffany, S. M., Bell, W. R., Jr., and Gutknecht, W. F. 1978a. Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 14:644-653. Graham, D. G., Tiffany, S. M., and Vogel, F. S. 1978b. The toxicity of melanin precursors. J. Invest. Dermatol. 70:113-116. Higgens, G. M. and Anderson, R. M. 1931. Experimental pathology of the liver: 1. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12:186-202. Ikeda, M. and Ohtsuji, H. 1971. Phenobarbital-induced protection against toxicity of toluene and benzene in the rat. Toxicol. Appl. Pharmacol. 20:30-43. Ikeda, M., Ohtsuji, H., and Imamura, T. 1972. In vivo suppression of benzene and styrene oxidation by co-administered toluene in rats and effects of phenobarbital. Xenobiotica 2:101-106. Jerina, D. M. and Daly, J. W. 1974. Arene oxides: A new aspect of drug metabolism. Science 185:573-582. Lee, E. W., Kocsis, J. J., and Snyder, R. 1974. Acute effects of benzene on 5 9 Fe incorporation into circulating erythrocytes. Toxicol. Appl. Pharmacol. 27:431-436. Lutz, W. K. and Schlatter, C. 1977. Mechanism of the carcinogenic action of benzene: Irreversible binding to rat liver DNA. Chem.-Biol. Interact. 18:241-245. McLeod, D. L., Shreeve, M. M., and Axelrod, A. A. 1974. Improved plasma culture system for production of erythrocytic colonies in vitro: Quantitative assay method for CFU-E. Blood 44:517-534. Moeschlin, S. and Speck, B. 1967. Experimental studies on the mechanism of action of benzene on the bone marrow (radioautographic studies using 3 H-thymidine). Acta Haematol. 38:104-111. Munro, H. M. and Fleck, A. 1966. The determination of nucleic acids. Methods Biochem. Anal. 14:113-176. Nomiyama, K. 1965. Studies on poisoning by benzene and its homologues. Ind. Health 3:53-57. Rondanelli, E. G., Gorini, P., Gerna, G., and Magliulo, E. 1970. Pathology of Erythroblastic Mitosis in Occupational Benzenic Erythropathy and Erythemia. In Vivo and in Vitro Studies, pp. 3-5. Basel: Karger. Snyder, R. and Kocsis, J. J. 1975. Current concepts of chronic benzene toxicity. Crit. Rev. Toxicol. 3:265-288. Snyder, R., Uzuki, F., Gonasun, L. M., Bromfeld, E., and Wells, A. 1967. The metabolism of benzene
in vitro. Toxicol. Appl. Pharmacol. 11:346-360. Snyder, R., Andrews, L. S., Lee, E. W., Witmer, C. M., Reilly, M., and Kocsis, J. J. 1977a. Benzene metabolism and toxicity. In Biological Reactive Intermediates, eds. D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio, pp. 286-301. New York: Plenum.
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Snyder, R., Lee, E. W., and Kocsis, J. J. 1977b. Bone marrow depressant and leukemogenic actions of benzene. Life Sci. 21:1701-1722. Snyder, R., Lee, E. W., and Kocsis, J. J. 1978. Binding of labeled benzene metabolites to mouse liver and bone marrow. Res. Commun. Chem. Pathol. Pharmacol. 20:191-194. Steinberg, B. 1949. Bone marrow regeneration in experimental benzene intoxication. Blood 4:550-556. Tunek, A., Platt, K. L., Bentley, P., and Oesch, F. 1978. Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modifications of this metabolism. Mol.
Pharmacol. 14:920-929.
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Received November 13, 1978 Accepted February 9, 1979
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Partial hepatectomy reduces both metabolism and toxicity of benzene D. Sammett , E. W. Lee , J. J. Kocsis & R. Snyder To cite this article: D. Sammett , E. W. Lee , J. J. Kocsis & R. Snyder (1979) Partial hepatectomy reduces both metabolism and toxicity of benzene, Journal of Toxicology and Environmental Health, 5:5, 785-792, DOI: 10.1080/15287397909529789 To link to this article: http://dx.doi.org/10.1080/15287397909529789
Published online: 15 Oct 2009.
Submit your article to this journal
Article views: 14
View related articles
Citing articles: 65 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Universite Laval]
Date: 26 September 2015, At: 23:34
PARTIAL HEPATECTOMY REDUCES BOTH METABOLISM AND TOXICITY OF BENZENE D. Sammett, E. W. Lee, J. J. Kocsis, R. Snyder
Downloaded by [Universite Laval] at 23:34 26 September 2015
Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania
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 59 Fe 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.
INTRODUCTION Chronic benzene exposure progressively depresses bone marrow function until eventually the marrow fails to produce normal circulating blood cells (Snyder and Kocsis, 1975). The mechanism by which benzene acts has not yet been completely established, but studies of red cell development indicate that benzene selectively damages the early proliferating cell forms—pronormoblasts and normoblasts—without affecting resting stem cells or reticulocytes (Lee et al., 1974; Snyder et al., 1977a, 1977b; Steinberg, 1949; Moeschlin and Speck, 1967; Rondanelli et al., 1970). Andrews et al. (1977) reported that the extent of benzene metabolism closely followed the degree of benzene-induced inhibition of erythrocyte 59 Fe uptake and suggested that a benzene metabolite mediates benzene toxicity. Although the liver is generally accepted as the major site of benzene metabolism, the observation that metabolite concentrations in marrow exceed those in blood and liver raises questions concerning the relative importance of liver and marrow in the production of toxic benzene metabolites. The studies described here were designed to further delineate the role of the liver in producing benzene-induced bone marrow depression. This work was supported by U.S. Public Health Service grant ES00322. Requests for reprints should be sent to Robert Snyder, Department of Pharmacology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania 19107. 785 Journal of Toxicology and Environmental Health, 5:785-792, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/040785-08$2.25
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MATERIALS AND METHODS Before surgery, male Sprague-Dawley rats (250-300 g) had free access to laboratory chow and tap water. After surgery, rats were maintained on 5% dextrose water, sugar cubes, and laboratory chow ad libitum. Partial hepatectomy was performed according to Higgens and Anderson (1931) and resulted in removal of 75 ± 8% (n = 16) of the liver. Sham operations were performed by making a midline incision reaching 1-1.5 cm posteriorly from the xiphoid process of the sternum, exposing the median and left lateral lobes of the liver, and then closing the area with no liver excision. All rats were sacrificed 72 h after surgery. Control rats received olive oil at a dose of 0.5% body weight and the benzene-treated animals received 2200 mg/kg (2.5 ml/kg) benzene sc as a 50% (v/v) benzene-olive oil solution. All injections were given 8 h after surgery. Generally labeled [ 3 H]benzene (approximately 80,000 dpm//imol, Amersham-Searle, Arlington Heights, III.) was administered to determine benzene metabolites in urine. After the injection, urine was collected at regular intervals from rats kept in metabolism cages, and urine samples were counted in a liquid scintillation counter. Total radioactivity in urine was reported as benzene equivalents in micromoles. To determine radioiron utilization, rats were given 59 Fe (1.0 nC\, 40-80 ng Fe) in the form of ferrous citrate (New England Nuclear, Boston, Mass.), ip 2 d after benzene administration. Blood (1.0 ml) was collected from the inferior vena cava 24 h after 59 Fe administration and analyzed in a gamma counter. The percentage of S9 Fe incorporated into erythrocytes was calculated, assuming a blood volume of 6% of the body weight. Protein was estimated by the biuret method (Gorrall et al., 1949), using bovine serum albumin as a standard. DNA was isolated from rat liver according to the Schmidt-Thannhauser method as modified by Munro and Fleck (1966) and estimated by a modified diphenylamine reaction, using calf thymus DNA as a standard. Liver cell nuclei were counted as a measure of cell number by using a modified orcinol reaction. A 10% (w/v) homogenate was prepared from fresh liver tissue in a 0.15 M KCI, 0.10 M phosphate buffer, pH 7.4. An aliquot of the homogenate (0.25 ml) was shaken with 5.0 ml orcinol reagent and then taken up to 10.0 ml with the reagent and shaken again. Nuclei were counted on a Neubauer-type hemocytometer and the results expressed as the number of nuclei per gram (wet weight) of liver. Tests of significance were performed by the Student f-test. RESULTS Effect of Partial Hepatectomy on S9 Fe Uptake into Erythrocytes To determine the importance of the liver in the production of benzene toxicity, the effect of partial hepatectomy on benzene-induced reduction
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TABLE 1. Effect of Partial Hepatectomy on Erythrocyte " F e Utilization in Control and Benzene-Treated Rats 59
Fe uptake in erythrocytes (%)°
Treatment
Sham-operated rats
Partially hepatectomized rats
Control Benzene (2200 mg/kg)
31.6 ± 13.3 (9)^ 15.4 ± 6.7 {\0)c>d
29.1 ± 6.5 (10) 36.4 ± 16.2 (iO) e
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"Based on administered dose. Mean ± SD; the number of animals is given in parentheses. c Significantly different from control sham-operated rats (p < 0.01). Significantly different from benzene-treated partially hepatectomized rats (p < 0.01). e Not significantly different from control partially hepatectomized rats.
of S9 Fe incorporation into erythrocytes was studied in rats given a single dose of benzene (Table 1). Although sham-operated and partially hepatectomized control rats showed no difference in 59 Fe uptake, benzene reduced 59 Fe uptake to about half of control values in sham-operated animals. No reduction in S9 Fe uptake, however, was observed in the benzene-treated partially hepatectomized rats, indicating that partial hepatectomy protected rats against benzene-induced bone marrow depression. Effect of Partial Hepatectomy on Benzene Metabolism Table 2 shows the results of two experiments in which partially hepatectomized and sham-operated rats were given a single dose of [ 3 H ] benzene (2200 mg/kg). For each animal the cumulative urinary excretion of labeled metabolites was monitored for a 36-h period. Partial hepatectomy markedly reduced benzene metabolism in both experiments, demonstrating the essential role of the liver in benzene metabolism. TABLE 2. Urinary Excretion of Labeled Metabolites after a Single Injection of [ 3 H] Benzene (2200 mg/kg, sc)° Benzene equivalents Treatment Sham operation Partial hepatectomy
132.2 ± 52.2 (4) 6 40.5 ± 19.8 (5) c
"Measured as 36-h cumulative urinary radioactivity. Mean ± SD; the number of animals is given in parentheses. c Significantly different from control sham-operated rats
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TABLE 3. Characteristics of Normal and Regenerated Liver in Control and Benzene-Treated Rats
Operation
Treatment0
Protein content (mg/g liver, wet weight)
DNA content (mg/g liver, wet weight)
Nucleated cell count (X 10*/g liver, wet weight)
Liver weight (g)
Sham Partial hepatectomy Sham Partial hepatectomy
Olive oil
189.9 ± 52.8 (3)b
1.5 ± 0.3 (3)
135.6 ± 17.7 (3)
13.2 ± 3.4 (3)
Olive oil Benzene
190.0 ± 57.6 (5) 186.0 ± 35.5 (4) c
3.1 ± 1.2 (5) 2.0 ± 0.3 (4) c
136.2 ± 10.6 (5) 143.5 ± 22.7 (4) c
9.8 ±1.3 (5) 12.6 ± 1.3 (4) c
Benzene
169.6 * 60.4 (4) c
2.9±1.4(4) c
134.6 ± 38.3 (4) c
6.8 ±1.1 (4)°
Injections were given approximately 8 h after surgery. Benzene was administered at a dose of 2200 mg/kg, sc; controls received olive oil at a dose of 0.5% body weight. Animals were sacrificed 72 h after surgery. "Mean ± SD; the number of animals is given in parentheses. c Not significantly different from respective controls. Significantly different from partially hepatectomized controls (p < 0.01).
Effect of Benzene on Liver Regeneration Benzene inhibits liver regeneration. Table 3 shows that the total amount of liver regenerated 72 h after hepatectomy was reduced in rats given a single dose of benzene compared with control rats. Protein and DNA concentrations and nucleated cells per gram of liver remained the same. After sham operation, no difference was found between the livers of control and benzene-treated rats with respect to protein content, DNA content, nucleated cell counts, or whole liver weights. The fact that there was no significant difference in nucleated cell counts between shamoperated and hepatectomized control and benzene-treated rats supports the concept that, during regeneration, hyperplasia is a more important factor than hypertrophy in restoring the liver (Bucher, 1963; Bresnick, 1971). DISCUSSION Previous studies have generally focused on the liver as the major site of benzene metabolism (Snyder et al., 1967; Gonasun et al., 1973; Drew and Fouts, 1974; Drew et al., 1975; Ikeda and Ohtsuji, 1971; Ikeda et al., 1972). The work of Andrews et al. (1977), however, suggested that the bone marrow itself may metabolize benzene and thus lead to hematotoxicity. Protection against bone marrow toxicity by partial hepatectomy supports the central role of the liver in the conversion of benzene to an ultimate toxic species. The covalent binding of benzene metabolites in the bone marrow indicates that benzene can be metabolically activated in marrow; however, a fully functional liver seems to be necessary for benzene to produce bone marrow depression. The covalent
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binding of benzene in liver suggests that reactive metabolites are formed there. It seems unlikely that reactive metabolites escape binding in the liver, survive transport in the circulation, and enter and covalently bind in the marrow. Therefore, a reasonable scheme for the production of toxic benzene metabolites (Fig. 1) is that a metabolite (I) of benzene is formed in the liver and transported to the marrow, where it is further metabolized to a reactive metabolite that causes benzene toxicity. Benzene is hydroxylated by the microsomal mixed function oxidase system to phenol and other hydroxylated benzene derivatives, probably through intermediate formation of benzene epoxide. The epoxide may rearrange nonenzymatically to phenol, conjugate with glutathione to produce a premercapturic acid, be hydrated by epoxide hydratase and oxidized to catechol, or react with various cellular constituents (Snyder and Kocsis, 1975). Phenol and its conjugates, as well as catechol and hydroquinone, are sufficiently stable to constitute, alone or in some combination, metabolite I. Autoxidation or superoxide-mediated oxidation of catechol and/or hydroquinone may lead to the formation of a reactive intermediate. Although this scheme is highly speculative, there is, by analogy to other compounds, theoretical support for the involvement of semiquinones or quinones. Covalent binding of estrogens appears to involve semiquinones or quinones as intermediates (Bolt and Kappus, 1974), and covalent binding of
Blood
Liver
Benzene other metabolites
Metabolite
reactive intermediate
covalent binding to nucleic acids (DNA.RNA.etc.) and protein
Bone Marrow (site of toxicity) Benzene
— Metabolite
Metabolite I
other metabolites
reactive intermediate
covalent binding to macromolecules
FIGURE 1. Postulated scheme for the production of toxic benzene metabolites and eventual toxicity.
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D. SAMMETTETAL.
methyldopa may proceed similarly (Dybing, 1976; Dybing et al., 1976). In addition, quinone intermediates such as 1,2- and 1,4-benzoquinone have been evaluated as cytotoxic through their reactivity as sulfhydryl reagents (Graham et al., 1978a, 1978b). Thus, although catechol and hydroquinone constitute a relatively small fraction of benzene metabolites in mice, they could still be the precursors (metabolite I) to the reactive intermediate that binds to macromolecules. So far, a number of metabolites have been proposed as mediators of benzene toxicity, including phenol (Tunek et al., 1978), catechol (Nomiyama, 1965), and benzene oxide (Jerina and Daly, 1974). However, experimental evidence to show that these compounds produce bone marrow toxicity is meager. The mechanism by which benzene metabolites produce bone marrow depression and possibly leukemia has been difficult to study because of the inaccessibility and relatively amorphous nature of the marrow and the small amounts available for study. The techniques of. bone marrow cell colony formation (McLeod et al., 1974) may provide a suitable method for monitoring the effect of toxic substances on cultured hematopoietic cells. It would also be of value to develop a model system in which the inhibition of cell proliferation in bone marrow could be mimicked with another organ. Although liver cells ordinarily undergo mitosis slowly, the regenerating liver cell shares with bone marrow the capacity for rapid cell division. The studies reported here indicate that benzene inhibits liver regeneration. Previous reports have shown that benzene covalently binds to DNA in liver (Lutz and Schlatter, 1977) and to solid residues of both liver and bone marrow (Snyder et al., 1978). Moeschlin and Speck (1967) and Boje et al. (1970) demonstrated inhibition of nucleic acid synthesis in bone marrow of benzene-treated animals. These observations suggest that benzene may interfere with nucleic acid synthesis, which is a critical factor in cell proliferation. It may now be possible to use the regenerating liver cell instead of the bone marrow as a model for studying the mechanism by which benzene inhibits cell proliferation.
REFERENCES Andrews, L. S., Lee, E. W., Witmer, C. M., Kocsis, J. J., and Snyder, R. 1977. Effects of toluene on the metabolism, disposition and hematopoietic toxicity of 3H-benzene. Biochem. Pharmacol. 26:293-300. Boje, V. H., Benkel, W., and Heiniger, H. J. 1970. Untersuchungen zur Leukopoese im Knochmark der Ratte nach Benzol-Inhalation. Blood 21:250-257. Bolt, H. M. and Kappus, H. 1974. Irreversible binding of ethynyl-estradiol metabolites to protein and nucleic acids as catalyzed by rat liver microsomes and mushroom tyrosinase. J. Steroid Biochem. 5:179-184. Bresnick, E. 1971. Regenerating liver: An experimental model for the study of growth. Methods Cancer Res. 6:347-397.
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Bucher, N. L. R. 1963. Regeneration of mammalian liver. Int. Rev. Cytol. 15:245-300. Drew, R. T. and Fouts, J. R. 1974. The lack of effects of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats. Toxicol. Appl. Pharmacol. 27:183-193. Drew, R. T., Fouts, J. R., and Harper, C. 1975. Species differences in benzene hydroxylation to phenol by pulmonary and hepatic microsomes. Drug. Metab. Dispos. 3:381-388. Dybing, E. 1976. Organ and species differences in microsomal activation of methyldopa. Drug. Metab. Dispos. 4:513-516. Dybing, E., Nelson, S. D., Mitchell, J. R., Sasame, H. A., and Gillette, J. R. 1976. Oxidation of α-methyl-dopa and other catechols by cytochrome P-450-generated superoxide anion: Possible mechanism of methyldopa hepatitis. Mol. Pharmacol. 12:911-920. Gonasun, L. M., Witmer, C. M., Kocsis, J. J., and Snyder, R. 1973. Benzene metabolism in mouse liver microsomes. Toxicol. Appl. Pharmacol. 26:398-406. Gorrall, A. G., Bardawill, G. J., and David, M. M. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-756. Graham, D. G., Tiffany, S. M., Bell, W. R., Jr., and Gutknecht, W. F. 1978a. Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol. Pharmacol. 14:644-653. Graham, D. G., Tiffany, S. M., and Vogel, F. S. 1978b. The toxicity of melanin precursors. J. Invest. Dermatol. 70:113-116. Higgens, G. M. and Anderson, R. M. 1931. Experimental pathology of the liver: 1. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12:186-202. Ikeda, M. and Ohtsuji, H. 1971. Phenobarbital-induced protection against toxicity of toluene and benzene in the rat. Toxicol. Appl. Pharmacol. 20:30-43. Ikeda, M., Ohtsuji, H., and Imamura, T. 1972. In vivo suppression of benzene and styrene oxidation by co-administered toluene in rats and effects of phenobarbital. Xenobiotica 2:101-106. Jerina, D. M. and Daly, J. W. 1974. Arene oxides: A new aspect of drug metabolism. Science 185:573-582. Lee, E. W., Kocsis, J. J., and Snyder, R. 1974. Acute effects of benzene on 5 9 Fe incorporation into circulating erythrocytes. Toxicol. Appl. Pharmacol. 27:431-436. Lutz, W. K. and Schlatter, C. 1977. Mechanism of the carcinogenic action of benzene: Irreversible binding to rat liver DNA. Chem.-Biol. Interact. 18:241-245. McLeod, D. L., Shreeve, M. M., and Axelrod, A. A. 1974. Improved plasma culture system for production of erythrocytic colonies in vitro: Quantitative assay method for CFU-E. Blood 44:517-534. Moeschlin, S. and Speck, B. 1967. Experimental studies on the mechanism of action of benzene on the bone marrow (radioautographic studies using 3 H-thymidine). Acta Haematol. 38:104-111. Munro, H. M. and Fleck, A. 1966. The determination of nucleic acids. Methods Biochem. Anal. 14:113-176. Nomiyama, K. 1965. Studies on poisoning by benzene and its homologues. Ind. Health 3:53-57. Rondanelli, E. G., Gorini, P., Gerna, G., and Magliulo, E. 1970. Pathology of Erythroblastic Mitosis in Occupational Benzenic Erythropathy and Erythemia. In Vivo and in Vitro Studies, pp. 3-5. Basel: Karger. Snyder, R. and Kocsis, J. J. 1975. Current concepts of chronic benzene toxicity. Crit. Rev. Toxicol. 3:265-288. Snyder, R., Uzuki, F., Gonasun, L. M., Bromfeld, E., and Wells, A. 1967. The metabolism of benzene
in vitro. Toxicol. Appl. Pharmacol. 11:346-360. Snyder, R., Andrews, L. S., Lee, E. W., Witmer, C. M., Reilly, M., and Kocsis, J. J. 1977a. Benzene metabolism and toxicity. In Biological Reactive Intermediates, eds. D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio, pp. 286-301. New York: Plenum.
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Snyder, R., Lee, E. W., and Kocsis, J. J. 1977b. Bone marrow depressant and leukemogenic actions of benzene. Life Sci. 21:1701-1722. Snyder, R., Lee, E. W., and Kocsis, J. J. 1978. Binding of labeled benzene metabolites to mouse liver and bone marrow. Res. Commun. Chem. Pathol. Pharmacol. 20:191-194. Steinberg, B. 1949. Bone marrow regeneration in experimental benzene intoxication. Blood 4:550-556. Tunek, A., Platt, K. L., Bentley, P., and Oesch, F. 1978. Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modifications of this metabolism. Mol.
Pharmacol. 14:920-929.
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Received November 13, 1978 Accepted February 9, 1979
