“Benzene Leukemogenesis – “Mode” of Action” ”Modalidade Carcinogénica da ação do Benzeno Terrence J. Monks, Ph.D. Professor e Cadeira Departmento de Farmacologia e Toxicologia Faculdade de Farmacia Universidade do Arizona, EUA
1. The postulated MOA: Is the weight of evidence sufficient toestablish a MOA in animals?2. Relevance to humans: Can human relevance of the MOA bereasonably excluded on the basis of fundamental, qualitativedifferences in key events between experimental animals and humans?3. Relevance to humans: Can human relevance of the MOA bereasonably excluded on the basis of quantitative differences in eitherkinetic or dynamic factors between experimental animals andhumans?4. Use of MOA analysis to modify the risk assessment: Are thereany quantitative differences in the key events such that defaultvalues for uncertainty factors for species or individual differencescould be modified? Are there significant data gaps in this context,which if filled, would permit more predictive assessment ofhuman risk?
Key Events (Meek & Klaunig, 2010)• Metabolism of benzene to a benzene oxide metabolite• Interaction of the benzene metabolite with target cells in the bone marrow• Formation of initiated, mutated bone marrow target cells• Selective clonal proliferation of mutated cells• Formation of the neoplasm (leukemia)
Benzene Metabolism - >145 years and Counting• Metabolic conversion of benzene to phenol -Schultzen and Naunyn, 1867.• Conjugation of phenol to sulphate - Baumann, 1876.• Anthracene, naphthalene, phenanthrene all converted to dihydrodiols - 1935 - 1950.• Boyland (1950) proposes intermediacy of an epoxide.• In 1949, Dennis Parke joins R.T. William’s laboratory and embarks on a study of “all known pathways” of benzene metabolism.
OH S-PHENYL-GSH 6-OH-tert,tert-HEXA- DIENOIC ACID SG CHO CHO COOH tert,tert-MUCONALDEHYDE OH + tert,tert-MUCONIC H ACID1-(GSyl)-CYCLOHEXA- 3,5-DIEN-2-OL H OHC HOOC HOOC GSH SG RING OPENING H EPOXIDE OH CYP2E1 HYDROLASE O O H NADPH OH BENZENE NADP BENZENE BENZENE OXIDE OXEPIN DIHYDRODIOL O OH OH OH DEHYDROGENASE OH [O] [O] [O]1,4-BENZOQUINONE OH PHENOL CATECHOL O HYDROQUINONE GSH OH GLUCURONIDE & SULFATE CONJUGATES SG + OH GS-HQ OH OH 1,2,4-BENZENETRIOL [O] OH O OH O OH O SG SG SG SG SG GSH [O] GSH [O] GS-1,4-BQ GS GS GS SG GS SG O OH O OH O 2,5-GS-1,4-HQ 2,5-GS-1,4-BQ 2,3,5-GS-1,4-HQ 2,3,5-GS-1,4-BQ (TGHQ)
O HO t,t-MUCONIC ACID 6-OXO-t,t-2,4-HEXA- DIENOIC ACID O OXIDATION HO OH O O O O ALDEHYDE O DEHYDROGENASE REDUCTION HO GS 6-OH-2,4-t,t-HEXA- SG + DIENOIC ACID O SG OO O ALCOHOL O DEHYDROGENASE OXIDATION OH t,t-MUCONALDEHYDE HO 6-OH-2,4-t,t-HEXADIENAL
Summary of the Metabolic Reactions of Benzene Oxide, and its Metabolites, That Consume Glutathione • Benzene oxide - Phenyl-GSH • Muconaldehyde - 2 GSH conjugates • Catechol - At least 1 GSH conjugate • Benzene triol - n= ? • Hydroquinone/1,4-benzoquinone- 1 mono-GSH conjugate 3 bis-GSH conjugates 1 tris-GSH conjugate 1 tetra-GSH conjugate Conclusion: Benzene likely causes hematotoxicity and leukemia through multiple reactive metabolites.
Stability vs Reactivity of EpoxidesDetermination of the Fraction of Bromobenzene-3,4-oxide Escaping Hepatocytes.
From: “Detection and half-life of bromobenzene-3,4-oxide in blood” Lau et al., Xenobiotica, 1984
Stability vs Reactivity of Epoxides ~90nM t1/2 = 7.9 minConcn (ng/mL) of benzene oxide in blood following a singleoral dose (400 mg/kg). Lindstrom et al., 1997.
Stability of Benzene-oxide in Aqueous Media Henderson et al., 2005.
Benzene Metabolites Identified in Bone MarrowBenzene (50 ppm for 6hrs):• Phenol and HQ glucuronides (B63CF1 mice, not rats [urinary metabolites]).• Phenol/catechol sulfate• trans,trans-muconic acid. (Sabourin et al., 1988).Benzene oxide-derived protein adducts in bone marrow:[14C13C6]Benzene (50-400 mg/kg)• 1,4-Benzoquinone - (Mice > Rat)• 1,2-Benzoquinone - (Rat > Mouse)• Benzene oxide – identified as phenyl cysteine. (McDonald et al., 1994)
Non-Occupational Sources of Benzene Metabolites (see MacDonald et al., Leukemia, 15, 10-20, 2001) • OTC medicinals - Phenol• Diet - Phenol, HQ, catechol • Smoke - HQ
Sorbic AcidPezzagno et al., Am. J. Ind. Med., 35:511-518, 1999
Identification & Quantitation of HQ-thioether Metabolites in Rat Bone MarrowHydroquinone glutathione conjugates (A), and mercapturic acid pathway metabolites (B) were quantified in rat bone marrowby HPLC-CEAS; (A) ( ) hydroquinone, ( ) 2-(glutathion-S-yl)hydroquinone, ( , dashed line) 2,5-bis-(glutathion-S-yl)hydroquinone, ( ) 2,6-bis-(glutathion-S-yl)hydroquinone, ( ) 2,3,5-tris-(glutathion-S-yl)hydroquinone; (B) ( ) 2-(glutathion-S-yl)hydroquinone, ( , dashed line) 2-(cystein-S-ylglycine)hydroquinone, ( , dashed line) 2-(cystein-S-yl)hydroquinone, ( , dashed line) 2-(N-acetylcystein-S-yl)hydroquinone. Each point represents the mean ± SEM (n= 3 ) .
Benzene induces leukemia-associated cytogenetic alterations in peripheralblood lymphocytes of benzene-exposed workers. • 5q-/-5, 7q-/-7, +8, t(8;21) • Aneuploidy – monosomy (5*, 6*, 7*, 10*, 16 & 19), trisomy (5, 6, 7, 8*, 10, 14, 16, 17, 21*, 22*) Limited evidence for benzene-induced mutations in humans, particularly mutations associated with AML (NPM1, AML1, FLT3, RAS, C/EBPα), but……….. Benzene and/or it’s metabolites generate reactive oxygen species and cause error-prone DNA repair.
Reactive Oxygen Species and Benzene Hematotoxicity No O2 consumption occurs in reactions in which the 1,4-benzosemiquinone free radical is formed enzymatically Ohnishi et al., 1969 1,4-Benzosemiquinone is so electron affinic, it’s rate of reduction by superoxide (9.6 x 108 M-1 sec-1;) is >4 orders of magnitude faster than the reverse reaction, the reduction of O 2 to O2•- (4.6 x 108 M-1 sec-1; Willson, 1971 Meisel, 1975, Sawada et al., 1975 Source of ROS?
Superoxide Generation by HQ and GS-HQ Conjugates 70 70 S u p e ro x id e A n io n G e n e ra tio n r 2 = 0 .8 0 60 60 50 40 50 (n m o l/m g /m in ) 30 40 20 10 30 0 -1 0 0 -8 0 -6 0 -4 0 -2 0 0 20 E 1 /2 ( m V ) 20 10 0 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 [M e ta b o lite ] m M Microsomes (0.5 mg/mL protein) were preincubated with acivicin (10 m M) for 15 min and then incubated with various concentrations of either phenol ( , dashed line), HQ ( ), 2-(GS-yl)HQ ( ),2,5-bis-(GS-yl)HQ ( , dashed line), BGHQ ( ), or TGHQ ( ), in the presence of succinoylated cytochrome C (12.5 M) and an NADPH generating system. Superoxide anion formation is expressed as nmol/mg protein/min. The inset shows the correlation between the oxidation potentials [E1/2 (mV)] for the HQ and its GSH conjuga tes, and their ability to catalyze superoxide anion formation. Each data point represents the mean ± SEM (n=3).
Base substitutions-G:C; Deletion; Mutations-G:C to A:T transitions and G:C to T:A; and G:C to C:G transversions.
Potential MOA’s of Benzene-Induced Leukemias (Adapted from McHale et al, 2012)
The Bone Marrow Niche, Stem Cells, and Leukemia: Impact of Drugs, Chemicals, and the EnvironmentMay 29 - 31, 2013 • New York City • www.nyas.org/BoneMarrow This meeting will bring together toxicology, hematology, and oncology research to explore bone marrow niche biology and the factors involved in spontaneous and chemically-induced bone marrow cancer and disease including AML and MDS. Call for Abstracts & Travel Fellowships Poster/Short Talk Abstract Deadline: April 5, 2013 Fellowship Application Deadline: April 5, 2013 Early Bird Discount Register by April 25, 2012 For more information and to register visit: www.nyas.org/BoneMarrow Presented by
Some Plausible Mechanisms by Which GS-HQ Conjugates Might Contribute to Benzene Hematotoxicity Potential targets?LTD4R: HQ mimicks the action of leukotriene D4 (LTD4) a downstream mediatorof G-CSF, to initiate terminal differentiation in IL-3-dependent murine myeloblasts.ABCTP: Functional roles for ATP-binding cassette (ABC) transporter proteins inhematopoietic stem cell function have recently been described. ABC transporterexpression/conformation/ function are modulated by ROS, which induce defects inhematopoietic stem cell homeostasis.
Some Plausible Mechanisms by Which GS-HQ Conjugates Might Contribute to Benzene Hematotoxicity • Generation of reactive oxygen species • Formation of covalent adducts with key proteins Potential targets?γ-GT: Tissues expressing very low levels of γ-GT usually possess a very active cystathionasepathway, in which cystathionine is deaminated and cleaved to form free cysteine and α-ketobutyrate.γ-GT activity in bone marrow is relatively low and the more immature, undifferentiated cellswithin the marrow (targets of benzene) express almost no cystathionase. Thus, inhibition of γ-GT in hematopoietic tissue dramatically reduces intracellular GSH levels.