Terrence J. Monks, Ph.D.Professor e Cadeira

Departmento de Farmacologia e Toxicologia

Faculdade de Farmacia Universidade do Arizona, EUA

“Benzene Leukemogenesis – “Mode” of Action”

”

Modalidade Carcinogénica da ação do Benzeno

1. The postulated MOA: Is the weight of evidence sufficient to establish a MOA in animals?

2. Relevance to humans: Can human relevance of the MOA be reasonably excluded on the basis of fundamental, qualitative differences in key events between experimental animals and humans? 3. Relevance to humans: Can human relevance of the MOA be reasonably excluded on the basis of quantitative differences in either kinetic or dynamic factors between experimental animals and humans?4. Use of MOA analysis to modify the risk assessment: Are there any quantitative differences in the key events such that default values for uncertainty factors for species or individual differences could be modified? Are there significant data gaps in this context, which if filled, would permit more predictive assessment of human 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.

BENZENE

CYP2E1

BENZENEOXIDE

BENZENEOXEPIN

RING OPENING

tert,tert-MUCONALDEHYDE

6-OH-tert,tert-HEXA-DIENOIC ACID

+tert,tert-MUCONIC ACID

DIHYDRODIOLDEHYDROGENASE

CATECHOL

EPOXIDEHYDROLASE

NADPNADPH

[O]

HYDROQUINONE

PHENOL

[O][O]1,4-BENZOQUINONE

GS-HQ

GSH

[O]

GSHGS-1,4-BQ

2,5-GS-1,4-HQ

[O] [O]GSH

2,5-GS-1,4-BQ 2,3,5-GS-1,4-BQ2,3,5-GS-1,4-HQ(TGHQ)

GSH

S-PHENYL-GSH

1-(GSyl)-CYCLOHEXA-3,5-DIEN-2-OL

GLUCURONIDE & SULFATECONJUGATES

1,2,4-BENZENETRIOL

+

OXIDATION

+

t,t-MUCONALDEHYDE

O

HO

OH

REDUCTION

6-OH-2,4-t,t-HEXA-DIENOIC ACID

OXIDATION

t,t-MUCONIC ACID

HO

ALDEHYDEDEHYDROGENASE

6-OXO-t,t-2,4-HEXA-DIENOIC ACID

ALCOHOLDEHYDROGENASE

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 conjugate3 bis-GSH conjugates1 tris-GSH conjugate1 tetra-GSH conjugate

Conclusion: Benzene likely causes hematotoxicity and leukemia through multiple reactive metabolites.

Determination of the Fraction of

Bromobenzene-3,4-oxideEscaping Hepatocytes.

Stability vs Reactivity of Epoxides

From: “Detection and half-life of bromobenzene-3,4-oxide in blood”Lau et al., Xenobiotica, 1984

Stability vs Reactivity of Epoxides

Concn (ng/mL) of benzene oxide in blood following a singleoral dose (400 mg/kg). Lindstrom et al., 1997.

~90nM

t1/2 = 7.9 min

Stability of Benzene-oxide in Aqueous Media

Henderson et al., 2005.

Benzene Metabolites Identified in Bone Marrow

Benzene (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

• Smoke - HQ

• Diet - Phenol, HQ, catechol

Arbutin (4-hydroxy--D-glucopyranoside)

Sesamol

Sorbic AcidPezzagno et al., Am. J. Ind. Med.,

35:511-518, 1999

Identification & Quantitation of HQ-thioetherMetabolites in Rat Bone Marrow

Benzene induces leukemia-associated cytogenetic

alterations in peripheral blood lymphocytes of benzene-

exposed workers.

Limited evidence for benzene-induced mutations in humans, particularly mutations associated with AML (NPM1, AML1, FLT3, RAS, C/EBP), but………..Benzene and/or it’s metabolites generate reactive oxygen species and cause error-prone DNA repair.

• 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*)

No O2 consumption occurs in reactions in which the1,4-benzosemiquinone free radical is formed enzymatically

Ohnishi et al., 1969

Reactive Oxygen Species and Benzene Hematotoxicity

1,4-Benzosemiquinone is so electron affinic, it’s rate of reductionby superoxide (9.6 x 108 M-1 sec-1;) is >4 orders of magnitude faster than the reverse reaction, the reduction of O2 to O2

•-

(4.6 x 108 M-1 sec-1; Willson, 1971 Meisel, 1975, Sawada et al., 1975

Source of ROS?

Redox Potentials of HQ and HQ-thioethers

Superoxide Generation by HQ and GS-HQ Conjugates

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 Stem Cell Niche

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 FellowshipsPoster/Short Talk Abstract Deadline: April 5, 2013Fellowship Application Deadline: April 5, 2013

Early Bird DiscountRegister by April 25, 2012

For more information and to register visit: www.nyas.org/BoneMarrow

The Bone Marrow Niche, Stem Cells, and Leukemia:Impact of Drugs, Chemicals, and the Environment

May 29 - 31, 2013 • New York City • www.nyas.org/BoneMarrow

Presented by

Some Plausible Mechanisms by Which GS-HQConjugates Might Contribute to Benzene Hematotoxicity

Potential targets?

LTD4R: HQ mimicks the action of leukotriene D4 (LTD4) a downstream mediator of G-CSF, to initiate terminal differentiation in IL-3-dependent murine myeloblasts.ABCTP: Functional roles for ATP-binding cassette (ABC) transporter proteins in hematopoietic stem cell function have recently been described. ABC transporter expression/conformation/ function are modulated by ROS, which induce defects in hematopoietic stem cell homeostasis.

Some Plausible Mechanisms by Which GS-HQConjugates Might Contribute to Benzene Hematotoxicity

• Generation of reactive oxygen species

• Formation of covalent adducts with key proteins

Potential targets?

-GT: Tissues expressing very low levels of -GT usually possess a very active cystathionase pathway, in which cystathionine is deaminated and cleaved to form free cysteine and -ketobutyrate.

-GT activity in bone marrow is relatively low and the more immature, undifferentiated cells within the marrow (targets of benzene) express almost no cystathionase. Thus, inhibition of -GT in hematopoietic tissue dramatically reduces intracellular GSH levels.