Seminário Nacional do Benzeno ( 5 e 6 dez/12) - NOVOS PARADIGMAS PARA O GERENCIAMENTO DO RISCO CARCINOGÊNICO - Terrence Monks

1. “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

3. 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?

4. 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)

5. 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.

6. OH
S-PHENYL-GSH 6-OH-tert,tert-HEXA-
DIENOIC ACID
SG CHO CHO COOH
tert,tert-MUCONALDEHYDE
OH +
tert,tert-MUCONIC
H ACID
1-(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)

7. 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
O
O O
ALCOHOL
O DEHYDROGENASE
OXIDATION OH
t,t-MUCONALDEHYDE
HO
6-OH-2,4-t,t-HEXADIENAL

8. 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.

9. Stability vs Reactivity of Epoxides
Determination of the Fraction of
Bromobenzene-3,4-oxide
Escaping Hepatocytes.

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

11. Stability vs Reactivity of Epoxides
~90nM
t1/2 = 7.9 min
Concn (ng/mL) of benzene oxide in blood following a single
oral dose (400 mg/kg). Lindstrom et al., 1997.

12. Stability of Benzene-oxide in Aqueous Media
Henderson et al., 2005.

13. 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)

15. Non-Occupational Sources of Benzene Metabolites
(see MacDonald et al., Leukemia, 15, 10-20, 2001)
• OTC medicinals - Phenol
• Diet - Phenol, HQ, catechol
• Smoke - HQ

16. Arbutin (4-hydroxy-β-D-glucopyranoside)

17. Sesamol

18. Sorbic Acid
Pezzagno et al., Am. J. Ind. Med., 35:511-518,
1999

19. Identification & Quantitation of HQ-thioether
Metabolites in Rat Bone Marrow
Hydroquinone glutathione conjugates (A), and mercapturic acid pathway metabolites (B) were quantified in rat bone marrow
by 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 ) .

20. Benzene induces leukemia-associated
cytogenetic alterations in peripheral
blood 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.

21. 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?

22. Redox Potentials of HQ and HQ-thioethers

23. 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).

24.  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.

26. Potential MOA’s of Benzene-Induced Leukemias
(Adapted from McHale et al, 2012)

27. The Stem Cell Niche

28. 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
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

29. 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 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.

30. 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 cystathionase
pathway, 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 cells
within the marrow (targets of benzene) express almost no cystathionase. Thus, inhibition of γ-
GT in hematopoietic tissue dramatically reduces intracellular GSH levels.