U.S. patent application number 14/153816 was filed with the patent office on 2014-12-25 for unifying mechanism and methods to prevent cancer and neurodegenerative diseases.
This patent application is currently assigned to Prevention, L.L.C.. The applicant listed for this patent is Prevention, L.L.C.. Invention is credited to Ercole L. Cavalieri, Eleanor G. Rogan.
Application Number | 20140378522 14/153816 |
Document ID | / |
Family ID | 28041935 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140378522 |
Kind Code |
A1 |
Cavalieri; Ercole L. ; et
al. |
December 25, 2014 |
UNIFYING MECHANISM AND METHODS TO PREVENT CANCER AND
NEURODEGENERATIVE DISEASES
Abstract
The present invention relates to methods for preventing the
development of cancer or neurodegenerative diseases by
administering N-Acetylcysteine (NAC), melatonin, or a combination
thereof. The present invention also relates to methods for
diagnosing cancer and/or neurdegenerative disease by detecting or
determining the amount of dopamine metabolites, 4-CE, 2-CE,
methylation of CE or CE-Q conjugates.
Inventors: |
Cavalieri; Ercole L.;
(Waterloo, NE) ; Rogan; Eleanor G.; (Omaha,
NE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Prevention, L.L.C. |
Waterloo |
NE |
US |
|
|
Assignee: |
Prevention, L.L.C.
Waterloo
NE
|
Family ID: |
28041935 |
Appl. No.: |
14/153816 |
Filed: |
January 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12533883 |
Jul 31, 2009 |
8629174 |
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14153816 |
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10940600 |
Sep 14, 2004 |
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12533883 |
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PCT/US03/07686 |
Mar 12, 2003 |
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10940600 |
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60364544 |
Mar 14, 2002 |
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Current U.S.
Class: |
514/419 ;
435/6.12; 514/440; 514/562 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/198 20130101; A61K 31/198 20130101; A61K 45/06 20130101;
Y10T 436/143333 20150115; A61K 31/40 20130101; A61K 31/05 20130101;
A61K 31/4045 20130101; A61K 31/4045 20130101; A61K 2300/00
20130101; A61K 31/195 20130101; A61K 31/195 20130101; A61K 31/05
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 31/381 20130101; C12Q
1/686 20130101; A61K 31/385 20130101; A61K 31/385 20130101 |
Class at
Publication: |
514/419 ;
435/6.12; 514/440; 514/562 |
International
Class: |
A61K 31/198 20060101
A61K031/198; A61K 45/06 20060101 A61K045/06; A61K 31/381 20060101
A61K031/381; A61K 31/05 20060101 A61K031/05; C12Q 1/68 20060101
C12Q001/68; A61K 31/4045 20060101 A61K031/4045 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The invention described herein was made with government
support under Grant Number PO1CA49210 and RO1CA49917 awarded by the
National Cancer Institute, NIH. The United States Government has
certain rights in the invention.
Claims
1. A method for preventing cancer in a mammal comprising
administering to the mammal an effective amount of N-acetylcysteine
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1 further comprising administering an
effective amount of melatonin, resveratrol, and lipoic acid, or
pharmaceutically acceptable salts thereof.
3. The method of claim 1 further comprising administering one or
more agents that induce quinone reductase.
4. The method of claim 1, further comprising administering an
effective amount of melatonin or a pharmaceutically acceptable salt
thereof.
5. The method of claim 1, further comprising administering an
effective amount of lipoic acid or a pharmaceutically acceptable
salt thereof.
6. The method of claim 1, further comprising administering an
effective amount of resveratrol or a pharmaceutically acceptable
salt thereof.
7. A method for preventing a pathological condition or symptom in a
mammal, such as a human, wherein the production of depurinating DNA
adducts from the action of endogenous quinones is implicated and
antagonism of such action is desired comprising administering to a
mammal in need of such preventive therapy, an effective amount of
N-Acetylcysteine, resveratrol, lipoic acid and melatonin or
pharmaceutically acceptable salts thereof.
8. The method of claim 7, wherein the pathological condition is
cancer.
9. A method for determining a mammal's risk of developing cancer
comprising: a) determining the amount of depurinating DNA adducts
present in a physiological sample from the mammal; b) comparing the
determined amount to an amount present in a control mammal, wherein
an elevated amount of depurinating DNA adducts correlates with the
risk of developing cancer.
10. The method of claim 9, wherein the physiological sample is a
tissue sample.
11. The method of claim 10, wherein the tissue is breast
tissue.
12. The method of claim 10, wherein the tissue is non-breast
tissue.
13. The method of claim 9, wherein the biological test sample is a
fluid.
14. The method of claim 13, wherein the fluid is whole blood or
blood serum.
15. The method of claim 13, wherein the fluid is urine.
Description
RELATED APPLICATION
[0001] This application is divisional of U.S. application Ser. No.
12/533,883, filed Jul. 31, 2009, which is a continuation of U.S.
application Ser. No. 10/940,600, filed Sep. 14, 2004, which is a
continuation-in-part of PCT/US03/07686, filed Mar. 12, 2003 (which
published in English on Sep. 25, 2003 as WO 03/077900) which claims
priority to U.S. Provisional Application Ser. No. 60/364,544 filed
Mar. 14, 2002, which applications and publication are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Cancer is a disease that begins with mutation of critical
genes: oncogenes and tumor suppressor genes. Mutation of critical
genes allows for a cancer cell to evolve and ultimately results in
pathogenic replication (a loss of normal regulatory control leading
to excessive cell proliferation) of various given types of cells
found in the human body. Conventional cancer treatments have
focused mainly on killing cancerous cells. Such treatments threaten
noncancerous cells, inherently are stressful to the human body,
produce many side effects, and are of uncertain efficacy. More
important, such treatment regimens are not necessarily directed
toward the actual root of the cancer problem or its prevention.
[0004] Other diseases are associated with excessive cell death. For
example, diseases associated with the loss of neurons in different
regions of the central nervous system (CNS), including, for
example, brain tissue and the spinal cord, such as Alzheimer's
disease, amyotrophic lateral sclerosis ("ALS" or "Lou Gehrig's
disease"), Parkinson's disease, Huntington's disease, brain aging,
Friedreich's ataxia, multiple sclerosis, diabetic necrosis,
ischaemia, and stroke. These types of diseases are exemplary of
diseases and disorders collectively referred to as
"neurodegenerative diseases." Treatment and prevention of
neurodegenerative disorders remains elusive in that many proposed
treatment methods are not practical since exogenous administration
of numerous putative therapeutics is not efficacious due to their
general inability to cross the blood-brain barrier.
[0005] Thus, there is a need in the art for therapeutic methods to
prevent or reduce the risk of the development of cancer and/or the
development of neurodegenerative diseases.
SUMMARY OF THE INVENTION
[0006] Applicant has discovered that oxidation of the carcinogenic
4-hydroxy catechol estrogens (CE) of estrone (E.sub.1) and
estradiol (E.sub.2) to catechol estrogen-3,4-quinones (CE-3,4-Q)
results in electrophilic intermediates that covalently bind to DNA
to form depurinating adducts at the N-7 of guanine and N-3 of
adenine by 1,4-Michael addition. The resultant apurinic sites in
critical genes can generate mutations that may initiate various
human cancers. As such, the endogenous quinones, including
CE-3,4-Q, may be endogenous tumor initiators. As yet, there are no
treatment methods available that are specifically directed toward
preventing the association of the metabolic intermediates, such as
endogenous (that which has originated or been produced within an
organism, tissue, or cell) quinones, with DNA, and thus, preventing
excessive cell growth and the development/formation of cancer.
[0007] Applicant has also discovered that the catecholamine
dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the
leukemogenic benzene can be oxidized to their quinones which react
with DNA to form predominantly analogous depurinating adducts. In
the case of depurinating adducts resulting from oxidization of
dopamine to its quinone, the resultant apurinic sites in critical
genes can generate mutations that may initiate brain cancer and/or
neurodegenerative diseases.
[0008] Therefore, Applicant has discovered that apurinic sites
formed by depurinating adducts are converted into tumor-initiating
or neurodegenerative-initiating mutations by error-prone repair.
Thus, Applicant has discovered a unifying molecular mechanism of
initiation for many cancers and neurodegenerative diseases. Using
this unifying molecular mechanism, Applicant has also designed
strategies to assess risk and to prevent such diseases.
[0009] Applicant has also discovered that N-acetylcysteine (NAC) is
capable preventing the formation of depurinating adducts
(endogenous tumor initiators and/or neurodegenerative initiators)
by aiding in the removal, detoxification and/or sequestration of,
for example, catechol quinones and/or the oxidation products of
benzene and dopamine prior to their association with DNA. Applicant
has further discovered that NAC may be useful to prevent the
formation of quinones.
[0010] Applicant has further discovered that melatonin is useful in
the prevention of the formation of depurinating adducts,
particularly those formed in the brain due to dopamine oxidation
and the resultant production of its quinone.
[0011] Thus, Applicant has discovered that the use of NAC and/or
melatonin can prevent or diminish the formation of depurinating
adducts, and therefore, prevent and/or treat cancer and/or
neurodegenerative disorders. Further, it is believed that practice
of the invention will, at least in part, influence and control
cellular mortality by allowing the cell to maintain a lower level
of endogenous quinones (that have the ability to bind DNA and form
depurinating adducts) and thus, allow the cell to follow a normal
apoptotic pathway (programmed cell death, such as that signaled by
the nuclei in normally functioning human and animal cells when age
or state of cell health and condition dictates).
[0012] Accordingly, the present invention provides pharmaceutical
compositions and methods to treat and/or prevent cancer and
neurodegenerative diseases and for reducing cancer and
neurodegenerative disease mortality. The present invention is
further directed to methods of utilizing N-acetylcysteine (NAC) and
melatonin to treat, prevent, and/or reduce the risk of cancer and
neurodegenerative diseases and disorders, to reduce the formation
of DNA adducts by reactive electrophilic estrogen metabolites,
and/or to reduce the formation of DNA adducts by reactive
electrophilic dopamine metabolites.
[0013] The present invention also relates to a therapeutic method
of preventing, treating or reducing the risk of a pathological
condition or symptom in a mammal, including a human, which is
suffering or may suffer from said condition, wherein production of
quinones is implicated and antagonism of such production or removal
of such quinones is desired, comprised of administering to a mammal
an effective amount of NAC, melatonin, a physiologically acceptable
salt thereof, or a combination thereof. Therefore, the present
invention provides a method for modulating quinone production
(e.g., CE-3,4-Q or the quinone of dopamine) or altering the amount
of quinones present in a mammal. Also provided is a therapeutic
method for preventing or treating a pathological condition or
symptom in a mammal, such as a human, wherein the production of
depurinating DNA adducts from the action of endogenous quinones is
implicated and antagonism of such action is desired, comprised of
administering to a mammal in need of such therapy, an effective
amount of NAC, melatonin, a pharmaceutically acceptable salt
thereof, or a combination thereof.
[0014] Further provided is a method of treating or preventing a
neoplastic or neurodegenerative condition or both conditions in a
subject comprising administering an effective amount of NAC,
melatonin, a physiologically acceptable salt thereof, or a
combination thereof. The combination treatment method provides for
simultaneous, sequential or separate use in treating such
conditions.
[0015] The present invention also relates to a method for
identifying an agent useful to prevent, reduce the risk, or treat
cancer comprised of contacting a host cell with E.sub.2 and a
candidate agent and determining whether the candidate agent reduces
the amount of CE-3,4-Q or depurinating adducts in the cell compared
to a control. Another method for identifying an agent useful to
prevent or reduce the risk of cancer or neurodegenerative disease
comprises incubating DNA with a catechol estrogen quinone or a
catecholamine dopamine quinone and a candidate agent and
determining whether the candidate agent reduces the association of
the quinone with DNA. The invention also provides agents identified
by such methods.
[0016] Also provided is a method for determining the risk of
developing cancer or a neurodegenerative disease in a mammal
comprising determining the amount of endogenous quinone present in
a biological test sample, such as blood, urine or other body fluid
(including spinal fluid) or a tissue biopsy, and comparing the
determined amount to an amount present in a normal sample, wherein
an increase in amount of quinone correlates with the risk of
developing cancer (e.g., breast cancer), and/or a neurodegenerative
disease.
[0017] The invention further provides a method for detecting cancer
and/or neurodegenerative disease in a mammal, preferably a human.
The method comprises subjecting a physiological sample from a human
to analytical detection to determine the presence or amount of
dopamine metabolites (such as dopamine quinones), 4-CE, 2-CE,
methylation of CE and/or CE-Q conjugates. The presence or amount of
dopamine metabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q
conjugates is then compared to an amount present in a control
sample, wherein an increase in the amount of dopamine metabolites,
4-CE, methylation of CE and/or CE-Q conjugates correlates to the
presence or absence of cancer and/or neurodegenerative disease.
Also provided is a diagnostic method for detecting dopamine
metabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q
conjugates.
[0018] The presence or amount of dopamine metabolites, 4-CE, 2-CE,
methylation of CE and/or CE-Q conjugates that changes over time can
indicate the progression or remission of cancer, such as breast
cancer, or neurodegenerative disease, as well as the presence of
previously undiagnosed metastatic or neurodegenerative disease.
Thus, the present invention provides a method for monitoring the
course, progression or remission of cancer, such as breast cancer,
and neurodegenerative disease. This method comprises analyzing a
physiological sample by analytical methods. The presence or amount
of dopamine metabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q
conjugates is detected or determined. At least one point later in
time, another sample is taken and the amount of dopamine
metabolites, 4-CE, 2-CE, methylation of CE and/or CE-Q conjugates
is determined. The amounts of dopamine metabolites, 4-CE, 2-CE,
methylation of CE and/or CE-Q conjugates, obtained at least at two
different time points, are compared.
[0019] The methods of the invention also optionally comprise
administering an agent that induces the protective enzyme quinone
reductase.
[0020] The methods of the invention also optionally comprise
administering an agent that inhibits CYP1B1.
[0021] The methods of the invention also optionally comprise
administering lipoic acid or a pharmaceutically acceptable salt
thereof.
[0022] The methods of the invention also optionally comprise
administering resveratrol or a pharmaceutically acceptable salt
thereof.
[0023] The methods of the invention also optionally comprise
administering lipoic acid and resveratrol or pharmaceutically
acceptable salts thereof.
[0024] The present invention also provides pharmaceutical
compositions which comprise an effective amount of NAC and an
effective amount of melatonin or a physiologically acceptable salt
thereof, together with one or more physiologically acceptable
carriers or excipients. Such a composition is useful, for example,
to treat and/or prevent cancer and/or neurodegenerative diseases,
as well as other diseases that are effected by the activity of
quinones (e.g., formation of genetic lesions). The invention also
provides a pharmaceutical composition comprising 1) NAC or a
physiologically acceptable salt thereof, 2) melatonin or a
physiologically acceptable salt thereof, 3) optionally one or more
agents that induce quinone reductase, and 4) one or more
physiologically acceptable carriers or excipients. The compositions
of the invention can also optionally comprise an agent that
inhibits CYP1B1.
[0025] The invention also provides the use of NAC and/or melatonin
to prepare a medicament useful to treat cancer and/or
neurodegenerative diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 depicts the formation of stable and depurinating DNA
adducts, and generation of apurinic sites.
[0027] FIG. 2 depicts the formation, metabolism, conjugation and
DNA adducts of estrogens.
[0028] FIG. 3 demonstrates redox cycling of catechol estrogen
semiquinones and quinones: DNA damage and formation of lipid
hydroperoxides.
[0029] FIGS. 4A-D depict H-ras mutations induced by DB[a,l]P or its
metabolite, anti-DB[a,l]PDE. Wild type sequences and nucleotide
numbers (GenBank accession No. U89950) are indicated below and
mutations are indicated above the line. (A) PCR artifact mutations
induced in untreated skin DNA and in a cloned H-ras gene (pWT)
treated with anti-DB[a,l]PDE or with acid. Under the treatment
conditions, anti-DB[a,l]PDE induces 1 adduct per 1000 bases and
acid induces 1 depurination per 170 bases (Chakravarti, D. Et al.,
Mutat. Res., 456, 17-32 (2000)). (B) H-ras mutations in mouse skin
DNA after treatment with 200 nmol DB[a,l]P in 100 .mu.L, acetone.
At 12 h-1 d, the spectra contained mostly A/T to G/C mutations. At
days 2 and 3, multiple codon 61 mutations were observed. At 4 d, no
clear pattern of mutations could be determined. At days 5 and 6,
multiple codon 52 (CTA to CCA) mutations were observed. Few
mutations were observed at day 9. .gradient., insertion. (C) H-ras
mutations in mouse skin DNA after treatment with 200 nmol of
anti-DB[a,l]PDE in 100 .mu.L, acetone. Fifty to sixty percent of
mutations between days 1 to 4 were A/T to G/C mutations. (D) H-ras
mutations after TDG treatment of DNA from anti-DB[a,l]PDE-treated
pWT and from DB[a,l]P- or anti-DB[a,l]PDE-treated mouse skin. TDG
treatment resulted in drastic reduction of A/T to G/C mutations and
the observation of multiple codon 61 (CAA to CTA) mutations at day
1. These mutations were also observed at days 2 and 3. In addition,
at days 2 and 3, multiple codon 13 (GGC to GTC) mutations were
observed.
[0030] FIG. 5 depicts a proposed pathway of formation of A to G
mutations by error-prone base excision repair of carcinogen-induced
apurinic sites and the detection of the resulting G.T
heteroduplexes by the TDG-PCR technique. The conversion of G.T
heteroduplexes into G. apyrimidinic sites results in a drastic
reduction in the formation of A/T to G/C mutations. G.T
heteroduplexes are converted into fixed mutations (G.C and A.T
pairs) by one round of replication.
[0031] FIG. 6 demonstrates sequence similarity among sites of
DB[a,l]P-induced mutations in H-ras DNA of mouse skin at day 1 (SEQ
ID NOs:1-11). A putative conserved sequence is shaded. The mutated
base is underlined. The italicized sequence (A.sup.314.fwdarw.G
mutation) is from the bottom strand.
[0032] FIGS. 7A-C depict H-ras mutations induced by E.sub.2-3,4-Q.
(A) PCR artifact mutations induced in untreated skin DNA and in a
cloned H-ras gene (pWT) treated with E.sub.2-2,3-Q or with
E.sub.2-3,4-Q. (B) H-ras mutations in mouse skin DNA after
treatment with 200 nmol E.sub.2-3,4-Q in 100 mL of acetone/ethanol
(70:30). The spectra contained mostly A/T to G/C mutations. (C)
H-ras mutations after TDG treatment of DNA from
E.sub.2-3,4-Q-treated mouse skin. TDG treatment resulted in drastic
reduction of A/T to G/C mutations in 6 h and 12 h samples, but not
in 1 d and 3 d samples. This suggests that these mutations were in
the form of G.T heteroduplexes between 6-12 h, but were converted
into fixed mutations after that.
[0033] FIGS. 8A-B demonstrate a unifying mechanism of activation
and formation of DNA adducts. (A) Natural and synthetic estrogens,
and (B) Benzene and dopamine.
[0034] FIG. 9 demonstrates metabolism of 4-OHE.sub.1(E.sub.2) and
formation of depurinating DNA adducts.
[0035] FIG. 10 depicts the synthesis of CAT-4-N7Gua and CAT-4-N3Ade
by reaction of CAT quinone with dG or Ade.
[0036] FIG. 11 demonstrates the synthesis of DA (NADA)-6-N7Gua and
DA (NADA)-6-N3Ade by reaction of DA (NADA) quinone with dG or
Ade.
[0037] FIG. 12 depicts the metabolism of DA to form neuromelanin or
depurinating DNA adducts.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention is based upon the discovery of a
unifying mechanism, namely, formation of catechol quinones and
reaction with DNA by 1,4-Michael addition to yield depurinating
adducts that could give rise to cancers and/or neurodegenerative
diseases. The present invention therefore provides pharmaceutical
compositions and methods to prevent and/or treat cancer and/or
neurodegenerative diseases resulting from the formation of quinones
and/or the reaction of such quinones with DNA by 1,4-Michael
addition yielding depurinating adducts.
Compositions of NAC and/or Melatonin for Therapeutic Use
[0039] Therapeutic and/or effective amounts of NAC, melatonin, or a
combination thereof are amounts which are effective to: prevent the
development, further development, or reduce the risk of development
of cancer and/or neurodegenerative diseases; reduce the formation
of DNA adducts by endogenous reactive electrophilic estrogen
metabolites; and/or reduce the formation of DNA adducts by
endogenous reactive electrophilic dopamine metabolites. Such
effects are achieved while exhibiting little or no adverse effects
on normal, healthy tissues or cells or while exerting negligible or
manageable adverse side effects on normal, healthy tissues or cells
of the mammal.
[0040] Administration of NAC and/or melatonin as salts may be
appropriate. Examples of pharmaceutically acceptable salts are
organic acid addition salts formed with acids which form a
physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate. Suitable inorganic salts may also be
formed, including hydrochloride, sulfate, nitrate, bicarbonate, and
carbonate salts.
[0041] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0042] NAC and/or melatonin can be formulated as pharmaceutical
compositions and administered to a mammalian host, such as a human
patient in a variety of forms adapted to the chosen route of
administration, i.e., orally or parenterally, by intravenous,
intramuscular, topical or subcutaneous routes.
[0043] Thus, NAC and/or melatonin may be systemically administered,
e.g., orally, in combination with a pharmaceutically acceptable
vehicle such as an inert diluent or an assimilable edible carrier.
They may be enclosed in hard or soft shell gelatin capsules, may be
compressed into tablets, or may be incorporated directly with the
food of the patient's diet. For oral therapeutic administration,
the active compound may be combined with one or more excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 2 to about 60% of the weight of a given unit dosage
form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0044] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0045] NAC and/or melatonin may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0046] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example,
aluminum monostearate and gelatin.
[0047] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilisation. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0048] For topical administration, NAC and/or melatonin may be
applied in pure form, i.e., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0049] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0050] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0051] Examples of useful dermatological compositions which can be
used to deliver NAC and/or melatonin to the skin are known to the
art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),
Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.
4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0052] Useful dosages of NAC and/or melatonin can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0053] Generally, the concentration of NAC and/or melatonin in a
liquid composition, such as a lotion, will be from about 0.1-25
wt-%, preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be
about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0054] The amount/preferred dose of NAC, melatonin, an active salt
or derivative thereof, or a combination thereof, required for use
in treatment will vary not only with the particular
salt/composition selected, but also with the route of
administration, the nature of the condition being treated and the
age, weight and condition of the patient. Importantly, the quantity
of NAC, melatonin, an active salt or derivative thereof, or a
combination thereof, used should be sufficient to prevent, inhibit,
reduce the risk of, or treat cancer and/or prevent, inhibit, reduce
the risk of, or treat neurodegeneration. Thus, a variety of
clinical factors will influence the preferred dosage ranges and
will be ultimately at the discretion of the attendant physician or
clinician.
[0055] In general, however, a suitable dose of NAC will typically
be in the range of from about 0.5 to about 10 mg/kg, e.g., from
about 2 to about 10 mg/kg of body weight per day, preferably in the
range of 5 to 9 mg/kg/day.
[0056] NAC can be conveniently administered in unit dosage form;
for example, containing 5 to 1000 mg, conveniently 10 to 750 mg,
most conveniently, 50 to 500 mg of active ingredient per unit
dosage form.
[0057] In general, however, a suitable dose of melatonin will
typically be in the range of from about 0.01 to about 0.2 mg/kg,
e.g., from about 0.1 to about 0.2 mg/kg of body weight per day,
preferably in the range of 0.05 to 0.15 mg/kg/day.
[0058] Melatonin can be conveniently administered in unit dosage
form; for example, containing 1 to 20 mg, conveniently 2 to 15 mg,
most conveniently, 3 to 10 mg of active ingredient per unit dosage
form.
[0059] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
Methods for Using NAC and/or Melatonin
[0060] Through research of the underlying mechanisms of the
formation of cancer and the development of neurodegenerative
disease or disorders, Applicant has made the unexpected discovery
of a unifying molecular mechanism of initiation for many cancers
and neurodegenerative diseases. This unifying mechanism involves
the oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE)
of estrone (E1) and estradiol (E2) to catechol
estrogen-3,4-quinones (CE-3,4-Q) resulting in electrophilic
intermediates that covalently bind to DNA to form depurinating
adducts at the N-7 of guanine and N-3 of adenine by 1,4-Michael
addition. It also involves the oxidation of the catecholamine
dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the
leukemogenic benzene to their quinones which react with DNA to form
predominantly analogous depurinating adducts. The resultant
apurinic sites in critical genes can generate mutations that may
initiate various human cancers and/or neurodegenerative diseases or
disorders.
[0061] Applicant has discovered that NAC and/or melatonin, both of
which may cross the blood-brain barrier, are two agents that reduce
the formation of such above-mentioned endogenous quinones. It has
also been discovered that NAC and/or melatonin reduce the formation
of depurinating adducts due to the action of quinones. As such,
administration of NAC or melatonin alone or in combination
surprisingly and unexpectantly offers a method for preventing
and/or reducing the risk of cancer and/or neurodegenerative
diseases.
[0062] In a preferred method, compositions comprising NAC and/or
melatonin are used for the prevention, inhibition, and/or treatment
of cancers such as primary or metastatic melanoma, thymoma,
lymphoma, sarcoma, lung cancer, liver cancer, brain cancer,
non-Hodgkin's lymphoma, Hodgkins lymphoma, leukemias, uterine
cancer, cervical cancer, bladder cancer, rectal cancer, kidney
cancer, colon cancer, and adenocarcinomas such as breast cancer,
prostate cancer, ovarian cancer, and pancreatic cancer.
[0063] In another preferred method, compositions comprising NAC
and/or melatonin are used for the prevention, inhibition and/or
treatment of neurodegenerative diseases such as diseases associated
with the loss of neurons in different regions of the central
nervous system (CNS), including, for example, brain tissue and the
spinal cord, such as Alzheimer's disease, amyotrophic lateral
sclerosis ("ALS" or "Lou Gehrig's disease"), Parkinson's disease,
Huntington's disease, brain aging, Friedreich's ataxia, multiple
sclerosis, diabetic necrosis, ischaemia, and stroke.
Other Agents
[0064] The unifying mechanism that has been discovered also
suggests other agents that will be useful for treating or
preventing cancer and neurodegenerative diseases. For example, it
has been determined that agents that induce the protective enzyme
quinone reductase, which reduces catechol estrogen quinones to
catechol estrogens, will also provide beneficial effects. Two such
agents are lipoic acid and resveratrol. Additionally, agents that
inhibit CYP1B1 will also provide a beneficial effect.
[0065] Lipoic acid (1,2-dithiolane-3-pentanoic acid) is an
antioxidant because the dithiolane structure is a strained
five-membered ring that is highly reactive. The relatively high
energy content of the disulfide group in lipoic acid makes it
reactive with oxidizing molecules. The reduced form of lipoic acid,
dihydrolipoic acid (the two forms are in equilibrium) has greater
antioxidant activity. Thus, the potent reducing capacity of
dihydrolipoic acid and the high reactivity of the disulfide groups
in lipoic acid make this couple important as an antioxidant defense
system in the cell. The LA/DHLA exhibits free radical (superoxide
anion radical and hydroxyl radicals) scavenging properties,
reducing oxidative stress. Because it is a strong reductant, it can
regenerate vitamin C, Vitamin E and GSH from their oxidized forms.
Lipoic acid readily crosses the blood-brain barrier. Finally, it is
believed that lipoic acid will induces the protective enzyme
quinone reductase, which reduces CEQ back to CE.
[0066] Resveratrol, a polyphenolic phytoalexin, is a natural
fungicide in more than 70 plant species. It is an antioxidant and
an antimutagen. Resveratrol scavenges hydroxyl radicals, superoxide
anion radicals and metal-induced radicals, thus protecting against
lipid peroxidation. It inhibits cytochrome P450 1A1, 1B1 and 3A4,
thus reducing oxidation of estrogens to catechol estrogens (CE). It
is also an inducer of quinone reductase, thus increasing the
reduction of CEQ to CE. Resveratrol inhibits dioxin-induced
expression of P450 1A1 and 1B1, as well as CE-mediated oxidative
damage to DNA in cultured human mammary epithelial cells.
Resveratrol also inhibits CYP1B1, the major enzyme that catalyzes
formation of 4-catechol estrogens in extrahepatic tissues (like the
breast, prostate, etc. Inhibition of CYP1B1 activity in the breast,
would be expected to lower amounts of 4-CE, lower mounts of
CE-3,4-quinones and reduce formation of depurinating 4-CE-DNA
adducts that generate mutations leading to the initiation of
cancer. Accordingly, the compositions of the invention can
optionally comprise one or more agents that induce quinone
reductase (e.g. lipoic acid and resveratrol). The compositions of
the invention can also optionally comprise one or more agents that
inhibit CYP1B1 (e.g. resveratrol). Additionally, the methods of the
invention can optionally comprise administering one or more agents
that induce quinone reductase. The methods of the invention can
also optionally comprise administering one or more agents that
inhibit CYP1B1.
Method for Detecting and/or Diagnosing Cancer and/or
Neurodegenerative Disease
[0067] As described herein below, 4-CE were 3.5 times more abundant
than the 2-CE and were 4 times higher than in women without breast
cancer, demonstrating that the amount of 4-CE present in a
physiological sample correlates with cancer. Additionally, a lower
level of methylation was observed for the CE cancer cases compared
to controls. Also, CE-Q conjugate levels were 3 times higher in
women with cancer than controls. Therefore, the determination of
the presence and/or amount of dopamine quinone, 4-CE, 2-CE,
methylation of CE and/or CE-Q conjugates may be useful in the
diagnosis, treatment and/or monitoring of the progression or
remission of cancer, such as breast cancer, and/or
neurodegenerative diseases. Thus, there is provided a method for
determining the risk of developing cancer and/or a
neurodegenerative disease in a mammal comprised of determining the
amount of endogenous quinone, dopamine quinone, 4-CE, 2-CE,
methylation of CE and/or CE-Q conjugates present in a physiological
sample from a mammal, such as blood, urine or other body fluid or a
tissue biopsy, and comparing the determined amount to an amount
present in a control sample, wherein an increase in amount of
quinone correlates with cancer, such as breast cancer, and/or
neurodegenerative disease. The presence or quantity of quinone in
the sample can be determined using any suitable analytical method,
such as IR, UV, NMR, Mass Spec or HPLC. A preferred method for
detecting or determining the presence or amount of estrogen
metabolites, dopamine metabolites, conjugates and depurinating DNA
adducts, including 4-CE, 2-CE, methylation of CE and/or CE-Q
conjugates, is by HPLC with electrochemical detection.
Screening Method for Identifying New Therapeutic Agents
[0068] The present invention also provides a screening method to
identify new therapeutic agents that inhibit the production and/or
the activity (e.g., the ability to associate with or bind DNA and
form depurinating DNA adducts) of endogenous quinones. Preferably,
the quinones have formed endogenously from the oxidation of the
carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E.sub.1)
and estradiol (E.sub.2) to catechol estrogen-3,4-quinones
(CE-3,4-Q) resulting in electrophilic intermediates that covalently
bind to DNA to form depurinating adducts at the N-7 of guanine and
N-3 of adenine by 1,4-Michael addition. The resultant apurinic
sites in critical genes can generate mutations that may initiate
various human cancers. Also, the quinones may form endogenously
from the oxidation of the catecholamine dopamine and the metabolite
catechol (1,2-dihydroxybenzene) of the leukemogenic benzene to
their quinones which can react with DNA to form predominantly
analogous depurinating adducts. In the case of depurinating adducts
resulting from oxidization of dopamine to its quinone, the
resultant apurinic sites in critical genes can generate mutations
that may initiate brain cancer and/or neurodegenerative
diseases.
[0069] The present invention provides an in vitro binding assay
comprising incubating DNA and catechol estrogen quinones or
catecholamine dopamine quinones (e.g., for about 2 hours at
37.degree. C.) with and without a candidate agent. After the
incubation period, stable adducts are quantified (e.g., by a
.sup.32P-postlabeling method, as used and described hereinbelow,
and the presence or quantity of depurinating adducts is analyzed
(e.g., by high pressure liquid chromatography, HPLC). The
components of the reaction mixture with the candidate agent are
compared to those of the reaction mixture without the candidate
agent (control) to determine whether the candidate agent is able to
inhibit or prevent the association/binding of DNA and quinones
and/or the formation of depurinating adducts. If the quantity of
DNA/quinone complexes and/or depurinating adducts formed in the
reaction mixture with the candidate agent is less than the mixture
without the candidate agent, then the candidate agent may be useful
in a method for preventing or reducing the risk of cancer and/or
neurodegenerative disease.
[0070] One cellular screening method comprises contacting a culture
of cells (e.g., mammalian cells) with E.sub.2, contacting a
duplicate culture of cells with a candidate agent and E.sub.2, and
measuring the effect of the candidate agent on the production of
CE-3,4-Q, or depurinating adducts. This screening method can
identify agents which block the production of endogenous quinones
and/or detoxify them (rendering them unable to produce depurinating
adducts).
[0071] Another cell model for screening for therapeutic agents
comprises contacting tissue cultured cells, such as cancerous
tissue (e.g., breast), which has been tested to contain relatively
higher than normal amounts of 4-CE or CE-3,4-Q (as determined by
analytical methods) with a candidate agent and measuring the effect
of the candidate agent on the production of 4-CE, CE-3,4-Q or
depurinating adducts. If the amount of 4-CE, CE-3,4-Q, or
depurinating adducts is reduced in the treated sample as compared
to a control sample, the candidate agent may be useful in a method
for preventing or reducing the risk of cancer and/or
neurodegenerative diseases.
[0072] According to the methods of the invention, a sample can be
compared to an appropriate control (e.g., a control mammal or a
control cell) the criteria for selecting an appropriate control are
well understood by those of skill in the art. For example, a
control mammal may be a similar mammal lacking the condition for
which you are testing (e.g., cancer (e.g., breast cancer) or
neurodegenerative disease).
[0073] The compositions and methods of the invention will now be
illustrated by the following non-limiting Examples.
Example I
Initiation of Cancer and Other Diseases by Catechol
Ortho-Quinones
A Unifying Mechanism
Introduction
[0074] One of the major obstacles in cancer research is that cancer
is a problem of 200 diseases. This viewpoint has impeded
researchers from looking at the etiology of cancers because the
search would be prohibitively complex. For this reason, the
etiology of breast, prostate and other human cancers remains
virtually unknown. While the expression of various cancers
coincides with the above concept, some scientists consider there to
be a common, but not yet elucidated, origin for many prevalent
types of cancer.
[0075] There is widespread agreement in the scientific community
that cancer is basically a genetic disease--not in the sense that
most cancers are inherited, but in the sense that cancer is
triggered by genetic mutations. Thus, cancer can be considered a
disease of mutated critical genes that modulate cell growth and
death. These include oncogenes and tumor suppressor genes, which
give rise to transformation and abnormal cell proliferation.
Understanding the origin of these mutations opens the door to
strategies for controlling and preventing cancer. (Chakravarti D.
et al., Mutation Res., 456, 17-32 (2000) and Oncogene, 20:7945-7953
(2001); Weinberg R. A., Sci. Am., 275, 62-77 (1996).)
[0076] A second barrier to the progress of cancer research is
related to the reluctance of the scientific community to recognize
that the natural estrogens, including estrone (E.sub.1) and
estradiol (E.sub.2), are true carcinogens, which induce tumors in
various hormone-dependent and independent organs of several animal
species and strains. (International Agency for Research on Cancer
Monographs, 6, 99-132 (1974); International Agency for Research on
Cancer Monographs, 21, 279-362 (1979); International Agency for
Research on Cancer Monographs, An updating of IARC monographs
volumes 1 to 42 (1987); IARC Monographs, Suppl. 7, 272-310).)
[0077] A third obstacle to the progress of research on breast and
other hormone-dependent cancers is related to the standard
paradigm, stated by Feigelson and Henderson, that estrogens,
through receptor-mediated processes, "affect the rate of cell
division and, thus, manifest their effect on the risk of breast
cancer by causing proliferation of breast epithelial cells.
Proliferating cells are susceptible to genetic errors during DNA
replication, which, if uncorrected, can ultimately lead to a
malignant phenotype". While there is no doubt that
estrogen-mediated control of cell proliferation plays a role in the
development of breast and other hormone-dependent cancers,
accumulating evidence suggests that specific oxidative metabolites
of estrogens, if formed, can be the endogenous ultimate
carcinogens. By reacting with DNA, they cause the mutations leading
to cancer. This initiating mechanism occurs in hormone-dependent
and independent tissues. (Feigelson H. S. and Henderson B. E.,
Carcinogenesis, 17: 2279-2284 (1996); JNCI Monograph 27, E.
Cavalieri and E. Rogan (eds.), Oxford Press, Washington
(2000).)
ABBREVIATIONS
[0078] Ade, adenine; BP, benzo[a]pyrene, CE, catechol estrogen(s);
CE-Q, catechol estrogen quinone(s); CE-SQ, catechol estrogen
semiquinone(s); COMT, catechol-O-methyltransferase; CYP, cytochrome
P450; DB[a,l]P, dibenzo[a,l]pyrene; anti-DB[a,l]PDE,
anti-dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide; DMBA,
7,12-dimethylbenz[a]anthracene; E.sub.1, estrone; E.sub.2,
estradiol; Gua, guanine; GSH, glutathione; H, Harvey; OHE.sub.2,
hydroxyestradiol; PAH, polycyclic aromatic hydrocarbon(s);
PCR-RFLP, polymerase chain reaction-restriction fragment length
polymorphism(s); TDG, T.G-DNA glycosylase.
Results and Discussion
[0079] Covalent Binding of Carcinogens to DNA: Stable and
Depurinating Adducts
[0080] Chemical carcinogens covalently bind to DNA to form two
types of DNA adducts: stable ones that remain in DNA unless removed
by repair and depurinating ones that are released from DNA by
destabilisation of the glycosyl bond (FIG. 1). Stable adducts are
formed when carcinogens react with the exocyclic N.sup.6 amino
group of adenine (Ade) or N.sup.2 amino group of guanine (Gua),
whereas depurinating adducts are obtained when carcinogens
covalently bind at the N-3 or N-7 of Ade or the N-7 or sometimes
C-8 of Gua. The loss of Ade or Gua by depurination leads to
formation of apurinic sites that can generate the mutations leading
to tumor initiation. (Cavalieri E. L. and Rogan E. G., Pharmacol.
Ther., 55, 183-99 (1992); Cavalieri E. L. and Rogan E. G.,
Mechanisms of tumor initiation by polycyclic aromatic hydrocarbons
in mammals, In: The Handbook of Environmental Chemistry: PAHs and
Related Compounds, 3J, pp. 81-117, Neilson A. H. (ed.), Springer,
Heidelberg, Germany (1998).)
[0081] Identification and quantification of polycyclic aromatic
hydrocarbon (PAH)-DNA adducts led to the discovery that there is a
correlation between depurinating adducts and oncogenic mutations,
suggesting that these adducts are the primary culprits in the tumor
initiating pathway. This discovery was made by identifying the DNA
adducts formed in mouse skin by dibenzo[a,l]pyrene (DB[a,l]P),
7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BP) and,
at the same time, determining the mutations in the Harvey (H)-ras
oncogene in mouse skin papillomas initiated by these three PAH
(Table 1).
TABLE-US-00001 TABLE 1 Correlation of depurinating adducts with
H-ras mutations in mouse skin papillomas H-ras Mutations No. of
mutations PAH Major DNA Adducts No. of mice codon DMBA N7Ade (79%)
4/4 CAA.fwdarw.CTA 61 DB[a,l]P N7Ade (32%) 10/12 CAA.fwdarw.CTA 61
N3Ade (49%) BP C8Gua + N7Gua (46%) 10/20 GGC.fwdarw.GTC 13 N7Ade
(25%) 5/20 CAA.fwdarw.CTA 61
[0082] These mutations correlate with the predominant formation of
depurinating Ade adducts by DMBA and DP[a,l]P and the two-to-one
ratio of depurinating Gua to Ade adducts formed by BP. This pattern
of ras mutations suggests that the oncogenic mutations in mouse
skin papillomas induced by these PAH are generated by misrepair of
the apurinic sites derived from loss of the depurinating adducts.
Because thousands of apurinic sites are formed by cells each day,
repair of apurinic sites induced by PAH might be expected. The
level of apurinic sites arising from treatment with PAH is,
however, 15-120 times higher than those formed spontaneously,
suggesting that this large increase in apurinic sites could lead to
misrepair. In summary, apurinic sites can generate the mutations
that play a critical role in the initiation of cancer, and
formation of depurinating adducts has become the common denominator
for recognizing the potential of a chemical to initiate cancer.
(Cavalieri E. L. and Rogan E. G., Mechanisms of tumor initiation by
polycyclic aromatic hydrocarbons in mammals. In: The Handbook of
Environmental Chemistry: PAHs and Related Compounds, 3J, pp.
81-117, Neilson A. H. (ed.), Springer, Heidelberg, Germany (1998);
Chakravarti D. et al., Proc. Natl. Acad. Sci. USA, 92, 10422-10426
(1995); Chakravarti D. et al., Mutation Res., 456, 17-32 (2000);
Lindahl T. and Nyberg B., Biochemistry, 11; 3610-3618 (1972);
Chakravarti D. et al., Oncogene, 16, 3203-3210 (1998).)
[0083] Formation, Metabolism and DNA Adducts of Estrogens
[0084] Evidence that depurinating PAH-DNA adducts play a major role
in tumor initiation provided the impetus for discovering the
estrogen metabolites that form depurinating DNA adducts and can be
potential endogenous initiators of cancer. Catechol estrogens (CE)
are among the major metabolites of E.sub.1 and E.sub.2. If these
metabolites are oxidized to the electrophilic CE quinones (CE-Q),
they may react with DNA. Specifically, the carcinogenic 4-CE are
oxidized to CE-3,4-Q, which react with DNA to form depurinating
adducts. These adducts generate apurinic sites that may lead to
oncogenic mutations, thereby initiating cancer. (Cavalieri E. L.
and Rogan E. G., Pharmacol. Ther., 55; 183-99 (1992); Cavalieri E.
L. and Rogan E. G. Mechanisms of tumor initiation by polycyclic
aromatic hydrocarbons in mammals. In: The Handbook of Environmental
Chemistry: PAHs and Related Compounds, 3J, 81-117, Neilson A. H.
(ed.), Springer, Heidelberg, Germany (1998); Chakravarti D. et al.,
Proc. Natl. Acad. Sci. USA, 92, 10422-10426 (1995); Cavalieri, et
al., Proc. Natl. Acad. Sci. USA, 94, 10937-10942 (1997); Liehr J.
G., et al., J. Steroid Biochem., 24, 353-356 (1986); Li J. J. and
Li S. A., Fed. Proc. 46, 1858-1863 (1987); Newbold R. R. and Liehr
J. G., Cancer Res., 60, 235-237 (2000); Li K. M., et al., Proc. Am.
Assoc. Cancer Res., 39, 636 (1998); Chakravarti D., et al.,
Mutation Res., 456, 17-32 (2000); Chakravarti D., et al., Oncogene,
16, 3203-3210 (1998); Chakravarti D., et al., Oncogene, 20,
7945-7953 (2001).)
[0085] Estrogen Metabolism.
[0086] E.sub.1 and E.sub.2 are obtained by aromatization of
4-androsten-3,17-dione and testosterone, respectively, catalyzed by
cytochrome P450 (CYP)19, aromatase (FIG. 2). The estrogens E.sub.1
and E.sub.2 are biochemically interconvertible by the enzyme
17.beta.-estradiol dehydrogenase. E.sub.1 and E.sub.2 are
metabolized via two major pathways: formation of CE and, to a
lesser, extent, 16.alpha.-hydroxylation (not shown in FIG. 2). The
CE formed are the 2- and 4-hydroxylated estrogens. The major
4-hydroxylase in extrahepatic tissues is CYP1B1. In general, the CE
are inactivated by conjugating reactions such as glucuronidation
and sulfation, especially in the liver (not shown in FIG. 2). The
most common pathway of conjugation in extrahepatic tissues,
however, occurs by O-methylation catalyzed by the ubiquitous
catechol-O-methyltransferase (COMT). (Spink D. C., et al., J.
Steroid Biochem. Mol. Biol., 51, 251-258 (1994); Hayes C. L., et
al., Proc. Natl. Acad. Sci. USA, 93, 9776-9781 (1996); Spink D. C.,
et al., Carcinogenesis, 19, 291-298 (1998); Mannisto P. T. and
Kaakola S., Pharmacol. Rev., 51, 593-628 (1999).)
[0087] A reaction that is competitive with the conjugation of CE is
their catalytic oxidation to CE-semiquinones (CE-SQ) and CE-Q (FIG.
2). CE-SQ and CE-Q can be neutralized by conjugation with
glutathione (GSH). A second inactivating pathway for CE-Q is their
reduction to CE by quinone reductase and/or cytochrome P450
reductase. If these two inactivating processes are insufficient,
CE-Q may react with DNA to form stable and depurinating adducts
(FIG. 2). The carcinogenic 4-CE are oxidized to form predominantly
the depurinating adducts 4-OHE.sub.1(E.sub.2)-1-N3Ade and
4-OHE.sub.1(E.sub.2)-1-N7Gua. Carcinogenic 2-CE are oxidized to
form predominantly stable adducts, 2-OHE.sub.1(E.sub.2)-6-N.sup.6dA
and 2-OHE.sub.1(E.sub.2)-6-N.sup.2dG, but also depurinating adducts
to a much lesser extent. (DT Diaphorase A quinone reductase with
special functions in cell metabolism and detoxification (Ernester
L, Estabrook R W, Hochstein P, Orrenius S., Eds.) Chemica Scripta
27A (1987); Roy, D. and Liehr J. G., J. Biol. Chem., 263, 3646-3651
(1988); Cavalieri E., et al., Estrogens as endogenous genotoxic
agents: DNA adducts and mutations. In: JNCI Monograph 27: Estrogens
as Endogenous Carcinogens in the Breast and Prostate, pp. 75-93, E.
Cavalieri and E. Rogan (eds.), Oxford Press, Washington (2000);
Liehr J. G., et al., J. Steroid Biochem., 24, 353-356 (1986); Li J.
J. and Li S., Fed. Proc., 46, 1858-1863 (1987); Newbold R. R. and
Liehr J. G., Cancer Res., 60, 235-237 (2000); Cavalieri E. L., et
al., Proc. Natl. Acad. Sci. USA, 94, 10937-10942 (1997); Li K. M.,
et al., Proc. Am. Assoc. Cancer Res., 39, 636 (1998), Stack D. E.,
et al., Chem. Res. Toxicol., 9, 851-859 (1996); Dwivedy I., et al.,
Chem. Res. Toxicol., 5, 828-833 (1992); Van Aerden C., et al.,
Analyst, 123, 2677-2680 (1998).)
[0088] Redox Cycling of Catechol Estrogen Semiquinones and
Quinones.
[0089] Redox cycling (FIGS. 2 and 3) generated by reduction of CE-Q
to CE-SQ, catalyzed by cytochrome P450 reductase, and subsequent
oxidation back to CE-Q by molecular oxygen forms superoxide anion
radicals (O.sub.2..sup.-). These O.sub.2..sup.- dismutate to
H.sub.2O.sub.2, either spontaneously or, even faster, when the
reaction is catalyzed by superoxide dismutase. H.sub.2O.sub.2 is
rather nonreactive, except in the presence of reduced transition
metal ions, namely Fe.sup.2+ and Cu.sup.+, which cause formation of
indiscriminate oxidants, the hydroxyl radicals. These reactive
species can damage DNA by formation of oxygenated bases.
Concurrently, hydroxyl radicals can initiate the lipid peroxidation
process, generating lipid hydroperoxides that can serve as
unregulated cofactors for oxidation of CE by cytochrome P450. In
contrast, under normal conditions nicotinamide dinucleotide
phosphate NADPH serves not only as a cofactor, but also regulates
cytochrome P450 in the oxidation of CE. Thus, once lipid
hydroperoxides are formed, the oxidation of CE to CE-SQ and CE-Q
can become a self-generating process that unbalances estrogen
homeostasis and leads to formation of CE-Q. (Liehr J. G. and Roy
D., Free Radical Biol. Med., 8, 415-423 (1990); Nutter L. M., et
al., Chem. Res. Toxicol., 7, 23-28 (1994); Malins D. C., et al.,
Cancer, 17, 3036-3043 (1993); Lavigne J. A., et al., Cancer Res.,
61, 7488-7494 (2001); Kappus H., Lipid peroxidation: Mechanisms,
analysis, enzymology and biological relevance. In: Sies, H., ed.,
Oxidative Stress, New York, Academic Press, 273-310 (1985).)
[0090] Binding of Catechol Estrogen Quinones to DNA.
[0091] To determine whether DNA adducts are formed in biological
systems, E.sub.2-3,4-Q or enzymatically-activated
4-hydroxyestradiol (4-OHE.sub.2) was reacted with DNA for 2 h at
37.degree. C. The stable adducts were quantified by the
.sup.32P-postlabeling method, and the depurinating adducts were
analyzed by high pressure liquid chromatography (HPLC) interfaced
with an electrochemical detector. When E.sub.2-3,4-Q reacted with
DNA, almost the same amount of the depurinating adducts
4-OHE.sub.2-1-N3Ade and 4-OHE.sub.2-1-N7Gua were obtained, and the
amount of stable adducts was 0.02% of the depurinating ones.
Activation of 4-OHE.sub.2 by horseradish peroxidase gave similar
results, whereas the mammalian lactoperoxidase produced a similar
amount of N3Ade adduct, but about 50% more N7Gua adduct. The same
two depurinating adducts were obtained in equal but smaller amounts
when 4-OHE.sub.2 was activated with tyrosinase or
phenobarbital-induced rat liver microsomes. In all cases, the level
of stable adducts was 0.02% or less compared to the depurinating
adducts (Li K. M., et al., Proc. Am. Assoc. Cancer Res., 39, 636
(1998); Cavalieri, et al., unpublished results).
[0092] DNA adducts were analyzed in vivo in rat mammary gland and
mouse skin after treatment of the animals with E.sub.2-3,4-Q or
4-OHE.sub.2. Female ACI rats, which are susceptible to
E.sub.2-induced mammary tumors, were treated by intramammillary
injection of E.sub.2-3,4-Q or 4-OHE.sub.2 (200 nmol in 20 .mu.L
DMSO/gland at four teats) for 1 h. The mammary tissue was excised,
extracted and analyzed for stable and depurinating adducts. N3Ade
and N7Gua adducts from both 4-OHE.sub.2 and 4-OHE.sub.1 were
detected in the range of 100-300 .mu.mol/mol DNA-P. The level of
stable adducts was not above the low level detected in untreated
mammary tissue. Similarly, female SENCAR mice were treated
topically on a shaved area of dorsal skin with E.sub.2-3,4-Q [200
nmol in 50 .mu.L acetone/DMSO (9:1)] for 1 h. The treated area of
skin was excised, extracted and analyzed for stable and
depurinating adducts. Equal amounts of 4-OHE.sub.2-1-N3Ade and
4-OHE.sub.2-1-N7Gua, approximately 12 .mu.mol/mol DNA-P, were
detected, and the amount of stable adducts was 0.02% of the
depurinating adducts. These results in rats and mice demonstrate
that the depurinating CE-DNA adducts are formed in vivo, generating
apurinic sites in the DNA that could lead to oncogenic mutations.
(Shull J. D., et al., Carcinogenesis, 18, 1595-1601 (1997);
Cavalieri, et al., unpublished results; Chakravarti D., et al.,
Oncogene, 20, 7945-7953 (2001).)
[0093] Depurinating Adducts and Induction of Mutations
[0094] Mouse skin provides a model system to study the conversion
of DNA lesions, such as carcinogen-induced depurinating and stable
DNA adducts, into mutations. In mouse skin, tumor initiation occurs
when these DNA lesions are converted into oncogenic mutations in
the H-ras gene. Previous studies indicated that stable adducts are
inefficiently removed by excision repair, and cells containing
these adducts enter the S-phase. In the S-phase, occasional
mutations are induced when replicative DNA polymerases go over
adducted templates. Therefore, it was concluded that adduct-induced
mutagenesis occurs in proliferating cells. These studies, however,
did not address the fate of apurinic sites formed by the
depurinating adducts. (Maher V. M., and McCormick J. J., Role of
DNA lesions and repair in the transformation of human cells. In: D.
Grunberger, S. P. Groff (eds) Mechanisms of Cellular Transformation
by Carcinogenic Agents. Pergamon Press, New York, 135-149 (1987);
Kaufman W. K., Cancer Metastasis Rev, 14, 31-41 (1995); Moriya M.,
et al., Biochemistry, 35, 16646-16651 (1996).)
[0095] Resting Cells are Greatly Susceptible to Tumor
Formation.
[0096] Mouse skin is most susceptible to tumor formation by
carcinogens during the telogen phase of the hair cycle. At telogen,
epidermal thickness is low, indicating that at these times
epidermal cells are in the resting phase. DMBA, which forms 99%
depurinating adducts and 1% stable adducts, induces several-fold
more tumors when applied to resting phase skin. This suggests that
apurinic sites induced by the depurinating DNA adducts may be most
efficiently converted into oncogene-activating mutations in G0-G1
phase cells. These questions were examined with DB[a,l]P, the
strongest among PAH carcinogens, which also forms 99% depurinating
adducts and 1% stable adducts in mouse skin DNA and induces the
H-ras codon 61 (CAA to CTA) mutation in tumors. (Devanesan P. D.,
et al., Chem. Res. Toxicol., 6, 364-371 (1993); Andreasen E., Acta.
Pathol. Scand., 32, 157-164 (1953); Cavalieri E. L. and Rogan E.
G., Mechanisms of tumor initiation by polycyclic aromatic
hydrocarbons in mammals. In: The Handbook of Environmental
Chemistry: PAHs and Related Compounds, 3J, 81-117, Neilson A. H.
(ed.), Springer, Heidelberg, Germany (1988); Chakravarti D., et
al., Proc. Natl. Acad. Sci. USA, 92, 10422-10426 (1995).)
[0097] Using a polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) technique, it was found that
treatment of mouse skin with 200 nmol of DB[a,l]P resulted in the
induction of these codon 61 mutations as early as one day after
treatment. In this technique, a segment of the H-ras gene is
PCR-amplified and the product is restricted with XbaI to examine
the induction of a RFLP from the codon 61 mutation. It was observed
that at one day, 0.1% of the H-ras genes in the treated area of
skin contained the codon 61 mutation, and then the population of
these mutations increased to a maximum of 5% between 3 and 4 days
after DB[a,l]P treatment. Subsequently, the level of mutations was
reduced to background levels (0.0001%). The early time of induction
of the codon 61 mutations (one day after DB[a,l]P treatment)
coincides with suppression of DNA synthesis and induction of
excision repair. Therefore, perhaps DB[a,l]P-induced DNA damage is
converted into mutations by error-prone excision repair in
pre-S-phase cells. (Chakravarti D., et al., Oncogene, 16, 3203-3210
(1998); Slaga T. J., et al., Cancer Res., 34a, 771-777 (1974);
Sawyer T. W., et al., Carcinogenesis, 9, 1197-1202 (1988); Gill R.
D., et al., Environ. Mol. Mutagen., 18, 200-206 (1991).)
[0098] Error-Prone Repair of Apurinic Sites is a Mechanism of Tumor
Initiation.
[0099] Evidence in support of this was obtained from a comparative
study of mutations in the mouse skin H-ras gene induced by 200 nmol
of DB[a,l]P or 200 nmol of
anti-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide (anti-DB[a,l]PDE).
Unlike DB[a,l]P, anti-DB[a,l]PDE forms 97% stable adducts and 3%
depurinating adducts in DNA. In these experiments, the types of
mutations that are induced in the early preneoplastic times was
identified (12 h to 9 days after treatment) and then analyzed
whether these mutations were induced as a result of error-prone
repair (FIG. 4). The mutations were identified by PCR amplifying a
segment of the H-ras gene from DNA extracted from mouse skin
treated with one of the carcinogens, cloning the PCR products in a
plasmid, isolating individual subclones and sequencing the H-ras
inserts to identify mutations. (Chakravarti D., et al., Mutation
Res., 456, 17-32 (2000); Cavalieri E. L. and Rogan E. G.,
Mechanisms of tumor initiation by polycyclic aromatic hydrocarbons
in mammals. In: The Handbook of Environmental Chemistry: PAHs and
Related Compounds, 3J, 81-117, Neilson A. H. (ed.), Springer,
Heidelberg, Germany (1998).)
[0100] The mutation spectra induced by DB[a,l]P contained 90% A/T
to G/C mutations at day 1. This correlated with the abundant
DB[a,l]P-Ade depurinating adducts (81% of total adducts) and
suggested that these A/T to G/C mutations were induced at Ade
depurinations. Thus, the adducts could be correlated with these
early preneoplastic mutations, as well as with the clonal H-ras
mutations found in the tumors (Table 1). If Ade depurinations
induce these A/T to G/C mutations, they may be A to G mutations
generated as G.T heteroduplexes by error-prone excision repair
(FIG. 5).
[0101] Using a novel technique, it was determined that these A to G
mutations in the H-ras gene are initially induced as G.T
heteroduplexes. In this technique, G.T heteroduplexes in skin DNA
are converted to G. apyrimidinic sites by treatment with T.G-DNA
glycosylase (TDG) (FIG. 5). (Chakravarti D., et al., Mutation Res.,
456, 17-32 (2000).)
[0102] Depurinated DNA templates are refractory to PCR
amplification. To demonstrate this point, a mixture of two plasmids
(one contained the wild type H-ras exon 1-2 segment (pWT) and the
other contained the same DNA with the codon 61 (CAA to CTA)
mutation (pMUTX)) was PCR amplified. The yield of pMUTX in the PCR
product was determined by XbaI digestion. When pMUTX was incubated
in an acidic buffer to induce a relatively small amount of
depurination that did not significantly degrade the plasmid
(.about.1 depurination/H-ras segment), mixed with untreated pWT and
PCR amplified, a drastically reduced amount of the product was
XbaI-digestible. This confirmed that depurinated templates are
refractory to PCR amplification. Failure to score mutations in pWT
depurinated either by acid-treatment (FIG. 4A) or through
depurinating adduct formation by E.sub.2-3,4-Q (FIG. 7A) may be
related to the unavailability of depurinated templates for PCR
amplification. (Chakravarti D., et al., Mutation Res., 456, 17-32
(2000); Fromenty B., et al., Nucl. Acids Res., 28, e50 (2000).)
[0103] Since abasic site-containing DNA molecules are refractory to
PCR amplification, the conversion of G.T heteroduplexes into G.
apyrimidinic sites makes H-ras molecules containing these
heteroduplexes unamplifiable. Under these circumstances, PCR
preferentially amplifies templates that do not contain G.T
heteroduplexes. As a result, PCR amplification of the H-ras gene
from TDG-treated skin DNA, followed by cloning the PCR product and
isolating and determining the sequence of individual subclones,
causes a specific, drastic reduction of A to G mutations in the
mutation spectra. In addition, the preferential PCR amplification
artificially enriches low-abundance mutations that are observed
only in TDG-treated spectra.
[0104] Following the entry of skin cells into S-phase, however, the
G.T heteroduplexes are converted into G.C and A.T base pairs by one
round of replication. At this stage, TDG treatment does not reduce
the frequency of A to G mutations in the spectra. Thus, the
specific reduction of A to G mutations in the mutation spectra by
the TDG-PCR procedure characterizes these mutations as G.T
heteroduplexes. The TDG-PCR procedure resulted in a drastic
reduction in the population of A/T to G/C mutations on day 1, but
did not make a significant change at days 2 and 3 (Table 2).
Therefore, A to G mutations remained as G.T heteroduplexes until
one day after DB[a,l]P treatment of the skin; beyond which they
were present as G.C and A.T mutations, presumably by replication.
Flow cytometric analysis of epidermal keratinocytes isolated from
DB[a,l]P-treated mouse skin confirms that cells begin to enter the
S-phase one day after the treatment. (Chakravarti D., et al.,
Mutation Res., 456, 17-32 (2000); Chakravarti et al., unpublished
results).
[0105] A major difference in mutation spectra induced by DB[a,l]P
and anti-DB[a,l]PDE was the presence or absence of multiple codon
61 (CAA to CTA) mutations in early preneoplastic skin. These
mutations were detectable one day after DB[a,l]P treatment by the
PCR-RFLP procedure, which indicated that they constitute 0.1% of
H-ras genes, and in the mutation spectrum obtained after TDG
treatment of skin DNA. Since these CAA to CTA mutations were
observed during the active repair period, it was hypothesized that
they were also induced by error-prone repair as T.T heteroduplexes.
Since these mutations were present in days 2-3 in a significantly
greater frequency relative to other mutations in the spectra, it
was hypothesized that the increase in frequency was due to a clonal
proliferation of codon 61-mutated (initiated) cells. Further
studies suggest that at days 2-3, these codon 61-mutated cells
express activated Ras protein (Chakravarti D., et al., Oncogene,
16, 3203-3210 (1998); Chakravarti D., et al., Mutation Res., 456,
17-32 (2000); Chakravarti et al., unpublished observations).
[0106] On the other hand, anti-DB[a,l]PDE formed approximately 50%
A/T to G/C mutations, which correlated with 48.5% formation of
anti-DB[a,l]PDE-Ade stable adducts in mouse skin DNA (Table 2). The
frequency of these mutations was not significantly reduced by the
TDG-PCR procedure, indicating that these mutations were not induced
by error-prone repair. Studies conducted in other laboratories also
indicate that nucleotide excision repair of bulky stable adducts is
error-free. When the pWT plasmid was treated with anti-DB[a,l]PDE
in vitro (97% bulky stable adducts) and subjected to PCR
amplification, A/T to G/C mutations was also found to constitute
50% of all mutations (5 out of 10) (FIG. 4A). These mutations are
induced by translesional synthesis over bulky stable adducts by the
PCR polymerases. If, as has been proposed by others, only a small
population of PAH-induced bulky stable adducts is removed by
pre-replication repair, a large fraction of anti-DB[a,l]PDE-induced
adducts would persist in the mouse skin DNA. It is, therefore,
possible that the mutations found in anti-DB[a,l]PDE-treated mouse
skin DNA are adduct-induced PCR artifacts. The similarity of the
frequencies of A/T to G/C mutations in vitro and in skin is
consistent with this idea.
TABLE-US-00002 TABLE 2 The frequency of changes in DB[a,l]P-induced
A/T to G/C mutations by TDG treatment followed by PCR A/T to B/C
mutations/total mutations PAH DNA Day -TDG +TDG anti-DB[a,l]PDE pWT
-- 5/10 (50%) 3/5 (60%) skin 1 5/8 (62.5%) 4/5 (80%) DB[a,l]P skin
1 10/11 (90%) 2/10 (20%) 2 11/35 (31%) 6/22 (27%) 3 7/22 (31%) 8/25
(32%)
(Watanabe M., et al., Mutat Res., 146, 285-294 (1985); Maher V. M.
and McCormick J. J., Role of DNA lesions and repair in the
transformation of human cells. In: D. Grunberger, S. P. Groff (eds)
Mechanisms of Cellular Transformation by Carcinogenic Agents.
Pergamon Press, New York, pp. 135-149 (1987).)
[0107] Four days after treating mouse skin with DB[a,l]P, no codon
61 mutations were observed in 48 plasmids that contained 15 other
mutations (FIG. 4B). No definite patterns of mutations were
recognized at this time. At days 5 and 6, the mutation spectra were
mainly limited to codon 52 (CTA to CCA) mutations. This coincided
with the early phase of DB[a,l]P-induced hyperplasia that starts at
day 5 and persists beyond day 10. The codon 52 mutation may be
oncogenic, but further study is required. (Casale, G. P., et al.,
Fund. Appl. Tox., 36, 71-78 (1997); Casale, G. P., et al., Mol.
Car., 27, 125-140 (2000).)
[0108] The repair error-induced A/T to G/C mutations in
DB[a,l]P-treated mouse skin frequently occurred 3' to a sequence
element, TGN-doublet (FIG. 6), whereas these mutations in
anti-DB[a,l]PDE-treated skin did not show a sequence context
preference (not shown in FIG. 6). This suggests that the sequence
context of the depurinated base may determine the erroneous base
incorporated by repair. It is also noted that DB[a,l]P induces
approximately 120-fold more depurinations through the depurinating
adducts than are formed by spontaneous base loss (10,000-20,000
depurinations/cell/day). This raised the possibility that abundant
depurination may be a factor in inducing infidelity in repair.
(Chakravarti D., et al., Mutation Res., 45, 17-32 (2000); Cavalieri
E. L. and Rogan E. G., Mechanisms of tumor initiation by polycyclic
aromatic hydrocarbons in mammals. In: The Handbook of Environmental
Chemistry: PAHs and Related Compounds, 3J, 81-117, Neilson A. H.
(ed.), Springer, Heidelberg, Germany (1998); Chakravarti D., et
al., Proc. Natl. Acad. Sci. USA, 92, 10422-10426 (1995); Lindahl T.
and Nyberg B., Biochemistry, 11, 3610-3618 (1972); Lindahl T.
Nature, 362, 709-715 (1993).)
[0109] Effect of a Burst of DNA Depurination.
[0110] Treatment of mouse skin with E.sub.2-3,4-Q provided evidence
that abundant depurination may induce errors in repair. Like
DB[a,l]P, E.sub.2-3,4-Q forms predominantly depurinating adducts in
mouse skin DNA, consisting of roughly equal amounts of two
depurinating adducts (4-OHE.sub.2-1-N3Ade and 4-OHE.sub.2-1-N7Gua).
The N3Ade adduct depurinates instantaneously after its formation,
whereas the N7Gua adduct depurinates slowly, with a half-life of 5
h. The difference in the rate of depurination of the two adducts
provided a way to examine the effect of abundant depurination on
repair fidelity. Briefly, E.sub.2-3,4-Q would challenge the mouse
skin repair machinery with a burst of Ade-specific depurination and
slow-release Gua-specific depurination. Should a burst of
depurination be a contributing factor in causing repair to be
error-prone, a greater frequency of Ade-specific mutations compared
to Gua-specific mutations would be expected in the mouse skin DNA.
(Chakravarti D., et al., Oncogene, 20, 7945-7953 (2001); Li K.-M.,
et al., Proc. Am. Assoc. Cancer Res., 40, 46 (1999).)
[0111] Treatment of mouse skin with 200 nmol of E.sub.2-3,4-Q
induced primarily A/T to G/C mutations in the H-ras gene (FIG. 7).
For example, 6 h after E.sub.2-3,4-Q treatment, 7 mutations were
identified among 29 H-ras inserts. Five of the seven were A/T to
G/C mutations. At 12 h, four out of the six mutations found in 30
H-ras inserts were A/T to G/C mutations. At day 1, seven out of the
11 mutations found among 50 plasmids were A/T to G/C mutations.
Cells do not have enough time to replicate by 6 h, but they may
undergo repair. The observation that E.sub.2-3,4-Q induces
mutations at 6 h is, therefore, a basis to propose that these
mutations are induced by error-prone repair. To confirm this,
TDG-PCR analysis of these mutations was conducted (FIG. 7).
Specifically, at 6 h, TDG treatment reduced the frequency of the
A/T to G/C mutations from 5 in 29 H-ras inserts to 0 in 33 H-ras
inserts. At 12 h, the change was from 4 in 30 H-ras inserts to 0 in
41 plasmids. These results suggest that at 6-12 h, A/T to G/C
mutations were in the form of G.T heteroduplexes. By day 1, a major
change in the frequency of A/T to G/C mutations was not observed,
following TDG treatment, suggesting that G.T heteroduplexes were
present as G.C and A.T pairs. The TDG-treated 1 day spectrum was
dominated by two clonal mutations of equal frequency (codon 16 AAG
to AGG and intronic C to T mutations). Because it is unlikely that
the intronic mutation would affect Ras activity and the two clonal
mutations were found in the same frequency, these mutations may be
allelic, belonging to a clonally proliferating population. In
contrast, TDG treatment of day 3 DNA did not make any perceptible
changes from the TDG-untreated spectrum. This suggests that the
mutations found at day 3 were double-stranded and could not be
affected by TDG treatment. (Chakravarti D., et al., Oncogene, 20,
7945-7953 (2001).)
[0112] E.sub.2-3,4-Q-induced early A/T to G/C mutations were
frequently found at Ade depurinations 5' to G residues. This
supports the hypothesis that the sequence context of depurination
influences the selection of which base is incorporated during
error-prone repair.
[0113] Although these studies suggest that depurinating adducts
play a major role in inducing transforming mutations to begin the
process of tumorigenesis, the stable adducts can also contribute to
these processes. Studies indicate that erroneous base incorporation
during replication over various bulky stable adducts contributes to
the induction of transforming mutations. For example, the
BP-7,8-dihydrodiol-9,10-epoxide-N.sup.2dG stable adduct induces A
incorporation, forming G to T mutations and the corresponding
N.sup.6dA stable adduct induces C incorporation, forming A to G
mutations. Similar studies indicate that E.sub.2-2,3-Q, which
induces primarily bulky stable adducts, is also mutagenic. The
2-OHE.sub.2-N.sup.6dA stable adducts cause mostly A to T mutations
and some A to G mutations, whereas 2-OHE.sub.2-N.sup.2dG stable
adducts cause mainly G to T mutations. (Moriya M., et al.,
Biochemistry, 35, 16646-16651 (1996); Chary P., et al., J. Biol.
Chem., 270, 4990-5000 (1995); Stack D. E., et al., Chem. Res.
Toxicol., 9, 851-859 (1996); Dwivedy I., et al., Chem. Res.
Toxicol., 5, 828-833 (1992); Van Aerden C., et al., Analyst, 123,
2677-2680 (1998); Terashima I., et al., Biochemistry, 37, 8803-8807
(1998); Terashima I., et al., Biochemistry, 37: 13807-13815 (1998);
Terashima I., et al., Biochemistry, 40: 8-14 (2001).)
[0114] Estrogen Homeostasis
[0115] There are several factors that unbalance estrogen
homeostasis, namely, the equilibrium between activating and
deactivating metabolic pathways with the scope of averting the
reaction of endogenous CE-Q with DNA (FIG. 2). The first critical
factor could be excessive synthesis of E.sub.2 by overexpression of
aromatase, CYP19, in target tissues and/or the presence of excess
sulfatase that converts stored E.sub.1 sulfate to E.sub.1. The
observation that breast tissue can synthesize E.sub.2 in situ
suggests that much more E.sub.2 is present in some sites of target
tissues than would be predicted from plasma concentrations. (Miller
W. R. and O'Neill J., Steroids, 50, 537-548 (1987); Simpson E. R.,
et al., Endocrine Rev., 15, 342-355 (1994); Yue W., et al., Cancer
Res., 58, 927-932 (1998); Yue W., et al., Cancer, 6, 157-164
(1999); Jefcoate C. R., et al., Tissue-specific synthesis and
oxidative metabolism of estrogens. In: JNCI Monograph 27: Estrogens
as Endogenous Carcinogens in the Breast and Prostate, pp. 95-112,
Cavalieri E. and Rogan E. (eds.), Oxford Press, Washington (2000);
Reed M. J. and Purohit A. Endocrine Review, 18, 701-715
(1997).)
[0116] A second critical factor in unbalancing estrogen homeostasis
might be the presence of high levels of 4-CE due to overexpression
of CYP1B1, which converts E.sub.2 predominantly to 4-OHE.sub.2
(FIG. 2). A relatively large amount of 4-CE could lead to more
extensive oxidation to CE-3,4-Q, with increased likelihood of
damaging DNA. (Spink D. C., et al., J. Steroid Biochem. Mol. Biol.,
51, 251-258 (1994); Hayes C. L., et al., Proc. Natl. Acad. Sci.
USA, 93, 9776-9781 (1996); Spink D. C., et al., Carcinogenesis, 19,
291-298 (1998).)
[0117] A third factor could be a lack or low level of COMT
activity. If this enzyme is insufficient, either through a low
level of expression or its low activity allele, 4-CE will not be
effectively methylated, facilitating their oxidation to the
ultimate carcinogenic metabolites CE-3,4-Q (FIG. 2).
[0118] Studies in Syrian Golden Hamsters.
[0119] The hamster provides an excellent model for studying
activation and deactivation (protection) of estrogen metabolites in
relation to formation of CE-Q. In fact, implantation of E.sub.1 or
E.sub.2 in male Syrian golden hamsters induces renal carcinomas in
100% of the animals, but does not induce liver tumors. Therefore,
comparison of the profiles of estrogen metabolites, conjugates and
DNA adducts in the two organs should provide information concerning
the imbalance in estrogen homeostasis generated by treatment with
E.sub.2. Hamsters were injected with 8 .mu.mol of E.sub.2 per 100 g
body weight, and liver and kidney extracts were analyzed for 31
estrogen metabolites, conjugates and depurinating DNA adducts by
HPLC interfaced with an electrochemical detector. Neither the liver
nor the kidney contained 4-methoxyCE, presumably due to the known
inhibition of COMT by 2-CE. More O-methylation of 2-CE was observed
in the liver, whereas more formation of CE-Q was detected in the
kidney (Table 3). (Li, J. J., et al., Cancer Res., 43, 5200-5204
(1983); Cavalieri E. L., et al., Chem. Res. Toxicol., 14, 1041-1050
(2001); Roy D., et al., Carcinogenesis, 11, 459-462 (1990).)
[0120] These results suggest less protective methylation of 2-CE
and more pronounced oxidation of CE to CE-Q in the kidney. To
further investigate the rationale behind this interpretation,
hamsters were first pretreated with L-buthionine (SR)-sulfoximine,
an inhibitor of GSH synthesis, to deplete GSH levels. The hamsters
were then treated with E.sub.2. Very low levels of CE and methoxyCE
were observed in the kidney compared to the liver, suggesting
little protective reduction of CE-Q to CE in the kidney (Table 3).
Most significantly, the 4-OHE.sub.1(E.sub.2)-1-N7Gua depurinating
adduct, arising from reaction of CE-3,4-Q with DNA, was detected in
the kidney, but not in the liver (Table 3). From these results, it
seems that tumor initiation in the kidney occurs because of poor
methylation of CE, which favors the competitive oxidation of CE to
CE-Q, and poor reductase activity to remove CE-Q. Thus, these two
effects lead to a large amount of CE-Q, which can react with
biological nucleophiles, including those in DNA. (Cavalieri E. L.,
et al., Chem. Res. Toxicol., 14, 1041-1050 (2001.)
TABLE-US-00003 TABLE 3 Selected estrogen metabolites, conjugates
and adducts formed in hamsters treated with E.sub.2 or E.sub.2 plus
BSO.sup.a nmol/g tissue Metabolites/conjugates.sup.b/ Kidney Liver
adducts E.sub.2 E.sub.2 + BSO E.sub.2 E.sub.2 + BSO
2-OHE.sub.1(E.sub.2) 2.66 1.02 4.75 10.27 4-OHE.sub.1(E.sub.2) 0.29
0.14 0.44 1.04 2-OCH.sub.3E.sub.1(E.sub.2) 1.13 0.42 4.16 4.46
E.sub.1(E.sub.2)-2,3-Q conjugates.sup.b 1.36 0.21 0.63 0.13
E.sub.1(E.sub.2)-3,4-Q conjugates.sup.b 0.30 0.09 0.06 0.01
E.sub.1(E.sub.2)-3,4-Q N7Gua <0.01 0.27 <0.01 <0.01
adducts .sup.aData are from Cavalieri et al., Chem Res. Toxicol.,
14: 10-41 (2001). BSO: L-buthionine (SR)-sulfoximine. The notation
E.sub.1(E.sub.2) indicates that the metabolites, conjugates or
adducts of both E.sub.1 and E.sub.2 are detected. .sup.bConjugates
include all compounds produced by reaction of CE-Q with GSH and
detected as GSH, cysteine or N-acetylcysteine conjugates.
Studies in Estrogen Receptor-.alpha. Knockout (ERKO)/Wnt-1
Mice.
[0121] A novel model for breast cancer was established by crossing
mice carrying the Wnt-1 transgene (100% of adult females develop
spontaneous mammary tumors) with the ERKO mouse line, in which the
mice lack estrogen receptor-.alpha. and estrogen receptor-.beta. is
not detected in the mammary tissue. Mammary tumors develop in these
mice despite the lack of functional estrogen receptor-.alpha.. To
begin investigating whether estrogen metabolite-mediated
genotoxicity may play an important role in the initiation of
mammary tumors, the pattern of estrogen metabolites and conjugates
was analyzed in ERKO/Wnt-1 mice. Extracts of hyperplastic mammary
tissue and mammary tumors were analyzed by HPLC interfaced with an
electrochemical detector. Picomole amounts of the 4-CE were
detected, but their methoxy conjugates were not. Neither the 2-CE
nor 2-methoxyCE were detected. 4-CE-GSH conjugates or their
hydrolytic products (conjugates of cysteine and N-acetylcysteine)
were detected in picomole amounts in both tumors and hyperplastic
mammary tissue, demonstrating the formation of CE-3,4-Q. These
preliminary findings indicate that estrogen homeostasis is
unbalanced in the mammary tissue, in that the normally minor 4-CE
metabolites were detected in the mammary tissue, but not the
normally predominant 2-CE. In addition, methylation of CE was not
detected, whereas formation of 4-CE-GSH conjugates was.
(Bocchinfuso W. P., et al., Cancer Res., 59, 1869-1876 (1999);
Devanesan P, et al., Carcinogenesis, 22, 1573-1576 (2001).)
Studies in Human Breast Tissue Specimens.
[0122] Imbalances in estrogen homeostasis were also observed in
women with breast carcinoma compared to women without breast cancer
(Table 4). Breast tissue specimens obtained from women undergoing
breast biopsy or surgery were analyzed for 31 estrogen metabolites,
conjugates and depurinating DNA adducts by HPLC with
electrochemical detection. In women without breast cancer, a larger
amount of 2-CE than 4-CE was observed. In women with breast
carcinoma, the 4-CE were 3.5 times more abundant than the 2-CE and
were 4 times higher than in the women without breast cancer.
Furthermore, a statistically lower level of methylation was
observed for the CE in cancer cases compared to controls. Finally,
the level of CE-Q conjugates in women with cancer was 3 times that
in controls, suggesting a larger probability for the CE-Q to react
with DNA in the breast tissue of women with carcinoma. These data
suggest that initiation of human breast cancer is due to imbalances
in estrogen homeostasis that result in excessive formation of the
electrophilic CE-Q. In particular, the CE-3,4-Q can react with DNA
to generate successively depurinating adducts, apurinic sites and
oncogenic mutations leading to breast cancer. (Badawi A. F., et
al., Proc. Amer. Assoc. Cancer Res., 42, 664 (2001).)
TABLE-US-00004 TABLE 4 Estrogen metabolites and conjugates in
breast tissue from women with and without breast cancer
Compounds,.sup.a pmol/g tissue 4-OHE.sub.1(E.sub.2) 4- + 2- CE-Q
Breast Tissue 4-OHE.sub.1(E.sub.2) 2-OHE.sub.1(E).sub.2
2-OHE.sub.1(E.sub.2) 4-OMeE.sub.1(E.sub.2) 2-OMeE.sub.1(E.sub.2)
OMeE.sub.1(E.sub.2) conjugates Controls.sup.b 3.6 .+-. 2.1
(10).sup.c 6.9 .+-. 6.1 (25) 0.52 4.9 .+-. 1.8 (24) 3.6 .+-. 2.3
(16) 8.5 2.6 .+-. 1.3 (29) Breast cancer 14.7 .+-. 11.5 (53) 4.2
.+-. 4.6 (46) 3.5 3.1 .+-. 2.3 (39) 1.7 .+-. 1.0 (29) 4.8 8.2 .+-.
6.4 (57) cases.sup.b p.sup.d 0.047 n.s..sup.e 0.049 0.050 0.003
.sup.aThe notation E.sub.1(E.sub.2) indicates that the metabolites,
conjugates or adducts of both E.sub.1 and E.sub.2 were detected.
.sup.bControls include 18 women with benign breast tissue and 31
with benign fibrocystic changes for a total of 49 women. Breast
cancer cases include 28 women with carcinoma of the breast.
.sup.cNumber in parentheses indicates the percentage of specimens
in which the compound was detected. .sup.dp was calculated by the
student's t-test. .sup.en.s.: not significant.
[0123] Unifying Mechanism of Initiation of Cancer and Other
Diseases
[0124] Oxidation of catechols to semiquinones and quinones is a
postulated pathway to initiate cancer not only with endogenous
estrogens but also with synthetic estrogens such as the human
carcinogen diethylstilbestrol and its hydrogenated derivative
hexestrol. In fact, these two compounds are also carcinogenic in
the kidney of Syrian golden hamsters, and the major metabolites are
their catechols. These catechols may be metabolically converted to
catechol quinones. The catechol quinone of hexestrol has chemical
and biochemical properties similar to those of CE-3,4-Q, namely, it
specifically forms N7Gua and N3Ade adducts by 1,4-Michael addition
after reaction with dG or Ade, respectively, as well as DNA (FIG.
8A). These data suggest that the hexestrol catechol quinone is the
electrophile involved in tumor initiation by hexestrol. In turn,
these results substantiate the hypothesis that CE-3,4-Q may be the
major endogenous tumor initiators. (Herbst A. L., et al., New Engl.
J. Med., 284, 878-881 (1971); Li, J. J., et al., Cancer Res., 43,
5200-5204 (1983); Liehr J. G., et al., Chem.-Biol. Interactions,
55, 157-176 (1985); Haaf H. and Metzler M., Pharmacol., 34,
3107-3115 (1985); Blaich, G., et al., J. Steroid Biochem., 35,
201-204 (1996); Metzler M. and McLachlan J. A., Adv. Exp. Med.
Biol., 136A, 829-837 (1981); Jan S.-T., et al., Chem. Res. Toxicol,
11, 412-419 (1998) and unpublished results).
[0125] The oxidation of phenols to catechols and then to
semiquinones and quinones is not only a mechanism of tumor
initiation for natural and synthetic estrogens, but it could also
be the mechanism of tumor initiation for the leukemogen benzene
(FIG. 8B). Certain metabolites of benzene may be responsible for
both its cytotoxic and genotoxic effects. Benzene is metabolized to
phenol in the liver by cytochrome P450 2E1. Other metabolites
include catechol, hydroquinone (1,4-dihydroxybenzene) and
muconaldehyde. Catechol and hydroquinone accumulate in the bone
marrow, where they can be oxidized by peroxidases, including
myeloperoxidase and prostaglandin H synthase. The resulting
quinones can yield DNA adducts. (Andrews, et al., Biochem.
Pharmacol., 26, 293-300 (1977); Sammett, D., et al., J. Toxicol.
Environ. Health, 5, 785-792 (1979); Snyder, R. and Kalf, G. F., CRC
Crit. Rev. Toxicol., 24, 177-209 (1994); Koop, D. R., et al.,
Toxicol. Appl. Pharmacol., 98, 278-288 (1989); Guengerich, F. P.,
et al., Chem. Res. Toxicol., 4, 168-179 (1991); Sabourin, P. J., et
al., Toxicol. Appl. Pharmacol., 99, 421-444 (1989); Latriano, L.,
et al., Proc. Natl. Acad. Sci. USA, 83, 8356-8360 (1986);
Schlosser, P. M., Carcinogenesis, 14, 2477-2486 (1993); Rickert, D.
E., et al., Toxicol. Appl. Pharmacol., 49, 417-423 (1979);
Greenlee, W. F., et al., Chem.-Biol. Interact., 33, 285-299 (1981);
Eastmond, D. A., et al., Mol. Pharmacol., 30, 674-679 (1986);
Subrahmanyam, V. V., Arch. Biochem. Biophys., 286, 76-84 (1991);
Sadler, A., et al., Toxicol. Appl. Pharmacol, 93, 62-71 (1988);
Schlosser, M. J., et al., Chem. Res. Toxicol., 3, 333-339 (1990);
Levay, G., et al., Carcinogenesis, 12, 1181-1186 (1991); Levay, G.
and Bodell, W. J., Proc. Natl. Acad. Sci. USA, 89, 7105-7109
(1992); Levay, G., et al., Carcinogenesis, 14, 2329-2334
(1993).)
[0126] In fact, catechol, one of the metabolites of benzene, when
oxidized to catechol quinone, reacts with dG and Ade to form the
catechol-4-N7Gua and catechol-4-N3Ade adducts in high yields,
respectively. Oxidation of catechol catalyzed by horseradish
peroxidase, tyrosinase or phenobarbital-induced rat liver
microsomes in the presence of DNA yielded the catechol-4-N7Gua
adduct, while the catechol-4-N3Ade adduct was obtained only with
tyrosinase. (Balu N., et al., Proc. Amer. Assoc. Cancer Res., 40,
46 (1999); Cavalieri, E. L., et al., Carcinogenesis, in press
(2002).)
[0127] Catecholamine neurotransmitters such as dopamine may produce
semiquinones and quinones via autoxidation, metal ion oxidation and
peroxidative enzyme or cytochrome P450 oxidation. This oxidative
process is similar to the one described for the benzene metabolite
catechol and the 4-CE, and it may initiate Parkinson's disease and
other neurodegenerative disorders. The etiology of Parkinson's
disease and the basic mechanism of loss of dopamine neurons are
unknown. One of the functions of dopamine is the synthesis of
neuronmelanin via oxidation of dopamine to its quinone. If
oxidation of dopamine to its quinone does not occur in a properly
controlled environment, dopamine quinone may react with DNA to
cause damage by formation of specific depurinating adducts. In
fact, N7Gua and N3Ade adducts (FIG. 8B) are obtained by reaction of
the dopamine quinone with dG or Ade, respectively, and the same
adducts are formed when dopamine is enzymatically activated in the
presence of DNA. The mutations generated by this damage may play a
role in the initiation of Parkinson's disease and other
neurodegenerative disorders. (Mattammal M. B., et al., J.
Neurochem., 64, 1845-1854 (1995); Kalyanaraman B., et al., Environ.
Health Perspect., 64, 185-194 (1985); Kalyanaraman B., et al., J.
Biol. Chem., 259, 7584-7589 (1984); Balu N., et al., Proc. Amer.
Assoc. Cancer Res., 40, 46 (1999); Cavalieri, E. L.,
Carcinogenesis, in press (2002)).
Conclusions
[0128] The carcinogenicity of estrogens in animal models led to an
investigation of the plausible estrogen metabolites that could
react with DNA and lead to mutations initiating cancer. The
electrophilic CE-3,4-Q can, indeed, react with DNA to form the
specific depurinating adducts bonded at the N-7 of Gua and N-3 of
Ade. The apurinic sites formed by depurinating adducts are
converted into tumor-initiating mutations by error-prone repair.
The specificity of the reaction of the electrophiles with DNA is
not limited to the natural estrogens, but also includes the
carcinogenic synthetic estrogens such as hexestrol. In this case
metabolic formation of its catechol and further oxidation to its
catechol quinone lead to formation of analogous specific
depurinating adducts at the N-7 of Gua and N-3 of Ade. In addition,
the metabolite catechol of the leukomogenic benzene and the
catecholamine neurotransmitter dopamine, when oxidized to quinone,
binds to DNA to form N7Gua and N3Ade adducts. (Cavalieri E., et
al., Estrogens as endogenous genotoxic agents: DNA adducts and
mutations. In: JNCI Monograph 27: Estrogens as Endogenous
Carcinogens in the Breast and Prostate, 75-93, E. Cavalieri and E.
Rogan (eds.), Oxford Press, Washington (2000); Chakravarti D., et
al., Mutation Res., 456: 17-32 (2000); Chakravarti D. et al.,
Oncogene, 20: 7945-7953 (2001); Jan S.-T., et al., Chem. Res.
Toxicol, 11: 412-419 (1998).)
[0129] Thus, a unifying mechanism, namely, formation of catechol
quinones and reaction with DNA by 1,4-Michael addition to yield
depurinating adducts, is at the origin of cancers induced by
oxidation of endogenous and synthetic estrogens, leukemia by
oxidation of benzene, and neurodegenerative diseases by oxidation
of dopamine. This unifying mechanism provides targets for disease
prevention and treatment, and methods to assess risk of developing
diseases and/or their progression.
Example II
Catechol Ortho-Quinones
The Electrophilic Compounds that Form Depurinating DNA Adducts and
could Initiate Cancer and Other Diseases
[0130] Introduction
[0131] An important pathway in the metabolism of catechol estrogens
(CE) and catecholamines is the oxidation to their respective
semiquinones and quinones. The basis of the biological activity of
catechol quinones is related to their ability to act both as
oxidants and electrophiles. As oxidants, catechol quinones redox
cycle with their semiquinones, producing an elevated level of
reactive oxygen species, a condition known as oxidative stress. As
electrophiles, catechol quinones can form covalent adducts with
cellular macromolecules, including DNA. These are stable adducts
that remain in DNA unless removed by repair and depurinating ones
that are released from DNA by destabilisation of the glycosyl bond.
Thus, DNA can be damaged by the reactive quinones themselves and by
reactive oxygen species (hydroxyl radicals). The formation of
depurinating adducts by CE quinones reacting with DNA may be a
major event in the initiation of breast and other human cancers.
The depurinating adducts are released from DNA, leaving apurinic
sites in the DNA that can generate mutations leading to cancer.
(Liehr, J. G. and Roy, D., Free Radic. Biol. Med., 8, 415-423
(1990); Cavalieri, E. L. and Rogan, E. G., The key role of catechol
estrogen-3,4-quinones in tumor initiation. In Creveling, C. R.
(ed). Role of Catechol Quinone Species in Cellular Toxicity, F. P.
Graham Pub. Co., Johnson City, Tenn., 247-260 (2000); Finley, K.
T., Quinones: The present state of addition and substitution
chemistry. In Patai, S. (ed.) The chemistry of hydroxyl, ether and
peroxide groups, John Wiley & Sons Ltd., Suppl. E, 1027-1134
(1993); Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA, 94,
10937-10942 (1997); Cavalieri, E., et al., Estrogens as endogenous
genotoxic agents: DNA adducts and mutations. In Cavalieri, E. and
Rogan, E. (eds.) JNCI Monograph: "Estrogens as endogenous
carcinogens in the breast and prostate", Oxford University Press,
75-93 (2000); Chakravarti, D., et al., Proc. Natl. Acad. Sci. USA,
92, 10422-10426 (1995); Chakravarti, D., Mutat. Res., 456, 17-32
(2000); Chakravarti, D., et al., Oncogene, 20, 7945-7953
(2001).)
[0132] An important metabolic pathway of the estrogens, estrone
(E.sub.1) and estradiol (E.sub.2), is formation of CE, namely, the
hydroxylated estrogens, 4-hydroxyestrone(estradiol)
[4-OHE.sub.1(E.sub.2)], which are carcinogenic in animals, and the
isomeric 2-OHE.sub.1(E.sub.2). Oxidation of 4-OHE.sub.1(E.sub.2) to
their quinones [E.sub.1(E.sub.2)-3,4-Q] and reaction with DNA form
the 4-OHE.sub.1(E.sub.2)-1-N7guanine (Gua) and
4-OHE.sub.1(E.sub.2)-1-N3adenine (Ade) adducts by depurination
(FIG. 9). (Liehr, J. G., et al., J. Steroid Biochem., 24, 353-356
(1986); Li, J. J. and Li, S. A., Fed. Proc., 46, 1858-1863 (1987);
Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA, 94,
10937-10942 (1997); Cavalieri, E., et al., Estrogens as endogenous
genotoxic agents: DNA adducts and mutations. In Cavalieri, E. and
Rogan, E. (eds.) JNCI Monograph: "Estrogens as endogenous
carcinogens in the breast and prostate", Oxford University Press,
pp. 75-93 (2000); Li, K.-M., et al., Proc. Amer. Assoc. Cancer
Res., 39, 636 (1998).)
Benzene
[0133] Benzene is carcinogenic and leukemogenic in rats and mice,
and epidemiological studies have established a relationship between
exposure to benzene and acute myelogenous leukemia in humans.
Several studies indicate that certain metabolites of benzene are
responsible for both its cytotoxic and genotoxic effects. High
levels of peroxidase and a lack of quinone reductase in the bone
marrow allow formation of toxic semiquinones and quinones without
the possibility of their being reduced. Benzene is initially
metabolized to phenol in the liver by cytochrome P450 2E1. Other
metabolites include catechol (CAT, 1,2-dihydroxybenzene),
hydroquinone (1,4-dihydroxybenzene) and muconaldehyde. Several
studies have shown that CAT and hydroquinone accumulate in bone
marrow, where they can be further activated to exert their
myelotoxic effects. (Cronkite, E. P., et al., Environ. Health
Perspect., 82, 97-108 (1989); Maltoni, C., et al., Environ. Health
Perspect., 82, 109-124 (1989); Huff, J. E., et al., Environ. Health
Perspect., 82, 125-163 (1989); (IARC, IARC Monographs on the
Evaluation of the Carcinogenic Risk of Chemicals to Humans 29,
93-148 (1982); Rinsky, R. A., et al., N. Engl. J. Med., 316,
1044-1050 (1987); Andrews, L. S., et al., Biochem. Pharmacol., 26,
293-300 (1977); Sammett, D., et al., J. Toxicol. Environ. Health,
5, 785-792 (1979); Kalf, G. F., CRC Crit. Rev. Toxicol., 18,
141-159 (1987); Snyder, R. and Kalf, G. F., CRC Crit. Rev.
Toxicol., 24, 177-209 (1994); Twerdok, L. E. and Trush, M. A., Res.
Commun. Chem. Pathol. Pharmacol., 67, 375-386 (1990); Koop, D. R.,
et al., Toxicol. Appl. Pharmacol., 98, 278-288 (1989); Guengerich,
F. P., et al, Chem. Res. Toxicol., 4, 168-179 (1991); Sabourin, P.
J., et al., Toxicol. Appl. Pharmacol., 99, 421-444 (1989);
Latriano, L., et al., Proc. Natl. Acad. Sci. USA, 83, 8356-8360
(1986); Schlosser, P. M., et al., Carcinogenesis, 14, 2477-2486
(1993); Rickert, D. E., et al., Toxicol. Appl. Pharmacol., 49,
417-423 (1979); Greenlee, W. F., et al., Chem.-Biol. Interact., 33,
285-299 (1981); Kalf, G. F., CRC Crit. Rev. Toxicol., 18, 141-159
(1987); Eastmond, D. A., Mol. Pharmacol., 30, 674-679 (1986);
Subrahmanyam, V. V., et al., Arch. Biochem. Biophys., 286, 76-84
(1991); Sadler, A., et al., Toxicol. Appl. Pharmacol., 93, 62-71
(1988); Schlosser, M. J., et al., Chem. Res. Toxicol., 3, 333-339
(1990); Levay, G., et al., Carcinogenesis, 12, 1181-1186 (1991);
Levay, G. and Bodell, W. J., Proc. Natl. Acad. Sci. USA, 89,
7105-7109 (1992); Levay, G., et al., Carcinogenesis, 14, 2329-2334
(1993).)
Dopamine
[0134] The neurotransmitter DA is formed in the cell bodies of the
dopaminergic neurons of the substantia nigra. Degeneration of the
nigrostriatal dopaminergic neurons and decreased production of DA
results in Parkinson's disease. The etiology of Parkinson's disease
and its underlying mechanism of loss of DA neurons are unknown.
There is evidence, however, that DA is involved in the etiology of
this disease, based on the observation by Graham, et al. that DA is
oxidized to the corresponding quinone. Injection of DA into
neostriatum generates toxicity to dopaminergic neurons, and the
toxicity correlates with protein binding. Glutathione and ascorbic
acid diminish the toxicity of protein binding. Covalent binding of
DA to DNA occurs upon incubating DA with HL-60 cells or human
glioblastoma cell lines, by copper-mediated oxidation of DA or by
oxidation of DA with prostaglandin H synthase. (Graham, D. G., et
al., Mol. Pharmacol., 14, 644-653 (1978); Hastings, T. G., et al.,
Proc. Natl. Acad. Sci. USA, 93, 1956-1961 (1996); Filloux, F. and
Townsend, J. J., Exper. Neurol., 119, 79-88 (1993); Hastings, T. G.
and Zigmond, M. J., J. Neurochem., 63, 1126-1132 (1994); Levay, G.
and Bodell, W. J., Carcinogenesis, 14, 1241-1245 (1993); Levay, G.,
et al., Exper. Neurol., 146, 570-574 (1997); Hastings, T. G., J.
Neurochem., 64, 919-924 (1995); Mattammal, M. B., J. Neurochem.,
64, 1845-1854 (1995).)
[0135] Oxidation of DA to its quinone and subsequent reaction with
DNA may cause DNA damage via formation of specific depurinating
adducts, and the mutations generated by that damage may play a
major role in initiating the series of events leading to
neurodegenerative disorders such as Parkinson's disease. In
general, catecholamine neurotransmitters such as DA can produce
semiquinones and quinones via autoxidation, metal ion oxidation,
and peroxidative enzyme or cytochrome P450 oxidation. This
oxidative process is similar to the one described above for the
benzene metabolite CAT and for the 4-OHE.sub.1(E.sub.2) formed by
the metabolism of E.sub.1 and E.sub.2. (Kalyanaraman, B., et al.,
Environ. Health Perspect., 64, 185-194 (1985); Kalyanaraman, B., et
al., J. Biol. Chem., 259, 7584-7589 (1984); Cavalieri, E. L., et
al., Proc. Natl. Acad. Sci. USA, 94, 10937-10942 (1997); Cavalieri,
E., et al., Estrogens as endogenous genotoxic agents: DNA adducts
and mutations. In Cavalieri, E. and Rogan, E. (eds.) JNCI
Monograph: "Estrogens as endogenous carcinogens in the breast and
prostate", Oxford University Press, 75-93 (2000).)
ABBREVIATIONS
[0136] Ade, adenine; o-BQ, ortho-benzoquinone; CAT, catechol or
1,2-dihydroxybenzene; CE, catechol estrogen(s); CE-Q, catechol
estrogen quinone(s); COMT, catechol-O-methyltransferase; DA,
dopamine; dG, deoxyguanosine; DMF, dimethylformamide; E.sub.1,
estrone; E.sub.2, estradiol; E.sub.1(E.sub.2)-3,4-Q,
estrone(estradiol)-3,4-quinones or catechol estrogen-3,4-quinones;
FAB, fast atom bombardment; Gua, guanine; MS/MS, tandem mass
spectrometry; NADA, N-acetyldopamine; OHE.sub.1(E.sub.2),
hydroxyestrone(estradiol); TFA, trifluoroacetic acid.
DEFINITIONS
[0137] The term catechol refers to an aromatic ring with vicinal
hydroxyl substituents. Herein catechol is spelled out when it is
used in a general sense. When it refers specifically to the
compound 1,2-dihydroxybenzene, normally called catechol, it is
abbreviated CAT.
Materials and Methods
[0138] Chemicals, Reagents and Enzymes:
[0139] CAT was obtained from ICN Pharmaceuticals Inc., Cleveland,
Ohio; Ag.sub.2O, NaIO.sub.4, Ade, thymidine, deuterated acetic acid
and trifluoroacetic acid (TFA) were purchased from Aldrich Chemical
Co., Milwaukee, Wis. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine and
2'-deoxycytidine were purchased from TCI Chemicals. DA, and
N-acetyldopamine (NADA), horseradish peroxidase (type VI) and
mushroom tyrosinase were purchased from Sigma Chemicals, St. Louis,
Mo. Liver microsomes from phenobarbital-induced female Wistar MRC
rats (Eppley Colony) were prepared by the previously published
method (Wong, A. K. L., et al., Biochem. Pharmacol., 35, 1583-1588
(1986).)
[0140] Instrumentation
[0141] UV:
[0142] The UV spectra were obtained during HPLC by using the
photodiode array detector (Waters 996, Milford, Mass.) for all
compounds synthesized. HPLC separations were monitored at 280
nm.
[0143] NMR:
[0144] Proton and homonuclear two-dimensional chemical shift
correlation spectroscopy NMR spectra were recorded in DMSO-d.sub.6
with one drop of D.sub.2O and one drop of CD.sub.3COOD on a Varian
Unity 500 instrument at 499.835 MHz at 25.degree. C. Chemical
shifts are reported relative to DMSO (2.5 ppm).
[0145] Mass Spectrometry:
[0146] Exact mass measurements of fast atom bombardment
(FAB)-produced ions were carried out on a Kratos MS-50 double
focusing mass spectrometer in a peak-match mode. Confirmation of
the presence of each adduct was by capillary HPLC coupled via
electrospray ionization with a Finnigan LCQ ion trap mass
spectrometer operating in the tandem mass spectrometry (MS/MS)
mode. The HPLC (Microtech Scientific) made use of a binary gradient
of solvent A [0.5% CH.sub.3COOH (v/v) in H.sub.2O] and solvent B
[0.5% CH.sub.3COOH (v/v) in CH.sub.3OH] at a flow rate of 40
.mu.L/min with a split of 10:1. The column was 0.3.times.100 Zorbax
C18 (Microtech Scientific) with a flow rate on the column of 4
.mu.L/min. The gradient was 95% A/5% B initially for 4 min, then
linearly adjusted to 60/40 over 14 min, and held at 60/40 for 20
min.
[0147] HPLC Methods for Synthetic Standards:
[0148] HPLC was conducted on a Waters (Milford, Mass.) 600 E system
equipped with a Waters 996 photodiode array detector interfaced
with an NEC-Powermate computer. Analyses and preparative
separations were carried out on reverse-phase C-18, YMC (Morris
Plains, N.J.) columns (5 .mu.m, 120 A, ODS-AQ (6.times.250 mm) and
ODS-AQ, 5 .mu.m, 120 A, (20.times.250 mm), respectively) using
specific mobile phases for the different compounds.
Synthesis of Standard Adducts
[0149] Catechol Adducts.
[0150] Because the ortho-benzoquinone (o-BQ, nascent quinone) is
rather unstable, various methods of synthesis were tested to obtain
its maximum yield. Oxidation of CAT using Ag.sub.2O in dry
dimethylformamide (DMF) was the best method. A solution of CAT (100
mg, 0.91 mmol) in dry DMF (7.5 mL) was stirred with Ag.sub.2O (842
mg, 3.60 mmol) for 30 min at 0.degree. C. The extent of formation
of o-BQ was followed by HPLC, using a linear analytical gradient
from 100% H.sub.2O (0.01% TFA, pH 2.6) to 30% CH.sub.3CN in 60 min
at a flow rate of 1 mL/min (monitored by UV absorbance at 300 nm on
a Waters 996 photodiode array detector). The yield of o-BQ was
>95%.
[0151] The dark red solution was immediately filtered into a
solution of dG (1.20 g, 4.54 mmol) or Ade (613 mg, 4.54 mmol) in
DMF/CH.sub.3COOH/H.sub.2O, 7.5 mL each (FIG. 10). The reaction
mixture was stirred for 8 h at room temperature, filtered and
washed with 10 mL of DMF/CH.sub.3COOH/H.sub.2O (2:1:1). The
reddish-brown filtrate was directly subjected to HPLC purification,
using a linear preparative gradient of 20% CH.sub.3CN in H.sub.2O
(0.01% TFA) to 80% CH.sub.3CN in H.sub.2O (0.01% TFA) over 60 min
at a flow rate of 9 mL/min with dG or 15% CH.sub.3CN in H.sub.2O
(0.01% TFA) to 60% CH.sub.3CN in H.sub.2O (0.01% TFA) over 60 min
at a flow rate of 9 mL/min with Ade. The products, isolated under
an argon atmosphere and stored at -20.degree. C. in 2 mL of
DMF/CH.sub.3COOH/H.sub.2O (2:1:1), were CAT-4-N7Gua and
CAT-4-N3Ade, the result of a 1,4-Michael addition between dG or Ade
and o-BQ.
[0152] For CAT-4-N7Gua, the yield was 59%; UV: .lamda..sub.max, 285
nm. .sup.1H NMR, .delta. (ppm): 6.79 (s, 2H, 5-H, 6-H), 7.08 (s,
1H, 3-H), 8.01 (s, 1H, 8-H [Gua]). FAB MS, [M+H].sup.+,
C.sub.11H.sub.9N.sub.5O.sub.3: calcd m/z 260.0785; obsd m/z
260.0783.
[0153] For CAT-4-N3Ade, the yield was 65%; UV: .lamda..sub.max, 279
nm. .sup.1H NMR, .delta. (ppm): 6.95 (bd, 2H, 5-H, 6-H), 7.08 (s,
1H, 3-H), 8.52 (s, 1H, 2-H [Ade]), 8.76 (s, 1H, 8-H [Ade]). FAB MS,
[M+H].sup.+, C.sub.11H.sub.9N.sub.5O.sub.2: calcd m/z 244.0836;
obsd m/z 244.0834.
[0154] N-Acetyldopamine Adducts.
[0155] A solution of NADA (9 mg, 0.047 mmol) in 1.5 mL of
CH.sub.3COOH/H.sub.2O (1:1) was stirred with NaIO.sub.4 (5 mg,
0.023 mmol) for 5 min at room temperature. To the resulting red
solution of the NADA quinone was added 5 equivalents of dG (59 mg,
0.23 mmol) in 1.5 mL of CH.sub.3COOH/H.sub.2O (1:1) (FIG. 11). The
reaction mixture was stirred for 3 h at room temperature and then
separated by HPLC, using a 45-min linear preparative gradient from
10% CH.sub.3CN in H.sub.2O (0.01% TFA) to 30% CH.sub.3CN in
H.sub.2O (0.01% TFA) at a flow rate of 10 mL/min. The yield of
NADA-6-N7Gua was 58%.
[0156] The NADA-6-N7Gua adduct was also synthesized following
oxidation of NADA by Ag.sub.2O. A solution of NADA (5 mg, 0.023
mmol) in 1 mL of dry DMF was stirred with Ag.sub.2O (43 mg, 0.19
mmol) for 30 min. The suspension was immediately filtered into a
solution of dG (29 mg, 0.12 mmol) in DMF/CH.sub.3COOH/H.sub.2O, 1
mL each. The reaction mixture was stirred for 10 h at room
temperature, and the product purified by HPLC, yielding 60%
NADA-6-N7Gua, UV: .lamda..sub.max, 245, 284 nm. .sup.1H NMR,
.delta. (ppm): 1.90 (s, 3H, CH.sub.3), 2.25 (t, 2H, J=6.7 Hz,
7-CH.sub.2), 3.00 (bt, 2H, 8-CH.sub.2), 6.62 (s, 1H, 5-H), 6.66 (s,
1H, 2-H), 7.85 (s, 1H, 8-H [Gua]). FAB MS, [M+H].sup.+,
C.sub.15H.sub.17N.sub.6O.sub.4: calcd m/z 345.1311; obsd m/z
345.1311.
[0157] To synthesize the Ade adduct, a solution of NADA (20 mg,
0.094 mmol) in 2 mL of CH.sub.3COOH/H.sub.2O (1:1) was oxidized
with NaIO.sub.4 (10 mg, 0.047 mmol) and reacted with Ade (63 mg,
0.47 mmol), as described above for the reaction with dG. The
product, NADA-6-N3Ade, was purified by HPLC, using a preparative
linear gradient from 5% CH.sub.3CN in H.sub.2O (0.01% TFA) over 60
min to 40% CH.sub.3CN in H.sub.2O (0.01% TFA) at a flow rate of 9
mL/min. The yield was 51%; UV: .lamda..sub.max, 275 nm. .sup.1H
NMR, .delta. (ppm): 1.90 (s, 3H, CH.sub.3), 2.15-2.30 (m, 2H,
7-CH.sub.2), 2.83-3.10 (m, 2H, 8-CH.sub.2), 6.81 (s, 1H, 5-H), 6.85
(s, 1H, 2-H), 8.45 (s, 1H, 2-H [Ade]), 8.65 (s, 1H, 8-H [Ade]). FAB
MS, [M+H].sup.+, C.sub.15H.sub.17N.sub.6O.sub.3: calcd m/z
329.1361; obsd m/z 329.1362.
[0158] Dopamine Adducts.
[0159] DA.HCl (50 mg, 0.264 mmol) and dG (622 mg, 2.6 mmol) were
dissolved in 13 mL of CH.sub.3COOH/H.sub.2O (1:1). To this mixture
a solution of NaIO.sub.4 (28 mg, 0.13 mmol) in 2 mL of
CH.sub.3COOH/H.sub.2O (1:1) was added dropwise over 10 min (FIG.
11). After 3 h at room temperature, the reaction was terminated,
and the product, DA-6-N7Gua, was purified by preparative HPLC,
using a linear gradient from 10% CH.sub.3CN in H.sub.2O (0.01% TFA)
to 30% CH.sub.3CN in H.sub.2O (0.01% TFA) over 60 min, then to 80%
CH.sub.3CN in 15 min at a flow rate of 8 mL/min. The colorless
semi-solid product was obtained in 46% yield. UV: .lamda..sub.max,
245 (sh), 283 nm. .sup.1H NMR, .delta. (ppm): 2.45 (m, 2H,
7-CH.sub.2), 2.75 (m, 2H, 8-CH.sub.2), 6.70 (s, 1H, 5-H), 6.70 (s,
1H, 2-H), 8.15 (s, 1H, 8-H [Gua]). FAB MS, [M+H].sup.+,
C.sub.13H.sub.14N.sub.6O.sub.3: calcd m/z 303.1207; obsd m/z
303.1205.
[0160] A solution of DA.HCl (50 mg, 0.26 mmol) and Ade (356 mg,
2.64 mmol) in 13 mL of CH.sub.3COOH/H.sub.2O (1:1) was treated with
a solution of NaIO.sub.4 (28 mg, 0.13 mmol) in 2 mL of
CH.sub.3COOH/H.sub.2O (1:1) in a manner similar to that used to
synthesize DA-6-N7Gua. After 3 h at room temperature, the reaction
was terminated. The mixture was subjected to preparative HPLC,
using H.sub.2O (0.01% TFA) at a flow rate of 5 mL/min for 20 min,
followed by a linear gradient to 80% CH.sub.3CN in H.sub.2O (0.01%
TFA) over 40 min at a flow rate of 10 mL/min. DA-6-N3Ade was
obtained in 40% yield. UV: .lamda..sub.max, 279 nm. .sup.1H NMR,
.delta. (ppm): 2.40 (m, 2H, 7-CH.sub.2), 2.82 (m, 2H, 8-CH.sub.2),
6.80 (s, 1H, 5-H), 6.86 (s, 1H, 2-H), 8.05 (s, 1H, 2-H [Ade]), 8.40
(s, 1H, 8-H [Ade]). FAB MS, [M+H].sup.+,
C.sub.13H.sub.14N.sub.6O.sub.2: calcd m/z 287.1258; obsd m/z
287.1256.
Enzymatically-Catalyzed Covalent Binding of Catechol and Dopamine
to DNA
[0161] CAT and DA were bound to DNA in 10-mL reaction mixtures
containing 3 mM calf thymus DNA in 0.067 M sodium-potassium
phosphate (pH 7.0), 0.8 .mu.M CAT or DA in 50 .mu.L DMSO and 1 mg
horseradish peroxidase plus 0.5 mM H.sub.2O.sub.2 or 1 mg mushroom
tyrosinase. CAT and DA (0.8 .mu.M) were also activated by 10 mg of
phenobarbital-induced rat liver microsomes in 150 mM Tris-HCl (pH
7.5), 150 mM KCl, 5 mM MgCl.sub.2, 1 mM cumene hydroperoxide and 3
mM DNA. The reactions were incubated for 2 h at 37.degree. C. A
1-mL aliquot was used for analysis of stable DNA adducts by the
.sup.32P-postlabeling method with 8 .mu.g of DNA. The DNA was
precipitated from the remaining reaction mixture with two volumes
of ethanol, and the supernatant was used for structure
determination of depurinating adducts. After evaporation under
vacuum, the residue was dissolved in 1 mL of DMSO/CH.sub.3OH. The
CAT adducts were first separated by HPLC on a preparative column
with a curvilinear gradient (CV 6) from 100% H.sub.2O (0.01% TFA)
to 15% CH.sub.3OH in H.sub.2O (0.01% TFA) in 60 min at a flow rate
of 3 mL/min. Fractions at the retention times of CAT-4-N3Ade (34.5
min) and CAT-4-N7Gua (40 min) were collected and analyzed by HPLC,
which was eluted with aqueous 50 mM (NH.sub.4).sub.3PO.sub.4, 5 mM
sodium dodecyl sulfate, 1% CH.sub.3COOH at a flow rate of 0.5
mL/min. The DA adducts were first separated by HPLC on a
preparative column eluted with a curvilinear gradient (CV 6) from
100% aqueous 50 mM (NH.sub.4).sub.3PO.sub.4, 5 mM sodium dodecyl
sulfate, 4% CH.sub.3COOH to 100% CH.sub.3CN at a flow rate of 0.5
mL/min. Fractions were collected at 34 min for DA-6-N3Ade and 37
min for DA-6-N7Gua and analyzed by HPLC as described above for the
CAT adducts. The remainder of the collected fractions was used to
confirm the structures of the adducts by MS. (Wong, A. K. L., et
al., Biochem. Pharmacol., 35, 1583-1588 (1986); Bodell, W. J., et
al., Chem. Res. Toxicol., 2, 312-315 (1989).)
Results
[0162] To demonstrate that the quinones of CAT and DA can react
with the nucleobases of DNA, standard adducts were synthesized by
reaction of o-BQ or DA quinone with dG or Ade. The syntheses
provided useful insights into the ability of these electrophilic
species to react with nucleophilic groups of deoxyribonucleosides.
Furthermore, the adducts served as standards to identify the
depurinating adducts formed when CAT and DA were oxidized in vitro
by various enzymes in the presence of DNA (see below).
Structure Elucidation of Adducts
[0163] Catechol Adducts.
[0164] The reaction of o-BQ with the deoxyribonucleoside bases to
afford the desired adducts is an acid-assisted 1,4-Michael addition
reaction analogous to that of CE quinones with nucleobases. With
o-BQ, however, the reaction in CH.sub.3COOH/H.sub.2O (1:1) did not
yield any products, due to the instability of o-BQ. To render this
reaction feasible a compromise was reached by conducting it in
DMF/CH.sub.3COOH/H.sub.2O (2:1:1). (Stack, D., et al., Chem. Res.
Toxicol., 9, 851-859 (1996); Jan, S.-T., et al., Chem. Res.
Toxicol., 11, 412-419 (1998).)
[0165] Reaction of o-BQ with dG afforded CAT-4-N7Gua (FIG. 10). The
structure was readily determined by both NMR and MS analysis. By
MS, the [M+H].sup.+ ion had an m/z 260, indicating that deoxyribose
had been lost. This implies that Gua is bonded to CAT at the N-7.
The NMR resonance of the 5-H and 6-H of the CAT moiety as a singlet
at 6.79 ppm and the 3-H as a singlet at 7.08 ppm indicates that the
bond to Gua in the CAT aromatic ring occurs at C-4.
[0166] For CAT-4-N3Ade, the structure was consistent with the NMR
spectrum, showing the aromatic protons 5-H and 6-H as a doublet at
6.95 ppm, and the singlet at 7.08 ppm that was assigned as 3-H.
Furthermore, the 2-H and 8-H of the Ade moiety were observed at
8.52 and 8.76 ppm, respectively. The mass of the FAB-produced ion
at m/z 244 corroborated the structure of this adduct.
[0167] Under the same conditions, o-BQ adducts of deoxyadenosine,
deoxycytidine and thymidine were not obtained. Furthermore,
reaction of the stable 1,4-benzoquinone (p-BQ) with dG, Ade,
deoxyadenosine, deoxycytidine or thymidine did not afford any
detectable adducts.
[0168] Dopamine Adducts.
[0169] The oxidation of DA and subsequent reaction with dG or Ade
were more difficult to accomplish because the amino group of the DA
quinone reacts intramolecularly by a 1,4-Michael addition to
produce a dihydroindole, a precursor to neuromelanin biosynthesis.
This reaction competes with the intermolecular acid-assisted
1,4-Michael addition of the nucleophilic groups of dG and Ade to
the DA quinone. To avoid the competitive cyclization reaction,
NADA, in which the nucleophilic amino group of DA is acetylated,
was oxidized to its quinone and reacted with deoxyribonucleosides
or nucleobases.
[0170] N-Acetyldopamine.
[0171] The structure of the NADA-6-N7Gua adduct (FIG. 11) was
consistent with MS results, which showed an m/z 345 ion, indicating
the loss of the deoxyribose moiety. It was also consistent with the
NMR results: the two aromatic protons of the NADA moiety, 5-H and
2-H, resonate at 6.62 and 6.70 ppm, respectively, assuming that the
reaction was a 1,4-Michael addition. If the reaction occurred by
1,6-addition, however, an adduct at C-2 and/or C-5 of NADA should
have been obtained. Reaction at C-2 can be disregarded on the basis
that the aromatic protons did not resonate as doublets. The adduct
with the Gua-NADA bond at C-5, formed by 1,6-addition, was
eliminated from consideration by a nuclear Overhauser enhancement
experiment in which the resonance of the 7-CH.sub.2 protons at 2.25
ppm was irradiated. This structure would entail that both the
resonance signals corresponding to 2-H and 6-H be enhanced. In
fact, only the signals corresponding to the 2-H of NADA at 6.70 ppm
and the 8-H of the Gua moiety at 7.85 ppm were enhanced. This
result unequivocally assigns the structure of the adduct as
NADA-6-N7Gua, proving that the reaction is a 1,4-Michael addition.
Following the same approach, the structure of NADA-6-N3Ade was
assigned.
[0172] Dopamine.
[0173] Reactions of DA.HCl were set at pH 1.2 in
CH.sub.3COOH/H.sub.2O (1:1) to minimize intramolecular 1,4-Michael
addition of DA and favor intermolecular 1,4-Michael addition of dG
or Ade to the 6 position of DA quinone. Although cyclization was
avoided under these conditions, owing to the extensive protonation
of the DA amino group, minor side reactions such as dimerization
and subsequent oligomerization of the resulting DA quinone could
not be eliminated. To minimize these competing reactions and obtain
the best yields, the DA o-quinone was generated in situ by adding a
solution of NaIO.sub.4 to a mixture of DA.HCl and dG or Ade.
[0174] The structures of the adducts obtained by reaction of DA
quinone with dG or Ade, DA-6-N7Gua and DA-6-N3Ade (FIG. 11), were
elucidated following the same criteria adopted for the NADA
adducts. Under the above conditions, DA quinone did not react with
deoxyadenosine, deoxycytidine or thymidine to form adducts to any
measurable extent.
[0175] In conclusion, the reaction of CAT quinone and DA quinone
with dG or Ade involves the specific nucleophilic sites of the N-7
of Gua and N-3 of Ade in the 1,4-Michael addition. The reactions of
E.sub.1(E.sub.2)-3,4-Q with dG or Ade by 1,4-Michael addition
exhibit the same specificity, forming N7Gua and N3Ade depurinating
adducts (Li, K.-M., et al., Proc. Amer. Assoc. Cancer Res., 39, 636
(1998); Stack, D., Chem. Res. Toxicol., 9, 851-859 (1996).)
Enzymatically-Catalyzed Covalent Binding of Catechol and Dopamine
to DNA
[0176] Conversion of CAT and DA to their quinones can generally
occur by autoxidation, metal-ion oxidation or cytochrome P450 or
peroxidase-catalyzed oxidation. In vivo the copper-containing
enzyme tyrosinase oxidizes DA to its quinone. To demonstrate
binding to DNA in vitro, CAT and DA were oxidized in reactions
catalyzed by horseradish peroxidase, tyrosinase or
phenobarbital-induced rat liver microsomes in the presence of DNA
(Table 5).
TABLE-US-00005 TABLE 5 Catechol- and Dopamine-DNA Adducts Formed In
Vitro. .mu.mol adduct/mol DNA-P.sup.a Phenobarbital- Horseradish
induced rat liver Adduct Peroxidase microsomes Tyrosinase Catechol
CAT-4-N7Gua 10 32 110 CAT-4-N3Ade nd.sup.b nd 2 Stable adducts 0.64
0.02 0.21 Dopamine DA-6-N7Gua 1 23 6 DA-6-N3Ade 5 9 3 Stable
adducts 0.30 0.24 0.35 .sup.aValues are the average of two
determinations that varied by 10-20%. .sup.bnd: not detected
All three enzymes catalyzed formation of detectable amounts of the
depurinating adducts of DA, DA-6-N3Ade and DA-6-N7Gua, as well as
the CAT-4-N7Gua depurinating adduct of CAT. In contrast, the
CAT-4-N3Ade adduct was detected only after activation by
tyrosinase. (Kalyanaraman, B., et al., Environ. Health Perspect.,
64, 185-194 (1985); Kalyanaraman, B., et al., J. Biol. Chem., 259,
7584-7589 (1984).)
[0177] Formation of the stable adducts of DA was low, less than 5%
of the total adducts formed with horseradish peroxidase, 4% of the
adducts formed with tyrosinase, and 1% of the adducts formed with
microsomes. Similarly, with CAT, stable adducts comprised less than
6% of the total adducts formed with horseradish peroxidase, 0.2% of
those formed with tyrosinase, and 0.1% of the adducts formed with
microsomes. With DA, the microsomes catalyzed formation of six to
seven stable adducts that were separated by the
.sup.32P-postlabeling method, whereas tyrosinase and horseradish
peroxidase catalyzed formation of the same stable adduct, which
appeared to be one of those formed by the microsomes. With CAT,
both the microsomes and horseradish peroxidase formed two adducts
separated by .sup.32P-postlabeling. One of these adducts was
detected with activation by both enzymes. This same adduct was the
only stable adduct detected when tyrosinase was used to catalyze
the binding of CAT to DNA.
[0178] Confirmation of the presence of each depurinating adduct
reported in Table 5 was by capillary HPLC/tandem mass spectrometry.
The unknowns have identical HPLC retention times as the standards
and give product-ion spectra of the [M+H].sup.+ ions containing the
same two or three intense signals as those of the standards. The
product-ion spectrum of the two modified guanine [M+H].sup.+ ions
showed that losses of 17 (NH.sub.3) and 42 (NC--NH.sub.2) occurred
for both. The CAT-modified Gua showed an additional loss of 24
(possible via formation of an ion-molecule product in the trap),
whereas the DA-modified Gua underwent a loss of 35 (NH.sub.3 and
H.sub.2O). The modified bases isolated from the in vitro
experiments showed these same ions. The [M+H].sup.+ ions of the
adenines modified with CAT or DA fragmented by losses of 17 and 46,
and the unknowns also showed signals for these processes. The
product-ion spectra of all the in vitro adducts showed other
comparable or weaker signals owing to coeluting interferences from
the reaction mixture.
[0179] In summary, enzymatic oxidation of CAT or DA in the presence
of DNA resulted in the formation of 94-99.9% depurinating CAT
adducts or 95-99% depurinating DA adducts.
Discussion
[0180] The catechol o-quinones derived from benzene and DA undergo
1,4-Michael addition with the N-7 and N-3 nucleophilic sites of Gua
and Ade in DNA, respectively, to form predominantly depurinating
adducts analogous to those formed by the E.sub.1(E.sub.2)-3,4-Q
(FIG. 9). These depurinating adducts are by far the major products
(94-99.9%) when the two o-quinones are enzymatically obtained from
the corresponding catechols, CAT and DA, in the presence of DNA
(Table 5). (Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA,
94, 10937-10942 (1997); Li, K.-M., et al., Proc. Amer. Assoc.
Cancer Res., 39, 636 (1998).)
[0181] The role of estrogens in causing DNA damage is better
understood than that of CAT and DA. The estrogens E.sub.1 and
E.sub.2, which are biochemically interconvertible, are metabolized
via two major pathways: formation of CE and, to a lesser extent,
16.alpha.-hydroxylation. In general, estrogens and CE are
inactivated by conjugating reactions, such as glucuronidation and
sulfation, especially in the liver. The most common pathway of CE
conjugation in extrahepatic tissues is O-methylation catalyzed by
the ubiquitous catechol-O-methyltransferase (COMT, FIG. 9).
Relatively high levels of cytochrome P450 1B1 and other
4-hydroxylases could cause the 4-OHE.sub.1(E.sub.2), which are
usually minor metabolites, to be the major ones, rendering
conjugation of 4-OHE.sub.1(E.sub.2) via methylation in extrahepatic
tissues insufficient. In this case, competitive catalytic oxidation
of CE to CE quinones could occur (FIG. 9). Redox cycling generated
by reduction of CE-Q to CE semiquinones, catalyzed by cytochrome
P450 reductase, and subsequent oxidation back to CE-Q by molecular
oxygen causes formation of superoxide anion radicals and,
subsequently, hydroxyl radicals (not shown in FIG. 9). This
process, which also occurs with the quinones of CAT and DA, may
constitute a significant source of reactive oxygen species.
Hydroxyl radicals can also react with DNA and contribute to total
DNA damage. (Cavalieri, E., et al., Estrogens as endogenous
genotoxic agents: DNA adducts and mutations. In Cavalieri, E. and
Rogan, E. (eds.) JNCI Monograph: "Estrogens as endogenous
carcinogens in the breast and prostate", Oxford University Press,
75-93 (2000); Cavalieri, E. L., et al., Proc. Natl. Acad. Sci. USA,
94, 10937-10942 (1997); Service, R., Science, 279, 1631-1633
(1998); Liehr, J. G. and Roy, D., Free Radic. Biol. Med., 8,
415-423 (1990).
[0182] CE-Q can be inactivated by conjugation with glutathione
(FIG. 9). A second inactivating pathway for CE-Q is their reduction
to CE by quinone reductase and/or cytochrome P450 reductase (FIG.
9). If the two inactivating processes are insufficient, CE-Q may
react with DNA to form predominantly stable adducts for the
2-OHE.sub.1(E.sub.2) (not shown in FIG. 9) and predominantly
depurinating adducts for the 4-OHE.sub.1(E.sub.2) (FIG. 9). The
depurinating adducts generate apurinic sites that may lead to
oncogenic mutations, thereby initiating a variety of human cancers,
including breast and prostate. In support of this hypothesis, a
burst of apurinic sites leads to mutations in the H-ras gene of
mouse skin treated with E.sub.2-3,4-Q. (DT Diaphorase--A quinone
reductase with special functions in cell metabolism and
detoxification. Ernester, L., Estabrook, R. W., et al. (eds.)
Chemica Scripta, 27A (1987); Roy, D. and Liehr, J. G., J. Biol.
Chem., 263, 3646-3651 (1988); Cavalieri, E. L., et al., Proc. Natl.
Acad. Sci. USA, 94, 10937-10942 (1997); Stack, D., et al., Chem.
Res. Toxicol., 9, 851-859 (1996); Dwivedy, I., et al., Chem. Res.
Toxicol., 5, 828-833 (1992); Li, K.-M., et al., Proc. Amer. Assoc.
Cancer Res., 39, 636 (1998); Chakravarti, D., et al., Proc. Natl.
Acad. Sci. USA, 92, 10422-10426 (1995); Chakravarti, D., et al.,
Mutat. Res., 456, 17-32 (2000); Chakravarti, D., Oncogene, 20,
7945-7953 (2001).)
[0183] The initiating mechanism of carcinogenesis for the synthetic
estrogen hexestrol may have a similar explanation. This compound,
which is carcinogenic in the kidney of Syrian golden hamsters, also
has catechol as a major metabolite, which can be metabolically
converted to catechol quinone. The catechol quinone of hexestrol
has chemical properties similar to those of E.sub.1(E.sub.2)-3,4-Q,
namely, it specifically forms an N7Gua adduct by 1,4-Michael
addition after reaction with dG or DNA. (Li, J J., Cancer Res., 43,
5200-5204 (1983); Liehr, J. G., et al., Chem.-Biol. Interactions,
55, 157-176 (1985); Metzler, M. and McLachlan, J. A., Adv. Exp.
Med. Biol., 136A, 829-837 (1981); Jan, S.-T., Chem. Res. Toxicol.,
11, 412-419 (1998).
[0184] The formation of depurinating adducts specifically at the
N-7 of Gua and N-3 of Ade by 1,4-Michael addition to CAT quinone,
analogously to those formed by E.sub.1(E.sub.2)-3,4-Q, suggests
that the metabolite CAT may play a major role in tumor initiation
by benzene. In fact, CAT is carcinogenic in mice and rats, inducing
glandular stomach tumors in these animals. The overall
leukemogenicity of benzene could result from a synergistic
genotoxic response to CAT quinone, which predominantly produces
depurinating DNA adducts, and 1,4-benzoquinone, which produces only
stable DNA adducts. (Hirose, M., et al., Carcinogenesis, 14,
525-529 (1993); Levay, G., et al., Carcinoges, 12, 1181-1186
(1991); Levay, G. and Bodell, W. J., Proc. Natl. Acad. Sci. USA,
89, 7105-7109 (1992); Robertson, M., et al., Mutat. Res., 249,
201-209 (1990); Smith, M. T., Environ. Health Perspect., 104:
Suppl. 6, 1219-1225 (1996).)
[0185] One of the functions of the neurotransmitter DA or its
precursor, L-Dopa, is the synthesis of neuromelanin. This occurs by
oxidation of DA to its o-quinone, followed by intramolecular
cyclization of the nucleophilic amino group via a 1,4-Michael
addition (FIG. 12). The product, leucochrome, is further oxidized
to aminochrome, which, after tautomerization to its quinone methide
and quinone imine, polymerizes to neuromelanin, the pigment of the
substantia nigra. Disregulation of DA compartmentalization may lead
to DA quinone formation by various oxidants. Under these
circumstances, intermolecular 1,4-Michael addition of the N-7 of
Gua or N-3 of Ade in DNA to DA quinone could compete successfully
with the intramolecular cyclization of DA quinone that leads to
dihydroindole derivatives (FIG. 12). In fact, DA cyclizes at a
slower rate than L-Dopa and epinephrine. Thus, if oxidation of DA
to its quinone does not occur in a properly controlled environment,
then perhaps the quinone will react with DNA to form depurinating
DNA adducts, generating mutations that could initiate
neurodegenerative disorders such as Parkinson's disease. (Hastings,
T. G., J. Neurochem., 64, 919-924 (1995); Mattammal, M. B., et al.,
J. Neurochem., 64, 1845-1854 (1995); Kalyanaraman, B., et al,
Environ. Health Perspect., 64, 185-194 (1985); Kalyanaraman, B., et
al., J. Biol. Chem., 259, 7584-7589 (1984); Pelizzetti, E., et al.,
J. Chem. Soc. Perkins II, 1651-1655 (1976).)
CONCLUSIONS
[0186] The o-benzoquinones formed in the metabolism of natural and
synthetic estrogens, benzene, and DA react with DNA via 1,4-Michael
addition to form specific depurinating adducts that may lead to
critical mutations responsible for initiating many cancers and
neurodegenerative diseases. Recognition of this proposed unifying
mechanism in the etiology of these diseases may provide unique
opportunities to develop strategies to assess risk and to prevent
diseases.
[0187] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. The invention has been described with
reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations
and modifications may be made while remaining within the spirit and
scope of the invention.
Sequence CWU 1
1
11111DNAArtificial SequenceA synthetic oligonucleotide 1ctggaggcgt
g 11211DNAArtificial SequenceA synthetic oligonucleotide
2tgtggacgag t 11311DNAArtificial SequenceA synthetic
oligonucleotide 3tgaccaaaca g 11411DNAArtificial SequenceA
synthetic oligonucleotide 4tggggtatga t 11511DNAArtificial
SequenceA synthetic oligonucleotide 5gtgcaagggt g
11611DNAArtificial SequenceA synthetic oligonucleotide 6tgcaaaacaa
c 11711DNAArtificial SequenceA synthetic oligonucleotide
7ttgcaggact c 11811DNAArtificial SequenceA synthetic
oligonucleotide 8tggggagaca t 11911DNAArtificial SequenceA
synthetic oligonucleotide 9atgtctactg g 111011DNAArtificial
SequenceA synthetic oligonucleotide 10agagtatagt g
111111DNAArtificial SequenceA synthetic oligonucleotide
11catcaacaac a 11
* * * * *