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1
QUANTIFICATION OF 4-AMINOBIPHENYL-INDUCED GENOTOXICITY BY
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY:
EVALUATION OF CHEMOPREVENTIVE AGENTS FOR THE INHIBITION OF
BLADDER CARCINOGENESIS
by
Kristen L. Randall
to
The Department of Chemistry and Chemical Biology
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the field of
Chemistry
Northeastern University
Boston, Massachusetts
May 2011
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QUANTIFICATION OF 4-AMINOBIPHENYL-INDUCED GENOTOXICITY BY
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY:
EVALUATION OF CHEMOPREVENTIVE AGENTS FOR THE INHIBITION OF
BLADDER CARCINOGENESIS
by
Kristen L. Randall
ABSTRACT OF DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Chemistry
in the Graduate School of Arts and Sciences of
Northeastern University, May 2011
3
ABSTRACT
This dissertation demonstrates the significance of liquid chromatography-mass
spectrometry (LC-MS) methods to the development of targeted early intervention
chemopreventive strategies against bladder cancer. Here, LC-MS techniques facilitate a
quantitative analysis of DNA adducts of the human bladder carcinogen 4-aminobiphenyl
(4-ABP). These DNA adducts are a measure of exposure and risk, and chemopreventive
agents can mitigate this risk by inhibition of adduct formation or promotion of adduct
repair. Accordingly, DNA adducts are effective biomarkers of the efficacy of
chemopreventive agents.
Chapter 1 provides an overview of the metabolism of 4-ABP and methods for quantifying
its genetic damage. Chapter 2 reviews the application of LC-MS in the quantitative
analysis of DNA adducts for the specific evaluation of chemopreventive agents. Many of
the findings in this dissertation contribute to this new and exciting field.
The development of a validated LC-MS/MS method for quantification of 4-ABP-induced
genotoxicity was crucial for this research and is described in chapter 3. This method
overcomes analytical challenges that are often encountered in the quantitative analysis of
DNA adducts from human samples, including limited sample availability and the need to
reach detection limits approaching the part-per-billion threshold. By operating at nano-
flow rates and incorporating a capillary analytical column in addition to an automated
4
sample enrichment step, we have developed a sensitive HPLC-MS/MS method
appropriate for quantifying dG-C8-4-ABP. The Limit of Detection (LOD) of 5 adducts
in 109 nucleotides (20 attomol dG-C8-4-ABP and 1.25 µg DNA nucleosides on column)
and low sample requirement of 5 µg DNA per sample makes this method an
improvement from the current methods. Subsequent chapters describe our efforts to
expand this research by considering DNA adducts not only as biomarkers of exposure but
as significant to the discovery of chemopreventive agents.
In Chapter 4, we investigate the widespread chemopreventive strategy of activating the
cytoprotective protein nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a potential
mechanism for 4-ABP DNA adduct inhibition in bladder cells and tissues. We show that
Nrf2 inhibits 4-ABP DNA adduct formation locally in human bladder RT-4 cells.
However, in vivo research comparing 4-ABP DNA adduct levels in Nrf2+/+
and Nrf2-/-
C57BL/6 mice revealed that in the process of detoxifying 4-ABP and its metabolites from
the liver, Nrf2 increased the bioavailability of 4-ABP in the bladder. Subsequent
experiments showed that UDP-glucuronosyltransferase (UGT) was at least partly
responsible for the detoxification and transport of 4-ABP from the liver to the bladder:
Nrf2 up-regulated UGT, promoted conjugation of 4-ABP with glucuronic acid in the
liver, and increased urinary excretion of the conjugate. We concluded that Nrf2
activation that doesn’t result in up-regulation of liver UGT should be considered and
investigated further as a chemopreventive strategy against 4-ABP induced bladder
carcinogenesis.
5
In Chapter 5, two cancer chemopreventive agents Sulforaphane (SF) and 5,6-
Dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) were evaluated for their
inhibition of 4-ABP DNA adducts. SF and CPDT, both derived from compounds found
in cruciferous vegetables, are well known chemopreventive agents that induce Nrf2 and
have been shown to be specific for bladder tissue. We found that SF and CPDT both
activate Nrf2 and the Nrf2 cytoprotective signaling pathway in human bladder carcinoma
RT-4 cells and C57BL/6 mouse bladder tissue. Both agents inhibit 4-ABP DNA adduct
formation in bladder cells and tissues and require Nrf2 to exert substantial cytoprotective
effects. Furthermore, neither agent induced UGT liver enzymes that are highly
significant in the transport of 4-ABP to the bladder. These results suggest that SF and
CPDT are promising chemopreventive agents against 4-ABP-induced bladder
carcinogenesis and provide mechanistic insight into their cytoprotective effects.
Future directions of this research are considered in Chapter 6. This includes
quantification of the isomers of 4-ABP DNA adducts, a comprehensive human smoke-
quit study involving the quantitative monitoring of 4-ABP DNA adducts in urothelial
cells over an extended period of time, an analysis of additional metabolites of 4-ABP, and
the evaluation of structural analogs of SF and CPDT.
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ACKNOWLEDGEMENTS
As I complete my degree requirements, I owe many thanks to many people and most
importantly, to my research advisor Dr. Paul Vouros. Dr. Vouros accepted me into his
well respected lab providing me with tremendous learning opportunities. He has given
me invaluable guidance and advice in my research and I have learned so much from him.
He truly cares about his students and I am thankful for his continuous patience and
compassion.
Much of the research in this dissertation has been completed as part of a collaboration
between Dr. Vouros’s lab and Dr. Yuesheng Zhang’s lab at Roswell Park Cancer
Institute, Buffalo, NY. I have learned a great deal from the many conference calls we
have had discussing new data and manuscripts. Dr. Zhang’s expertise in
chemoprevention research has made this dissertation significantly more comprehensive.
Joe Paonessa and Yi Ding in Dr. Zhang’s lab have conscientiously prepared samples and
shipped them to me for further processing and analysis. They contributed significantly to
many of the positive and insightful findings described in this dissertation.
Special thanks is reserved for the members of my thesis committee: Dr. Penny Beuning,
Dr. David Forsyth, and Dr. Roger Giese.
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I’d like to express my sincere gratitude to Dr. Jim Glick who spent a lot of time training
me on the use and proper maintenance of the mass spectrometers in our lab and on
sample processing methods. Jim was always happy to help me move forward with my
research, whether that involved getting an instrument working or offering constructive
comments and advice.
I’d like to thank the very inspiring alumni of the Vouros research group including: Drs.
Christine Andrews, Dayana Argoti, Susan Bird, Terrence Black, Caroline Flarakos,
Jimmy Flarakos, Jim Glick, Dennis Szymanski, Tom Trainor, and John Williams. Thank
you also to Drs. Steve Coy, Roger Kautz, and Ravi Kc. Thank you to the current
members: Rose Gathungu, Adam Hall, Amol Kafle, Josh Klaene, Vaneet Sharma, and
Samantha Sokup.
I’d also like to acknowledge the Department of Chemistry and Chemical Biology at
Northeastern University. Thank you to Andrew Bean, Jean Harris, Felicia Hopkins,
Jeffrey Kesilman, Shari Khalil, Bill O’Neill, Rich Pumphrey, Cara Shockley, Nancy
Weston and Jana Wolf. Thank you to the faculty who taught me relevant and practical
chemistry in their courses. Thank you to Dr. Kevin Millea and Alex Henriksen who
helped the labs I was a T.A. for run smoother. Thank you to the graduate students and
postdocs who have been wonderful colleagues and friends.
8
From my days as an undergraduate student, I’d like to thank the Carleton College
Chemistry department for teaching me so much. I’d also especially like to thank Dr.
Stanley May and the Chemistry Department at the University of South Dakota for a very
enriching REU experience. Special thanks to Lisa Tilkens who was a wonderful mentor.
Finally, I would like to thank my family. Without the support of my parents Barbara and
Robert, to whom this dissertation is dedicated, the completion of this degree would not
have been possible. I am extremely grateful for their love and encouragement. I’d also
like to thank my best friend and sister Lauren and her boyfriend Scott, my brother Mark
and sister-in-law Eva, and my boyfriend Joe for their love, support, and understanding.
Uncle Paul and Aunt Ardys, thank you for your inspiration and fun times in Minnesota.
Aunt Mary, Uncle Jim, Aunt Sue, Uncle Pat, Stephanie, Jimmy, Danielle, Steve, Melissa
and Josh, thank you for the family get-togethers that have meant so much to me through
the years.
Thank you to everyone who has helped me along the way.
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to my mother and father, Barbara and Robert
10
TABLE OF CONTENTS
Abstract 2
Acknowledgements 6
Dedication 9
Table of Contents 10
List of Figures 14
List of Tables 19
List of Abbreviations 20
Chapter 1: DNA adducts of the human bladder carcinogen 4-
aminobiphenyl as biomarkers of exposure
26
1.1 Bladder cancer and its causes 27
1.2 Metabolism of 4-ABP 30
1.3 Formation of 4-ABP DNA adducts 36
1.4 4-ABP mutagenicity and link to bladder cancer 38
1.5 Quantification of 4-ABP DNA adducts 40
1.6 Significance of dose-response studies as representations of
real-life exposure
49
1.7 4-ABP DNA adducts in smokers versus non-smokers 50
1.8 Conclusions 51
11
Chapter 2: Quantification of bulky DNA adducts by liquid
chromatography-mass spectrometry for the evaluation of early
intervention chemopreventive agents
66
2.1 Early intervention chemoprevention 67
2.2 LC-MS/MS of modified nucleotides 71
2.3 Mass spectrometric detection of modified bases 75
2.4 Additional applications of LC-MS to chemopreventive
agent analysis
83
2.5 Conclusions 84
Chapter 3: An improved liquid chromatographty-tandem mass
spectrometry method for the quantification of 4-aminobiphenyl
DNA adducts in urinary bladder cells and tissues
91
3.1 Introduction 92
3.2 Project goals 97
3.3 Experimental 97
3.4 Results and Discussion 105
3.5 Conclusions 134
12
Chapter 4: Activation of Nrf2 as a chemopreventive strategy
against 4-ABP-induced bladder cancer
141
4.1 Introduction 142
4.2 Project goals 147
4.3 Experimental 147
4.4 Results 152
4.5 Discussion 166
4.6 Conclusions 171
Chapter 5: Inhibition of 4-ABP DNA adduct formation by
Sulforaphane and 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-
thione in bladder cells and tissues
178
5.1 Introduction 179
5.2 Project goals 187
5.3 Experimental 187
5.4 Results 191
5.5 Discussion 206
5.6 Conclusions 211
13
Chapter 6: Summary and Future Directions 215
6.1 Summary 216
6.2 Detection and quantification of dG-ABP isomers 217
6.3 Investigation into 4-ABP metabolism 220
6.4 Comprehensive human study 221
6.5 Evaluation of analogs of SF and CPDT 222
Biographical data 225
14
LIST OF FIGURES
Chapter 1
Figure 1.1 The chemical structures of arylamines associated
with human bladder cancer.
29
Figure 1.2 4-ABP and some of its metabolites. 31
Figure 1.3 Schematic of 4-ABP metabolism. 33
Figure 1.4 4-ABP DNA adducts. 37
Figure 1.5 A good correlation was observed in a comparison of
4-ABP DNA adduct levels between a GC/MS
analysis and an immunohistochemical method with
fluorescence detection.
48
Chapter 2
Figure 2.1 Selected phytochemicals that have been identified as
potential chemopreventive agents.
69
Figure 2.2 MS/MS spectrum of dG-C8-4-ABP showing the loss
of deoxyribose [M+H-116]+.
73
Figure 2.3 Three isomers from the B[a]P treated MCF-7 cells
were detected in an LC-MS/MS analysis.
74
Figure 2.4 Co-treatment of cells with B[a]P and chrysoeriol
reduced BPDE-DNA adduct levels.
75
Figure 2.5 LC-MS of aflatoxin-N7-guanine isolated from urine. 77
Figure 2.6 Calibration curve of AFB1 dose versus AFB1-N7-gua
detected in rat urine.
78
Figure 2.7 LC-MS detection of AFB1-N7-gua in rat urine of rats
treated with AFB1.
79
Figure 2.8. Chemical structures of Resveratrol, NAcCys,
melatonin, and lipoic acid.
81
15
Chapter 3
Figure 3.1 Typical offline SPE workflow. 93
Figure 3.2 A. Isolation of precursor ion, no fragmentation. B.
Fragmentation amplitude: 0.10 volt. C.
Fragmentation amplitude: 0.15 volt. D. MS3
435�319 �302.
106
Figure 3.3 The proposed fragmentations for dG-C8-4-ABP. 107
Figure 3.4 HPLC-MS of dG-C8-4-ABP standards. 108
Figure 3.5 Typical workflow in DNA adduct analysis by LC-
MS.
111
Figure 3.6 A detailed view of the microfluidic chip used in our
automated sample enrichment method.
113
Figure 3.7 A detailed view of the Agilent valve of the
microfluidic chip used in our automated sample
enrichment method.
114
Figure 3.8 Heat treatment stability assessment. 115
Figure 3.9 Internal standard degradation during digest. 117
Figure 3.10 Standard curve dG-C8-4-ABP of fmol analyte on
column versus the ratio of analyte to internal
standard peak areas were generated in triplicate over
an 8-month period.
119
Figure 3.11 Zoom of the lowest five points of the calibration
curve.
121
Figure 3.12 LOD and LOQ. Extracted ion chromatograms
(435�319) of the LOD, 20 amol on column in 1.25
µg DNA or approximately 5 adducts in 109
nucleosides, the LOQ, 70 amol on column in 1.25 µg
DNA or 2 adducts in 108 nucleosides, and the
procedure blank, 1.25 µg calf-thymus DNA.
122
16
Figure 3.13 Logarithmic scale plot of dG-C8-4-ABP levels
observed in dosed RT-4 human bladder carcinoma
cells.
125
Figure 3.14 Plot of 4-ABP dose versus detected dG-C8-4-ABP
quantity in bladder tissue from rats treated with 4-
ABP.
126
Figure 3.15 Zoom in of the EIC (435�319 for the analyte and
444�328 for the IS) from the lowest dose rat and
control samples.
127
Figure 3.16 Analysis of dG-C8-4-ABP adducts by digesting 1 µg
of DNA.
132
Chapter 4
Figure 4.1 Structure of 4-ABP-N-glucuronide. 145
Figure 4.2 Schematic of glucuronidation in the metabolic
pathways of 4-ABP induced human bladder cancer.
146
Figure 4.3 An example EIC in the time course study. 153
Figure 4.4 4-ABP-induced DNA damage in human bladder
carcinoma RT-4 cells in a dose duration study.
154
Figure 4.5 4-ABP-induced DNA damage in human bladder
carcinoma RT-4 cells in a dose concentration study.
155
Figure 4.6 A western blot of Nrf2 in whole cell lysates from
RT-4 cells treated with control siRNA and Nrf2
siRNA for 48 hours.
156
Figure 4.7 dG-C8-4-ABP DNA adduct levels in human bladder
carcinoma RT-4 cells.
157
Figure 4.8 dG-C8-4-ABP adduct levels in wild type and Nrf2
knockout mouse bladder tissue following treatment
with 50 mg/kg 4-ABP.
158
17
Figure 4.9 dG-C8-4-ABP adduct levels in wild type and Nrf2
knockout mouse liver tissue following treatment
with 50 mg/kg 4-ABP.
159
Figure 4.10 Western blots of liver homogenates from Nrf2+/+
mice and Nrf2-/-
mice.
160
Figure 4.11 S9 fractions were prepared from liver tissues of
Nrf2+/+
mice and Nrf2-/-
mice and measured for UGT
activity in catalyzing 4-ABP conjugation with
glucuronic acid.
161
Figure 4.12 Total amounts of 4-ABP-N-glucuronide were
measured in 24 h urine collected from Nrf2+/+
mice
and Nrf2-/-
mice given 50 mg/kg 4-ABP and were
adjusted by urinary creatinine levels.
162
Figure 4.13 Full scan of 1 ng 4-ABP-N-glucuronide synthetic
standard. A. Full scan. B. EIC 346.1 C. Mass
spectrum.
163
Figure 4.14 LC-MS/MS of 4-ABP-N-glucuronide standard. The
top panel shows the extraction ion chromatogram
m/z 346 �170 and the bottom panel shows the mass
spectrum under this peak.
164
Figure 4.15 Injection of mouse urine sample. 200 µL of urine
from two wild type mice that were administered 50
mg/kg 4-ABP was taken through an acetonitrile
precipitation and injected for detection of 4-ABP-N-
glucuronide.
165
Chapter 5
Figure 5.1 Chemical structures of SF and CPDT. 180
Figure 5.2 Conversion of glucoraphanin to SF or the SF nitrile. 181
Figure 5.3 SF and PEITC reduced AFB1 DNA adduct
formation in human hepatocyte cultures.
184
18
Figure 5.4 The structures of three dithiolethiones D3T,
Olitpraz, and ADT.
186
Figure 5.5 SF activates Nrf2 and the Nrf2 signaling pathway in
human bladder carcinoma RT-4 cells.
192
Figure 5.6 CPDT activates Nrf2 and the Nrf2 signaling
pathway in human bladder carcinoma RT-4 cells.
193
Figure 5.7 Wild-type C57BL/6 mice and Nrf2 knocked out
C57BL/6 mice were treated with vehicle or SF by
gavage once a day for 5 days.
194
Figure 5.8 Nrf2+/+
mice and Nrf2-/-
mice were treated with
CPDT or vehicle by gavage once daily for 5 days; 24
h later after the last CPDT dose, animals were killed,
and phase 2 enzymes in the bladders and livers were
measured by Western blotting.
196
Figure 5.9 4-ABP-induced DNA damage in human bladder
cells and the protective effect of SF.
198
Figure 5.10 4-ABP-induced DNA damage in human bladder
cells and the protective effect of SF in sprout form.
199
Figure 5.11 4-ABP-induced DNA damage in rat bladder cells
and the protective effect of SF in sprout form.
200
Figure 5.12 CPDT inhibits 4-ABP DNA adduct formation in
human bladder carcinoma RT-4 cells.
201
Figure 5.13 SF requires Nrf2 to inhibit 4-ABP DNA adduct
formation in mouse bladder tissue.
203
Figure 5.14 CPDT inhibits 4-ABP DNA adduct formation in
wild type mouse bladder tissue.
204
Figure 5.15 CPDT does not inhibit 4-ABP DNA adduct
formation in mouse liver tissue.
205
19
LIST OF TABLES
Chapter 3
Table 3.1 LC-MS methods for quantifying dG-ABP adducts. 96
Table 3.2 Dose–response data in human cells and rat tissue. 128
20
ABBREVIATIONS AND CONVENTIONS
°C Degree Centigrade
1,6-DNP 1,6-dinitropyrene
3H Tritium
4-ABP 4-aminobiphenyl
4-OHE2 4-hydroxyestradiol
ACN Acetonitrile
ADT 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione
AFB1 Aflatoxin B1
AFB1-N7-gua Aflatoxin B1-N
7-guanine
AFB2 Aflatoxin B2
amol Attomole
AMS Accelerator mass spectrometry
APCI Atmospheric pressure chemical ionization
ARE Antioxidant response element
ATP Adenosine triphosphate
B[a]P Benzo[a]pyrene
BBI Bowman-Birk Inhibitor
BBN N-butyl-N-(4-hydroxybutyl)nitrosamine
BPDE Benzo[a]pyrene diol epoxide
CE Capillary electrophoresis
21
CH3OH Methanol
CHL Chlorophyllin
CIA Chemiluminescence immunoassay
CID Collision induced dissociation
CNL Constant neutral loss
CPDT 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione
ct DNA Calf thymus DNA
CYP1A2 Cytochrome P450 1A2
CYP450 Cytochrome P450
D3T 3H-1,2-dithiole-3-thione
dA Deoxyadenosine
Da Dalton
dA-C8-4-ABP N-(deoxyadenosin-8-yl)-4-aminobiphenyl
dC Deoxycytosine
DELFIA Dissociation-enhanced lanthanide fluoroimmunoassay
dG Deoxyguanosine
dG-C8-4-ABP N-(deoxyguanosin-8-yl)-4-aminobiphenyl
dG-N2-4-ABP 3-(deoxyguanosin-N
2-yl)-4-aminobiphenyl
dG-N2-N
4-4-ABP N-(deoxyguanosin-N
2-yl)-4-aminobiphenyl
DMBA 7,12-dimethylbenz[a]anthracene
DMS Differential mobility spectrometry
22
DMSO Dimethyl sulfoxide
dN deoxynucleotide
DNA Deoxyribonucleic acid
DNaseI Deoxyribonuclease 1
E2-3,4-Q Estradiol-3,4-quinone
EDTA Ethylenediaminetetraacetic acid
EGRF-TK Epidermal growth factor receptor-tyrosine kinase
EIC Extracted ion chromatogram
ELISA Enzyme-linked immunosorbent assay
EROD Ethoxyresorufin-O-deethylase
ESI Electrospray ionization
ESP Epithiospecifier protein
fmol Femtomole
GADPH Glyceraldehyde 3-phosphate dehydrogenase
GC Gas chromatography
GCSc Catalytic subunit of glutamate cysteine ligase
GCSm Regulatory subunit of glutamate cysteine ligase
GSH Glutathione
GST Glutathione S-transferase
HPLC High performance liquid chromatography
i.p. Intraperitoneal injection
23
IARC International Agency for Research on Cancer
ID Internal diameter
IQ 2-amino-3-methylimidazo[4,5-f]quinoline
IS Internal standard
Keap1 Kelch-like ECH-associated protein 1
kg Kilogram
LC Liquid chromatography
LC-MS Liquid chromatography-mass spectrometry
LIF Laser-induced fluorescence
LIT Linear ion trap
LOD Limit of detection
LOQ Limit of quantification
m/z Mass to charge ratio
mg Milligram
mM Millimolar
mol Mole
MS Mass spectrometry
MS/MS Tandem mass spectrometry
N-acetylcysteine NAcCys
NADP Nicotinamide adenine dinucleotide phosphate
NAT1 N-acetyltransferase 1
24
NAT2 N-acetyltransferase 2
NCI National Cancer Institute
nESI Nanoelectrospray
N-OH-4-ABP N-hydroxy-4-ABP
N-OH-AABP N-hydroxy-4-acetyl-aminobiphenyl
NQO1 NAD(P)H:quinone oxidoreductase
Nrf2 NF-E2 related factor 2
NSAID Nonsteroidal anti-inflammatory drugs
PAH Polycyclic aromatic hydrocarbon
PBS Phosphate buffered saline
PEITC Phenylethyl isothiocyanate
PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
ppb Part per billion
QIT Quadrupole ion trap
QR NAD(P)H-quinone reductase
R2
Correlation coefficient
RF Radio frequency
RIA Radioimmunoassay
RNA Ribonucleic acid
ROS Reactive oxygen species
RP Reverse phase
25
RSD Relative standard deviation
RT Retention time
S/N Signal to noise ratio
SD Standard deviation
SERMS Selective estrogen receptor modulators
SF Sulforaphane
SIM Single ion monitoring
SPE Solid phase extraction
SRM Selective reaction monitoring
SULT Sulfotransferase
TLC Thin Layer Chromatography
UGT Uridine 5’-diphospho (UDP)-glucuronosyltransferase
UPLC Ultra performance liquid chromatography
UV Ultraviolet
v/v Volume to volume ratio
µg Microgram
µL Microliter
µM Micromolar
26
CHAPTER 1
DNA ADDUCTS OF THE HUMAN BLADDER CARCINOGEN
4-AMINOBIPHENYL AS BIOMARKERS OF EXPOSURE
27
1.1 Bladder cancer and its causes
Bladder cancer is a widespread health problem. The National Cancer Institute (NCI)
estimates that as one of the most common types of malignancies, the United States alone
saw 70,530 new cases and 14,680 deaths in 2010 (1). This prevalence, compounded by
the frequently prolonged treatments and high recurrence rate, makes it the most
expensive cancer to treat and monitor in the United States (2). Thus, there is a need for a
greater understanding of the causes of bladder cancer, its progression, and methods for
treatment and prevention.
Smoking is the primary cause of bladder cancer but other risk factors include age,
occupation, race, gender, certain infections, treatment with cyclophosphamide or arsenic,
family history, personal history, low fluid intake, and diets low in fruits and vegetables
and high in fat (1-7). Certain polyaromatic hydrocarbons and arylamines have been also
implicated in bladder cancer (8-11). For most individuals, the primary source of
exposure to arylamines is through cigarette smoke. Certain industries are also associated
with high levels of exposure to arylamines, including rubber, plastics, cable, and wood
manufacturing, but scientists and technicians who directly handle arylamines are at the
greatest risk for occupational exposure (12). Additional sources of arylamine exposure
include colorants, pesticides, fumes from heated oils and fuels, drugs, fugitive emissions,
and the environment as a result of the combustion of nitrogen containing organic material
(5, 11, 13-21).
28
Several arylamines have been assessed by the International Agency for Research on
Cancer (IARC) and categorized based on carcinogenicity. 4-aminobiphenyl (4-ABP), 2-
naphthylamine, and benzidine are known human bladder carcinogens and classified in
Group I as carcinogenic to humans (22-24). Other arylamines are strongly associated
with bladder cancer: O-toluidine is classified in Group 2A as probably carcinogenic to
humans and 2,6 dimethylaniline and 4,4’-methylenebis(2-chloroaniline) are classified in
Group 2B as possibly carcinogenic to humans (25). The structures of these arylamines
are shown in Figure 1.1.
Certain structural characteristics of an arylamine can be an indication of the extent of its
carcinogenicity. All arylamines contain at least one aromatic hydrocarbon and one
amine, but the positions of the amine substituents and the number of aromatic rings vary,
affecting hydrophobicity and electronic and steric properties. Arylamines that have more
amine substituents and that are more hydrophobic are more likely to be absorbed and
permeate biological membranes for faster distribution to the target tissues (26). The
placement of amine group in the ortho or para position of phenyl compounds and the β or
2 position of polycyclic aromatic compounds increases the chance that the arylamine will
be N-oxidized and activated (27). 4-ABP for example, which has an amine at the para
position is more carcinogenic than 3-ABP and 2-ABP (28, 29).
29
H2N
4-aminobiphenyl
H2N
2-naphthylamine
NH2H2N
benzidine
H2N
o-toluidine
H2N
2,6-dimethylaniline
H2N
Cl
NH2
Cl
4,4'-methylenebis(2-chloroaniline)
Figure 1.1. The chemical structures of arylamines associated with human bladder
cancer.
30
1.2 Metabolism of 4-ABP (17)
4-ABP is a procarcinogen and is not genotoxic and mutagenic until it is converted to an
active metabolite that will react with DNA. The resulting DNA adducts are recognized
as an initiating step in carcinogenesis (21). The structures of 4-ABP and some of its
metabolites are shown in Figure 1.2. The metabolism of 4-ABP can be described as a
series of activating and deactivating reactions, several of which are shown in Figure 1.3
(17). Activating reactions lead to the formation of the active metabolite and ultimate
carcinogen while detoxifying and deactivating reactions lead to the excretion of un-
reactive metabolites of 4-ABP.
4-ABP is activated to N-hydroxy-4-ABP (N-OH-4-ABP) primarily in the liver. The
hepatic enzymes that catalyze this reaction are cytochrome P450 1A2 (CYP1A2) in rats
and humans (30) and another unidentified form of cytochrome P450 (CYP450) in mice
(31, 32). Following activation, N-OH-4-ABP then either remains in the liver or is
transported through the blood to other organs. N-OH-4-ABP however is also known to
form in the bladder from 4-acetylaminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, and
acetylCoA (33-36). N-OH-4-ABP can then undergo further activation via Phase 2
conjugation reactions such as sulfation, acetylation or glucuronidation. Sulfation of N-
OH-4-ABP is catalyzed by hepatic sulfotransferases (SULT) enzymes and is associated
with liver DNA adducts. In the bladder, N-acetyltransferase 1 (NAT1) catalyzes the O-
31
N H 2
4 - a m i n o b i p h e n y l
N H
O
4 - a c e t y l a m i n o b i p h e n y l
N H
O H N - h y d r o x y - 4 - a m i n o b i p h e n y l
N
O
O H N - h y d r o x y - 4 - a c e t y l- a m i n o b i p h e n y l
N H
O
O
N - a c et o x y - 4 - a m i n o b i p h e n y l
N
O
O
O
N - a c e t o x y - 4 - a c e t y l a m i n o b i p h e n y l
H O
O H
O H
O
O
O H H N
1 - ( [ 1 , 1 - B i p h en y l ] - 4 - y l a m i n o )- 1 - d e o x y - g l u c o p y r a n u r o n i c A c i d
N H +
B i p h e n y l - 4 - y l- a m m o n i u m
N H
O S
O
O
O H
N - s u l f o x y - 4 - a m i n o b i p h e n y l
Figure 1.2. 4-ABP and some of its metabolites.
4-aminobiphenyl nitrenium ion
32
or N-acetylation of N-OH-4-ABP to form N-acetyoxy-4-aminobiphenyl and N-hydroxy-
4-acetyl-aminobiphenyl (N-OH-AABP). N-OH-AABP and N-acetoxy-4-
acetylaminobiphenyl can be then converted to N-acetyoxy-4-aminobiphenyl by an N,O-
acetyltransferase (33, 37). Under acidic conditions the acetyl group of N-acetyoxy-4-
aminobiphenyl will be oxidized to form the nitrenium ion, the reactive electrophilic ion
that ultimately adducts to DNA.
These 4-ABP activation reactions are in constant competition with detoxification
reactions. Early detoxification reactions of 4-ABP in the liver, such as ring
hydroxylation, N-acetylation, and N-glucuronidation involve formation of metabolites
that can not be directly converted to N-OH-4-ABP by the activating CYP450 enzymes.
Although N-acetyl-4-ABP metabolites can be oxidized to N-OH-AABP and metabolized
to an O-glucuronide which is transported to the bladder, this is not acid labile and is
excreted. Thus, these 4-ABP detoxification reactions compete with the activating N-
oxidation of 4-ABP. If N-OH-4-ABP is formed in the liver there are deactivation
reactions that can occur and these include Phase 2 reactions such as acetylation,
glucuronidation, sulfation, and glutathione addition. These conjugates can be transported
from the liver to the bladder for excretion. However, many of the resulting metabolites
may be converted back to the active metabolite or ultimate carcinogen before excretion
occurs. For example, N-glucuronides of 4-ABP that are transported to the bladder are
acid labile and can result in the formation of DNA adducts (36).
33
Fi gure 1.3. Schematic of 4-ABP metabolism. (Reproduced from (32))
NH+
34
Differences in response to 4-ABP exposure explain variations in cancer risk. While the
target organ in humans, rabbits and dogs is the bladder, 4-ABP targets the liver and
bladder in mice, and mammary glands and intestine in rats (12, 38-40). Differences in 4-
ABP metabolism however, are apparent not only among species but also among
individuals within a species. This is due to a number of factors that affect the activation
and deactivation of 4-ABP including genetic polymorphisms, variations in conjugation
reactions, urinary pH, and urothelial cell proliferation.
Rate of 4-ABP acetylation has a large impact on whether or not the procarcinogen will be
activated and is determined by genetic polymorphism. Humans either have the
phenotype of a rapid or slow acetylator, and this is referring specifically to the activity of
N-acetyltransferase-2 (NAT2). NAT2 is expressed primarily in the liver and intestinal
epithelium (41) and catalyzes the formation of 4-acetylaminobiphenyl from 4-ABP
before it can undergo CYP450 catalyzed oxidation to N-OH-4-ABP. In slow acetylators,
4-ABP remains in the liver for a relatively long time, increasing the possibility of the
formation of the active metabolite. Indeed, slow acetylators have been identified as being
more susceptible to bladder cancer than rapid acetylators because they are exposed to
more active carcinogens in the bladder (42, 43). Genetic polymorphisms of NAT1,
another acetylator but one which is primarily expressed in the bladder and catalyzes 4-
ABP detoxification reactions, have not been identified as having significant effects on
bladder cancer development.
35
Variations in the activities of Phase 2 enzymes also affect the outcome of 4-ABP
exposure. These enzymes catalyze competing reactions where inhibition of one type of
conjugation reaction may result in enhancement of the others, and vice-versa (44). Phase
2 metabolism of N-OH-4-ABP is typically thought of as a detoxification but the resulting
conjugates may be oxidized to form the nitrenium ion that adducts to DNA and some are
more susceptible to this than others. Additionally, certain Phase 2 reactions are
associated with organ-specific tumors. Sulfation of N-OH-4-ABP for example, is linked
to liver cancer. Human sulfotransferases will catalyze the formation of DNA adducts
from N-OH-4-ABP in the liver, possibly even reducing adduct formation in the bladder
(45). Glucuronidation of N-OH-4-ABP is associated with bladder cancer (46, 47). In the
liver, uridine 5’-diphospho (UDP)-glucuronosyltransferase (UGT) catalyzes the
formation of a 4-ABP N-glucuronide conjugate, a metabolite that is more water soluble
than 4-ABP. It is transported from the liver to the urinary bladder where it is either
excreted or at low pH, dissociates to form the 4-ABP nitrenium ion capable of adducting
to DNA. Hence, competing activities of Phase 2 enzymes can influence whether or not
4-ABP will be excreted or genotoxic, and if genotoxic, which tissues are affected.
If 4-ABP metabolites are transported to the bladder, urinary pH, mostly a consequence of
diet, affects the dissociation of 4-ABP esterified compounds and whether or not the
nitrenium ion will form. If other bladder carcinogens are present, there may be a
synergistic response resulting in a greater risk of bladder cancer development.
Furthermore, exposure to these carcinogens may be even more damaging if cells are
36
already proliferating. Risk of developing bladder cancer therefore, is not only dependent
on exposure but also on a number of modulating factors specific to an individual.
1.3 Formation of 4-ABP DNA adducts
The 4-ABP nitrenium ion is electrophilic and covalently binds to nucleophilic sites on
DNA. More than 70% of these 4-ABP DNA adducts are located at the C8 position of
guanine to produce N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) (48-55),
but other adducts have been identified and characterized: N-(deoxyadenosin-8-yl)-4-
aminobiphenyl (dA-C8-4-ABP) and a hydrazo linked adduct, N-(deoxyguanosin-N2-yl)-
4-aminobiphenyl (dG-N2-N
4-4-ABP) were observed in hepatic cells that were treated with
4-ABP (56) and 3-(deoxyguanosin-N2-yl)-4-aminobiphenyl (dG-N
2-4-ABP) was
observed in human uroepithelial cells treated with N-OH-4-ABP (57). The structures of
these 4-ABP DNA adducts are shown in Figure 1.4.
37
Fig
ure
1.4
. 4
-AB
P D
NA
ad
du
cts.
NH
OH
OHO
N
N
H2N
N
N
N-(
deo
xyaden
osi
n-8
-yl)
-4-a
min
obip
hen
yl
NH
OH
OH
ON
N
O
H N
NH
2
N
N-(
deo
xy
gu
ano
sin
e-8
-yl)
-4-a
min
ob
iphen
yl
H2N
HO
HO
O
N
NO N
H
HN
N
3-(
deo
xy
gu
an
osi
n-N
2-y
l)-4
-am
ino
bip
heny
l
N H
OH
OH
O
N
N
O
HN
H N
N
N-(
deo
xyguanosi
n-N
2-y
l)-4
-am
inobip
henyl
38
1.4 4-ABP mutagenicity and link to bladder cancer
If these 4-ABP-DNA adducts are not repaired, mutations can occur that ultimately may
lead to bladder cancer (58-65). The genetic events of carcinogenesis have been classified
into the three distinct phases of initiation, promotion and progression, although this
process may not actually transpire in such a well defined sequence. The initiation phase
is usually associated with carcinogen binding and damage to DNA. We are constantly
exposed to carcinogens and DNA adducts are constantly forming but the majority of
DNA damage is repaired. However, over time genetic mutations accumulate and the cell
enters the promotion phase. Cell proliferation begins and the cancer advances to the
progression phase. While 4-ABP DNA adducts form within hours of exposure, 4-ABP-
induced mutations arise within two weeks of exposure in vivo and urinary bladder tumors
take years to develop (66-68).
Some researchers have focused on the effect of 4-ABP on the highly significant tumor
suppressor gene p53. 4-ABP has been shown to induce p53 mutations in the bladder (20,
69-72) and several studies have found a link between 4-ABP induced p53 mutations and
bladder tumor grade and invasiveness (73). Additionally, common codons of mutational
hotspots in the p53 gene were observed in human bladder cancer and cell cultures treated
with 4-ABP (74). For example, codon 285 on the p53 gene is a mutational hot spot in
human bladder cancer and also the preferential binding site for 4-OH-ABP (75).
39
However, the relationship between 4-ABP exposure, the resulting genetic damage, and
bladder cancer risk is not completely understood and results have been conflicting (12).
In some cases, a clear relationship was identified between 4-ABP adduct levels and
tumorogenesis in bladder tissue (19, 76, 77) and several studies have shown that after
cases were adjusted for exposures, higher levels of DNA adducts were observed in cancer
patients than non-cancer patients (78-82). However, in another study adduct levels were
not correlated with tumor grade (83). These researchers hypothesized that the time
between the adduct formation and the initiation of tumors may account for these differing
results; the quantified adduct levels might reflect the average 4-ABP exposure at the
moment the measurement is made while the tumor grade reflects the accumulation of
genetic damage (83).
40
1.5 Quantification of 4-ABP-DNA adducts (84)
4-ABP DNA adducts are important biomarkers in 4-ABP-induced bladder cancer.
Quantitative relationships can be evaluated between mechanisms of carcinogenesis
ranging from initiation to promotion to progression, but the measurement of DNA
adducts has several advantages over others as a gauge of exposure and risk. For example,
because DNA adduct formation is the initiating step in carcinogenesis, their
quantification is an early detection measurement that can be made within hours of
exposure. Other measurements of later stages of carcinogenesis are not early detection
measurements and might only be detectable weeks or years after the initiation stage (85,
86). Another advantage of 4-ABP DNA adduct quantification is that the source of the
DNA damage is inherent in the measurement, while in DNA mutagenesis studies or
tumor grade analyses the cause may not be as clear. Indirect measurements of 4-ABP
exposure can only serve as estimates of potential DNA damage but are often easier to
make because of the relative high concentrations of the analyte. Such measurements
include the quantification of 4-ABP in cigarette smoke or the quantification of 4-ABP
hemoglobin adduct levels. Direct measurements of 4-ABP DNA adducts account for
inconsistencies in adsorption, metabolism, detoxification, and DNA repair and are
consequently more relevant to assessing the effect of exposure on bladder cancer (87).
Accordingly, DNA adducts serve as both biomarkers of exposure and susceptibility to
cancer and for that reason their measurement has significant implications for disease risk
assessment (88).
41
DNA adducts also have a specific niche as biomarkers of the efficacy of
chemopreventive and chemotherapeutic agents. As a measurement of genotoxic risk and
present in all individuals in at least background levels, DNA adducts can be used to
identify agents that decrease this risk by inhibition of their formation or promotion of
their repair. Chemoprevention studies are aimed at preventing carcinogenesis and require
biomarkers of cancer initiation rather than progression and DNA adducts meet this
criterion (40). Chemotherapeutic agents are evaluated based on their efficacy in
decreasing malignancy (85) and as DNA adducts have been detected in tumor tissue, they
can also serve as biomarkers of therapeutic intervention in later stages of carcinogenesis.
Several techniques appropriate for the quantification of 4-ABP DNA adducts are
discussed below (84). Important factors to consider include sensitivity, selectivity, and
amount of DNA required. In the analysis of DNA adducts from human samples, adduct
levels are expected to be in the range of 1 adduct in 108 nucleosides. Furthermore, these
adducts will likely be present in complex mixtures and typically only µg quantities of
DNA are available. As an example of one human study the GC-MS analysis of bladder
cancer biopsies had a limit of detection of 0.1 fmol of 4-ABP DNA adduct per ug DNA
and DNA adducts were detected in 37 out of 75 patients. The average DNA adduct level
was 2.7 ± 0.7 fmol/ug DNA adducts (86 ± 22 adducts in 108 nucleosides) (76). These
adduct quantities have been observed in a number of studies employing a wide array of
42
distinct analytical methodologies that include 32
P-postlabeling, immunoassays,
fluorimetry, and mass spectrometry (89).
1.5.1 32
P-postlabeling (90)
32P-postlabeling involves radiolabeling adducted nucleosides and measuring the
32P-
decay. In this method, DNA is enzymatically digested to 3’ mononucleotides and
adducted nucleotides are isolated and radiolabeled at the 5’-position with [γ-32
P]ATP by
polynucleotide kinase-mediated phosphorylation. The resulting [5’-32
P]-3’-biphosphates
are chromatographically separated and 32
P-decay is measured.
32P-postlabeling can be combined with High Performance Liquid Chromatography
(HPLC) or Thin Layer Chromatography (TLC) for greater specificity. These methods
also have high sensitivity, requiring only 10 µg DNA or less for the analysis of samples
containing only 1 adduct in 1010
nucelotides. The technique is particularly useful when
analyzing adducted nucleosides in complex mixtures.
However, 32
P-postlabeling requires working with radioactive material and does not
provide structural information. In addition, the technique generally requires extensive
sample preparation. Errors can occur in quantification: false negatives reflect the loss of
adducts during sample preparation, incomplete digestion, and inefficiency of labeling and
false-positive results result from polynucleotide kinase labeling of non-nucleic acid
43
components and contamination of DNA with RNA. Improvements in sensitivity are
limited because the technique is dependent on the specific activity of [32
P]-ATP.
1.5.2 Immunoassays (91)
In immunoassay based DNA adduct quantification, antibodies react with specific DNA
adducts and are subsequently detected by radiometry, fluorometry, coulorometry, or
chemiluminesence (92-94). There are a large variety of immunoassay techniques for the
analysis of DNA adducts each with their own advantages and drawbacks.
Immunohistochemical techniques for example can be used to detect adducts in specific
cell types in small amounts of tissue, either frozen or paraffin preserved. However, these
assays have low sensitivity and quantification is relative. Absolute quantification is
possible using immuno-dot/slot-blot methods, which involve detection of enzyme-labeled
secondary antiserium by colorimetric or chemoluminescent techniques. However,
although less than a µg of DNA per sample is analyzed, the assay is not sensitive enough
for the analysis of human samples. Radioimmunoassays (RIAs) are sensitive and
reproducible but have been largely replaced by the enzyme-linked immunosorbent assay
(ELISA), which doesn’t require the use of handling radioactive material and is
considered to be sensitive enough for the analysis of human samples. Dissociation-
enhanced lanthanide fluoroimmunoassay (DELFIA) uses the same antisera as ELISA and
has greater sensitivity. The chemiluminescence immunoassay (CIA) is even more
sensitive than DELFIA and is almost as sensitive as 32
P-postlabelling. Furthermore, there
is a lower background, higher signal-to-noise, and standard curves are more reproducible.
44
In general, immunoassays have the advantage of being high-throughput, rapid,
straightforward, reproducible and highly sensitive.
However, immunoassays have a number of drawbacks. These techniques require raising
specific antibodies from rabbits or mice, characterizing the antiserum, and validating the
assay. They rely on antibodies to react specifically with certain DNA adducts, but in
practice these antibodies cross-react with different structurally similar DNA adducts and
are therefore considered to be class specific rather than adduct specific. Furthermore,
those antibodies that react with DNA adducts also react with the corresponding RNA
adducts.
Monoclonal antibodies and rabbit sera have already been developed for 4-ABP DNA
adducts (95). The monoclonal antibody 3D6, for example is specific for dG-C8-4-ABP
(96) and has been used as an enrichment step before LC-MS/MS analysis of human
bladder samples (97).
1.5.3 Fluorescence spectroscopy
Fluorescence assays have been developed that either exploit the fluorescent
characteristics of some adducts or attach a fluorescent label to non-fluorescent adducts.
HPLC combined with fluorescence detection allows for the detection of stereoisomers.
They are fast and inexpensive, and although they require large quantities of DNA (about
100 to 1000 µg), the method has a detection limit of about 1 adduct in 108 nucleotides. In
45
synchronous fluorescence spectrophotometry, excitation and emission wavelengths are
scanned at a fixed wavelength difference. Theoretically, this technique can be applied to
resolve multiple components in mixtures without separation. For the detection of non-
fluorescent adducts, capillary electrophoresis and laser-induced fluorescence (CE-LIF)
can be an ultrasensitive assay. Fluorescent dyes can also be chemically linked to DNA
adducts and then separated and detected by CE-LIF. This approach achieved a detection
limit of 2 adducts in 106 nucleotides for PAHs. Electrostacking further improved the
detection limit to about 1 adduct in 107 nucleotides. Hydroxyl-specific fluorescence
labeling of dG-ABP has been achieved with BODIPY (98).
1.5.4 Mass spectrometry
The main advantage of mass spectrometry in DNA adduct quantification is that in
addition to the quantitative determination of adduct levels, it provides structural
characterization of the analyte. This is useful for confirmation that the correct compound
is being analyzed, identification of unknown compounds, and structural characterization
of standards. These standards may then be used to determine adduct levels by mass
spectrometry or other methods. Mass spectrometry methods can also have high
sensitivity and low sample requirements (99). Furthermore, this technology is
continually improving in sensitivity due to advances in efficiency of ionization,
transmission and detection of ions. Accurate quantification of DNA adduct levels is
achieved with stable isotope internal standards which account for losses during sample
preparation and variations in response of the mass spectrometer, including ion
46
suppression. Typical detection limits are at about 1 adduct in 108 nucleotides using about
50 µg of DNA. Gas chromatography and liquid chromatography are separation methods
commonly performed before mass spectrometric analysis. The use of these methods
decreases interferences in the mass spectrometric analysis of the analyte and also
provides an important characteristic for identification of the analyte, retention time.
GC-MS requires DNA adducts to be volatile and sample preparation therefore involves
de-glycosylation and derivatization. Because de-glycosylation is required however, RNA
interferences can be a problem. Chemical derivatization methods, such as silylation and
electrophore labeling introduce problems such as the production of artifacts, interferences
and sample to sample variability in derivatization (100).
In LC-MS analysis, DNA adducts can be analyzed in their nucleoside, nucleotide, and
oligonucleoide forms. LC however, usually has lower peak resolution than GC. In LC
analysis, the sample must be only soluble in the mobile phase before injection, rather than
be thermally labile as in GC-MS analysis. Mass spectrometric analysis requires that the
analyte is in the gas phase, and two ionization methods that are commonly used are ESI
and APCI (99). In the analysis of bulky DNA adducts such as 4-ABP, reversed phase
separation is often employed. If this is too hydrophilic, ion-pairing methods may used
but this introduces ion suppression effects in electrospray ionization, decreasing overall
sensitivity.
47
1.5.4.a. Accelerator mass spectrometry (AMS) (101)
AMS measures isotopes with a low natural abundance and a long half-life and is the most
sensitive analytical method for detecting DNA adducts. Measurements are precise and
detection limits can be as low as 1 adduct in 1012
nucleotides. However, cross-
contamination of isotopes between samples is a huge problem. In addition, the technique
may not differentiate between different metabolites of the same labeled carcinogen so
sample preparation must isolate the analyte from possible interferences.
1.5.5 Summary of techniques for the quantification of 4-ABP DNA adducts
Sensitivity, selectivity, and the amount of DNA required for an analysis are significant
parameters in a method for the quantitative analysis of DNA adducts; the most sensitive
technique is AMS, the most selective methods are mass spectrometry based, and the
method requiring the lowest quantity of DNA for an analysis is usually 32
P-postlabeling.
One study compared absolute 4-ABP DNA adduct levels detected between ESI-LC-MS,
32P-postlabeling, DELFIA, and specific binding of
3H-radiolabeling. In this research,
32P-postlabeling underestimated adduct levels (3-5% relative level), DELFIA
overestimated adduct levels (260-550% relative level) and ESI-LC-MS gave levels of
adducts similar to those obtained in the specific binding of the 3H-radiolabel assay (102).
Furthermore, they found that LC-MS was the most precise method.
48
However, it can be difficult to compare methods based on the absolute number of adducts
detected because often the recovery of the adduct during sample preparation is unknown
and the accuracy in quantification can not always be determined. One study was
designed to compare immunohistochemistry-fluorescence and GC-MS methods for
detection of 4-ABP DNA adducts in liver tissue of 4-ABP treated mice. A good
correlation was observed and is shown in Figure 1.5. Rather than comparing absolute
adduct levels, it focused on relative increases in fluorescence intensity or adduct levels
within each method (103).
Figure 1.5. A good correlation was observed in a comparison of 4-ABP DNA adduct
levels between a GC/MS analysis and an immunohistochemical method with
fluorescence detection. (Reproduced from (103))
49
1.6 Significance of dose-response studies as representations of real-life exposure
(104, 105)
Cell and animal dose response studies are useful models for carcinogen-induced
genotoxicity and tumor formation in humans. In DNA adduct dose response studies,
cells or animals are treated with a carcinogen and the resulting formation of DNA adducts
is measured. All of the pathways involved in the formation and repair of DNA adducts
can be saturated. If no pathway is saturated, a linear dose-response relationship is
expected. However, if activation is saturated, a supralinear response occurs and if
detoxication or DNA repair is saturated, a sublinear response occurs. Exposure in
humans generally occurs chronically and at low doses reaching steady-state
concentrations. Therefore, a linear relationship is observed between carcinogen exposure
and DNA adduct formation (106). Steady-state levels can also be reached under
experimental conditions if exposure continues at low levels for several weeks allowing
adducts to accumulate (39, 107-110).
The synergistic effect of exposure to multiple carcinogens can result in greater damage in
real-life exposure than in experimental models at the same exposure levels. For example,
other chemicals in cigarette smoke increase the genotoxicity of 4-ABP; reactive oxygen
species (ROS) or free radicals hydroxylate DNA bases, causing single strand breaks or
oxidative DNA damage. Furthermore, fifty of the approximately 4000 hazardous
chemicals in cigarette smoke have been directly implicated in cancer (IARC) (111).
50
Increased cell proliferation caused by the many chemicals in cigarette smoke provides
more DNA on which 4-ABP can cause damage, potentiating its genotoxicity.
1.7 4-ABP DNA adducts in smokers versus non-smokers
Although several studies have not established a relationship between detected DNA
adducts and the formation of tumors, many have found that adduct levels are higher in
smokers than nonsmokers (49, 76, 112-117). Exposure to cigarette smoke is the main
source of 4-ABP and the main cause of bladder cancer (118-120). Cigarette smoke is
also known to contribute to many cancers including lung, larynx, pharynx, esophagus,
bladder, uterine, kidney, cervical and pancreatic cancers (120, 121). Mainstream
cigarette smoke, composed of both inhaled and exhaled smoke, contains 2.4 to 4.6 ng 4-
ABP per filtered cigarette and 0.2 to 23 ng per filtered cigarette and sidestream smoke
emitted directly from the end of a lit cigarette, contains up to 140 ng 4-ABP per cigarette
(122).
51
1.8 Conclusions
4-ABP is implicated in bladder cancer, a serious health problem. Its DNA adducts reflect
exposure and tumor development and can be used as a biomarker for risk assessment and
for the evaluation of chemopreventive agents. LC-MS/MS is one of the most sensitive
and specific techniques available for the measurement of DNA adducts. In the next
chapter, LC-MS techniques in chemoprevention research involving DNA adducts will be
discussed. This is followed by a record of our own research developing a highly sensitive
LC-MS/MS method for 4-ABP DNA adduct formation, the evaluation of a
chemopreventive strategy for inhibition of DNA adduct formation, and the evaluation of
two chemopreventive agents against 4-ABP-induced bladder cancer.
52
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66
CHAPTER 2
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRIC ANALYSIS OF
BULKY DNA ADDUCTS FOR THE EVALUATION OF EARLY
INTERVENTION CHEMOPREVENTIVE AGENTS
67
2.1 Early intervention chemoprevention
The significance of cancer prevention research is well recognized and this is manifested
in its high priority level in research funding by the National Cancer Institute (NCI) (1).
Although many studies are focused on the later stages of carcinogenesis for slowing,
preventing, or reversing tumor progression (2), measures taken before cancer initiates
may actually have the greatest potential for reducing cancer incidences and costs (3).
While minimizing exposure to known carcinogens is one effective approach to
decreasing the likelihood of cancer initiation, carcinogens are ubiquitous and impossible
to completely avoid (4-6). Accordingly, an additional precaution against cancer initiation
may involve administration of a chemopreventive agent to inhibit genetic damage caused
by carcinogens in people at high risk for tumor development.
An ideal chemopreventive agent is inexpensive, effective, easily and infrequently
administered and has minimal side effects (7, 8). This is especially important in primary
prevention in which subjects have no symptoms of cancer development. Agents can be
either natural or synthetic but those that are in foods consumed by people are often
desired because it is already known that they are safe at least at the levels at which they
are typically consumed. Fruits and vegetables contain many potential chemopreventive
agents and in fact the International Agency for Research on Cancer (IARC) has
associated their consumption with reduced risk of cancer (9). Furthermore, researchers
have identified specific compounds in these foods that are responsible for their anti-
68
carcinogenic properties. Many anticancer studies are focusing on non-traditional
nutrients like phytochemicals and other bioactive plant chemicals that have potential
chemopreventive activity, such as those shown in Figure 2.1 (10). Plant derived agents
being investigated for their chemopreventive potential include the Bowman-Birk
Inhibitor (BBI), carotenoids, curcumin, indoles, isothiocyanates, tea polyphenols,
resveretrol, retinoids, soy isoflavones, selenium compounds, sulfur containing
antioxidants, and vitamin D. Other agents being investigated include anti-androgens,
epidermal growth factor receptor-tyrosine kinase (EGRF-TK) inhibitors, NO-releasing
NSAIDS, NSAIDS, ornithine decarboxylase, PPAR gamma agonists, p53 suppressors or
gene modulators, resquimod, rexanoids, selective estrogen receptor modulators
(SERMS), and statins (11).
The analysis of carcinogen-DNA adduct biomarkers is one approach that has been taken
for measuring the efficacy of early therapeutic intervention. The formation of DNA
adducts is recognized as an initiating step in the development of carcinogenesis and
therefore mitigation of adduct quantities by a chemopreventive agent may demonstrate its
potential to reduce risk of cancer development. Adducts present in tissue from healthy
individuals may indicate risk of cancer development and need for chemopreventive
intervention. The biological relevance to DNA adducts in chemoprevention has been
reviewed (12, 13).
69
Figure 2.1. Selected phytochemicals that have been identified as potential
chemopreventive agents. (Reproduced from (14))
70
Many of these agents exert their protective effects through multiple mechanisms which
can be elucidated through various biochemical techniques combined with DNA adduct
quantification. These biochemical techniques include knocking out key genes, analyzing
multiple analogs of a class of chemopreventive agents to determine the important
structural features, or exposing samples to several carcinogen metabolites to determine
the stage at which the chemopreventive agent acts. Mechanisms by which
chemopreventive agents can decrease DNA adduct levels include detoxification of the
carcinogen, eliciting antioxidant activity or electrophile scavenging activity, inducing
Phase 2 carcinogen-detoxification enzymes, inhibiting Phase 1 carcinogen-activating
enzymes, enhancing DNA repair systems, or inducing cell apoptosis (15, 16).
Understanding these mechanisms is useful in the discovery of other chemopreventive
agents and in drug modification and development.
Sensitivity, selectivity, and low sample requirement are important parameters in the
quantification of DNA adducts. Many preliminary studies on chemoprevention of DNA
adducts incorporate high doses of carcinogens and chemopreventive agents. While these
studies are meaningful and may reveal important characteristics about chemopreventive
agents, those that involve low amounts of chemical treatments resembling typical human
exposure are most relevant and are often the best indicators of an agent’s
chemopreventive capacity. Several studies have shown that the degree of protection by a
chemopreventive agent is dependent on the carcinogen dose and that often greater
protection is observed at lower carcinogen doses (17). Furthermore, in the analysis of
71
human samples, limited sample may be available, necessitating not only high sensitivity
but also a low sample requirement. Widely used techniques for the analysis of DNA
adducts include 32
P-postlabeling, immunoassays, fluorimetry, accelerator mass
spectrometry and mass spectrometry and comparisons between them is the focus of many
reviews (12, 18-24).
2.2. LC-MS/MS of modified nucleotides
LC-MS/MS quantification of DNA adduct formation is generally achieved by detection
of the adducted monomeric nucleoside. In this type of analysis, DNA is enzymatically
digested to mononucleosides and the adducted nucleosides are enriched, often through
solid phase extraction (SPE). Quantification of adduct levels is accomplished through the
addition of a stable isotope internal standard prior to enzymatic digestion and the
subsequent analysis of a standard curve. Samples prepared in the absence of
chemopreventive agents are used as a positive control and percent inhibition of a
particular chemopreventive agent can be calculated based on the adduct levels of these
samples. The analyte is identified by retention time and MS/MS loss of deoxyribose
[M+H-116]+ (25).
Tandem mass spectrometry provides increased specificity and due to the universal
MS/MS behavior of the nucleosides’ loss of the deoxyribose group, it is well suited for
72
their analysis. MS/MS quantification of these monomeric nucleosides is usually
performed on a triple quadrupole because of these instruments’ large dynamic range, high
duty cycle, sensitivity, precision, and accuracy, especially with multiple analytes.
Selective reaction monitoring (SRM) is typically used for quantification. In this case, the
protonated adduct [M+H]+ is selectively transmitted through the first analyzer Q1,
fragmented in Q2 by collision-induced dissociation (CID), and the fragment exhibiting
the loss of the deoxyribose, [M+H-116]+ is selectively transmitted in Q3.
An example mass spectrum of the MS/MS analysis of dG-C8-4-ABP is shown in Figure
2.2. Here, the loss of the deoxyribose group from the molecular ion m/z 435.1 results in
the transmission of the aglycone ion m/z 319.1. Higher collision-energies can further
fragment the aglycone ion to obtain structural information. Constant neutral loss (CNL)
can be used to screen a complex sample for an overall study of the adducts present.
However due to the slow scanning rate of the triple quadrupole, approximately 100-fold
lower sensitivity is observed in a CNL analysis than in an SRM analysis and lower
abundance adducts are less likely to be detected. For this same reason, lower sensitivity
is also observed in product ion scans.
73
Figure 2.2. MS/MS spectrum of dG-C8-4-ABP showing the loss of deoxyribose [M+H-
116]+. (Reproduced from (26))
A sensitive LC-MS/MS method based on the characteristic loss of deoxyribose was
developed for detection of benzo[a]pyrene (B[a]P) DNA adduct formation and this was
applied to the evaluation of methoxy flavone chrysoeriol (3’-methoxy-4’,5,7-
trihydroxyflavone) as an inhibitor of these adducts. In this analysis, three isomers from
the B[a]P treated MCF-7 cells were detected and are shown in the chromatogram in
Figure 2.3 as one major peak and two smaller peaks 1 and 2. The major peak was
identified as (+)-trans-BPDE-N2-dG. This was known because it was the standard used
in the study. The other smaller peaks were assumed to be (+)-anti-cis-BPDE-N2-dG
(peak 1) and syn-BPDE-N2-dG (peak 2). The retention times of the three isomers
74
differed, but all showed the characteristic loss of m/z 116 (m/z 570�454). Interestingly,
co-treatment of cells with 2 µM B[a]P and 5 or 10 µM chrysoeriol inhibited (+)-trans-
BPDE-N2-dG and (+)-anti-cis-BPDE-N
2-dG formation but not syn-BPDE-N
2-dG
formation (Figure 2.4). The dominant adduct formed, (+)-trans-BPDE-N2-dG is
considered to be the most mutagenic and was the adduct most inhibited by chyrsoeriol
(27). However, it has been hypothesized that the high abundance isomer is not always
the isomer with the greatest contribution to the development of cancer, possibly because
high abundance isomers are more likely to be repaired (28-32). Accordingly, the analysis
and inhibition of (+)-anti-cis-BPDE-N2-dG and syn-BPDE-N
2-dG is of great interest.
Figure 2.3. Three isomers from the B[a]P treated MCF-7 cells were detected in an LC-
MS/MS analysis. (Reproduced from (27))
75
Figure 2.4. Co-treatment of cells with B[a]P and chrysoeriol reduced BPDE-DNA
adduct levels. Solid bars represent adduct levels in cells treated only with 2 µM B[a]P.
Lined bars represent adduct levels in cells treated with 2 µM B[a]P and 5 µM chrysoeriol
and dotted bars represent cells that were treated with 2 µM B[a]P and 10 µM chrysoeriol.
While (+)-trans-BPDE-N2-dG and (+)-anti-cis-BPDE-N
2-dG levels decreased
significantly following treatment with chrysoeriol, syn-BPDE-N2-dG levels did not
significantly change. (Reproduced from (27))
2.3. Mass spectrometric detection of modified bases
DNA damage can also be quantified for the purposes of chemopreventive agent
evaluation by a surrogate method of detecting modified bases that have been excised
from DNA. When a carcinogen binds to nucleophilic sites of adenine or guanine, the
carcinogen-purine conjugate may be hydrolyzed from the DNA strand leaving a
76
depurinated site. This depurinated site can then contribute to mutations, including error
prone repair. Accordingly, depurinating adducts can play a major role in the initiation of
cancer making them valuable biomarkers of the need for chemopreventive intervention.
A major advantage of using depurinating adducts as biomarkers is that samples are
readily accessible; rather than having to isolate genomic DNA from tissues, modified
bases can be extracted from urine or blood. A drawback however is that the
measurement of the carcinogen-purine conjugate is removed from genetic damage. It is
not known whether or not the DNA is repaired and if it is repaired correctly.
Furthermore, detection of depurinating adducts is only a measure of recent exposure and
the base may not necessarily be from DNA but could be from RNA.
Before LC-MS/MS analysis of depurinating adducts, DNA is usually removed from the
sample by precipitation and the carcinogen-purine conjugates are concentrated by SPE.
If the sample source is urine, adduct levels can be normalized to creatinine levels. Just
like in the quantitative analysis of nucleoside adducts, the use of calibration curves and
internal standards enable quantification and the analytes are characterized based on
retention time and fragmentation patterns. Both absolute amount of DNA adducts and
percent inhibition information can be obtained.
An example LC-MS/MS of a depurinating adduct, aflatoxin-N7-guanine is shown in
Figure 2.5. This analysis was done on a quadrupole ion trap and the ions from both the
base (m/z 152) and the aflatoxin adduct (m/z 329) are visible in the mass spectrum.
77
Figure 2.5. LC-MS/MS of aflatoxin-N7-guanine. (Reproduced from (33))
LC-MS/MS quantification of depurination by the human liver carcinogen aflatoxin B1
(AFB1) was used to determine the efficacy of Oltipraz inhibition. Rats were treated with
AFB1 and their urine was analyzed for depurinated products. The two major compounds
they observed were aflatoxin B1-N7-guanine (AFB1-N
7-gua) and its imidazole ring
opened derivative 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl
formamido)-9-hydroxy-aflatoxin B1 (AFB-FAPyr). Aflatoxin B2 (AFB2) was used as an
internal standard for recovery and for quantification because it is not naturally produced.
The calibration curve for this study is shown in Figure 2.6 and plots AFB1 dose as a
function of pg AFB1-N7-gua/mg creatinine. For increased analyte enrichment following
SPE, the samples were purified on a monoclonal antibody immunoaffinity column. MS
analysis was performed on an LCQ. LC-MS and LC-MS/MS chromatograms and mass
spectra of their detection of AFB1-N7-gua are shown in Figure 2.7. As expected, a
78
significantly better signal-to-noise ration was observed in the tandem MS experiment.
Treatment with Oltipraz resulted in significantly reduced adduct levels (34).
Figure 2.6. Calibration curve of AFB1 dose versus AFB1-N7-gua detected in rat urine.
(Reproduced from (34))
79
Figure 2.7. Panel A shows the LC-MS detection of AFB1-N7-gua in rat urine of rats
treated with AFB1. Panel B shows the LC-MS/MS scan of the same sample. Panel C
shows the full MS scan from the chromatogram of panel A and panel D shows the
MS/MS scan from the chromatogram of panel B. (Reproduced from (34))
80
A study measuring depurinating estrogen adducts was designed to evaluate several
chemopreventive agents, the structures of which are shown in Figure 2.8. Several
studies have found that healthy women have lower estrogen-purine adducts in their urine
than women at high risk or with breast cancer. Accordingly, decreasing these adduct
levels by chemopreventive agents may reflect a decrease in risk or occurrence of breast
cancer. Depurination of DNA following exposure to estrogens has been shown to be
inhibited by several natural compounds using an LC-MS based technique. In this case,
calf-thymus DNA was treated with estradiol-3,4-quinone (E2-3,4-Q) or lactoperoxidase
(LP)-activated 4-hydroxyestradiol (4-OHE2) and a potential chemopreventive agent.
Following DNA precipitation and SPE purification, the most predominant adducted bases
4-OHE2-1-N3Ade and 4-OHE2-1-N
7-Gua were detected and quantified by
Ultraperformance LC-MS/MS analysis with a triple quadrupole mass spectrometer. N-
acetylcysteine (NAcCys) and reduced lipoic acid reduced the formation of E2-3,4-Q
adducts but resveratrol and melatonin did not. Adduct formation by LP-activated 4-
OHE2 was inhibited by all of the compounds, the greatest by resveratrol followed by
NAcCys, reduced lipoic acid, and finally melatonin (35). In a subsequent manuscript,
this same group showed that resveratrol also inhibited E2-3,4-Q or 4-OHE2-induced
adduct formation in human breast epithelial cells (MCF-10F) (36).
Comparison between estrogen-purine adduct levels resulting from E2-3,4-Q or 4-OHE2
enabled the researchers to gain insight into the mechanisms of action of chemopreventive
agents. They speculated that the chemopreventive agents could either prevent the
81
formation of E2-3,4-Q and its reaction with DNA or modulate estrogen activating or
deactivating enzymes. However, as their research was with calf thymus DNA and did not
include these enzymes, they were essentially evaluating the antioxidant effects of the
chemopreventive agents on E2-3,4-Q or 4-OHE2. NAcCys’s or dihydrolipoic acid’s
inhibition of depurinating adducts increased more significantly with increasing
concentration of the chemopreventive agent when the DNA was treated with 4-OHE2
than E2-3,4-Q. In contrast, melatonin or resveratrol only reduced depurination when
DNA was treated with 4-OHE2 but not E2-3,4-Q. This indicated that all four agents were
involved in reducing the E2-3,4-semiquinone to 4-OHE2, but only NAcCys and
dihydrolipoic acid directly interacted with E2-3,4-Q (35). In later work, Resveratrol was
shown to inhibit depurination when MCF-10F cells were treated with E2-3,4-Q but this
was attributed to its induction of quinone reductase (36).
HO
OH
OH
Resveratrol
HN
HN
O
O
melatonin
O
OH
S
S
lipoic acid
O
OH
HS
O
HN
NAcCys
Figure 2.8. Chemical structures of Resveratrol, NAcCys, melatonin, and lipoic acid.
82
Clinical trials involving LC-MS analysis of depurination by AFB1 have been carried out
to investigate the chemopreventive potential of chlorophyllin (CHL) and SF. These
studies were done in an area where people are at a high risk for the development of
hepatocellular carcinoma due to consumption of foods contaminated with aflatoxins.
Both studies measured the urinary levels of AFB1-N7-gua and used AFB2 as an internal
standard. SPE enriched the aflatoxins and this was followed by further enrichment on an
AFB1-specific preparative mAb immunoaffinity column. AFB1-N7-gua and AFB2 were
quantified by measuring the peak area of specific MS/MS daughter fragments MH+
152.1 and 259.1 from the parent ions 480.1 and 315.1.
CHL was found to reduce urinary levels of these adducts. This CHL intervention trial
involved 180 healthy adults who ingested 100 mg CHL or a placebo three times a day for
4 months. AFB-N7-gua was detected in 105 of 169 samples and Chlorophyllin
consumption led to 55% lower urinary adduct levels. The researchers concluded that
consumption of CHL could reduce the development of hepatocellular carcinoma (33).
In the SF inhibition clinical trial, 200 healthy adults drank hot water infusions of 3-day-
old broccoli sprouts every night for two weeks. The hot water infusions of one group
contained 400 µmol glucoraphanin while those of the placebo group contained less than 3
µmol glucoraphanin. Participants were not allowed to eat any green vegetables that
contain glucosinolates. Although there was not a significant difference in AFB1-N7-gua
between people taking broccoli sprouts and people taking the placebo, this was attributed
83
to the large variation in the bioavailability of SF in the broccoli sprout preparation.
Bioavailability of SF was monitored through the urinary levels of dithiocarbamates,
which are metabolites of SF. The researchers did observe an inverse association between
dithiocarbamate and AFB1-N7-gua concentrations in the group taking the broccoli sprouts
but not in the placebo group (8).
2.4. Additional applications of LC-MS to chemopreventive agent analysis.
In addition to DNA adduct quantification research, LC-MS is useful for characterizing
chemopreventive agents, determining their bioavailability, identifying and quantifying
their metabolites, and investigating their interaction with carcinogens. Unlike UV,
fluorescence, and electrochemical detection, mass spectrometry-based methods provide
structural information about a compound which is especially important in the analysis of
metabolites of chemopreventive agents.
Many chemopreventive agent metabolites exist in biological fluids as Phase 2
metabolites, including glucuronide and sulfonate conjugates and their detection would
provide insight into their metabolism and bioavailability. An analysis of the carcinogen
metabolites can be used to determine the ability of a chemopreventive agent to modulate
the carcinogen’s metabolism. Typical sample preparation for this analysis involves
incorporation of an isotopically labeled internal standard and analyte enrichment, such as
84
by SPE. However, dilution-only methods have also been shown to be effective and
eliminate both time-consuming sample preparation steps and the chance that metabolites
were lost during these steps. Hydrolysis of these metabolites would be useful in
understanding their bioavailability, while isolation and analysis of the entire conjugate
would provide insight into their metabolism. Metabolite profiling usually involves a full
scan which provides information on the high abundance metabolites, but lower
abundance metabolites are not detected. Constant neutral-loss is useful for detecting
glucuronides (loss of 176), sulfates (loss of 80) and glutathione (GSH) adducts (loss of
129). Furthermore, the neutral loss of 56 Da (2 x CO) is a common feature of all
isoflavones (37).
2.5. Conclusions
DNA adducts are valuable biomarkers of the efficacy of chemopreventive agents. As a
measurement of genotoxic risk and present in all individuals, they can be used to identify
agents that decrease this risk by inhibition of their formation or promotion of their repair
(12, 38). Furthermore, in the evaluation of chemopreventive agents, it is important to
have a biomarker that can be measured before cancer develops (13). In the case where
carcinogenesis has been initiated, DNA adducts can potentially serve as biomarkers of
chemopreventive intervention (12).
85
Comprehensive detection of these adducts for chemoprevention presents several
analytical challenges. A sensitive method is required for research that involves the
detection of low abundance isomers. The importance of a highly sensitive method is
even greater in research that most closely resembles typical human exposure, an
important feature of an experiment as carcinogen dose affects the metabolism and
formation of adducts. Moreover, if the detection of adducts is for the evaluation of
chemopreventive agents, adduct levels will be decreased even further. LC-MS based
techniques are well suited for these analyses because it provides structural information
and sensitivity is continually increasing.
These analyses in combination with others are useful in identifying and evaluating
chemopreventive agents. Other important measurements include measuring antioxidant
activity, inhibition of Phase 1 enzymes such as Cyp450 carcinogen activating enzymes
through the EROD assay, measuring the induction of Phase 2 enzymes, protein adduct
analysis and metabolite analysis.
86
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CHAPTER 3
AN IMPROVED LIQUID CHROMATOGRAPHY-
TANDEM MASS SPECTROMETRY METHOD FOR THE QUANTIFICATION
OF 4-AMINOBIPHENYL DNA ADDUCTS IN URINARY BLADDER CELLS
AND TISSUES
92
3.1 Introduction
A major challenge in the analysis of bulky DNA adducts such as 4-ABP has been the
relatively low levels at which they typically occur in vivo. They are found in a complex
matrix of protein, ribonucleic acid (RNA), and salt as well as excess unmodified bases,
which in the case of 4-ABP is a million to a billion-fold (1, 2). Moreover, often only a
small quantity of DNA may be obtainable for analysis from in vivo samples,
necessitating minimal analyte loss during sample handling and a sensitive analytical
method. Detection at the part-per-billion threshold has become achievable with
impressive advances in the trace level detection of bulky DNA adducts by liquid
chromatography-mass spectrometry (LC-MS); improvements in sample preparation
techniques and in instrumentation have led to reduced sample requirements and greater
overall sensitivity.
An enrichment step prior to LC-MS analysis is desired for maximum sensitivity in the
measurement of DNA adducts because the matrix in which these adducts are found will
interfere with their detection (2). Typical techniques employed for purifying adducted
nucleosides following isolation and digestion of genomic DNA include liquid-liquid
partitioning, solid-phase extraction (SPE), HPLC, further enzymatic digestion and
immunoaffinity chromatography. Liquid-liquid extraction involves handling hazardous
organic materials and immunoaffinity chromatography is expensive. SPE has been
successfully used in our laboratory to extract DNA adducts from their complex matrix.
93
A typical manual SPE workflow is demonstrated in Figure 3.1. The cartridge is first
washed, generally with the same solvent used for elution. The sample is then loaded onto
the column and washed to remove unadducted nucleosides and salts. Finally, the analyte
is eluted and prepared for analysis.
Figure 3.1. Typical manual SPE workflow. (Reproduced from (3))
There are a number of drawbacks to manual SPE. The procedure can be labor-intensive
and time-consuming, especially if a large number of samples are involved. Impurities
from the extracts, which were either in the sample initially or added to the sample during
SPE, can lead to pressure build-up on HPLC columns in the LC-MS analysis, ion
suppression, and reduced recoveries (4). Analyte loss and the hydrolysis of modified
nucleosides both contribute to measurement errors that may also be introduced. A major
limitation of many studies using manual SPE is the requirement of relatively large (100
µg) quantities of DNA in order to compensate for the inevitable analyte losses during
sample processing.
94
For these reasons, automated enrichment, which minimizes sample handling by
incorporating a trapping column before the analytical column, is preferred. This requires
two HPLC pumps and a valve. Because enrichment is carried out within the system, less
analyte is lost. Not only are results more reproducible, sample preparation time is
significantly decreased and the method can be high throughput (5).
The overall sensitivity of the analysis can be improved by reducing flow rates to the
nanoliter (nL) per minute range and accordingly, incorporating smaller internal diameter
(ID) analytical columns and ionizing by nanoESI (6). Sources under atmospheric
pressure, such as in ESI, are particularly susceptible to ion loss during transfer of ions
from the source to the analyzer and decreasing the flow rate leads to more efficient
transfer. Because only a very small fraction of the molecules are actually ionized, it is
important to maximize ion transfer. Use of nanoESI, instead of ESI or microESI,
improves ionization efficiency and allows for a smaller sample volume without
decreasing signal to noise ratio; Because mass spectrometry is concentration dependent,
low concentrations in low volumes and low concentrations in high volumes produce the
same signal to noise ratio (7).
Developments in mass analyzer technology have also led to improvements in sensitivity
by reducing ion losses, more efficiently transferring ions from the source to the detector,
and improving detection sensitivities. Various devices such as radio frequency (RF) ion
95
guides, lenses, and ion funnels have been developed to facilitate efficient ion transfer and
reduce losses in ion current between major components in a mass spectrometer (7).
Developments in technology that improve efficiency and therefore sensitivity have been
made in ionization, transmission and detection of ions (4).
With the increase in sensitivity and decrease in sample loss during analyte enrichment
and analysis, the amount of DNA required per analysis is reduced. This is particularly
relevant in the analysis of in vivo samples where limited sample is available. In many
cases only limited sample is available in experimental systems too, such as those that
involve small rodents with small tissues. The amount of DNA in different types of
tissues vary; from 20 mg of liver tissue, one may expect to isolate 60 µg DNA while 20
mg of bladder tissue may only yield 20 µg DNA and 200 mL of urine may only yield 1
µg urothelial cell DNA. With lower yields of DNA, it is particularly important that the
method used for detection is sensitive.
As quantitative LC-MS methods have been developed for the analysis of 4-ABP DNA
adducts, there is a general trend of decreasing sample requirement and limit of detection
(LOD). This can be attributed to the optimization of enrichment conditions or use of an
automated clean-up step, use of reduced flow rates and capillary ID columns coupled to
nanoESI, and development of more sensitive mass analyzer technology. Some of these
methods are presented in Table 3.1 (2, 8-13).
96
Pa
per
Qu
an
tity
of
DN
A
pro
cess
ed (µ
g)
Flo
w r
ate
(µL
/min
)
LO
Q (
ad
du
cts/
nu
cleo
sid
es)
E
nri
chm
ent
met
hod
A
naly
tica
l
colu
mn
ID
(m
m)
Typ
e o
f m
ass
spec
tro
met
er
[2]
10
0
200
2
/10
7
Onli
ne
trap
co
lum
n
2
Sin
gle
qu
adru
po
le
[8]
50
0
250
4
/10
8
Liq
uid
-liq
uid
extr
acti
on
2
QIT
[9]
30
0
0.3
6
/10
9
pro
tein
pre
cip
itat
ion,
SP
E
0.3
2
Tri
ple
qu
adru
po
le
[10
] 20
0
0.3
8
/10
9
pro
tein
pre
cip
itat
ion,
SP
E
0.0
75
T
rip
le
qu
adru
po
le
[11
] 20
0
200
3
/10
9
Imm
uno
aff
init
y
cle
an-u
p,
onli
ne
trap
colu
mn
2.1
T
rip
le
qu
adru
po
le
[12
] 10
6
1/1
08
SP
E
0.3
2
LIT
[13
] 5
0
.3
2/1
08
Pro
tein
pre
cip
itat
ion,
onli
ne
trap
co
lum
n
0.0
75
Q
IT
Ta
ble
3.1
. L
C-M
S m
etho
ds
for
qu
anti
fyin
g d
G-A
BP
ad
du
cts.
97
The method we present here integrates a 75 µm i.d. analytical column with automated
sample enrichment on a trap column prior to analysis, reducing both the quantity of DNA
required and detection levels. This improved DNA adduct quantification methodology
has a detection level of 5 adducts per 109
nucleosides using a total of 5 µg of DNA and
the equivalent of only 1.25 µg of DNA per analysis.
3.2 Project Goals
The goal of this project was to develop a sensitive LC-MS/MS method for the
quantification of dG-C8-4-ABP in human cells and tissues. Furthermore, this method
was designed to quantify dG-C8-4-ABP adducts from genomic DNA in human urothelial
cells to establish a procedure for a future comprehensive human smoke-quit study.
3.3 Experimental
All cell and animal dosing was done in Dr. Yuesheng Zhang’s laboratory at Roswell Park
Cancer Institute, Buffalo, NY by Joseph Paonessa and Yi Ding.
98
3.3.1 Chemicals and Standards
McCoy’s 5A medium supplemented with 10% v/v fetal bovine serum from Life
Technologies (Grand Island, NY), rat liver S9 from Moltox (Boone, NC), Nicotinamide
adenine dinucleotide phosphate (NADP) from Amresco, Inc. (Solon, OH) and Harlan
7012 Nature Ingredient diet from Harlan Laboratories (Bartonsville, IL) were required
during cell and animal dosing periods. The following chemicals were obtained from
Sigma Aldrich Chemical Co. (St. Louis, MO): D-Glucose-6-phospate disodium hydrate,
calf thymus DNA, nuclease p1 from penicillium citrinium, deoxyribonuclease 1 (DNase
I) type 2 from bovine pancreas, alkaline phosphatase from Escherichia coli (type IIIs),
ethanol, magnesium chloride, dimethyl sulfoxide (DMSO), and 4-Aminobiphenyl (4-
ABP). Hydrochloric acid was purchased from Fisher Scientific (Pittsburgh, PA, USA).
Phosphodiesterase 1 (crotalus adamanteous venom) was purchased from USB
Corporation (Cleveland, OH). For HPLC-MS/MS analysis, acetic acid (glacial, 99.99+%)
was acquired from Aldrich Chemical Co. (Milwaukee, WI), and Burdick and Jackson
solvents (methanol, acetonitrile, and water) were obtained from Thermo Fischer
Scientific (Pittsburgh, MA) and were HPLC grade. N-(2-deoxyguanosine-8-yl)-4-ABP
(dG-C8-4-ABP) was acquired from Toronto Research Chemicals (North York, ON) and
the deuterium labeled internal standard dG-C8-4-ABP-D9 was previously synthesized and
characterized in our laboratory (14).
99
3.3.2 Cell study
RT-4 human bladder carcinoma cells were grown in McCoy’s 5A medium supplemented
with 10% v/v fetal bovine serum and maintained in a humidified incubator at 37ºC with
5% CO2. Cells were plated at a concentration of 2.0 million cells per 10-cm dish with 10
mL growth medium overnight and then treated with 0, 0.5, 5.0, or 50. µM 4-ABP
dissolved in DMSO for 3h in the presence of 6% rat liver S9, 10 mM D-Glucose-6-
phospate disodium hydrate, and 5 mM NADP. Triplicate cultures were completed at
each condition. Following treatment, cells were harvested by trypsinization, washed once
with ice cold PBS and stored at -80°C until analysis. DNA was extracted using Qiagen
Blood and Cell Culture DNA Midi Kits according to the manufacturer’s instructions. Cell
cultures of roughly 5 million cells yielded 20 to 40 µg of nuclear DNA.
3.3.3 Animal study
Twelve two-month old male F/344/NHsd rats (approximately 180 g of body weight) were
purchased from Harlan Sprague Dawley (Indianapolis, IN). The protocol was reviewed
and approved by the Animal Care and Use Committee at Roswell Park Cancer Institute.
Animals were acclimated for one week and were fed the Nature Ingredient diet (Harlan
7012) and water ad libitum. 4-ABP was prepared in DMSO and administered by IP
injection at doses of 0, 25, 100, or 250 mg/kg of body weight in a final volume of 0.1 mL
per rat. Cell and animal dosings were completed in triplicate at each concentration.
Animals were sacrificed 24 h after the dosing, and the urinary bladders were obtained
immediately and stored at -80°C. DNA was extracted with an Invitrogen Easy-DNA kit
100
according to the manufacturer’s instructions. Approximately 10 to 30 µg of DNA was
isolated from rat bladder specimens of about 30 mg each.
3.3.4 Exfoliated human urothelial cell study
Four 150 - 250 mL urine samples from a lifetime non-smoker were collected and stored
at -80°C. Samples were thawed and urothelial cells were pelleted by centrifugation at
8,000 x g for 10 minutes at 4°C (Thermo Scientific, Sorvall RT-1). DNA was isolated
using a Qiagen Blood and Cell Culture DNA Midi Kit with the following yields: 0, 1.3,
2.2 and 2.8 µg. One µg of DNA was removed from each sample, digested according to
the procedure described below and reconstituted in 20 µL 10% methanol for three 5 µL
injections per sample. DNA from two of the samples was pooled for a 1 µg digest to be
spiked with IS and 2.24 fmol dG-C8-4-ABP before protein precipitation. The remainder
of DNA from the third sample was reserved for testing digestion efficiency. Specifically,
1 µg urothelial cell DNA was pooled with 10 ng DNA isolated from 4-ABP dosed RT-4
cells. For comparison, 1 µg calf thymus DNA was also pooled with 10 ng of the same
adducted RT-4 cell DNA. These two samples were digested according to the protocol
described below and reconstituted in 20 µL 10% methanol for three 5 µL injections.
3.3.5 DNA quantification, enzymatic digestion and protein precipitation
DNA isolated from cells and tissues was dissolved in 10 mM MgCl2/5 mM Tris-HCl
buffer (pH 7.2) to about 1 mg/mL. An Invitrogen Corporation (Carlsbad, CA) Quant-
IT™ double strand (ds) DNA BR Assay kit with a Qubit fluorometer was used for DNA
101
quantification. One or 5 µg aliquots were removed for digestion and analysis, and the
remainder was stored at -80°C. DNA was hydrolyzed according to a previously described
procedure (15) with some modifications. Specifically, samples were incubated at 98°C
for 3 to 5 minutes and chilled on ice during the addition of 0.3 units of nuclease P1 (0.3
units µL-1
solution of 5 mM tris(hydroxymethyl)amino methane (Tris-HCl, pH 7.4) and
3.1 Kunits of DNase I (1 µg µL-1
solution in 5 mM Tris-HCl/10 mM MgCl2, pH 7.4) per
µg of DNA. Following a 5-hour incubation at 37°C, 0.003 units of phosphodiesterase
(100 ng µL-1
in 5 mM Tris-HCl/10 mM MgCl2, pH 7.4), and 0.002 units of alkaline
phosphatase per µg of DNA were added and the reaction mixture was held at 37°C for 18
hours. At the end of the incubation period, 6 fmol of internal standard, dG-C8-4-ABP
was added per µg DNA. The digestion was terminated with the addition of five volumes
of ice-cold ethanol (-20°C). Proteins were pelleted by centrifugation at 7500 x g (Thermo
Scientific, Sorvall RT-1) for 15 min at 4°C. Following lyophilization, samples were
reconstituted in 20 µL of 10% methanol. Samples at replicate doses were pooled and
analyzed in triplicate.
3.3.6 Preparation of dG-C8-4-ABP standard curves
Calf-thymus DNA was weighed out and dissolved in 10 mM MgCl2/5 mM Tris-HCl
buffer (pH 7.2) to an approximate concentration of 1 mg/mL. DNA concentration was
verified using the Invitrogen Corporation (Carlsbad, CA) Quant-IT™ double strand (ds)
DNA BR Assay kit with the Qubit fluorometer. Following DNA digestion but before
protein precipitation, dG-C8-4-ABP was spiked into 5 µg aliquots of calf-thymus DNA in
102
the following amounts: 0, 0.27, 0.54, 1.1, 2.2, 4.3, 8.6, 17., 35., 70., 140, and 280 fmol.
Each aliquot was also spiked with 6 fmol dG-C8-4-ABP-D9 per µg DNA. For accurate
quantification, the calibration curve was prepared in triplicate.
3.3.7 Preparation of standards for heat treatment stability assessment
To determine the stability of 4-ABP adducts during heat treatment at 98ºC for three
minutes, four sets of mixtures were prepared in which 8.4 fmol of dG-C8-4-ABP and 30
fmol dG-C8-4-ABP-D9 were added prior to or post-heat treatment to a final volume of 20
µL in 10% methanol. Triplicate mixtures were completed for each set and samples within
each set were pooled before analysis.
3.3.8 Capillary liquid chromatography and nESI-MS/MS
Liquid chromatography was performed using ChemStation for LC 3D Systems operating
an Agilent 1100 Series system (Agilent Technologies, Wilmington, DE) equipped with a
capillary pump, a nano pump, a 1200 series micro well-plate autosampler, and a chip
cube interface. Chromatographic separations were performed on an Agilent Technologies
small molecule microfluidic chip containing a 40 nL trap and 75 µm i.d. x 43 µm
analytical column of reverse phase Zorbax SB-C18 and 5 µm particles. Each injection
loaded 5 µL of sample onto the trap column at a flow rate of 4.00 µL min-1
to be washed
for 4 minutes with 93% mobile phase A (3% acetonitrile, 0.1% acetic acid in water) and
7% mobile phase B (0.1% acetic acid in methanol). Chromatographic separations were
conducted at a flow rate of 300 nL min-1
with mobile phase A (water with 0.1% acetic
103
acid) and mobile phase B (methanol with 0.1% acetic acid). Mobile phase B was held at
10% for 4.21 min, then linearly increased to 90% over 2 min, held for 2 min, then
stepped down to 10% for a 5 min re-equilibration period.
To reduce carryover and prevent plugging of the narrow i.d. capillaries used for
connections between the autosampler and chip cube, several cleaning procedures were
implemented. An inline prefilter (0.5 µm) was placed between the autosampler and the
loading capillary to prevent particulates in the sample from plugging the loading capillary
and enrichment column inlet screen. During analysis mode, a needle wash program
cleaned the needle seat, the needle seat capillary, and rotor seal with acetonitrile,
methanol, and water while the loading capillary was washed with 90% B. One methanol
blank and two 10% methanol blanks were injected between samples. In addition, nano
pump and capillary pump pressures, system suitability standards and internal standards
were used to monitor system performance.
Mass spectrometric data were acquired on an Agilent 6330 Ion Trap mass spectrometer
(Santa Clara, CA) operated with 6300 Series Ion Trap HPLC-MS Software Ver. 6.1 and
6300 Series Trap Control Software Ver. 6.1 from Bruker Daltonik GmbH (Bremen,
Germany). The mass spectrometer was calibrated by infusing tuning solution and 10
ng/mL dG-C8-4-ABP. For all analyses, the N2 drying gas was set to 3.0 L min-1
at 325°C
and the capillary voltage was held at -1900 V with an end-plate offset of -500 V. Ion
optics and trap parameters were as follows: skimmer 1: 40.5 V, capillary exit: 163.5 V,
104
trap drive: 54.2 V, ultra scan mode (24,000 m/z per scan), and positive ion mode. Ion
charge control parameters were set to a maximum accumulation of 50 ms or 500,000 ions
and 3 spectral averages per scan. CID pressure was 3 mT He and voltage ramped from
0.45 to 3 V. MS/MS spectra were collected within a scan window of 290-475 m/z and
precursor ions 435.4 ± 1.0 and 444.0 ± 1.5 Da were isolated and fragmented with a cutoff
fragmentation of 27% of the precursor ion mass.
Quant Analysis (Ver. 1.8) and Data Analysis (Ver 3.4) for 6300 Ion Trap LC/MS (Bruker
Daltonik GmbH) were used for data processing. Extracted ion chromatograms selected
to monitor for the characteristic loss of deoxyribose were 435�319 for the analyte, dG-
C8-4-ABP and 444�328 for the internal standard, dG-C8-4-ABP-D9. Gaussian
smoothing was set to a width of 0.65 m/z.
3.3.9 Quantification of dG-C8-4-ABP in biospecimens
The standard curve of dG-C8-4-ABP plots the mass of dG-C8-4-ABP vs. the mean
analyte to internal standard peak area ratio and was generated in triplicate over an 8
month period. Triplicate analyses were also conducted for every point in each calibration
plot. Linear regression data derived from the average of all three plots enabled the
quantification of dG-C8-4-ABP in 4-ABP-exposed human bladder cells and rat bladder
tissues.
105
3.4. Results and Discussion
3.4.1 Mass spectrometric analysis of dG-C8-4-ABP synthetic standard
A 10 ng mL-1
solution of dG-C8-4-ABP in 70:30:0.1 (v%) methanol:water:acetic acid
was infused at 300 nL/min to optimize the trap parameters and ion optics for the dG-C8-
4-ABP 435�319 transition. This fragmentation is due to the characteristic loss of
deoxyribose, [M+H]+�[M+H-116]
+.
Fragmentation amplitude was optimized and its adjustment provided further structural
confirmation. Figure 3.2 shows the mass spectra of the infusion of the synthetic standard
at various fragmentation amplitudes. In the first panel, the mass spectrometer was set to
isolate the precursor ion 435.1 ± 1.0 and the fragmentation voltage was turned off,
resulting in a mass spectrum showing only the parent ion. A fragmentation amplitude of
0.1 volts only fragmented some of the analyte, and a significant precursor ion peak was
still present. Increasing the voltage to 0.15 fragmented most of the analyte. At 1.5 V, the
precursor ion was no longer present in the mass spectrum and the ion of greatest
abundance was 319. The MS3 spectrum, in which the precursor ion 435.1 was isolated
and fragmented followed by the isolation and fragmentation of the fragment ion 319, is
shown in the bottom panel. The major fragment ion shown is m/z 302. The proposed
fragmentations are shown in Figure 3.3 (16).
106
A.
B.
C.
D.
Figure 3.2. A. Isolation of precursor ion, no fragmentation. B. Fragmentation
amplitude: 0.10 volt. C. Fragmentation amplitude: 0.15 volt. D. MS3 435�319 �302.
319.1
356.9396.1
435.2
+MS2(435.0), 0.1-2.5min #(5-136)
0.0
0.2
0.4
0.6
0.8
5x10
Intens.
200 250 300 350 400 450 500 550 m/z
154.9249.0
302.1
+MS3(435.4->319.0), 0.7-1.7min #(2-6)
0
20
40
60
80
100
Intens.
100 200 300 400 m/z
319.1
356.9396.1
435.1
+MS2(435.0), 0.1-2.1min #(4-114)
0
1
2
3
4
4x10
Intens.
200 250 300 350 400 450 500 550 m/z
435.1 +MS2(i435.0), 0.1 -2.1min #(3 -110)
0.0 0.5 1.0 1.5 2.0
4 x10 Intens .
200 250 300 350 400 450 500 550 m/
435.1 +MS2(i435.0), 0.1 -2.1min #(3 -110)
0.0 0.5 1.0 1.5 2.0
4 x10 Intens .
200 250 300 350 400 450 500 550 m/
107
NH
OH
OHO
N
N O
NH
NH2
N
H+
m/z 435
H
NH
N
N O
NH
NH2
N
H+
m/z 319
NH
N
N
O
NH
N
m/z 302
H+
Figure 3.3. The proposed fragmentations for dG-C8-4-ABP.
3.4.2 LC-MS and LC-MS/MS of synthetic standard.
Following infusion, both the dG-C8-4-ABP standard and dG-C8-4-ABP-D9 internal
standard were injected on column and the retention time was determined to be 8.9 min.
The chemical structures and extracted ion chromatograms (435�319 and 444�328 for
the analyte and IS respectively) of 1.89 fmol dG-C8-4-ABP (in red) and 1.74 fmol IS (in
blue) are shown in Figure 3.4. The chromatograms are shown as time (min) versus
relative peak intensity. The internal standard is isotopically labeled with deuterium
around the biphenyl moiety.
108
24
68
10
12
Tim
e [m
in]
0.0
0.2
0.4
0.6
0.8
1.0
Inte
ns.
x1
06
dG
-AB
P
dG
-AB
P-D
9 Inte
rnal S
tan
da
rd
319.1
012345
Inte
ns.
x1
05
300
320
34
0360
380
40
042
0440
460
m/z
dG
-AB
P M
as
s S
pe
ctr
um
32
8.2
02468
Inte
ns.
x1
05
300
320
34
03
60
380
400
42
04
40
460
m/z
dG
-AB
P-D
9 In
tern
al
Sta
nd
ard
Ma
ss
Sp
ec
tru
m
NH
NH
N
N
O
NH
2
N
O
HO
H
HH
HH
HO
m/z
31
9
24
68
10
12
Tim
e [m
in]
0.0
0.2
0.4
0.6
0.8
1.0
Inte
ns.
x1
06
24
68
10
12
Tim
e [m
in]
0.0
0.2
0.4
0.6
0.8
1.0
Inte
ns.
x1
06
dG
-AB
P
dG
-AB
P-D
9 Inte
rnal S
tan
da
rd
319.1
012345
Inte
ns.
x1
05
300
320
34
0360
380
40
042
0440
460
m/z
dG
-AB
P M
as
s S
pe
ctr
um
319.1
012345
Inte
ns.
x1
05
300
320
34
0360
380
40
042
0440
460
m/z
dG
-AB
P M
as
s S
pe
ctr
um
32
8.2
02468
Inte
ns.
x1
05
300
320
34
03
60
380
400
42
04
40
460
m/z
dG
-AB
P-D
9 In
tern
al
Sta
nd
ard
Ma
ss
Sp
ec
tru
m
32
8.2
02468
Inte
ns.
x1
05
300
320
34
03
60
380
400
42
04
40
460
m/z
dG
-AB
P-D
9 In
tern
al
Sta
nd
ard
Ma
ss
Sp
ec
tru
m
NH
NH
N
N
O
NH
2
N
O
HO
H
HH
HH
HO
m/z
31
9
109
Fig
ure
3.4
. H
PL
C-M
S o
f d
G-C
8-4
-AB
P s
tan
dar
ds.
T
he
top
pan
el s
ho
ws
the
chem
ical
str
uct
ure
of
the
dG
-C8
-4-A
BP
add
uct
in
red
. T
he
dG
-C8
-4-A
BP
-D9 i
nte
rnal
sta
nd
ard
(IS
) in
blu
e is
iso
top
ical
ly l
abel
ed w
ith
deu
teri
um
aro
un
d t
he
bip
hen
yl
mo
iety
. T
he
extr
acte
d i
on
ch
rom
ato
gra
ms
(43
5�
31
9 a
nd
44
4�
32
8 f
or
the
anal
yte
an
d I
S r
esp
ecti
vel
y)
of
1.8
3 f
mo
l d
G-C
8-4
-AB
P a
nd
1.7
4 f
mo
l IS
are
sh
ow
n a
bo
ve.
R
eten
tio
n t
ime
for
the
stan
dar
d a
nd
th
e in
tern
al s
tan
dar
d i
s
8.9
min
. T
he
MS
/MS
sp
ectr
um
of
dG
-C8
-4-A
BP
is
dis
pla
yed
in
th
e m
idd
le p
anel
. T
he
MS
/MS
sp
ectr
um
of
the
IS i
s
dis
pla
yed
in
th
e b
ott
om
pan
el.
110
As expected, the internal standard elutes very slightly ahead of the standard but the peaks
overlap considerably. It is not uncommon for the internal standard to co-elute with the
analyte in this type of analysis. Because the analyte dG-C8-4-ABP and internal standard
dG-C8-4-ABP-D9 co-elute, we know that they are subject to identical conditions during
analysis, making quantification more accurate than if they were chromatographically
resolved.
The retention time of the analyte was 8.9 minutes. The gradient begins 4.2 minutes into
the run, reaches 90% methanol at 6.2 minutes and is held at 90% methanol until 8.2
minutes. During the first four minutes, the capillary pump loads the analyte onto the trap
column, sending whatever is not retained to waste. At four minutes, the valve in the chip
switches so that the nano pump can wash the analyte from the trap column to the
analytical column for separation. The dead volume on the nano pump is 0.27 µL, which
takes 1.1 minutes at a flow rate of 0.3 µL/minute. Including the 40 nL trap column and
the 160 nL analytical column, the beginning of the gradient is detected by the mass
spectrometer 1.6 minutes after it has started. The gradient should reach the mass
spectrometer from 5.8 to 7.8 minutes and stay at 90% methanol until 9.8 minutes. The
dG-C8-4-ABP analyte is eluting at 8.9 minutes at the top of the gradient.
dead volume: 0.11 µL (25 µm x 23 cm) + .0.16 µL (15 µm x 90 cm) = 0.27 µL
column volume: 0.04 µL trap + 0.16 µL analytical (75 µm x 4.3 cm) = 0.20 µL
111
3.4.3 SPE matrix interference and cartridge comparisons: recovery of analyte in matrix
Sample preparation can have a substantial impact on sensitivity in the quantification of
DNA adducts by mass spectrometry (1). A typical workflow in the analysis of DNA
adducts is shown in Figure 3.5. Injection of the dG-C8-4-ABP standard in the matrix
from an SPE cartridge resulted in a loss in signal. A 70% loss was observed when the
matrix was from Isolute C18 cartridges and and 84% loss was observed with Waters tC18
cartridges. By designing the experiment this way, possible loss of the analyte was
eliminated and it is known that the lower signal is caused by interference from the SPE
matrix. This is most likely due to ion suppression from materials introduced from the
SPE cartridge. Because the loss of analyte during SPE sample preparation would
contribute to an even lower signal, it was necessary to find an alternative method for
sample enrichment.
Figure 3.5. Typical workflow in DNA adduct analysis by LC-MS.
Isolation of
nuclear DNA
Enzymatic
digestion down to
nucleosides
Protein
Precipitation
Removal of
unmodified
nucleosides
LC-MS/MS
Analysis
Isolation of
nuclear DNA
Enzymatic
digestion down to
nucleosides
Protein
Precipitation
Removal of
unmodified
nucleosides
LC-MS/MS
Analysis
112
3.4.4 Automated adduct enrichment
The automated sample clean-up method presented here eliminates the need for solid-
phase extraction (SPE), reducing sample preparation time, sample handling, and analyte
loss as well as eliminating the introduction of SPE related artifacts which can cause ion
suppression. The microfluidic chip (Figure 3.6) used in this method incorporates both a
trap and analytical column, enabling the automated adduct enrichment step. The sample
was loaded onto the 40 nL reversed phase C18 trap column and salts and unmodified
nucleosides were washed off with 93% water and 7% methanol for four minutes before
placing the trap column inline with the analytical column during analysis mode. The
analyte was then washed from the trap column to the analytical column for separation.
The chip integrates a 40 nL trap column, a 75 µm i.d. x 43 µm analytical column of
reverse phase Zorbax SB-C18 and 5 µm particle size, and a nanospray emitter. In
enrichment mode, the sample was washed with 7% methanol, removing unwanted salts
and excess unmodified nucleosides. In analysis mode, the nano pump moved the analyte
from the trap column to the analytical column for separation. The chip can be set in
back-flush or forward-flush mode, but back-flushing sharpens the peak profile. The
valve configuration in enrichment mode and analysis mode is illustrated in Figure 3.7
113
Figure 3.6 A detailed view of the microfluidic chip used in our automated sample
enrichment method. The chip integrates a 40 nL trap column, a 75 µm id x 43 mm
analytical column of reverse phase Zorbax SB-C18 and 5 µm particle size, and a
nanospray emitter (courtesy of Agilent Technologies, Inc.).
Micro-
filter
Sprayer-tip Analytical
column
Inert
polyimide
114
Figure 3.7. A detailed view of the valve of the Agilent microfluidic chip used in our
automated sample enrichment method (courtesy of Agilent Technologies, Inc.). The top
panel shows the valve in enrichment mode, during which the sample was washed with
7% methanol, removing unwanted salts and excess unmodified nucleosides. The bottom
panel shows the valve in analysis mode, during which the analyte was removed from the
trap column to the analytical column for separation.
115
3.4.5 dG-C8-4-ABP stability assessment during heat treatment
Samples are subject to heat treatment during the digestion procedure to denature the
DNA for efficient hydrolysis by Nuclease P1. Therefore, to determine the stability of 4-
ABP adducts towards heat treatment at 98ºC for three minutes, we compared standards
that were exposed to heat treatment with standards that were not. Four sets of triplicate
mixtures were evaluated in which 8.4 fmol of dG-C8-4-ABP and 30 fmol dG-C8-4-ABP-
D9 were added prior to or post-heat treatment. The precision within each set was below
20% RSD. As shown in Figure 3.8, the standard error bars of all sets overlap, indicating
that their difference is not statistically significant.
Figure 3.8. Heat treatment stability assessment. Four sets of mixtures were compared in
which 8.4 fmol of dG-C8-4-ABP and 30 fmol dG-C8-4-ABP-D9 were added prior to or
post-heat treatment at 98°C for 3 min. Each set was taken through the protocol in
triplicate and the precision of all sets was below 20% RSD. The standard error bars of all
sets overlap, indicating that their difference is not statistically significant.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Analyte and Internal
Standard added prior to
heat treatment
Analyte and Internal
Standard added post-
treatment
IS added prior to heat
treatment, Analyte added
post-treatment
Analyte added prior to heat
treatment, IS added post-
treatment
Pe
ak
Are
a R
ati
o o
f A
na
lyte
/In
tern
al S
tan
da
rd
116
3.4.6 Assessment of internal standard degradation during digest
In our method, the internal standard was added immediately following the enzymatic
digestion of DNA. Although addition of the internal standard post-digestion does not
account for oxidation/hydrolysis during digestion, we have found that degradation of the
dG-C8-4ABP adduct during the digestion is minimal. To evaluate degradation of the
analyte and internal standard during the DNA digestion procedure, we compared four sets
of digests in which the standards were added prior to or post- digestion. 8.4 fmol of dG-
ABP and 39.6 fmol dG-C8-4-ABP-D9 were added to each digest. 5 µg aliquots of calf-
thymus DNA were digested in triplicate for each set and taken through a protein
precipitation before analysis. The standard error bars of all sets overlap with each set
having a precision under 15% RSD (Figure 3.9). It can therefore be concluded that their
difference is not statistically significant.
117
Figure 3.9. Internal standard degradation during digest. To evaluate degradation of the
analyte and internal standard during the DNA digestion procedure, we compared four sets
of digests in which the standards were added prior to or post- digestion. 8.4 fmol of dG-
C8-4-ABP and 30 fmol dG-C8-4-ABP-D9 were added to each digest. 5 µg aliquots of
calf-thymus DNA were digested in triplicate for each set and taken through a protein
precipitation before analysis. The standard error bars of all sets overlap with each set
having a precision under 15% RSD. It can therefore be concluded that their difference is
not statistically significant.
0
10
20
30
40
50
60
70
80
Analyte + Internal
Standard added post-
digestion
Analyte + Internal
Standard added prior
to digestion
Internal Standard
added prior to
digestion, Analyte
added post-digestion
Analyte added prior to
digestion, Internal
Standard added post-
digestionNum
ber
of dG
-AB
P a
dducts
in 1
0̂8 n
ucle
osid
es
118
3.4.7. Calibration curve and linearity
The standard curve of the number of fmol dG-C8-4-ABP versus the mean analyte to
internal standard peak area ratios was prepared in triplicate in a matrix of 5 µg calf-
thymus DNA to simulate the conditions of the real samples. Every point from each
calibration curve was also analyzed in triplicate. The isotopically labeled internal
standard was added to correct for variations in sample preparation and instrument
operation. A linear regression analysis of the data incorporating all three curves was
generated in Microsoft Excel using the least squares fit to the line y= mx + b. Each point
was equally weighted and the line was not forced through the origin. Values derived
from the standard curves (Figure 3.10) were as follows: slope = 0.100 ± 0.001, y-
intercept = 0.05 ± 0.03, and correlation coefficient R2 = 0.99 ± 0.08. A zoom of the low
end of the calibration curve is shown in Figure 3.11. The LOD was determined to be 20
amol on column or approximately 5 adducts in 109 nucleosides and the LOQ, the lowest
point on the calibration curve, was 70 amol on column or 2 adducts in 108 nucleosides.
Representative chromatograms of dG-C8-4-ABP analysis at the LOD and LOQ levels are
shown in Figure 3.12. Extracted ion chromatograms (435�319) of the LOD, 20 amol
on column in 1.25 µg of DNA or approximately 5 adducts in 109 nucleosides and the
LOQ, 70 amol on column in 1.25 µg of DNA or 2 adducts in 108 nucleosides. The
extracted ion chromatogram (435�319) of 1.25 µg calf thymus DNA on column is also
shown as the procedure blank.
119
120
Fig
ure
3.1
0.
Sta
nd
ard
cu
rve
dG
-C8
-4-A
BP
of
fmo
l an
aly
te o
n c
olu
mn
ver
sus
the
rati
o o
f an
aly
te t
o i
nte
rnal
sta
nd
ard
pea
k a
reas
wer
e g
ener
ated
in
tri
pli
cate
ov
er a
n 8
-mo
nth
per
iod
. E
ver
y p
oin
t fr
om
eac
h c
alib
rati
on
cu
rve
was
als
o
anal
yze
d i
n t
rip
lica
te.
Th
e li
nea
r re
gre
ssio
n l
ine
inco
rpo
rate
s al
l th
ree
curv
es a
nd
has
a s
lop
e o
f 0
.10
0±
0.0
01
, a
y-
inte
rcep
t o
f 0
.05
±0
.03
, an
d a
co
rrel
atio
n c
oef
fici
ent
R2 o
f 0
.99
±0
.08
.
121
Figure 3.11. Zoom of the lowest five points of the calibration curve. dG-C8-ABP
calibration curves of fmol analyte on column versus the ratio of analyte to internal
standard peak areas were generated in triplicate over an 8 month period. Every point from
each calibration curve was also analyzed in triplicate. The linear regression line
incorporates all three curves and has a slope of 0.100 ± 0.001, a y-intercept of 0.05 ±
0.03, and a correlation coefficient R2 of 0.99 ± 0.08.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.0 0.2 0.4 0.6 0.8 1.0 1.2
fmols of dG-ABP on column
An
aly
te/IS
Peak A
rea
Sep-08
Jan-09
Apr-09
Linear (Average)
122
Figure 3.12. LOD and LOQ. Extracted ion chromatograms (435�319) of the LOD, 20
amol on column in 1.25 µg DNA or approximately 5 adducts in 109 nucleosides, the
LOQ, 70 amol on column in 1.25 µg DNA or 2 adducts in 108 nucleosides, and the
procedure blank, 1.25 µg calf-thymus DNA.
3.4.8 Reproducibility
Reproducibility was determined with replicate doses of RT-4 cell and rat bladder DNA
digests. RT-4 cells were treated in triplicate with 4-ABP plus an S9 activation system,
taken through the sample preparation procedure that was previously described, and each
sample was analyzed twice. Subsequently, 6 µL aliquots from each sample were pooled
and injected three times. To test reproducibility in the rat specimens, 5 µg aliquots of rat
bladder DNA from three rats dosed at 25 mg/kg 4-ABP were taken through the sample
preparation procedure and the equivalent of 1.25 µg of DNA from each digest was
injected three times. The average number of adducts was determined to be 6 ± 2 adducts
123
in 107 nucleosides when the samples were run individually and 8 ± 3 adducts in 10
7
nucleosides when pooled. The precision of the pooled and individually run methods was
determined to be statistically the same by the F test (the calculated F-value 1.83 was less
than the tabulated value of 4.45 at the 95% confidence level). Applying the T test, there
was no statistical difference between running the samples individually or pooled (the
calculated value of T, 1.43 was less than the tabulated value of 2.23 at the 95%
confidence level). Because replicate dose concentrations resulted in similar adduct
quantities in both cells and rats, samples were pooled.
3.4.9 Quantification of dG-C8-4-ABP adducts in human bladder RT-4 cells and rat
bladder tissue
The quantity of dG-C8-4-ABP adducts detected in dosed cells and tissues was calculated
from the peak area ratio of dG-C8-4-ABP to IS and the standard curve linear regression
values (Figure 3.13, Figure 3.14). These findings are summarized in Table 3.2. Plots of
4-ABP dose versus observed adduct content and extracted ion chromatograms of the
lowest dose rat sample and the control sample are displayed in Figure 3.15. As indicated,
no adduct was detected in control samples. Because the ratio of analyte to IS by peak
area of the highest dosed cell sample was greater than that of the last point on the
calibration curve, the regression values were linearly extrapolated to calculate the adduct
quantity in this sample. The observation that adduct content increased with increasing 4-
ABP dose in the RT-4 cell study reinforces the implication of this carcinogen in DNA
adduct formation (17, 18). This trend may have been more apparent in the rat study had
124
the 4-ABP dose extended over a wider range, for example two orders of magnitude as in
the cell experiment, rather than only one. The ambiguity of this trend may also be due to
the complicated processes involved in DNA adduction; the quantification of DNA
adducts is not simply a measurement of exposure, but also the metabolism of the
carcinogen, including activation and deactivation, enzymatic esterification processes, and
detoxification and repair pathways, which may differ among species, tissues, and even
phenotypes (1, 19). In comparison to an experimental cell line, the steps leading to
adduction are even more complex in an organism, where certain processes are specific to
distinct tissues. For example, following exposure to 4-ABP, and certainly with many
competing reactions along the way, it is generally accepted that the carcinogen is
converted to N-hydroxy-4-ABP in the liver and then to a reactive electrophile in the
target tissue (20).
125
Figure 3.13. Logarithmic scale plot of dG-C8-4-ABP levels observed in dosed RT-4
human bladder carcinoma cells. Cell dosings were completed in triplicate at each
concentration 5 µg DNA was digested from each sample and samples from the same dose
concentration were pooled before triplicate analysis. Only 1.25 µg DNA was utilized per
injection.
1
10
100
1000
10000
0.5 5 50
Concentration of 4-ABP Dose (uM)
Nu
mb
er
of
dG
-AB
P A
dd
ucts
De
tec
ted
in
10^
8 N
ucle
os
ide
s
126
Figure 3.14. Plot of 4-ABP dose versus detected dG-C8-4-ABP quantity in bladder
tissue from rats treated with 4-ABP. Animal dosings were completed in triplicate at each
concentration 5 µg DNA was digested from each sample and samples from the same dose
concentration were pooled before triplicate analysis. Only 1.25 µg DNA was utilized per
injection.
0
20
40
60
80
100
120
140
160
25 mg/kg 4-ABP IP inection 100 mg/kg 4-ABP IP
inection
250 mg/kg 4-ABP IP
inection
Concentration of 4-ABP Dose
Nu
mb
er
of
dG
-AB
P
ad
du
cts
in
10
^8
No
rma
l N
uc
leo
sid
es
127
8.6 8.8 9.0 Time [min]
0.0
0.5
1.0
1.5
2.0
Intens.
x104
IS
25 mg/kg
4-ABP IP
inj
DMSO inj
8.6 8.8 9.0 Time [min]
0.0
0.5
1.0
1.5
2.0
Intens.
x104
IS
25 mg/kg
4-ABP IP
inj
DMSO inj
Figure 3.15. Zoom in of the EIC (435�319 for the analyte and 444�328 for the IS)
from the lowest dose rat and control samples.
128
A.
4-A
BP
Do
sed
Hu
ma
n B
lad
der
RT
-4 C
ells
Co
nce
ntr
ati
on
of
4-A
BP
in
DM
SO
(µ
M)
An
aly
te/I
nte
rnal
Sta
nd
ard
by
Pea
k A
rea
fmo
l d
G-A
BP
on
colu
mn
Nu
mb
er
of
Ad
du
cts
Det
ecte
d i
n
10
8 N
ucl
eosi
des
0
No
t det
ecte
d
No
t det
ecte
d
Not
det
ecte
d
0.5
0.0
65 ±
0.0
05
*
*
5.0
0
.14 ±
0.0
3
0.9
± 0
.5
20
± 1
0
50.
4.0
± 0
.8
40 ±
8
11
00 ±
20
0
*E
rro
r d
iscr
edit
s valu
e
B.
4-A
BP
Do
sed
F/3
44/N
Hsd
Ra
ts
Co
nce
ntr
ati
on
of
4-A
BP
in
DM
SO
IP
In
ject
ion
(m
g 4
-
AB
P/k
g r
at)
An
aly
te/I
nte
rna
l S
tan
da
rd b
y
Pea
k A
rea
fmo
l d
G-A
BP
on
colu
mn
Nu
mb
er o
f A
dd
uct
s D
etec
ted
in 1
08 N
ucl
eosi
des
0
Not
det
ecte
d
Not
det
ecte
d
No
t det
ecte
d
25
0.3
4 ±
0.0
9
3 ±
1
80 ±
30
100
0.4
9 ±
0.0
8
4.4
± 0
.9
120
± 2
0
250
0
.5 ±
0.1
4 ±
1
120
± 3
0
129
Ta
ble
3.2
. D
ose
–re
spo
nse
dat
a in
hu
man
cel
ls a
nd
rat
tis
sue.
(A
) d
G-A
BP
ad
du
ct c
on
ten
t an
d a
bso
lute
sta
nd
ard
dev
iati
on
fro
m R
T-4
hu
man
bla
dd
er c
arci
no
ma
cell
s th
at w
ere
trea
ted
wit
h 4
-AB
P d
isso
lved
in
DM
SO
. C
ells
wer
e
cult
ure
d i
n t
rip
lica
te,
po
ole
d,
and
an
aly
zed
in
tri
pli
cate
. (B
) A
dd
uct
qu
anti
ty a
nd
ab
solu
te s
tan
dar
d d
evia
tio
n f
rom
bla
dd
er t
issu
e D
NA
ex
trac
ted
fro
m t
wel
ve
2-m
on
th o
ld F
/34
4/N
Hsd
rat
s th
at w
ere
adm
inis
tere
d 4
-AB
P p
rep
ared
in
DM
SO
. D
NA
fro
m r
epli
cate
do
ses
was
po
ole
d b
efo
re a
nal
ysi
s an
d i
nje
ctio
ns
wer
e co
mp
lete
d i
n t
rip
lica
te.
130
3.4.10 Analysis of Urothelial Cell DNA adducts for a smoker quit study
Given the well established propensity of 4-ABP to target the bladder urothelium and the
implications associated with bladder cancer, an ultimate objective would be the
application of the methodology described here to the analysis of urothelial cell DNA. A
particular challenge presented here is the low quantity of exfoliated urothelial cell DNA
typically isolated from urine samples. This quantity may vary widely, with often only 1
µg or less extracted from about 200 mL of urine (20-23). In view of this, we scaled down
the method to be more suitable for the analysis of these samples by digesting only 1 µg of
DNA from these samples rather than 5 µg. This approach would then consume the
equivalent of 250 ng rather than 1.25 µg DNA per analysis. Assuming the mass LOD
remains at 20 amol, this method would correspond to a detection limit of 2.5 adducts in
108 nucleotides and would still be well suited for the analysis of human samples.
Initially, the 1 µg digestion process was tested by analyzing a bladder DNA sample from
a rat exposed to the lowest level of 4-ABP (25 mg/kg 4-ABP). The amount of adduct in
the 1 µg digest was found to be 7 ± 2 adducts in 107 nucleotides (Figure 3.16, A), which
was not significantly different from that determined previously using a 5 µg digest (the
calculated value of T, 0.7 was less than the tabulated value of 2.5 at the 95% confidence
level).
Following confirmation of capability of our method to handle reduced amounts of DNA,
three 1 µg aliquots of urothelial cell DNA from the lifetime non-smoker were digested
131
and the equivalent of 250 ng DNA was analyzed three times per sample. As shown in the
representative chromatogram of Figure 3.16, B, no dG-C8-4-ABP adduct was detected in
these samples and this should not be surprising since they were obtained from a never-
smoker; several studies have linked smoking to 4-ABP adducts in bladder biopsy
specimens or exfoliated urothelial cells (21-23). Significantly, however, the response of
the internal standard did not appear to be compromised by the urothelial cell matrix.
Therefore, the remainder of the DNA isolated from two of the exfoliated urothelial cell
samples was pooled for a 1 µg digest and spiked with IS and 2.24 fmol dG-C8-4-ABP. A
chromatogram of the analysis showing the detection of 14 ± 3 adducts in 107 nucleotides
is shown in Figure 3.16, C. Based on back-calculating the mass of dG-C8-4-ABP from
the ratio of analyte to internal standard peak areas, this value corresponds to an accuracy
of 113%.
Furthermore, to test the digestion efficiency in exfoliated urothelial cell DNA, 4-ABP-
modified DNA was mixed with exfoliated urothelial cell DNA prior to digestion.
Accordingly, digests of 10 ng 4-ABP dosed RT-4 cell DNA mixed with 1 µg of urothelial
cell DNA and 10 ng of 4-ABP dosed RT-4 cell DNA mixed with 1 µg of calf thymus
DNA were compared. In each case, approximately 1.5 adducts in 106 nucleotides were
detected indicating similar digestion efficiencies. These results are summarized in a bar
graph form in Figure 3.16, D. These experiments demonstrate that this LC-MS method is
capable of measuring dG-C8-4-ABP from exfoliated urothelial cells.
132
24
68
10
12
14
Tim
e [
min
]0123
Inte
ns
x 1
04
24
68
10
12
14
Tim
e [
min
]0123
Inte
ns
x 1
04
24
68
10
12
Tim
e [
min
]0246
Inte
ns.
x 1
04
24
68
10
12
Tim
e [
min
]0246
Inte
ns.
x 1
04
24
68
10
12
Tim
e [
min
]01234
x1
04
Inte
ns.
24
68
10
12
Tim
e [
min
]01234
x1
04
Inte
ns.
0.0
0.5
1.0
1.5
2.0
2.5
Urothelial C
ell D
NA
+
R
T-4 cell D
NA
Calf Thym
us D
NA
+
R
T-4 cell D
NA
Number of adducts in 10̂7 nucleosides
D.
A.
B.
C.
133 F
igu
re 3
.16
A
nal
ysi
s of
dG
-C8
-4-A
BP
ad
du
cts
by
dig
esti
ng
1 µ
g o
f D
NA
. I
n e
ach
ch
rom
ato
gra
m,
the
inte
rnal
stan
dar
d i
s sh
ow
n i
n b
lue
and
th
e an
aly
te i
s sh
ow
n i
n r
ed.
A.
Ch
rom
ato
gra
m o
f d
G-C
8-4
-AB
P f
rom
th
e p
roce
ssin
g
of
1 µ
g D
NA
(eq
uiv
alen
t o
f 2
50
ng
in
ject
ed o
n-c
olu
mn
) in
bla
dd
er s
amp
le o
f a
rat
trea
ted
wit
h 2
5 m
g/k
g 4
-AB
P.
Sig
nal
co
rres
po
nd
s to
7±
2 a
dd
uct
s in
10
7 n
ucl
eoti
des
, w
hic
h i
s th
e sa
me
as t
hat
ob
tain
ed f
rom
th
e an
aly
sis
of
a 5
µg
dig
est.
B
. R
epre
sen
tati
ve
chro
mat
og
ram
fro
m t
he
anal
ysi
s o
f a
1 µ
g u
roth
elia
l ce
ll D
NA
dig
est
of
a li
feti
me
no
n-
smo
ker
. C
. A
nal
ysi
s o
f d
G-C
8-4
-AB
P f
rom
tw
o p
oo
led
uro
thel
ial
cell
sam
ple
s; 1
µg
D
NA
was
dig
este
d a
nd
sp
iked
wit
h I
S a
nd
2.2
4 f
mo
l dG
-C8
-4-A
BP
. O
n c
olu
mn
in
ject
ion
of
25
0 n
g c
orr
esp
on
ds
to i
nje
ctio
n o
f 1
4±
3 a
dd
uct
s in
10
7 n
ucl
eosi
des
. D
. D
eter
min
atio
n o
f d
iges
tio
n e
ffic
ien
cy o
f u
roth
elia
l ce
ll D
NA
. C
om
par
iso
n o
f d
G-C
8-4
-AB
P
add
uct
lev
els
in 1
µg
ex
foli
ated
uro
thel
ial
cell
DN
A a
nd
1 µ
g c
alf-
thy
mu
s D
NA
eac
h s
pik
ed w
ith
10
ng
4-A
BP
-
mo
dif
ied
DN
A p
rio
r to
dig
esti
on
. N
o s
tati
stic
al d
iffe
ren
ces
are
ob
serv
ed.
134
3.5 Conclusions
The HPLC-MS/MS method presented in this paper is suitable for the quantification of 4-
ABP DNA adducts in human samples. High sensitivity was accomplished with nano-flow
rates, capillary columns and automated sample enrichment prior to HPLC-MS/MS
analysis. Only 1.25 µg of DNA is needed per analysis to reach the detection limit of 20
amol (5 adducts in 109 nucleosides) and the linear range spanned from 70 amol to 70
fmol.
Although high reproducibility, accuracy, and precision have been demonstrated on an ion
trap mass spectrometer, we expect the use of a triple quadrupole mass analyzer would
even further improve the method’s robustness. Traditionally, triple quadrupole mass
spectrometers have been used for quantitative measurements because they are more
robust than ion trap instruments for reproducibility and precision for trace, quantitative
analytical work. In an ion trap, the number of scans acquired is restricted by the slow
duty cycle of the scanning events and because fewer data points are acquired for each
peak, quantitative measurements may be imprecise. However, adjustments can be made
to find a balance between sensitivity, precision and reproducibility and we were able to
achieve sufficient sensitivity with high reproducibility in our analyses. Although the
linear dynamic range of a triple quadrupole is greater than that of an ion trap, we have
produced a linear calibration curve that will allow for quantification. Additionally, by
using an ion trap, we were able to perform MS3 for characterization of the analyte. There
135
are several examples of the use of ion traps in quantitative DNA adduct measurements
(24-27).
Once the methodology was established, the assay was applied to the quantification of dG-
C8-4-ABP adducts in human bladder cells and rat bladder tissue. Human bladder
carcinoma cells were treated with 0.5 to 50 µM 4-ABP and rats were administered 25 to
250 mg/kg 4-ABP, with analytical replicates at each dose. Replicate doses were shown to
be precise and reproducible, and the direct relationship between dose and DNA adduct
quantity confirmed that 4-ABP exposure led to adduct formation. The sample
requirements were further reduced to the equivalent of 250 ng DNA per injection in the
analysis of exfoliated human urothelial cells and it was demonstrated that low levels of
dG-C8-4-ABP could be detected in this matrix.
The described method can be applied to explore the relevance of 4-ABP DNA adducts to
bladder cancer and its relationship to smoking or other exposures. In combination with
related research projects such as those analyzing carcinogen metabolites (28),
determining the impact of sequence context on the formation of DNA adducts (29-31),
profiling gene expression (32, 33), elucidating complex 3-D nucleic acid structures (34)
or evaluating chemopreventive agents (35-37), this method can have a considerable
impact on the current understanding of carcinogenesis induced by 4-ABP or other
structurally related carcinogens.
136
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140
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141
CHAPTER 4
ACTIVATION OF NRF2 AS A CHEMOPREVENTIVE STRATEGY
AGAINST 4-ABP-INDUCED BLADDER CANCER
142
4.1 Introduction
4.1.1 Nrf2 induction as a chemoprevention strategy (1)
Activation of the ubiquitous transcription factor NF-E2 related factor 2 (Nrf2) is a
widespread strategy for cancer chemoprevention. Nrf2 stimulates the expression of genes
involved in many aspects of cytoprotection and many potential chemopreventive agents
mediate their cytoprotective effects through Nrf2. Nrf2 has protected cells against
carcinogens, including N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a bladder
carcinogen in rodents (2). It has also been shown to reduce the formation of carcinogen
DNA adducts including inhibition of the formation of 7,12-dimethylbenz[a]anthracene
(DMBA) DNA adducts in MCF-7 cells (3) and Benzo[a]pyrene (B[a]P) (4-6) and 2-
amino-3-methylimidazo[4,5-f]quinoline (IQ) (7) DNA adducts in mice. Accordingly, we
investigated Nrf2 as a target for the inhibition of 4-ABP DNA adduct formation in
bladder cells and tissues.
Nrf2 induces Phase 2 carcinogen detoxifying genes. In the basal state, Nrf2 is bound to a
Keap1 dimer, ubiquitinated, and targeted for proteasomal degradation. However,
chemopreventive and pharmacological agents can activate Nrf2: their interaction with
the sulfhydryl groups of Keap1 bring about a conformational change of Keap1 so that
Nrf2 is no longer ubiquitinated and therefore no longer targeted for proteasomal
degradation. Nrf2 then accumulates in the cytosol and moves into the nucleus. There, it
binds as a heterodimer to the antioxidant response element (ARE) in the 5’-untranslated
143
region of its target genes (8, 9). This results in the transcription of upstream genes which
include Phase 2 genes. Some Phase 2 enzyme catalyzed reactions include glutathione
addition, glucuronidation, and sulfation. These are often detoxification reactions of
carcinogens such as 4-ABP (10, 11).
There is a great deal of evidence suggesting that Phase 2 enzyme induction is crucial for
cytoprotection against carcinogens by many chemopreventive agents. That a
chemopreventive agent can detoxify a wide variety of carcinogens and that a carcinogen
can be detoxified by a wide variety of chemopreventive agents suggests that low
specificity mechanisms, such as induction of Phase 2 enzymes are involved.
Furthermore, greater efficacy from the chemopreventive agent is usually observed when
it has been administered prior to or at the same time as carcinogens instead of after,
suggesting that inhibition of DNA damage is occurring rather than repair. Finally, Phase
2 enzymes have been shown to both inactivate and alter the metabolism of carcinogens
and be inducible by chemopreventive agents.
Probing Phase 2 enzymes and their activities can therefore facilitate the identification and
evaluation of chemopreventive agents. Extent of carcinogen conjugation with Phase 2
metabolites can be quantified to provide an estimation of carcinogen detoxification. The
approximate efficacy of a chemopreventive agent can then be gauged based on the
relative change in levels of these conjugates following treatment with the
144
chemopreventive agent. Measurement of enzyme activities is a more direct measurement
of Phase 2 enzyme induction by chemopreventive agents (12-15).
4.1.2 Glucuronidation of 4-ABP is implicated in bladder cancer (16)
The Nrf2-regulated Phase 2 enzymes, Uridine 5'-diphospho (UDP)-
glucuronosyltransferases (UGTs), catalyze the conjugation of 4-ABP or its metabolites
with glucuronic acid (17-19). Although an elimination reaction, glucuronidation of 4-
ABP has been implicated in bladder cancer and has been shown to facilitate transport of
the active metabolite from the liver to the bladder (20). The structure of the 4-ABP-N-
glucuronide is shown in Figure 4.1. The glucuronidation reaction involves the transfer
of the glucuronosyl group of uridine 5'-diphospho-glucuronic acid (UDPGA) to an
oxygen, nitrogen, sulfur or carboxyl moieties of its substrates. UGTs metabolize many
compounds, endogeous and xenobiotic, but there is a specific human UGT that catalyzes
the formation of 4-ABP glucuronide conjugates (21). UGT enzymes are classified into
two families, UGT1 and UGT2 (22). In humans, the UGT1A family incorporates nine
isoforms (UGT1A1, UGT1A3-UGT1A10) and the UGT1A contributes more to the
metabolism of arylamines than the UGT2B family. 4-ABP in particular is glucuronidated
mostly by UGT1A4 and UGT1A9 (23, 24). There are species and individual differences
in glucuronidation based on individual genes, type of isoforms, quantities of UGTs,
substrate specificities, age and extent of exposure to inducers. Many UGT isoforms that
are expressed in mice are not detected in humans, or vice versa (25, 26). A recent study
showed that multiple UGT isoforms might be up-regulated by Nrf2 in mouse liver,
145
including UGT1A6, UGT1A7, UGT2B1, UGT2B35 and UGT2B36 (2, 17). A different
set of UGT isoforms appear to be up-regulated by Nrf2 in humans, including UGT1A1,
UGT1A8 and UGT1A10 (19, 27).
OH
OH
HO
O
O
HO
HN
Figure 4.1. Structure of 4-ABP-N-glucuronide.
The glucuronidation pathways that affect 4-ABP metabolism and the formation of 4-ABP
DNA adducts are illustrated in Figure 4.2 (16). 4-ABP and its oxidized metabolite N-
OH-4-ABP may be N-glucuronidated becoming more water soluble and therefore more
easily transported out of the liver and through the blood stream to the bladder. There,
low pH results in hydrolysis and the formation of DNA adducts (16, 20). Although N-
hydroxy-N-acetyl-4-aminobiphenyl will also undergo glucuronidation, this glucuronide
conjugate is not acid labile and will not result in bladder cancer.
146
Figure 4.2. Schematic of glucuronidation in the metabolic pathways of 4-ABP induced
human bladder cancer. (Reproduced from (16))
147
4.2 Project Goals
We investigated Nrf2 as a target for inhibition of 4-ABP DNA adduct formation. The
interesting effect of Nrf2 on 4-ABP DNA adduct formation in bladder tissue initiated an
investigation into Nrf2 expression and the Nrf2 signaling pathway. This led us to
consider Nrf2-induced glucuronidation of 4-ABP as a significant mechanism for
increasing the bioavailability of 4-ABP in bladder tissue.
4.3 Experimental
All cell and animal dosings, western blots, HPLC-UV measurements, and enzyme
activity experiments were carried out and analyzed in Dr. Yuesheng Zhang’s laboratory
at Roswell Park Cancer Institute, Buffalo, NY by Joseph Paonessa and Yi Ding.
4.3.1 Chemicals and Standards
4-ABP and N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP) were purchased from
LKT Laboratories (St. Paul, MN), Sigma (St. Louis, MO), and Midwest Research
Institute (Kansas City, MO), respectively. Rat liver S9 (36-43 mg protein/ml) was
purchased from Moltox (Boone, NC). Antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA), including antibodies specific for Nrf2, and from
Millipore (Billerica, MA) for the antibody for glyceraldehyde 3-phosphate
148
dehydrogenase (GAPDH). 4-ABP-glucuronide and the corresponding internal standard
were purchased from Toronto Research Chemicals (North York, Ontario).
4.3.2 Cell Study
RT-4 cells were purchased from American Type Culture Collection (ATCC), stored in
liquid nitrogen, and passaged in Dr. Zhang’s lab at Roswell Park Cancer Institute for
fewer than 6 months (28). The cell line was characterized by ATCC by antigen
expression, DNA profile, cytogenetic profile and isoenzymes. RT-4 cells were cultured
and treated with 4-ABP (plus 6% S9) or N-OH-AABP (29, 30). To silence Nrf2, cells
were treated with a Nrf2 siRNA or a control siRNA for 48 h (30). To induce DNA
damage, RT-4 cells (2-3 x 106 cells) were grown in each 10-cm dish with 10 ml medium
24-48 h, followed by treatment with either 4-ABP or N-OH-AABP for 3 h in 5 ml fresh
medium per dish. In cases where cells were co-treated with 4-ABP and S9, the medium
also contained 6% S9 (v/v), 10 mM glucose-6-phosphate and 5 mM NADP. Cells were
harvested by trypsin treatment and low-speed centrifugation, and cell pellets were washed
with ice-cold PBS and used for DNA adduct analysis and Western blotting of Phase 2
proteins.
4.3.3 Mouse Study
Wild type C57BL/6 mice (6 weeks of age) (NCI, Frederick, MD) and their Nrf2-deficient
counterparts (31) (bred at the Roswell Park Cancer Institute animal facility; the breeders
were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins Bloomberg School of
149
Public Health) (31)) were treated with a single dose of 4-ABP i.p. and sacrificed 24 h
later for quantification of dG-C8-4-ABP in the liver and bladder tissues (29). Groups of
3-4 mice were randomized and treated with a single dose of 4-ABP or vehicle i.p. The
mice were sacrificed 24 h later, from which the livers and bladders were promptly
removed for measurement of dG-C8-4-ABP. Liver and bladder tissues were stored at -
80oC. To measure urinary levels of 4-ABP-glucuronide, mice were given a single dose of
4-ABP i.p. and immediately moved to metabolism cages (2 mice/cage) for 24-h urine
collection. The specimens were stored at -80oC. All animal protocols and procedures
were approved by the Roswell Park Cancer Institute Animal Cancer and Use Committee.
4.3.4 Measurement of dG-C8-4-ABP
Procedures of DNA isolation from bladder cells and tissues and the detailed protocol for
the quantitative analysis of dG-C8-4-ABP by capillary liquid chromatography and nESI-
MS/MS have been described in chapter 3 of this dissertation and a recent publication (29,
32).
4.3.5 Western blot analysis
Cellular and tissue levels of Nrf2 and/or several Phase 2 enzymes were measured by
Western blotting (30). All antibodies were purchased from Santa Cruz Biotechnology,
except for anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which was
purchased from Millipore.
150
4.3.6 Measurement of liver UGT activity and urinary levels of 4-ABP-glucuronide and
creatinine
The assay protocols for UGT activity and 4-ABP-glucuronide were based on those by Al-
Zoughool and Talaska (24) with some modifications. Briefly, liver tissues were
homogenized in ice-cold 50 mM potassium phosphate buffer in a glass homogenizer; the
homogenates were cleared by centrifugation for 20 min at 9,100g at 4oC to prepare the S9
fraction. UGT activity toward ABP was measured by an HPLC-based biochemical assay
coupled with reverse phase HPLC. Each liver S9 sample (0.35 mg protein) was first
incubated with 17.5 µg alamethicin in 0.171 ml of 50 mM potassium phosphate buffer
(pH7.4) containing 8.75 µL methanol on ice for approximately 20 min. The entire
mixture was then transferred to a final 1 ml reaction solution, containing 50 mM
potassium phosphate (pH 7.4), 10 mM magnesium chloride, 5 mM saccharalactone and
0.5 mM 4-ABP (containing 4 µL methanol). The reaction was initiated by adding 5 mM
UDP-glucuronic acid and continued for 30 min in a 37oC water bath. The reaction was
stopped by adding 0.2 ml methanol to each reaction solution. A preliminary experiment
showed that the formation of 4-ABP-glucuronide in the reaction was linear for at least 60
min. The solutions were then centrifuged at 16,000g for 5 min at 4ºC, and the
supernatant portions were analyzed by HPLC. HPLC analysis of 4-ABP-glucuronide was
carried out using an Agilent system with a diode-array detector. The mobile phase
consisted of 50 mM potassium phosphate (pH 6.8) and methanol. The system was
operated at a flow rate of 1.75 ml/min and a partisil 10 ODS-2 reverse phase column (4.6
mm x 250 mm, Whatman) was used. A linear gradient gradually increased methanol
151
concentration: from 0-7 min, 35% to 55% and from 7-14 min, 55% to 80%. The 4-ABP-
glucuronide was monitored at 280 nm and eluted at 5.1 min, and the peak area was
integrated using the Agilent’s ChemStation software. A calibration curve of 4-ABP-
glucuronide was established using a pure standard (Y = 0.421X + 12.266, R2 = 1.0, where
Y is the peak area and X is pmol of 4-ABP-glucuronide injected to HPLC). Urinary
creatinine was measured using a creatinine kit purchased from Kaymen, according to
manufacturer’s instruction.
4.3.7 Capillary liquid chromatography and nESI-MS/MS of 4-ABP glucuronide
The presence of 4-ABP-N-glucuronide in the urine of mice treated with 4-ABP was
confirmed by LC-MS. 4-ABP-N-glucuronide was first enriched in 200 uL of mixed urine
from two wild type mice treated with 50 mg/kg 4-ABP. Briefly, 1 mL of acetonitrile was
added and the sample was centrifuged (Thermo Scientific, Sorvall RT-1) at 10,000 x g
for 10 minutes at 4ºC. The supernatant was removed, lyophilized, and reconstituted in 10
µL of 10 mM ammonium acetate. 10 pg of the 4-ABP-N-glucuronide standard was taken
through the same sample processing steps to verify minimal loss of the analyte. Capillary
liquid chromatography and nESI-MS/MS was performed using the same instrumentation
as previously described in chapter 3(32) but with modifications to the method as
follows: Following 1 µL sample injections, mobile phase A (10 mM ammonium acetate)
was held at 100% for 4.3 minutes. Mobile phase B (acetonitrile) was then linearly
increased to 65% over 2 min, held for 0.5 min, and stepped back down to 0% for a 5 min
re-equilibration period. MS/MS spectra were collected within a scan window of m/z 100-
152
400 and the precursor ion m/z 346 ± 1.0 was isolated and fragmented. In data analysis,
the extracted ion chromatogram selected to monitor for the loss of the glucuronide was
m/z 346�170.
4.3.8 Statistical analysis
The numerical results are expressed as means ± SD. Unpaired two-tailed Student t-test
was used for data analysis, with a P value <0.05 being considered significant (GraphPad
Version 5.00, GraphPad Software, San Diego, CA).
4.4 Results
We began this research by establishing the experimental dose conditions in human
bladder cells and mouse bladder tissue. To determine the effect of Nrf2 on 4-ABP DNA
adduct levels, we compared these levels in cells and tissues with Nrf2 to those without
Nrf2. Our findings that adduct levels were higher in wild type mice than Nrf2 knockout
mice initiated an investigation into the expression of Nrf2-regulated proteins, with a
focus on UGT enzymes that are implicated in bladder carcinogenesis.
153
4.4.1 Nrf2 protects human bladder carcinoma RT-4 cells from 4-ABP and N-OH-AABP
4.4.1 a Establishing experimental dosing conditions
In a dose-duration study, human bladder carcinoma RT-4 cells were treated with 50 µM
4-ABP plus S9 for 3, 6, or 24 hours and 4-ABP DNA adduct levels were measured. An
example extracted ion chromatogram of the standard dG-C8-4-ABP 435�319 and
isotopically labeled internal standard 444�328 from the 6 h set is shown in Figure 4.3.
In the first three hours, 4-ABP DNA adduct levels reached 430±40 adducts in 108
nucleosides, after 6 hours, the adduct level was measured to be 590±50 adducts in 108
nucleosides and after 24 hours, adduct levels fell back down to 51±4 adduct in 108
nucleosides (Figure 4.4).
Figure 4.3. An example EIC in the time course study. 4-ABP-induced DNA damage in
human bladder carcinoma RT-4 cells. RT-4 cells were treated with 4-ABP for 6 hours
and harvested for measurement of dG-C8-4-ABP levels by LC-MS/MS. The standard is
shown in red and the internal standard in blue.
2.5 5.0 7.5 10.0 Time [min]
0.0
0.5
5x10
Intens.
154
0
100
200
300
400
500
600
700
800
900
3 6 24
Duration of 50 µM 4-ABP treatment (h)
Nu
mb
er
of
dG
-AB
P a
dd
uc
ts
de
tec
ted
in
10
^8
nu
cle
os
ide
s
Figure 4.4. 4-ABP-induced DNA damage in human bladder carcinoma RT-4 cells in a
dose duration study. RT-4 cells were treated with 4-ABP and harvested for measurement
of dG-C8-4-ABP levels by LC-MS/MS.
Human bladder carcinoma RT-4 cells were exposed to 4-ABP at 0.05, 0.1, 0.25 or 0.5
mM for 3 h in the presence of a rat liver S9 and adduct levels were measured (Figure
4.5). The adduct level was below the limit of detection in cells not treated with S9 in the
presence 0.5 mM 4-ABP. Dose dependent DNA damage was detected: adduct levels at
0.05, 0.1, 0.25, and 0.5 mM 4-ABP were measured to be 520±50, 2530±200, 5100±800,
and 20000±3000 in 108 nucleotides, respectively.
155
Figure 4.5. 4-ABP-induced DNA damage in human bladder carcinoma RT-4 cells in a
dose concentration study. RT-4 cells were treated with 4-ABP and harvested for
measurement of dG-C8-4-ABP levels by LC-MS/MS.
4.4.1 b Effect of Nrf2 specific siRNA treatment
Nrf2 was silenced in RT-4 cells by treatment with a Nrf2-specific siRNA for 48 h.
Western blots of whole cell lysates from these Nrf2 siRNA treated cells were compared
to those from control siRNA treated RT-4 cells. GAPDH served as the loading control.
This western blot, shown in Figure 4.6 confirms that the control cells are expressing Nrf2
while the Nrf2 siRNA cells are not.
0
5000
10000
15000
20000
25000
500
(no S9)
50 100 250 500
4-ABP treatment (µM)
Nu
mb
er
of
dG
-AB
P a
dd
ucts
dete
cte
d i
n 1
0^
8 N
ucle
osid
es
156
Nrf2
GAPDH
Co
ntr
ol s
iRN
A
Nrf
2 s
iRN
A
Figure 4.6. A western blot of Nrf2 in whole cell lysates from RT-4 cells treated with
control siRNA and Nrf2 siRNA for 48 hours. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) is a loading control.
Both Nrf2 siRNA treated cells and control siRNA treated cells were then exposed to 4-
ABP at 0.5 mM (plus S9) or N-OH-AABP at 0.1 mM for 3 h and adduct levels were
quantified (Figure 4.7). As expected, in both cases higher levels of dG-C8-4-ABP were
detected in cells treated with Nrf2 siRNA. Number of adducts detected in 108
nucleosides in cells treated with 0.5 mM 4-ABP were 29000±5000 in Nrf2 siRNA cells
and 16000±1000 in control cells. Number of adducts detected in 108 nucleosides in cells
treated with 0.1 mM N-OH-AABP were 5600±400 in Nrf2 siRNA cells and 3200±200 in
control cells.
157
0
10000
20000
30000
40000
control Nrf2 siRNA
Nu
mb
er
of
dG
-AB
P a
dd
uc
ts
de
tec
ted
in
10^
8 n
uc
leo
sid
es
500 uM 4-ABP
100 uM N-OH-AABP
Figure 4.7. dG-C8-4-ABP DNA adduct levels in human bladder carcinoma RT-4 cells.
After 48 h treatment with control siRNA or Nrf2 siRNA, RT-4 cells were exposed to 4-
ABP plus S9 or N-OH-AABP for 3 h before dG-C8-4-ABP measurement.
4.4.2 Nrf2 potentiates 4-ABP DNA adduct formation in mouse bladder but inhibits 4-ABP
DNA adduct formation in mouse liver
Wild type and Nrf2 knockout C57BL/6 mice (6-8 weeks of age) were administered a
single dose of 50 mg/kg 4-ABP by i.p. injection and 4-ABP-DNA adduct levels in
bladder and liver tissues were measured (Figure 4.8, Figure 4.9). Bladder tissue dG-C8-
4-ABP levels in terms of adducts in 108 nucleotides were as follows: female wild type:
158
1100±200; female Nrf2-/-
: 1900±300; male wild type: 4500±200; male Nrf2-/-
: 2500±300.
Liver tissue dG-C8-4-ABP levels in terms of adducts in 108 nucleotides were as follows:
female wild type: 1900±300; female Nrf2-/-
: 2700±300; male wild type: 480±50; male
Nrf2-/-
: 830±40. Dose-response data from male mice administered a single dose of 0, 5,
10, and 50 mg/kg was linear and for each dose, wild type mice had higher adduct levels
than Nrf2 knockout mice.
0
1000
2000
3000
4000
5000
Female Male
Nu
mb
er
of
dG
-AB
P a
dd
uc
ts
de
tec
ted
in
10
^8
nu
cle
os
ide
s
WT
Nrf2 KO
Figure 4.8. dG-C8-4-ABP adduct levels in wild type and Nrf2 knockout mouse bladder
tissue following treatment with 50 mg/kg 4-ABP. After 24 hours, they were sacrificed
and dG-C8-4-ABP levels in bladder tissue were measured by LC-MS/MS.
159
0
500
1000
1500
2000
2500
3000
3500
female male
mouse gender
Nu
mb
er
of
dG
-AB
P a
dd
ucts
dete
cte
d i
n 1
0^
8 n
ucle
osid
es
wild type
Nrf2 ko
Figure 4.9. dG-C8-4-ABP adduct levels in wild type and Nrf2 knockout mouse liver
tissue following treatment with 50 mg/kg 4-ABP. After 24 hours, they were sacrificed
and dG-C8-4-ABP levels in bladder tissue were measured by LC-MS/MS.
Our observations that adduct levels in the bladder tissue of male wild type mice were
higher than those in Nrf2 knockout mice required further study. Accordingly, the next
phase of this project involved an examination of the expression of key Nrf2-target genes
with particular attention to those that are known to be involved in 4-ABP metabolism. As
glucuronidation facilitates transport of 4-ABP from the liver to the bladder and is
associated with 4-ABP-induced bladder carcinogenesis, it was not surprising to find that
Nrf2-induced UGT enzymes may be partially responsible for increasing 4-ABP DNA
adduct levels in bladder tissue of male mice.
160
4.4.3 Nrf2 promotes UGT-mediated Phase 2 metabolism and urinary excretion of 4-
ABP
Western blot analyses of the liver enzymes N-acetyltransferases (NAT1 and NAT2) and
UGTs were compared between male wild type mice and Nrf2 knockout mice (Figure
4.10). Concentrations of NAT1 and NAT2 were not affected by the presence of Nrf2.
One UGT1A and one UGT2B were up-regulated in Nrf2+/+
mice compared to Nrf2-/-
mice.
NAT1
NAT2
UGT1A
UGT2B
Nrf2+/+
Nrf2-/-
GAPDH
Figure 4.10. Western blots of liver homogenates from Nrf2+/+
mice and Nrf2-/-
mice.
Arrows point to the UGT isozymes that were up-regulated by Nrf2. GAPDH is a loading
control.
161
In view of the liver homogenate western blot analysis, UGT enzymes were further
examined for their response to Nrf2 in an enzyme activity assay. Enzymatic activity in
catalyzing the conjugation of 4-ABP with glucuronic acid was 1.94 fold higher in wild
type mice than in Nrf2-deficient mice (Figure 4.11).
0
Nrf2+/+
Nrf2-/-
Liv
er
UG
T a
cti
vit
y
(nm
ol/m
g/m
in)
0.08
0.16
0.24
P=0.0066
Figure 4.11. S9 fractions were prepared from liver tissues of Nrf2+/+
mice and Nrf2-/-
mice and measured for UGT activity in catalyzing 4-ABP conjugation with glucuronic
acid.
162
Wild type and Nrf2 deficient mice were treated with 4-ABP at 50 mg/kg and 24-h urinary
level of 4-ABP-N-glucuronide (adjusted by urinary creatinine) was measured. 4-ABP-N-
glucuronide level was 34% higher in the wild type mice than in the Nrf2-deficient mice
(Figure 4.12).
A
BP
-G/c
rea
tin
ine
in u
rine
(µm
ol/m
g)
Nrf2+/+
Nrf2-/-
2
4
6
0
P=0.0225
Figure 4.12. Total amounts of 4-ABP-N-glucuronide were measured in 24 h urine
collected from Nrf2+/+
mice and Nrf2-/-
mice given 50 mg/kg 4-ABP and were adjusted
by urinary creatinine levels.
163
The 4-ABP-N-glucuronide was analyzed by LC-MS for structural confirmation. The full
scan of 1 ng 4-ABP-N-glucuronide standard (Figure 4.13) showed one obvious peak at
8.8 min with a mass spectrum base peak of m/z 346.1.
A. 0 2 4 6 8 10 Time [min]
0.0
0.5
1.0
1.5
2.0
8x10
Intens.
0 2 4 6 8 10 Time [min]
0.0
0.5
1.0
1.5
2.0
8x10
Intens.
B. 0 2 4 6 8 10 Time [min]
0.00
0.25
0.50
0.75
1.00
1.25
8x10
Intens.
0 2 4 6 8 10 Time [min]
0.00
0.25
0.50
0.75
1.00
1.25
8x10
Intens.
C.170.1
214.0
346.1
360.1
1. +MS, 8.7-8.9min #(2221-2297)
0
1
2
3
4
7x10
Intens.
100 150 200 250 300 350 m/z
Figure 4.13. Full scan of 1 ng 4-ABP-N-glucuronide synthetic standard. A. Full scan.
B. EIC 346.1 C. Mass spectrum.
164
The LC-MS/MS of the 4-ABP-N-glucuronide standard is seen in Figure 4.14 in which
the mass spectrometer was set to isolate and fragment the precursor ion m/z 346.1 ± 1.0.
The extracted ion chromatogram 346�170 represents the loss of the glucuronide,
[M+H]+�[M+H-176]
+.
Figure 4.14. LC-MS/MS of 4-ABP-N-glucuronide standard. The top panel shows the
extraction ion chromatogram m/z 346 �170 and the bottom panel shows the mass
spectrum under this peak.
170.1
194.0
212.1266.1
310.1
328.2
1. +MS2(346.1), 8.7-8.9min #(821-842)
0
2
4
4x10
Intens.
150 200 250 300 350 m/z
1
2 4 6 8 Time [min]
0
2
4
6
4x10
Intens.
165
The chromatogram of the mouse urine sample had a major peak at 8.8 minutes, matching
the retention time of the neat standard (Figure 4.15). Furthermore, the mass spectrum
under this peak showed the same major fragments as those observed in standard mass
spectrum.
1
2 4 6 8 Time [min]
0
2
4
4x10
Intens.
170.0
194.1
212.2 234.2 264.1
310.2328.1
+MS2(346.1), 8.8-8.8min #(826-827)
0
1
2
3
4
4x10
Intens.
150 175 200 225 250 275 300 325 350 m/z
Figure 4.15. Injection of mouse urine sample. 200 µL of urine from two wild type mice
that were administered 50 mg/kg 4-ABP was taken through an acetonitrile precipitation
and injected for detection of 4-ABP-N-glucuronide.
166
4.5 Discussion
4.5.1 Dose-response
High 4-ABP DNA adduct levels were preferred in these experiments because
forthcoming chemoprevention research will use high concentrations of chemopreventive
agents. Although such levels of exposure are not typically observed in humans, the same
level of exposure by one carcinogen in real life leads to greater DNA damage than would
be observed in an experimental setting. This is because typical human exposure occurs
over a much longer time period, allowing for an accumulation of genetic damage.
Furthermore, in real life, exposure to one carcinogen is combined with exposure to other
carcinogens, instigating a synergistic effort to cause DNA damage. Based on our dose-
response results, for the majority of our experiments we chose to treat cells with 500 µM
4-ABP plus S9 for 3 hours.
4-ABP treatment of human bladder carcinoma RT-4 cells for 3, 6 and 24 hours led to
rapid dG-C8-4-ABP formation followed by rapid repair of these adducts. In the first 3
hours, DNA adduct levels reached 5 adducts in 106 nucleotides but only increased 1.4
fold to 8 adducts in 106 nucleotides. This indicated that the majority of the adducts had
already formed in the first three hours. However, after 24 hours, adduct levels fell
substantially to 1 adduct in 106 nucleotides, 9% of the level observed at the 6 hour mark.
We attributed this decrease in adduct level to repair as RT-4 cells carry the normal p53
167
(33) and p53 has been shown to be required for repair of 4-ABP-induced genomic DNA
damage in human bladder cells (34).
Dose dependent DNA damage was observed in human bladder carcinoma RT-4 cells
after exposure to 4-ABP at 0.05, 0.1, 0.25, or 0.5 mM for 3 hours in the presence of rat
liver S9 activation system. Adduct levels were below the limit of detection in cells that
were treated with 0.5 mM 4-ABP not in the presence of S9. This confirmed that the
bladder cells were deficient in the enzymes that activate 4-ABP and that S9 was required
for 4-ABP activation. The occurrence of a linear dose-response curve suggests that
sufficient S9 was being added to activate 4-ABP in the 3 hour dosing experiment and that
neither activation, detoxification nor repair was saturated.
4.5.2 Nrf2 protects bladder cells in vitro
Nrf2 provided protection against 4-ABP and N-OH-AABP in human bladder carcinoma
RT-4 cells. Compared to control cells containing Nrf2, dG-C8-4-ABP levels in Nrf2-
silenced cells following treatment with either 4-ABP or N-OH-AABP were about 2-fold
higher. This suggests that Nrf2 is involved in the detoxification of both 4-ABP and N-
OH-AABP.
4.5.3 In vivo detection of dG-C8-4-ABP in mouse liver and bladder tissue
The C57Bl/6 mouse strain used in the research presented in this dissertation is a rapid
acetylator strain. Therefore, a significant portion of the 4-ABP dose is expected to be
168
acetylated and detoxified from the liver before activation occurs. This acetylated
conjugate can be oxidized, glucuronidated, and transported to the bladder where it is
excreted even in acidic conditions (20). In wild type mice, however we still observed
high adduct levels ranging from about 5 to 50 adducts in 106 nucleotides.
Our animal study involved comparisons between adduct levels in male and female mice
because gender may affect bladder cancer risk. Epidemiological studies have indicated
that men are more likely to develop bladder cancer than women, and this has been found
to be true even among smokers who are exposed to more bladder carcinogens such as 4-
ABP (24, 35). In one study, male mice that were chronically exposed to 4-ABP
developed bladder cancer and had higher levels of DNA adduct levels in the bladder than
females that developed liver cancer and had higher levels of DNA adducts in the liver
(36). 4-ABP targets the liver in female BALB/c mice and the bladder in BALB/c male
mice (36-38). It has been suggested that this difference may be due to variations in N-
glucuronidation or N-acetylation, leading to differences in effective dose delivered to the
bladder. Because 4-ABP targets the liver in female mice and because 4-ABP is primarily
metabolized and activated in the liver in both genders, we chose to examine the effect of
Nrf2 on dG-C8-4-ABP levels in liver tissue in addition to bladder tissue.
As expected, dG-C8-4-ABP levels were higher in bladder tissue of male mice than
female mice, and higher in liver tissue of female mice than male mice. Furthermore,
more adducts formed in the bladder than in the liver in male mice, and more adducts
169
formed in the liver than in the bladder in female mice. The presence of Nrf2 had a
protective effect in both types of tissue in female mice, but only in the liver in male mice.
Interestingly, higher levels of dG-C8-4-ABP were detected in bladder tissue of wild type
male mice than Nrf2-/-
male mice. This is the opposite effect that was observed in female
mice and human bladder RT-4 cells. This reversed effect from bladder cells indicates
that Nrf2 is acting outside the bladder to potentiate 4-ABP-induced DNA damage in the
bladder. Knowing that 4-ABP is metabolized in the liver and that N-glucuronidation and
N-acetylation are important in the metabolism of 4-ABP, subsequent experiments
examined the effect of Nrf2 on the key catalyzing enzymes in these reactions.
Nrf2 activating compounds have in fact been shown to shift organ carcinogenicity from
the liver to the bladder in previous studies. For example, treating rats with the Nrf2-
activating compound butylated hydroxytoluene (BHT) has been shown to reduce N-2-
fluorenylacetamide (FAA)-induced liver tumors but increase FAA-induced bladder
tumors (39). Moreover, only liver tumors were observed in mice not treated with BHT.
Accordingly, the observation that Nrf2 is involved in bladder carcinogenesis is not
unprecedented. However, that Nrf2 potentiates DNA adduct levels caused by 4-ABP
specifically is novel.
170
4.5.4 Nrf2 promotes UGT-mediated Phase 2 metabolism and urinary excretion of 4-
ABP
Western blot experiments were carried out to investigate the contrasting in vitro and in
vivo results in bladder cells of male mice. Because knocking out Nrf2 in male mice
treated with 4-ABP resulted in fewer DNA adducts than their wild-type counterpart, we
examined the effect of Nrf2 on NAT and UGT enzymes in male mice. Western blots of
liver homogenates from male Nrf2+/+
mice and Nrf2-/-
mice showed that Nrf2 did not
have an effect on the concentrations of NAT1 and NAT2 but that it up-regulated one
UGT1A enzyme and one UGT2B enzyme. While neither NAT1 nor NAT2 have been
shown to be regulated by Nrf2, a number of UGT isoforms are up-regulated by Nrf2 (17-
19). The specific UGT isoforms that were up-regulated by Nrf2 in our research were not
identified, but this is important to consider in future research in view of the potentially
important role of UGT in bladder cancer. We confirmed that UGT activity and 4-ABP-
N-glucuronide levels were increased in Nrf2+/+
mice compared to Nrf2-/-
mice. These
results support the hypothesis that UGT enzymes are induced by Nrf2 and are involved in
the detoxification and transport of 4-ABP in male mice.
Similarly, it was suggested that glucuronidation of benzidine was involved in benzidine-
induced bladder cancer. The authors proposed that the N-glucuronide of benzidine in the
liver resulted in the transport and accumulation of benzidine in the bladder (40).
171
4.6 Conclusions
Nrf2 is known as a cytoprotective protein involved in defense against chemical
carcinogens and oxidants and as a promising target in chemoprevention, it is widely
studied. Our research has shown that Nrf2 locally protects cells against DNA damage
cause by 4-ABP. In vivo systems are complex, however, and our results have indicated
that Nrf2-induced 4-ABP detoxification in one tissue may lead to an increase in its
bioavailability in another. In fact, we found that Nrf2 induction of liver glucuronosyl
transferases leads to high levels of bladder DNA adduct formation.
We observed differences in 4-ABP DNA adduct formation based on gender, which has
previously been reported in the literature (36-38). In female mice, the effect of Nrf2 on
4-ABP DNA adduct formation in the bladder and liver was similar to that in bladder
cells; Nrf2 protected against 4-ABP-induced damage and thus its activation would be
expected to be an effective strategy against 4-ABP. While this was also true in liver
tissue of male mice, the opposite effect occurred in male bladder tissue. This may be due
in part to Nrf2 induced up-regulation of UGT enzymes that metabolize and transport 4-
ABP.
Therefore, induction of liver UGT may be harmful to the bladder in humans exposed to
4-ABP, particularly in smokers who are exposed to greater quantities of the carcinogen.
It is important that a cancer chemopreventive agent that activates Nrf2 does not
172
significantly induce liver UGT. Accordingly, activation of Nrf2 is a suitable
chemopreventive strategy against 4-ABP provided that the chemopreventive agent is
specific to bladder tissue or does not up-regulate UGT in liver tissue.
173
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178
CHAPTER 5
INHIBITION OF 4-ABP DNA ADDUCT FORMATION BY SULFORAPHANE
AND 5,6-DIHYDROCYCLOPENTA[C][1,2]-DITHIOLE-3(4H)-THIONE IN
BLADDER CELLS AND TISSUES
179
5.1 Promising chemopreventive agents from cruciferous vegetables
In chapter 4 we concluded that activation of Nrf2 should be considered as a strategy for
chemoprevention of 4-ABP-induced bladder cancer, provided that the UGT enzymes in
the liver tissue are not up-regulated. The promising chemopreventive agents
Sulforaphane (4-methylsulphinylbutyl isothiocyanate, SF) and 5,6-
dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) have been shown to mediate
some of their cytoprotective effects through Nrf2 and moreover they have shown
specificity to the bladder. Furthermore, it is well recognized that SF and CPDT both
induce Phase 2 enzymes which are involved in carcinogen detoxification. We
hypothesized that SF and CPDT inhibit 4-ABP DNA adduct formation and act through
mechanisms involving Nrf2 to carry out these cytoprotective effects.
SF, an isothiocyanate and CPDT, a dithiolethione are derived from compounds found in
cruciferous vegetables (plants of the family Brassicaceae or Cruciferae) (1). Their
chemical structures are shown in Figure 5.1. Both have shown promise as anticancer and
anticarcinogenic compounds, acting on all stages of carcinogenesis and interfering with
several cell-signaling pathways (2, 3).
180
CH3
S
O
N
S
S
S
S
Figure 5.1. Chemical structures of SF and CPDT.
5.1.1 Sulforaphane, an isothiocyanate (4)
SF is a well known chemopreventive phytochemical derived from the glucosinolate
Glucaraphanin (β-thioglucoside N-hydroxysulfate), in which broccoli sprouts are
especially rich (~ 1000 µmol SF per kg fresh broccoli). The conversion of
Glucuraphanin to SF is shown in Figure 5.2. When damage to the plant occurs, such as
that from food processing, chewing, or digestion, glucaraphanin comes in contact with
myrosinase (thioglucoside glucohydrolase) and is deglycosylated. Glucuraphanin that
reaches the intestinal tract without coming in contact with myrosinase can be hydrolyzed
by microflora. Following hydrolysis, the intermediate thiohydroximate-O-sulfonate can
either be spontaneously converted to the chemopreventive agent SF or catalytically
converted to the SF nitrile by Epithiospecifier protein (ESP).
The glucuraphanin content of a particular vegetable depends on a number of factors
including variety, age, and growth conditions (5). Broccoli sprouts, for example are
181
much richer in glucuroaphanin than mature florets. In addition to this, the SF yield varies
depending on storage conditions, cooking conditions, metabolism of the devouring
individual, and enteric bacteria in an individual (4, 6, 7). High heat, such as that during
steaming or boiling denatures myrosinase and decomposes SF. Heating at 60-70ºC,
however, can denature ESP without inactivating myrosinase, resulting in a high SF yield.
The sulfur in the sulfinyl functional group is a chiral center of Sulforaphane, but R-SF
and S-SF show the same chemopreventive properties. Broccoli however, yields only R-
SF (8).
CH3
S
O
N
S
CH3
S
O
N
CH3
S
O
SH
N
O
S
O
OO
-
CH3
S
O N
O
S
O
OO
-
S-β-D-glucoseGlucoraphanin
Myrosinase
ESPSpontaneous
SFSF nitrile
Figure 5.2. Conversion of glucoraphanin to SF or the SF nitrile. (Reproduced from (4))
182
SF has been reported to have a number of chemopreventive properties; it has been shown
to inhibit carcinogen-activating enzymes, induce carcinogen deactivating enzymes,
induce apoptosis, arrest cell cycle progression, inhibit inflammation and angiogenesis,
inhibit the growth of cancer cells, prevent cancer development, reduce damage caused by
carcinognens, induce cell death, and affect the cell cycle, invasion and metastasis (4, 9,
10).
SF may also be particularly important in bladder cancer chemoprevention and several
studies have shown that SF is selectively delivered to bladder tissue through urinary
excretion (9, 11, 12). Furthermore, epidemiological research has found a link between
consumption of SF-rich foods and decreased human bladder cancer risk (12-14).
5.1.2 SF inhibits DNA adduct formation and induces Phase 2 enzymes
Several studies have shown that SF inhibits the formation of DNA adducts in a dose-
dependent manner and induces Phase 2 enzymes (15-19). In one study, SF inhibited 2-
amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adduct formation in both
human HepG2 cells and human hepatocytes as measured by Accelerator Mass
Spectrometry and induced UDP-glucuronosyltransferase (UGT) 1A1 and glutathione S-
transferase (GST) A1 mRNA expression. In this study, SF did not appear to affect DNA
repair (15). In another study, SF was found to inhibit the formation of benzo[a]pyrene
(B[a]P) and 1,6-dinitropyrene (1,6-DNP) DNA adducts in MCF-10F cells as measured by
32P-postlabeling and increase the expression of NAD(P)H-quinone reductase (QR) and
183
GST proteins (16). In addition, several studies have shown that SF inhibits DNA adducts
of the human liver carcinogen Aflatoxin B1 (AFB1) and these observations have also been
made in conjunction with experiments showing its induction of Phase 2 enzymes.
In vitro, SF inhibited AFB1 DNA adduct formation in human hepatocyte cultures as
observed by liquid scintillation counting, and GSTM1 was shown to be involved in AFB1
DNA adduct inhibition. SF did not increase GSTM1 expression but it did decrease
CYP3A4 mRNA. SF, however was only effective in inhibiting the formation of AFB1
DNA adducts when cells were treated with it before the carcinogen rather than at the
same time or after, indicating that its protective effects were dependent on changes in
gene expression rather than inhibition of catalytic activity. Compared to its analog
phenylethyl isothiocyanate (PEITC), SF was more effective in reducing AFB1 DNA
adduct levels, although both were effective inhibitors (Figure 5.3) (19).
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Figure 5.3. SF and PEITC reduced AFB1 DNA adduct formation in human hepatocyte
cultures. Hepatocytes were treated with the phytochemical and then were co-incubated
with 3H-AFB1. The mean control value from cells only treated with
3H-AFB1 was 5.9
adducts in 107 nucleotides. (Reproduced from (19))
In vivo, SF reduced AFB1 DNA adduct formation and increased GST levels in two
strains of rats. In this study, AFB1-N7-gua was hydrolyzed from liver tissue DNA and
quantified by UPLC-MS. Interestingly, both chemopreventive agents had a greater effect
on Sprague Dawley rats, which are more resistant to AFB1 adduct formation than Fischer
rats in reducing both adduct formation and increasing GST levels. Although the
researchers also investigated gender-specific responses to SF, no differences were
observed (17).
185
A human study was also conducted to measure the chemopreventive potential of SF in
people at high risk of developing AFB1-induced hepatocellular carcinoma. Participants
consumed 3-day-old broccoli sprouts or a placebo and their urinary levels of AFB1-N7-
gua were measured by LC-MS/MS. Although no correlation between broccoli sprout
consumption and reduced levels of AFB1-N7-gua was found, this was attributed to the
large variation in glucosinolate concentration in the broccoli sprout mixtures. An inverse
correlation however was found between the SF metabolites dithiocarbamates and AFB1-
N7-gua (18).
5.1.3 CPDT, a dithiolethione (3, 20)
Many dithioloethiones have shown promise as chemopreventive agents, particularly in
their induction of cytoprotective proteins that are involved in the detoxification of
carcinogens. 3H-1,2-dithiole-3-thione (D3T) represents the basic structure of these
chemopreventive dithiolethiones and a number of its analogs have been synthesized.
Both 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (ADT) and 4-methyl-5-pyrazinyl-
3H-1,2-dithiole-3-thione (Oltipraz) are well studied and are shown in Figure 5.4.
Oltipraz has been shown to decrease N-nitrosobutyl(4-hydroxybutyl)amine (BBN)
induced bladder cancer in vivo and is effective against AFB1, B[a]P, and PhIP. A
number of clinical trials have also been conducted on Oltipraz but it has exhibited limited
efficacy with substantial side effects.
186
SS
S
D3T
N
N
SS
CH3
S
SS
S
O
CH3
Oltipraz ADT
Figure 5.4. The structures of three dithiolethiones D3T, Olitpraz, and ADT.
Several different dithiolethiones were compared based on their mitigation of AFB1 DNA
adduct levels. In this study, rats were pre-treated with dithiolethiones followed by
administration of AFB1, and AFB1 DNA adducts in liver tissue were measured by
radiometric methods. All dithiolethiones, including CPDT reduced adduct levels
compared to the control levels from rats that were not pre-treated with a dithiolethione
(21).
CPDT was shown to be a highly effective Nrf2 activator and Phase 2 enzyme inducer in
cultured bladder cells, and its induction of Phase 2 enzymes was shown to depend on
Nrf2. In a rat study in vivo, CPDT was particularly effective in the bladder, and more
significantly, Nrf2 activation and induction of Phase 2 enzymes in the bladder by CPDT
187
occurred exclusively in the epithelium, which is the principal site of bladder cancer
development (22). Its activation of Nrf2, induction of Phase 2 enzymes and bladder
tissue specificity was confirmed in a separate study (23).
5.2 Project Goals
The ultimate goal of this research was to determine whether or not SF and CPDT inhibit
4-ABP DNA adduct formation in bladder cells and tissues. Because we have found that
Nrf2 is a promising target for inhibition of 4-ABP DNA adducts, we first investigated
whether or not we could observe an inductive effect of SF and CPDT on Nrf2. Following
our observations that Nrf2 was induced by both SF and CPDT, we examined the effect of
these agents on adduct levels in cells and animals with and without Nrf2.
5.3 Experimental
All cell and animal dosings and western blots were carried out and analyzed in Dr.
Yuesheng Zhang’s laboratory at Roswell Park Cancer Institute, Buffalo, NY by Joseph
Paonessa and Yi Ding.
188
5.3.1 Chemicals
SF, 4-ABP, and N-hydroxy-N-acetyl-4-aminobiphenyl (N-OH-AABP) were purchased
from LKT Laboratories (St. Paul, MN), Sigma (St. Louis, MO), and Midwest Research
Institute (Kansas City, MO), respectively. CPDT was synthesized (24). Rat liver S9 (36-
43 mg protein/ml) was purchased from Moltox (Boone, NC). Antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA), including antibodies specific for the
catalytic subunit and the regulatory subunit of glutamate cysteine ligase (GCSc and
GCSm), NAD(P)H:quinone oxidoreductase 1 (NQO1) and Nrf2, from Alpha Diagnostic
International (San Antonio, TX), including antibodies specific for the α isoform of
glutathione S-transferase (GST), and from Millipore (Billerica, MA) for the antibody of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Another antibody against NQO1
purchased from Cell Signaling Technology (Danvers, MA) was used in some of the
Western blot analyses.
5.3.2 Cell study
Human bladder carcinoma RT-4 cells were cultured (25). The cell line was purchased
from ATCC (Manassas, VA), stored in liquid nitrogen, and passaged in our laboratory for
fewer than 6 months. To induce DNA damage, RT-4 cells (2-3 x 106 cells) were grown in
each 10-cm dish with 10 ml medium 24-48 h, followed by treatment with either 4-ABP or
N-OH-AABP for 3 h in 5 ml fresh medium per dish. In cases where cells were co-treated
with 4-ABP and S9, the medium also contained 6% S9 (v/v), 10 mM glucose-6-
phosphate and 5 mM NADP. In experiments involving pretreatment with a
189
chemopreventive agent, cells were first treated with SF, CPDT or vehicle and after 24 h
or 48 h, exposed to the carcinogen. All test agents were dissolved in DMSO, and the
final concentration of DMSO in medium was ≤ 5%. Cells were harvested by trypsin
treatment and low-speed centrifugation, and cell pellets were washed with ice-cold PBS
and used for DNA adduct analysis.
5.3.3 Animal study
Wild type and Nrf2-/-
C57BL/6 mice (male, 6 weeks of age) were used. The wild type
mice were purchased from National Cancer Institute (Frederick, MD). The Nrf2-deficient
mice were bred at the animal facility at Roswell Park Cancer Institute; the breeders were
kindly provided by Dr. Thomas W. Kensler (Johns Hopkins Bloomberg School of Public
Health) (26). Groups of 3-4 mice were randomized and treated with SF or vehicle by
gavage once daily for 5 days. Three hours after the last SF dose, the mice were each
administered a single dose of 4-ABP i.p. and were sacrificed 24 h after 4-ABP treatment
to collect the livers and bladders for measurement of dG-C8-4-ABP. For CPDT
intervention, the mice were treated with CPDT (in soy oil) or the vehicle by gavage once
daily for 5 days; 4-ABP was given 3 h after the last CPDT dose. To determine the effect
of SF or CPDT on Phase 2 enzymes by Western blotting, mice were treated with SF or
CPDT by gavage once daily for 5 days and sacrificed 24 h later. Liver and bladder tissues
were stored at -80oC. All animal protocols and procedures were approved by the Roswell
Park Cancer Institute Animal Care and Use Committee.
190
5.3.4 Quantification of dG-C8-4-ABP
Procedures of DNA isolation from bladder cells and tissues and the detailed protocol for
the quantitative analysis of dG-C8-4-ABP by capillary liquid chromatography and nESI-
MS/MS have been described in Chapter 3 of this dissertation and in a recent publication
(27).
5.3.5 Western blot analysis
Cellular and tissue levels of Nrf2 and/or several Phase 2 enzymes were measured by
Western blotting (22). All antibodies were purchased from Santa Cruz Biotechnology,
except for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which was purchased
from Millipore.
5.3.6 Statistical analysis
The numerical results are expressed as means ± SD. Unpaired two-tailed Student t-test
was used for data analysis, with a P value <0.05 being considered significant (GraphPad
Version 5.00, GraphPad Software, San Diego, CA).
191
5.4 Results
Before investigating the effect of SF and CPDT on 4-ABP DNA adduct levels, we
examined their effect on the expression of Nrf2 and Nrf2-regulated proteins. After
observing that both agents did indeed induce Nrf2 and the Nrf2 signaling pathway, we
compared the effect of these agents on cells with and without Nrf2 with the goal of
evaluating the compounds as chemopreventive agents and determining the function of
Nrf2 in their chemopreventive activities.
5.4.1 SF and CPDT activate Nrf2 and the Nrf2 signaling pathway in human bladder
cells and mouse bladder tissue
The effect of SF treatment on Nrf2 and Nrf2-regulated enzymes was examined in human
bladder carcinoma RT-4 cells. Western blot analyses of RT-4 cells that were treated with
0, 2, and 4 µM SF for 24 h were conducted on Nrf2 and several Phase 2 enzymes
including the catalytic subunit of glutamate cysteine ligase (GCSC), the regulatory
subunit of GCS (GCSM) and NAD(P)H:quinone oxidoreductase 1 (NQO1) (Figure 5.5).
Treatment with SF increased expression of Nrf2 and Nrf2-regulated GCSC, GCSM and
NQO1. Western blots of UDP-glucuronosyltransferases (UGTs) and N-acetyltransferases
(NATs) however, did not indicate a change in expression levels following treatment with
SF.
192
SF (µM) 0 2 4
GCSM
GAPDH
Nrf2
GCSC
NQO1
Figure 5.5. SF activates Nrf2 and the Nrf2 signaling pathway in human bladder
carcinoma RT-4 cells. RT-4 cells were treated with SF for 24 h and then harvested for
measurement of Nrf2 and selected Phase 2 proteins by Western blotting. GAPDH is a
loading control. The data are representative of two experiments.
Human bladder carcinoma RT-4 cells were then treated with 0, 12.5, and 25 µM CPDT
for 48 h and activation of Nrf2 and Nrf2-regulated proteins was measured by western blot
analyses (Figure 5.6). Treatment with CPDT resulted in a significant increase in the
expression of Nrf2, NQO1, GCSc and GCSm. Two UGT1A bands and four UGT2B
bands were detected in RT-4 cells, but CPDT had no effect on UGT1A and only slightly
elevated two UGT2B isoforms. The identities of these isoforms have not been
determined.
193
Figure 5.6. CPDT activates Nrf2 and the Nrf2 signaling pathway in human bladder
carcinoma RT-4 cells. RT-4 cells were treated with CPDT for 24 h. Nrf2 and Phase 2
enzymes in whole cell lysates were measured by Western blotting. GAPDH is a loading
control. The arrows indicate two UGT2B bands (isoforms) induced by CPDT.
Wild-type C57BL/6 mice and Nrf2 knocked out C57BL/6 mice were treated with vehicle
or SF by gavage once a day for 5 days. The mice were killed 24 h later for measurement
of selected Phase 2 proteins by Western blotting (Figure 5.7). Mice treated with 0
µmol/kg SF showed significantly lower Phase 2 enzyme expression in both the bladder
and liver tissue in Nrf2-/-
mice compared to Nrf2+/+
mice. In both the bladder and liver of
wild type mice, SF treatment resulted in increased expression of glutathione S-transferase
CPDT (µM) 0 12.5 25
Nrf2
NQO1
GAPDH
GCSc
GCSm
UGT1A
UGT2B
194
(GST), GCS, and NQO1 but this did not occur in Nrf2-/-
mice. SF treatment did not
affect the expression levels of UGTs and NATs in either tissue as indicated by western
blots.
Figure 5.7. Wild-type C57BL/6 mice and Nrf2 knocked out C57BL/6 mice were treated
with vehicle or SF by gavage once a day for 5 days. The mice were killed 24 h later for
measurement of selected Phase 2 proteins by Western blotting. GAPDH is a loading
control. The data are representative of mice in each group.
195
Nrf2+/+
mice and Nrf2-/-
mice were treated with CPDT or vehicle by gavage once daily
for 5 days; 24 h later after the last CPDT dose, animals were killed, and Phase 2 enzymes
in the bladders and livers were measured by Western blotting (Figure 5.8). Mice treated
with 0 µmol/kg CPDT showed significantly lower Phase 2 enzyme expression in both the
bladder and liver tissue in Nrf2-/-
mice compared to Nrf2+/+
mice. In the bladder of wild
type mice, CPDT activated Nrf2, GCSc, GCSm, NQO1, one isoform of UGT1A, and one
isoform of UGT2B. In the liver of wild type mice, CPDT induced NQO1, one isoform of
UGT1A, several isoforms of UGT2B, but not GCSc, GCSM, and the remaining isoforms
of UGT1A and UGT2B. CPDT did not induce any of the enzymes examined in both the
bladder and liver of Nrf2-/-
mice. GCSc and GCSm were undetectable in the bladder of
Nrf2-/-
mice but significant levels of these enzymes were detected in their liver tissues.
Multiple bands of both UGT1A and UGT2B were detected in the bladder and liver,
reflecting their multi-isomeric nature, but only one band in each family was up-regulated
by Nrf2. The arrows indicate the UGT bands (isoforms) that were regulated in an Nrf2-
dependent manner. The Nrf2-regulated UGT bands were slightly elevated by CPDT in
the bladder tissues, and to a lesser extent in the liver tissues.
196
Figure 5.8. Nrf2+/+
mice and Nrf2-/-
mice were treated with CPDT or vehicle by gavage
once daily for 5 days; 24 h later after the last CPDT dose, animals were killed, and Phase
2 enzymes in the bladders and livers were measured by Western blotting. GAPDH is a
loading control. The arrows indicate the UGT bands (isoforms) that were regulated in an
Nrf2-dependent manner.
GCSc
CPDT
(µmol/kg)0 5 20 80 0 5 20 80
Nrf2+/+ mice Nrf2-/- mice
NQO1
GAPDH
GCSm
0 5 20 80 0 5 20 80
Nrf2+/+ mice Nrf2-/- mice
Bladder Liver
UGT1A
UGT2B
197
5.4.2 SF inhibits dG-C8-4-ABP formation in bladder carcinoma cells
RT-4 cells were pretreated with 0, 2 or 4 µM SF for 24 h and then exposed to 0.5 mM 4-
ABP plus S9 for 3 h. They were harvested and dG-C8-4-ABP levels were examined
(Figure 5.9). Samples prepared in the absence of SF are used as a positive control and
percent inhibition is measured based on the adduct levels of these samples. Compared to
the positive control, cells treated with 2 and 4 µM SF had 70±6% and 59±9% lower
adduct levels, respectively. RT-4 cells that were pretreated with SF for 24 h and then
exposed to 0.5 mM 4-ABP plus S9 for only 1 h showed 52±3% and 45±6% lower adduct
levels than the positive control following 2 and 4 µM SF pretreatment. When RT-4 cells
were pretreated with only 1 and 2 µM SF for 24 h and then exposed to 0.5 mM 4-ABP
plus S9 for 3 h, 68±4% and 67±4% lower adduct levels were observed than the positive
control following 1 and 2 µM SF pretreatment.
198
-100
-50
0
50
100
0 2 4
SF treatment (µM)%
ch
an
ge i
n a
dd
uc
t le
ve
l
-70%-59%
-100
-50
0
50
100
0 2 4
SF treatment (µM)
% c
ha
ng
e i
n a
dd
uc
t le
ve
l
-52% -45%
-100
-50
0
50
100
0 1 2
SF treatment (µM)
% c
ha
ng
e i
n a
dd
uc
t le
ve
l
-68%-67%
Figure 5.9. 4-ABP-induced DNA damage in human bladder cells and the protective
effect of SF. RT-4 cells were pretreated with SF for 24 h and then exposed to 0.5 mM 4-
ABP plus S9 before measurement of dG-C8-4-ABP levels. In the top panel cells were
exposed to 2 and 4 µM SF pretreatment and followed by 4-ABP treatment for 3 hours. In
the middle panel cells were exposed to 2 and 4 µM SF pretreatment and followed by 4-
ABP treatment for 1 hours. In the bottom panel, cells were exposed to 1 and 2 µM SF
pretreatment and followed by 4-ABP treatment for 3 hours.
199
Human bladder carcinoma RT-4 cells and rat bladder carcinomoa NBT-II cells were
treated with 0, 2, or 4 µM SF in sprout form for 24 h and then exposed to 0.5 mM 4-ABP
plus S9 for 3 h. Subsequent measurement of dG-C8-4-ABP showed a decrease in adduct
levels following SF treatment in both cell types (Figure 5.10, Figure 5.11). Compared to
the positive control, RT-4 cells treated with 2 and 4 µM SF in sprouts had 55±7% and
45±2% lower adduct levels, respectively. Compared to the positive control, NBT-II cells
treated with 2 and 4 µM SF in sprouts had 39±7% and 51±7% lower adduct levels,
respectively.
-100
-50
0
50
100
0 2 4
SF sprout treatment (µM)
% c
ha
ng
e i
n a
dd
uc
t le
ve
l
-55% -45%
Figure 5.10. 4-ABP-induced DNA damage in human bladder cells and the protective
effect of SF in sprout form. RT-4 cells were pretreated with SF in sprout form for 24 h
and then exposed to 0.5 mM 4-ABP plus S9 for 3 h before measurement of dG-C8-4-
ABP levels.
200
-100
-50
0
50
100
0 2 4
SF sprout treatment (µM)
% c
ha
ng
e i
n a
dd
uc
t le
ve
l
-39% -51%
Figure 5.11. 4-ABP-induced DNA damage in rat bladder cells and the protective effect
of SF in sprout form. NBT-II rat bladder carcinoma cells were pretreated with SF in
sprout form for 24 h and then exposed to 0.5 mM 4-ABP plus S9 for 3 h before
measurement of dG-C8-4-ABP levels.
5.4.3 CPDT inhibits dG-C8-4-ABP formation in RT-4 bladder carcinoma cells
RT-4 cells were pretreated with CPDT at 12.5 and 25 µM for 48 h; cells were then
washed with fresh medium and exposed to 0.5 mM 4-ABP plus rat liver S9 for 3 h. dG-
C8-4-ABP levels in these cells were then measured. The CPDT concentrations as well as
the treatment conditions for 4-ABP were based on previous findings (22, 28). Treatment
with 12.5 and 25 µM CPDT to significant and dose-dependent inhibition of dG-C8-4-
ABP formation: 24±6% and 61±6% inhibition respectively (Figure 5.12).
201
Figure 5.12. CPDT inhibits 4-ABP DNA adduct formation in human bladder carcinoma
RT-4 cells. RT-4 cells were treated with CPDT or solvent for 48 h and then exposed to
4-ABP plus S9 for 3 h, followed by dG-C8-4-ABP analysis by LC-MS/MS.
5.4.4 SF and CPDT inhibit dG-C8-4-ABP formation in mouse bladder tissue and Nrf2 is
essential for this chemopreventive activity
To assess the effect of SF on 4-ABP in vivo, both wild type mice and Nrf2 knockout
mice were administered SF at 0, 10, 40 µmol/kg by gavage once daily for 5 days. A
single i.p. dose of 4-ABP at 50 mg/kg was given 3 h after the last SF dose. The mice were
sacrificed 24 h after 4-ABP treatment, and the bladders were processed for measurement
-100
-50
0
50
100
0 12.5 25
CPDT treatment (µmol/kg)
% c
han
ge i
n a
dd
uct
level
(µM)
-61%
-24%
202
of DNA damage. SF caused dose-dependent inhibition of dG-C8-4-ABP formation in the
bladder of the wild type mice, achieving 50±10% and 61±7% inhibition at 10 and 40
µmol/kg dose levels, respectively, as compared to the positive control, mice treated only
with 4-ABP. SF did not inhibit adduct formation in the Nrf2 knockout mice (Figure
5.13).
The chemopreventive activity of CPDT was also assessed in mouse bladder and liver
tissue based on its inhibition of 4-ABP DNA adducts. Wild type and Nrf2 knockout mice
were administered CPDT at 0, 5, 20 and 80 mg/kg by gavage once daily for 5 days; 3 h
after the last CPDT dose, each mouse was given 4-ABP at 50 mg/kg i.p.; the mice were
killed 24 h after 4-ABP injection for measurement of tissue dG-C8-4-ABP levels.
27±4%, 59±2%, and 67±4% lower adduct levels were observed in the bladder tissue of
wild type mice pretreated with 5, 20, and 80 µmol/kg CPDT respectively as compared to
the positive control (Figure 5.14). 11±6%, 9±6%, and 33±6% lower adduct levels were
observed in the bladder tissue of Nrf2 knockout mice pretreated with 5, 20, and 80
µmol/kg CPDT respectively as compared to the positive control.
203
Fig
ure
5.1
3.
SF
req
uir
es N
rf2
to
in
hib
it 4
-AB
P D
NA
ad
du
ct f
orm
atio
n i
n m
ou
se b
lad
der
tis
sue.
W
ild
-ty
pe
and
Nrf
2-k
no
cked
ou
t C
57
BL
/6 m
ice
wer
e tr
eate
d w
ith
veh
icle
or
SF
by
gav
age
on
ce a
day
fo
r 5
day
s.
Th
ree
ho
urs
afte
r th
e la
st S
F d
ose
, ea
ch m
ou
se w
as g
iven
a s
ing
le i
.p.
do
se o
f 4
-AB
P.
Th
e m
ice
wer
e k
ille
d 2
4 h
lat
er f
or
mea
sure
men
t o
f d
G-C
8-4
-AB
P i
n b
lad
der
tis
sues
.
-10
0
-500
50
10
0
01
04
0
SF
tre
atm
en
t (µ
mo
l/kg
)
% change in adduct levelC
57B
L/6
-50
%-6
1%
-10
0
-500
50
10
0
01
04
0
SF
tre
atm
en
t (µ
mo
l/kg
)
% change in adduct levelC
57B
L/6
-50
%-6
1%
-10
0
-500
50
10
0
01
04
0
SF
tre
atm
en
t (µ
mo
l/kg
)
% change in adduct level
Nrf
2 K
O-/
-
-10
0
-500
50
10
0
01
04
0
SF
tre
atm
en
t (µ
mo
l/kg
)
% change in adduct level
Nrf
2 K
O-/
-
204
-100
-50
0
50
100
0 5 20 80
CPDT treatment (µmol/kg)
% c
ha
ng
e i
n a
dd
uc
t le
ve
lC57BL/6
-27%
-59%-67%
-100
-50
0
50
100
0 5 20 80
CPDT treatment (µmol/kg)
% c
han
ge i
n a
dd
uct
level
Nrf2 KO -/-
-11% -9% -33%
Figure 5.14. CPDT inhibits 4-ABP DNA adduct formation in wild type mouse bladder
tissue. Nrf2+/+
mice and Nrf2-/-
mice were treated with CPDT or vehicle by gavage once
daily for 5 days; 3 h after the last CPDT dose, each mouse was given 4-ABP i.p.; 24 h
later, animals were killed, and dG-C8-4-ABP levels in the bladders and livers were
quantified by LC-MS/MS.
205
CPDT did not inhibit 4-ABP DNA adduct formation in the livers of wild type mice
(Figure 5.15).
-100
-50
0
50
100
0 5 20 80
CPDT treatment (µmol/kg)
% c
ha
ng
e i
n a
dd
uc
t le
vel
C57BL/6
Figure 5.15. CPDT does not inhibit 4-ABP DNA adduct formation in mouse liver tissue.
Nrf2+/+
mice were pretreated with CPDT or vehicle by gavage once daily for 5 days; 3 h
after the last CPDT dose, each mouse was given 4-ABP i.p.; 24 h later, animals were
killed, and dG-C8-4-ABP levels in the bladders and livers were quantified by LC-
MS/MS.
206
5.5 Discussion
5.5.1 SF and CPDT activate Nrf2 and the Nrf2 signaling pathway
The effect of treatment by the chemopreventive agents SF and CPDT on Nrf2 and Nrf2-
regulated enzymes was examined by western blot analyses. Expression levels of GCSC,
GCSM, NQO1, UGTs, NATs and GSTs were evaluated. Although UGTs and NATs are
known to be involved in 4-ABP metabolism and NQO1 and GCS may modulate 4-ABP
detoxification (29), changes in their expression levels in these experiments only indicates
Nrf2 transactivation and is not necessarily related to 4-ABP metabolism.
SF and CPDT treatment led to significant increases in Nrf2 levels in human bladder
carcinoma cells and this is consistent with previous reports (22, 30). As both agents
induced Nrf2, increased expression levels of the Nrf2-regulated GCSC,GCSM and NQO1
were observed, indicating activation of the Nrf2 signaling pathway. Interestingly, SF did
not induce two Phase 2 enzymes that are thought to be very significant in 4-ABP-induced
cancer, UGTs and NATs. CPDT induced expression of one isoform of UGT1A and one
isoform of UGT2B out of several UGTs that were observed.
Effect of SF and CPDT on Nrf2 and Nrf2 regulated proteins was also examined in mouse
bladder tissue. In general, lower enzyme levels were observed in western blots of Nrf2-/-
mice than Nrf2+/+
mice indicating that these enzymes are at least partly regulated by Nrf2.
Importantly, both SF and CPDT clearly elevated expression of GST, GCS and NQO1 of
207
wild type mice, but not Nrf2 knockout mice, showing activation of the Nrf2
cytoprotective signaling pathway in the bladder. This is consistent with the results from
bladder cells. Also in agreement with bladder cell results was that CPDT pretreatment
resulted in higher levels of one isoform of UGT1A and one isoform of UGT2B and that
SF pretreatment did not affect the levels of UGT or NAT.
4-ABP is known to be metabolized mainly in the liver and therefore hepatic levels of
these Phase 2 enzymes were also analyzed. Because hepatic levels of GST, GCS and
NQO1 were significantly elevated in SF-treated wild type mice, but not in the Nrf2
knockout mice, SF was determined to be not bladder specific. This is inconsistent with
previous reports showing that SF is selectively delivered to the bladder through urinary
excretion (9, 11, 12). However, the fact that SF did not induce UGTs which are
important for 4-ABP transport to the bladder, indicates that its induction of other Phase 2
enzymes in the liver may not necessarily increase the bioavailability of 4-ABP in the
bladder. Therefore, despite the apparent lack of organ specificity, SF should not be
counted out as a potential chemopreventive agent against 4-ABP-induced bladder cancer.
On the other hand, CPDT appears to target the bladder but not the liver; it only induced
NQO1 out of the three Nrf2 regulated genes examined and its inducing effect on UGT in
the liver was limited. CPDT therefore seems to be relatively bladder-specific and this is
in agreement with previous data (22).
208
Interestingly, while both GCSc and GCSm were undetectable in the bladder of Nrf2-/-
mice, significant levels of these enzymes were detected in their liver tissues, implying
that these genes are subjected to regulation by Nrf2-independent mechanisms in the liver.
In view of these positive western blot results indicating the SF and CPDT activate Nrf2,
we proceeded with our research examining the effect of these agents on 4-ABP DNA
adduct formation.
5.5.3 SF and CPDT inhibit 4-ABP DNA adduct formation in bladder cells and tissues
Inhibition by chemopreventive agents in these studies is not likely to be caused by the
chemopreventive agent preventing S9 from activating 4-ABP or by direct interaction
between the chemopreventive agent and 4-ABP. This is because cells are pretreated with
the chemopreventive agent which is removed before exposure to the carcinogen. The
occurrence of adduct inhibition is therefore more likely to be caused by induction of
Phase 2 cytoprotective proteins.
Both CPDT and SF were determined to be highly effective in inhibiting 4-ABP-induced
genotoxicity in bladder cells. Compared to positive control cells that were only treated
with 4-ABP, cells treated with 2 and 4 µM SF had 70% and 59% lower adduct levels,
respectively. Interestingly, the low SF dose was somewhat more effective. CPDT
treatment led to dose-dependent inhibition of dG-C8-4-ABP formation; 24% and 61%
209
fewer adducts were measured in cells treated with 12.5 and 25 µM CPDT compared to
the positive control.
The decrease in adduct levels following SF in sprout form was unexpectedly less
pronounced than that following pure SF treatment. A study involving SF in sprout form
more closely resembles typical human consumption of broccoli, and cruciferous
vegetables containing bioactive components other than glucosinolates that may play a
role in anti-carcinogenesis. For example, brussel sprouts which contain a large variety of
glucosinolates, also contain a number of protective compounds including glucobrassicin,
crambene, S-methyl cysteine sulfoxide, and dithiolethione. In broccoli however, SF only
has been shown to be responsible for the up-regulation of detoxification enzymes. It is
possible that another compound in the sprout mixture is interfering with SF’s induction of
cytoprotective enzymes or is inducing 4-ABP activating enzymes (31).
5.5.4 SF and CPDT inhibit dG-C8-4-ABP formation in mouse bladder tissue but not in
mouse liver tissue
Pretreatment with either SF or CPDT resulted in dose-dependent inhibition of dG-C8-4-
ABP formation in the bladder of wild type mice that were treated with 4-ABP: 10 and 40
µmol/kg SF pretreatment led to 50% and 61% inhibition of 4-ABP DNA adducts,
respectively and 5, 20, and 80 µmol/kg CPDT pretreatment led to 27%, 59%, and 67%
inhibition of 4-ABP DNA adducts respectively. SF did not inhibit dG-C8-4-ABP
formation in the bladder tissue of Nrf2 knockout mice that were treated with 4-ABP and
210
therefore Nrf2 is required by SF to inhibit 4-ABP-induced DNA damage in the bladder.
CPDT only slightly inhibited adduct formation in Nrf2 knockout mice treated with 4-
ABP. These very much reduced chemopreventive effects of CPDT in Nrf2 knockout
mice indicate that the main cytoprotective pathway for this agent is through Nrf2.
However, the occurrence of some chemopreventive activity by CPDT in Nrf2 knockout
mice suggests that CPDT may act through Nrf2-idependent pathways as well.
Liver tissue from the mice treated with CPDT was also examined for 4-ABP DNA adduct
formation and in contrast to the inhibition that was observed in the bladder tissue, CPDT
pretreatment had virtually no effect on 4-ABP-induced dG-C8-4-ABP formation in the
livers of wild type mice. Previously, researchers have shown that CPDT and several
other dithiolethiones are specific for bladder tissue (23). Our observations that CPDT did
not inhibit 4-ABP DNA adduct formation in mouse liver tissue supports this prior
research. The inability of CPDT to protect liver tissue against 4-ABP in Nrf2 knockout
mice suggests that detoxification and therefore glucuronidation of 4-ABP and its
metabolites is diminished without Nrf2.
211
5.6 Conclusion
We have shown that SF and CPDT are highly effective in blocking 4-ABP-induced DNA
damage in human bladder cells in vitro and in mouse bladder tissue in vivo.
Furthermore, we have shown that not only do these agents activate the well-known
cytoprotective protein Nrf2 and the Nrf2 signaling pathway, but they require Nrf2 to
substantially inhibit 4-ABP DNA damage. Although SF was not bladder specific, it did
not induce UGTs which were determined to be significant enzymes in the transport of 4-
ABP from the liver to the bladder. Showing even more promise as a bladder
chemopreventive agent, CPDT did not induce Nrf2 in the liver nor did it protect the liver
against the formation of 4-ABP DNA adduct formation.
SF and CPDT are promising cancer chemopreventive agents, as they strongly protect
cells and tissues against 4-ABP. Both agents have high potential as bladder
chemopreventive agents because they do not induce UGTs in the liver. Accordingly, our
research suggests that SF and CPDT should continue to be investigated as
chemopreventive agents against 4-ABP induced bladder cancer.
212
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216
CHAPTER 6
SUMMARY AND FUTURE DIRECTIONS
217
6.1 Summary
Quantification of DNA adducts can have significant implications in risk assessment.
Moreover, they are important biomarkers in the analysis of chemopreventive agents.
Detection of these adducts in healthy tissue can indicate the need for administration of a
chemopreventive agent. A decrease in adduct levels following treatment with a
chemopreventive agent can reflect the efficacy of the agent in reducing bladder cancer
risk. Detection of 4-ABP DNA adducts in human samples is a challenging undertaking
due to the minimal sample often available and the low adduct levels expected. In the
case of chemoprevention research these levels may be even lower and a sensitive method
for detection is therefore required. Cell and animal experiments in this field of research
are particularly important for elucidating the mechanisms of action of these agents and to
achieve high enough adduct levels for detection and clear signs of inhibition. In view of
this, we developed an LC-MS/MS method sensitive enough for detection of adducts in
human samples and designed cell and animal experiments for the purpose of evaluating
chemoprevention strategies and agents.
This LC-MS/MS method enabled quantification of dG-C8-4-ABP in human bladder cells
and animal tissues, and most significantly human urothelial cells. Our chemopreventive
research focused on targeting the cytoprotective protein Nrf2. The findings that Nrf2
potentiated 4-ABP DNA adduct formation in mouse bladder tissue in vitro but protected
mouse liver tissue and human bladder cells against 4-ABP initiated an investigation into
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the effect of Nrf2 on key Phase 2 enzymes involved in 4-ABP metabolism. We observed
that UTG, a protein that has already been implicated in 4-ABP-induced bladder
carcinogenesis, is at least partly responsible for increasing the bioavailability of Nrf2 in
the bladder. Nrf2 protects cells locally against 4-ABP and accordingly, is an effective
target for a chemopreventive agent against bladder carcinogenesis provided that the
chemopreventive agent does not up-regulate UGT in the liver. In support of these results,
SF and CPDT were shown to induce Nrf2 and the Nrf2 signaling pathway and inhibit 4-
ABP DNA adduct formation in human bladder cells and mouse bladder tissue. Neither
compound induced UGT enzymes in the liver, and both were determined to be possible
chemopreventive agents against bladder cancer.
This study provided convincing evidence that administration of chemopreventive agents
can result in lower DNA adduct levels and therefore reduce risk of bladder
carcinogenesis. These promising results in conjunction with the limitations of our study
provide a direction for future research in this area. The ultimate goal is to reduce risk of
4-ABP-induced bladder carcinogenesis, and to do this a greater understanding of 4-ABP
metabolism and the mechanisms of action of potential chemopreventive agents is
important. What follows are some suggestions for future research.
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6.2 Detection and quantification of dG-ABP isomers
Our research focused on detection and quantification of dG-C8-4-ABP, but 4-ABP binds
to DNA to form several different isomers and these isomers may be highly significant.
Researchers have hypothesized that the biological effect of exposure to 4-ABP is not
shown just in the quantification of the major adduct, but that the minor low abundance
adducts may have a greater effect on cancer initiation; a high abundance adduct may be
the preferred substrate for a repair enzyme leaving the minor adduct to cause mutations
and possibly make it the greatest contributor to cancer initiation (1, 2). Indeed, some
studies on other small molecule-induced genetic damage have found that the C8 adduct is
not as biologically important as the N2 adduct (3-5). We have detected both the dA-C8-4-
ABP DNA and the dG-N2-4-ABP adduct in 4-ABP dosed human bladder cells and mouse
bladder tissue but further confirmation with characterized standards is required.
Furthermore, dose-response curves should be generated and the effect of
chemopreventive agents on their abundances should be investigated.
Detection of these minor adducts presents several analytical challenges. The importance
of a highly sensitive method is even greater as they are often present in significantly
lower quantities. Moreover, if their detection is for the evaluation of chemopreventive
agents, the chemopreventive agent will decrease these already low adduct levels even
further.
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6.3 Investigation into 4-ABP metabolism (6, 7)
An analysis of the metabolites of 4-ABP is important to understanding 4-ABP DNA
adduct formation and inhibition. A comprehensive scan of 4-ABP metabolites in urine of
experimental animals treated with 4-ABP may point to key detoxifying reactions. The
extent of carcinogen conjugation with Phase 2 metabolites can be quantified to provide
an estimation of carcinogen detoxification. Furthermore, the approximate efficacy of a
chemopreventive agent can be gauged based on the relative change in levels of these
conjugates following treatment with the chemopreventive agent. In chapter 4, we
examined the effect of Nrf2 on urinary glucuronidation of 4-ABP in mice treated with 4-
ABP. Glucuronidation, however is only one significant reaction in the metabolism of 4-
ABP and an analysis of sulfation, glutathione conjugation, and acetylation might provide
more insight into the detoxification of 4-ABP.
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6.4 Comprehensive human study
Following the development of our validated and sensitive LC-MS/MS method
appropriate for the quantification of dG-C8-4-ABP in human samples, our laboratory, in
conjunction with the Roswell Park Cancer Institute will be conducting a comprehensive
human smoke-quit study. In this study, the variation in 4-ABP DNA adduct levels in
urothelial cells will be monitored as smokers quit smoking. This will be an extensive 7
week study involving 100 subjects and therefore this research will require a high
throughput method. A limiting factor of the methodology in this dissertation is the time
required for sample enrichment and separation. If SPE is involved in this next study, use
of SPE plates could significantly expedite sample preparation. An ultrafast separation
method such as that achieved with the use of differential mobility spectrometry (DMS)
may also be feasible.
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6.5 Evaluation of analogs of SF and CPDT
In chapter 5, we evaluated SF and CPDT as potential chemopreventive agents against
bladder carcinogenesis based on their inhibition of 4-ABP DNA adduct formation and
induction of Nrf2 and Nrf2 regulated genes. Activation of Nrf2 in bladder tissue but not
liver tissue was determined to be a promising strategy against bladder carcinogenesis in
chapter 4. Although our research suggests that both SF and CPDT are promising
chemopreventive agents against 4-ABP-induced bladder carcinogenesis, it would be
interesting to compare their cytoprotective characteristics to those of their analogs which
may be even more effective in inhibiting 4-ABP DNA adduct formation. Indeed,
derivatives of 3H-1,2-dithiole-3-thione (D3T) have been evaluated for effectiveness in
inducing Phase 2 enzymes and some were found be more effective than CPDT (8).
Analogs of SF have also been investigated, including phenylethyl isothiocyanate (PEITC)
(9, 10). Furthermore, it might also be interesting to measure the effect of these
chemopreventive agents on other carcinogens implicated in bladder carcinogenesis, such
as 2-naphthylamine or in rodents, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (11).
223
References
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(2005) Mutagenic specificity of 2-acetylaminonaphthalene-derived DNA adduct
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phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence
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M. P., and Kadlubar, F. F. (1994) Glucuronidation of N-hydroxy heterocyclic
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J., Gange, S. J., Kensler, T. W., and Sutter, T. R. (2003) Evaluation of the cancer
chemopreventive potency of dithiolethione analogs of oltipraz, Carcinogenesis
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9. Kumar, A., and Sabbioni, G. (2010) New biomarkers for monitoring the levels of
isothiocyanates in humans, Chem Res Toxicol 23, 756-765.
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C., and Eaton, D. L. (2010) Sulforaphane- and phenethyl isothiocyanate-induced
inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of
GSTM1 genotype and CYP3A4 gene expression, Toxicol Sci 116, 422-432.
11. Iida, K., Itoh, K., Kumagai, Y., Oyasu, R., Hattori, K., Kawai, K., Shimazui, T.,
Akaza, H., and Yamamoto, M. (2004) Nrf2 is essential for the chemopreventive
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BIOGRAPHICAL DATA
EDUCATION Doctorate of Philosophy in Analytical Chemistry, May 2011
Barnett Institute of Chemical and Biological Analysis
Department of Chemistry and Biological Chemistry
Northeastern University, Boston, MA
Bachelor of Arts in Chemistry, June 2004
Carleton College, Northfield, MN
PROFESSIONAL Graduate Research Assistant, Sept 2006 – Present
EXPERIENCE Barnett Institute / Dept. of Chemistry and Biological Chemistry,
Northeastern University, Boston, MA
Graduate Teaching Assistant, Sept 2005-August 2006
Dept. of Chemistry and Biological Chemistry,
Northeastern University
Technical Support Specialist, August 2004-March 2005
Systems 3000, Eatontown, NJ
Research Experience for Undergraduates (REU) 6/2003 – 8/2003
Department of Chemistry
University of South Dakota, Vermillion, SD
Laboratory Teaching Assistant September 2002 – June 2003
Department of Chemistry
Carleton College, Northfield, MN
Technical Aide Intern 2000, 2001
Avaya, Inc., Lincroft, NJ
PROFESSIONAL American Chemical Society (ACS), Student Affiliate
AFFILIATIONS American Society of Mass Spectrometry (ASMS), StudentMember
Greater Boston Mass Spectrometry Discussion Group (GBMSDG)
Massachusetts Separation Science Discussion Group (MASSEP)
HONORS NEU Graduate Dissertation Completion Fellowship Spring 2011
Gustel and Ernst Giessen Memorial Award in Advanced Research 2010
Gustel and Ernst Giessen Memorial Award in Advanced Research 2009
Douglas and Irene DeVivo Award for Continued Excellence 2008
Sigma Xi Scholar 2004
George Walter Davis Scholarship 2000-2004
N. J. Bloustein Distinguished Scholar 2000
226
LIST OF PRESENTATIONS
(presenter in bold)
Kristen L. Randall, Joseph D. Paonessa, Yi Ding, Dayana Argoti, Yuesheng Zhang,
Paul Vouros. Activation of Nrf2 as a chemopreventive strategy against 4-ABP-induced
bladder cancer. 59th American Society for Mass Spectrometry and Allied Topics
Conference (ASMS), Denver, CO, 2011, Small Molecules: Quantitative Analysis.
Kristen L. Randall, Yi Ding, Joseph D. Paonessa, Dayana Argoti, Rex Munday,
Yuesheng Zhang, Paul Vouros. Inhibition of 4-Aminobiphenyl-induced DNA Damage by
Sulforaphane and 5,6-Dihydrocyclopenta[c]-dithiole-3(4H)-thione in Bladder Cells and
Tissues. 240th
American Chemical Society (ACS) National Meeting, Boston, MA, 2010,
Division of Chemical Toxicology.
Kristen L. Randall, Yi Ding, Joseph D. Paonessa, Dayana Argoti, Rex Munday,
Yuesheng Zhang, Paul Vouros. Inhibition of 4-Aminobiphenyl-induced DNA Damage by
Sulforaphane and 5,6-Dihydrocyclopenta[c]-dithiole-3(4H)-thione in Bladder Cells and
Tissues. 58th American Society for Mass Spectrometry and Allied Topics Conference
(ASMS), Salt Lake City, UT, 2010, Mass Spectrometry in Environmental Toxicology.
(Oral)
Kristen L. Randall, Dayana Argoti, Joseph D. Paonessa, Yi Ding, Zachary Oaks,
Yuesheng Zhang, Paul Vouros. LC-MS Detection of 4-ABP-DNA Adduct Formation in
Bladder Cells and Tissues. Conference on Small Molecule Science (COSMOS), Boston,
MA, 2009.
Kristen L. Randall, Dayana Argoti, Joseph D. Paonessa, Yi Ding, Zachary Oaks,
Yuesheng Zhang, Paul Vouros. LC-MS Detection of 4-ABP-DNA Adduct Formation in
Bladder Cells and Tissues. 57th American Society for Mass Spectrometry and Allied
Topics Conference (ASMS), Philadelphia, PA, 2009, Toxicology 591.
Dayana Argoti, Kristen L. Randall, Yuesheng Zhang, Paul Vouros. 4-ABP-DNA
Adducts in Human Bladder Cells and the Role of Isothiocyantes as a Chemopreventive
Phytochemical. 56th American Society for Mass Spectrometry and Allied Topics
Conference (ASMS), Denver, CO 2008, Toxicology 212.
Lisa Tilkens, Kristen Randall, Jian Sun, Mary T Berry, P Stanley May; Spectroscopic
Evidence for Equilibrium between Eight- and Nine-Coordinate Eu3+
(aq) Species in 0.1 M
EuCl3(aq). National Conferences on Undergraduate Research (NCUR), Indianapolis, IN
2004.
227
LIST OF PUBLICATIONS
Randall, K. L.; Zhang, Y.; Vouros, P. Quantification of bulky DNA adducts by liquid
chromatography-mass spectrometry for the evaluation of early intervention
chemopreventive agents. Manuscript in Preparation.
Paonessa, J. D. *; Ding, Y. *; Randall, K. L. *; Munday, R.; Argoti, D.; Vouros, P.;
Zhang, Y. Identification of an unintended consequence of Nrf2-directed cytoprotection
against a key tobacco carcinogen plus a counteracting chemopreventive intervention.
Cancer Research, 2011, Manuscript in press.
*contributed equally to this work
Ding, Y.*; Paonessa, J. D.*; Randall, K. L.*; Argoti, D.; Chen, L.; Zhang, Y.; Vouros,
P. Sulforaphane Inhibits 4-Aminobiphenyl-induced DNA Damage in Bladder Cells and
Tissues. 2010, Carcinogenesis, 2010, 31(11), 1999-2003.
*contributed equally to this work
Randall, K. L.; Argoti, D.; Paonessa, J. D.; Ding, Y.; Oaks, Z.; Zhang, Y.; Vouros, P. An
Improved Liquid Chromatography–Tandem Mass Spectrometry Method for the
Quantification of 4-aminobiphenyl DNA Adducts in Urinary Bladder Cells and Tissues.
J. Chrom. A. 2009, 1217(25), 4135-4143.
Tilkens, L.; Randall, K.; Sun, J.; Berry, M. T.; May, P. S.; Toshihiro, Y. Spectroscopic
Evidence for Equilibrium between Eight- and Nine-Coordinate Eu3+
(aq) Species in 0.1 M
EuCl3(aq). J. Phys. Chem. A. 2004, 108, 6624-6628.