Dihydromethysticin (DHM) Blocks Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Induced O6-Methylguanine in a Manner Independent of the Aryl Hydrocarbon Receptor (AhR) Pathway in C57BL/6 Female Mice

Article abstract of DOI:10.1021/acs.chemrestox.6b00203

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a key carcinogen responsible for tobacco smoke-induced lung carcinogenesis. Among the types of DNA damage caused by NNK and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), O⁶-methylguanine (O⁶-mG) is likely the most carcinogen in A/J mice. Results of our previous studies showed that levels of O⁶-mG and other types of NNAL-derived DNA damage were preferentially reduced in the lung of female A/J mice upon dietary treatment with dihydromethysticin (DHM), a promising lung cancer chemopreventive agent from kava. Such a differential blockage may be mediated via an increased level of NNAL glucuronidation, thereby leading to its detoxification. The potential of the aryl hydrocarbon receptor (AhR) as an upstream target of DHM mediating these events was evaluated herein using Ahr^+/- and Ahr^-/- C57BL/6 female mice because DHM was reported as an AhR agonist. DHM (0.05, 0.2, and 1.0 mg/g of diet) and dihydrokavain (DHK, an inactive analogue, 1.0 mg/g of diet) were given to mice for 7 days, followed by a single intraperitoneal dose of NNK at 100 mg/kg of body weight. The effects of DHM on the amount of O⁶-mG in the lung, on the urinary ratio of glucuronidated NNAL (NNAL-Gluc) and free NNAL, and on CYP1A1/2 activity in the liver microsomes were analyzed. As observed in A/J mice, DHM treatment significantly and dose-dependently reduced the level of O⁶-mG in the target lung tissue, but there were no significant differences in O⁶-mG reduction between mice from Ahr^+/- and Ahr^-/- backgrounds. Similarly, in both strains, DHM at 1 mg/g of diet significantly increased the urinary ratio of NNAL-Gluc to free NNAL and CYP1A1/2 enzymatic activity in liver with no changes detected at lower DHM dosages. Because none of these effects of DHM were dependent on Ahr status, AhR clearly is not the upstream target for DHM.

Full text of DOI:10.1021/acs.chemrestox.6b00203

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Dihydromethysticin (DHM) blocks tobacco carcinogen 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-
induced O6-methylguanine independent of aryl
hydrocarbon receptor (AhR) in C57BL/6 female mice
Sreekanth C Narayanapillai, Shang-Hsuan Lin, Pablo Leitzman,
Pramod Upadhyaya, Carolyn J. Baglole, and Chengguo Xing
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00203 • Publication Date (Web): 11 Oct 2016
Downloaded from http://pubs.acs.org on October 12, 2016
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Dihydromethysticin (DHM) blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (NNK)-induced O6-methylguanine independent of aryl hydrocarbon
receptor (AhR) pathway in C57BL/6 female mice
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Sreekanth C. Narayanapillai,† ShangꢀHsuan Lin,† Pablo Leitzman,† Pramod Upadhyaya,║
Carolyn J. Baglole,§ and Chengguo Xing*,†,║,#
† Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota,
Minneapolis, MN 55455
║Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
§MeakinsꢀChristie Laboratories, McGill University, Montreal, Québec, Canada
#Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville,
FL 32610
*To whom correspondence should be addressed: Department of Medicinal Chemistry, College of
Pharmacy, University of Florida, Gainesville, FL 32610, USA. Phone: 352ꢀ294ꢀ8511; Eꢀmail:
chengguoxing@cop.ufl.edu
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ABSTRACT
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4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK) is a key carcinogen responsible for
tobacco smokeꢀinduced lung carcinogenesis. Among DNA damages caused by NNK and its
metabolite 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol (NNAL), O6ꢀmethylguanine (O6ꢀmG)
is likely the most carcinogenic in A/J mice. Results of our previous studies showed that O6ꢀmG
and other NNALꢀderived DNA damages were preferentially reduced in the lung of female A/J
mice upon the dietary treatment with dihydromethysticin (DHM), a promising lung cancer
chemopreventive agent from kava. Such a differential blockage may be mediated via increased
NNAL glucuronidation and thereof leading to its detoxification. The potential of the aryl
hydrocarbon receptor (AhR) as an upꢀstream target of DHM mediating these events was
evaluated herein using Ahr+/ꢀ and Ahrꢀ/ꢀ C57BL/6 female mice because DHM was reported as an
AhR agonist. DHM (0.05, 0.2 and 1.0 mg/g of diet) and dihydrokavain (DHK, an inactive analog,
1.0 mg/g of diet) were given to mice for seven days, followed by a single i.p. dose of NNK at
100 mg/kg of bodyweight. The effects of DHM on the amount of O6ꢀmG in the lung, on the
urinary ratio of glucuronidated NNAL (NNALꢀGluc) and free NNAL, and on CYP1A1/2 activity
in the liver microsomes were analyzed. As observed in A/J mice, DHM treatment significantly
and doseꢀdependently reduced O6ꢀmG in the target lung tissue, but there were no significant
differences in O6ꢀmG reduction between mice from Ahr+/ꢀ and Ahrꢀ/ꢀ backgrounds. Similarly, in
both strains, DHM at 1 mg/g of diet significantly increased the urinary ratio of NNALꢀGluc to
free NNAL and CYP1A1/2 enzymatic activity in liver with no changes detected at lower DHM
dosages. Since none of these effects of DHM were dependent of Ahr status, AhR clearly is not
the upꢀstream target for DHM.
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INTRODUCTION
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As the leading cause of cancerꢀrelated death, lung cancer accounted for about 160,000 deaths
in the United States and more than 1.6 million deaths worldwide annually.1 Due to the lack of
robust early diagnosis and effective treatment, the fiveꢀyear survival rate of lung cancer patients
has been around 15 – 18% for decades.2 Although developing effective therapy and early
diagnosis is important in the clinical management of lung cancer, it is also essential to develop
chemopreventive agents against this disease.
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Tobacco use accounts for 85 – 90% of lung cancer incidence.3 Many chemicals detected in
tobacco smoke have been classified as human carcinogens.4 Among these, 4ꢀ
(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK, a tobaccoꢀspecific nitrosamine) is a wellꢀ
studied pulmonary carcinogen.5
Upon cytochrome P450 (CYP450) enzymeꢀmediated
hydroxylation, NNK generates reactive intermediates that can form DNA damages.
Alternatively, NNK can be metabolically reduced to 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀ
butanol (NNAL), which will be bioactivated by CYP450 to form DNA damages (Scheme 1).
These DNA damages, if not repaired, can lead to gene mutations and initiate lung
tumorigenesis.6 Therefore, chemical entities that can block NNK/NNALꢀinduced DNA damages
have the potential to prevent lung carcinogenesis.
Our earlier studies showed that dihydromethysticin (DHM), a natural product from Piper
methysticum (kava), could block NNKꢀinduced lung tumorigenesis in A/J mice; provided it was
given during the tumor initiation phase.7,8 DHM at a dose of 0.05 mg/g in diet was able to
reduce lung adenoma multiplicity by 97%, with a significant reduction in DNA adduct O6ꢀmG
(likely the most carcinogenic DNA damage in A/J mice9). Further studies suggested that the
reduction in O6ꢀmG by DHM may originate from the increased detoxification of NNAL.10
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Specifically, dietary DHM increased the relative abundance of 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀ
pyridyl)ꢀ1ꢀ(OꢀβꢀDꢀglucopyranuronosyl)butane (NNALꢀOꢀGluc) in A/J mouse urine, which is
likely mediated via the enhanced NNAL glucuronidating activity.10
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NNAL glucuronidation is catalyzed by UDPꢀglucuronosyltransferases (UGTs).11 UGTs are
typically regulated by several transcriptional factors, including pregnane X receptor (PXR),
constitutive androstane receptor (CAR)12,13 and aryl hydrocarbon receptor (AhR).14 Recently Li
et al. reported that DHM and methysticin, another structurally similar kavalactone in kava that
also blocks NNK/NNALꢀinduced O6ꢀmG formation in A/J mice,8 could activate the AhR
pathway in vitro.15 AhR is a ligandꢀdependent transcriptional factor highly expressed in the
lung.16 Upon the binding of its agonist, AhR translocates to the nucleus, dimerizes with ARNT,
followed by the binding to the xenobioticꢀresponse element (XRE) of drugꢀmetabolizing genes to
activate their transcription.17 Besides UGTs and other phase II enzymes, phase I enzymes,
particularly CYP1A1, are also dominantly regulated by AhR. While being essential for normal
metabolism and other regular cellular functions,18 CYP1A1/2 enzymes may also be involved in
the bioactivation of certain carcinogens, such as benzo[a]pyrene (BaP)19 and 2ꢀaminoꢀ1ꢀmethylꢀ
6ꢀphenylimidazo(4,5ꢀb)pyridine (PhIP).20 Due to these complicated roles, the induction of
CYP1A1/2 has been carefully evaluated in many herbꢀdrug or drugꢀdrug interactions.21 Indeed,
Li et al. demonstrated that DHM and methysticin could induce CYP1A1 at mRNA, protein and
function levels in Hepa1c1c7 cells in an AhRꢀdependent manner.15 Several in vivo studies also
reported that high dosage of kava extract led to increased mRNA and protein of hepatic CYP1A1
in rodents.22,23 These results overall suggest that AhR might be one possible upꢀstream target of
DHM, which may activate UGTs and result in enhanced detoxification of NNAL that would
account for its effect against NNK/NNALꢀinduced DNA damage in A/J mice. At the same time,
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DHM may enhance the metabolic activation of other carcinogens and increase the risk of herbꢀ
drug interaction upon activating AhR and inducing CYP1A1/2.24
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Given DHM’s outstanding efficacy in blocking NNK/NNALꢀinduced DNA damage and lung
tumorigenesis, there is an urgent need to elucidate its mechanism of action. In this study, we
investigated the role of AhR as a potential target of DHM using Ahr+/ꢀ and Ahrꢀ/ꢀ C57BL/6 mice.
We analyzed whether AhR deficiency has any effect on 1) DHMꢀmediated reduction in O6ꢀmG
in target lung tissues, 2) increased NNALꢀOꢀglucuronidation in urine, and 3) induction of
CYP1A1/2 in the liver. Our results revealed that AhR status has no influence on any of these
DHMꢀmediated effects, demonstrating that AhR is not the upstream target.
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REAGENTS AND METHODS
Caution: NNK and NNAL are human carcinogens. Personnel handlings are expected to have
appropriate safety measures.
Chemicals and reagents NNK, [13C6]NNK, [CD3]O6ꢀmG and [4ꢀCD2,CD3]NNALꢀOꢀGluc were
purchased from Toronto Research Chemicals (Toronto, ON, Canada). NNAL and [13C6]NNAL
were synthesized from NNK or [13C6]NNK via sodium borohydride reduction.10 (±)ꢀDHM and
(±)ꢀDHK were synthesized inꢀhouse with a slight modification of a reported procedure.25 AINꢀG
powdered diet was purchased from Harlan Teklad (Madison, WI). Recombinant βꢀglucuronidase
was purchased from SigmaꢀAldrich (St. Louis, MO). Ethoxyresorufin was purchased from
SigmaꢀAldrich. NADPH was purchased from RPI (Mount Prospect, IL). The qPCR primers
were purchased from IDT (Coralville, IA) and the sequences are as follows, Gapdh sense 5’ꢀ
AACTTTGGCATTGTGGAAGGꢀ3’, antisense 5’ꢀACACATTGGGGGTAGGAACAꢀ3’; Ahr
sense
5’ꢀAGCCGGTGCAGAAAACAGTAAꢀ3’,
antisense
5’ꢀ
AGGCGGTCTAACTCTGTGTTCꢀ3’; Cyp1a1 sense 5’ꢀGGTTAACCATGACCGGGAACTꢀ3’,
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antisense
5’ꢀTGCCCAAACCAAAGAGAGTGAꢀ3’;
Cyp1a2
sense
5’ꢀ
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TGGAGCTGGCTTTGACACAGꢀ3’, and antisense 5’ꢀCGTTAGGCCATGTCACAAGTAGCꢀ3’.
All other chemicals or solvents were purchased from either Fisher Scientific (Fairlawn, NJ) or
SigmaꢀAldrich (St. Louis, MO), unless stated otherwise.
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Diet preparation and characterization (±)ꢀDHM and (±)ꢀDHK supplemented AINꢀG
powdered diets were prepared as per our previously reported procedures.8 Briefly, (±)ꢀDHM or
(±)ꢀDHK was reconstituted in absolute ethanol (50 mL) and then mixed with the AINꢀ93 G
powdered diet (150g). Absolute ethanol (50 mL) was mixed with the AINꢀ93 G powdered diet
(150g) for the control diet. The reconstituted diets were dried under vacuum to remove ethanol
and then ground into fine powders. All diets were then mixed well with additional AINꢀ93 G
powdered diet to the desired dose. The abundance of DHM and DHK in the diet was analyzed in
triplicate by HPLC and confirmed to be within ±10% of the specified dose.
Animal Studies The animal studies performed herein were approved by the University of
Minnesota Institutional Animal Care and Use Committee and conducted following the National
Institutes of Health guidelines. Fiveꢀ to sixꢀweek old C57BL/6 Ahr+/ꢀ and Ahrꢀ/ꢀ female mice
were procured from McGill University, Montreal, Canada and housed in the core animal
facilities of the Research Animal Resources, University of Minnesota.26 Ahr+/ꢀ mice are
phenotypically indistinguishable from wildꢀtype (Ahr+/+) mice and are often used as controls
when examining the physiological, pathophysiological and toxicological parameters of the AhR
rendering AhR+/ꢀ mice as suitable controls for this study.27–29 After one week of acclimation, 18
Ahr+/ꢀ and 18 Ahrꢀ/ꢀ mice were randomized into six groups each (n=3). From Day 1 to Day 7,
they were fed with the corresponding diet. Bodyweight of the mice was measured every two or
three days and their food intake was monitored twice a week. On Day 8, except for mice in the
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negative control groups, all other mice received a single dose of NNK in saline (100 µL) at 100
mg/kg of body weight via ip injection. Mice in the negative control groups were given saline.
Based on the results from our previous studies, four hours after NNK exposure, mice were
euthanized with CO2 overdosing.10 Mouse sera, urine, lung and liver tissues were collected and
stored at ꢀ80 °C following our established procedures.10 For lung and liver tissues, small
portions were stored in RNA stabilization solution.
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Quantification of O6-mG DNA adduct in the lung tissues DNA isolation from the lung tissues
and LCꢀMS/MS quantification were performed following the standard procedure.7 Briefly DNA
was isolated from half of the whole lung tissue of each individual mouse, following Puregene
DNA isolation protocol (Qiagen Corp). O6ꢀmG was quantified by liquid chromatographyꢀ
electrospray ionization/tandem mass spectrometry (LCꢀESIꢀMS/MS) with [CD3]O6ꢀmG as the
internal standard.
Urinary NNAL-O-Gluc and free NNAL Quantification Urinary NNALꢀOꢀGluc and free
NNAL were quantified via an established LCꢀMS/MS method.10 Briefly urine samples were
diluted 105 times with saline. The diluted samples (0.1 mL each) were mixed with [4ꢀCD2,
CD3]NNALꢀOꢀGluc and [13C6]NNAL at a final concentration of 5 and 10 ng/mL respectively.
LCꢀMS/MS analysis was performed using an Agilent 1100 series capillary highꢀpressure liquid
chromatography system (Agilent Technologies, Palo Alto, CA) interfaced to a TSQ Quantum
Discovery Max triple quadrupole mass spectrometer (Thermo Electron, San Jose, CA). NNAL
and NNALꢀOꢀGluc were analyzed simultaneously on a Phenomenex Luna C18 (150 × 0.5 mm, 3
microns) capillary column, under conditions reported before.10
Mouse Liver Microsome Preparation and CYP1A1/2 Enzymatic Assay (EROD) Mouse liver
microsomes were prepared following the standard protocol.10 Briefly, liver tissue (~250 mg)
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was manually homogenized in ice cold microsome buffer (1 mL, 50 mM Tris·HCl, 1.15% KCl, 1
mM EDTA, pH 7.0). The resulting homogenate was centrifuged at 10,000 RPM at 4°C for 30
minutesusing 70.1 Ti rotor (Beckman Coulter, Brea, CA). The supernatant was collected and
further centrifuged at 40,000 RPM at 4°C for 90 minutes. The pellet was resuspended in ice cold
microsome buffer (100 µL). Upon protein quantification by BCA method, microsome was
diluted to a concentration of 5 mg/mL total protein, aliquoted and stored at ꢀ80°C until use.
Aliquoted microsomes were thawed on ice right before the enzymatic assay. 7ꢀEthoxyꢀ
resorufinꢀOꢀdeethylation (EROD) enzymatic assays were conducted following the standard
protocols in 96ꢀwell plates.30 Briefly, microsome was incubated with 7ꢀethoxyꢀresorufin under
37°C for 10 min followed by the addition of NADPH. With a final reaction volume of 100 uL,
each well contained 0.2 mg/mL microsomal protein, 1 mM NADPH, and 1 µM 7ꢀ
ethoxyresorufin in microsome buffer. Fluorescence intensity in each well was continuously
monitored by a Tecan microplate reader (GENios Pro) with an excitation wavelength of 535 nm
and an emission wavelength of 590 nm (20 measurements each well in 15min). During this
period of time, the increase in fluorescence intensity was linear, suggesting minimal changes of
the enzymatic activity and substrate concentration (data not shown). Standard curve used for
unit conversion was generated with resorufin purchased from Santa Cruz Biotechnology (Dallas,
TX).
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Western Blotting Analyses Liver (~50mg) and lung (~10mg) tissue samples were first
homogenized using TissueRuptor from Qiagen with TꢀPER Tissue Protein Extraction Reagent
from Thermo Fisher, supplemented with protease inhibitors (Thermo Fisher) and phosphatase
inhibitors cocktail (Cell Signaling, Danvers, MA). Protein concentration was determined by
BCA method. Western blotting was conducted with 50 µg liver tissue lysates or 12.5 µg lung
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tissue lysates per well. Primary antibody for AhR was purchased from Enzo Life Sciences
(Farmingdale, NY) (cat. BMLꢀSA210), 1:5000. HRPꢀlinked βꢀactin antibody was obtained from
SigmaꢀAldrich (cat. A3854), 1:25000. HRPꢀlinked antiꢀrabbit IgG from Cell Signaling was used
as secondary antibody for AhR with a dilution factor of 1:3000. βꢀactin served as loading
control and protein bands were visualized by the chemiluniscence method.
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Quantitative Reverse Transcription PCR Liver and lung tissue samples were saved in RNA
stabilization solution. Total RNA was isolated using RNeasy Mini kit (Qiagen) following
manufacturer’s protocol. cDNA was synthesized from 2 µg total RNA each using SuperScript
Vilo cDNA Synthesis kit (Invitrogen, Carlsbad, CA). qPCR was done on a StepOnePlus™ Realꢀ
Time PCR System using SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA) with the
following thermal cycling: 95°C for 5 minutes, proceeded by 45 cycles of 95°C for 15 seconds,
and 60°C for 1 minute. Gapdh served as a reference gene.
Statistical Analyses Data shown here represent 3 biological repeats (3 mice) with their mean ±
standard deviation. Statistical significance is denoted by asterisks with the following definition:
not significant, pꢀvalue > 0.05; *, pꢀvalue ≤ 0.05; **, pꢀvalue ≤ 0.01; ***, pꢀvalue ≤ 0.001; ****,
pꢀvalue ≤ 0.0001. If not specified with any asterisks, there was no significant difference between
compared groups. Except for the results of qRTꢀPCR, differences between treated and control
groups were analyzed by oneꢀway ANOVA, with Dunnett’s method used for multiple
comparison, at 95% confidence interval. For qRTꢀPCR results, mRNA expressions between
Ahr+/ꢀ and Ahrꢀ/ꢀ groups were compared with twoꢀtailed student’s tꢀtest, at 95% confidence
interval.
RESULTS AND DISCUSSION
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Characterization of Ahr Status and Health Monitoring Before randomization and after
euthanasia, all mice were genotyped for Ahr, following an established procedure (data not
shown).26 In addition, Ahr mRNA and protein were analyzed via qRTꢀPCR and Western blotting
respectively in the lung and liver tissues from representative animals (Figure S1). These results
validated the Ahr status of mice used herein. During the study, there were no significant
differences in bodyweight changes or food intake among mice with different treatments (data not
shown).
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The Effect of Ahr Status on Reduction in O6-mG by DHM in Mouse Lung Tissues O6ꢀmG is
likely the most carcinogenic DNA damage relative to other NNK/NNALꢀinduced DNA damages
in A/J mice9 and it was effectively reduced by DHM in the target lung tissue.8 Therefore,
pulmonary O6ꢀmG was analyzed as the representative DNA damage in the Ahr+/ꢀ and Ahrꢀ/ꢀ mice.
As shown in Figure 1, NNK treatment resulted in similar levels of O6ꢀmG adduct formation
irrespective of the Ahr status and DHM doseꢀdependently reduced O6ꢀmG levels in both Ahr+/ꢀ
and Ahrꢀ/ꢀ mice. As expected, the extent of reduction in O6ꢀmG by DHK was much less, if any,
relative to DHM in both genotypes. These results unambiguously demonstrated that the
reduction in O6ꢀmG by DHM is AhR independent.
The Effect of Ahr Status on DHM-induced NNAL Glucuronidation Our earlier work
revealed that dietary DHM increased the abundance of urinary NNALꢀOꢀGluc relative to free
NNAL in A/J mice and enhanced the NNAL glucuorinidating activity in the lung and liver
tissues.10 Mechanistically, increase in UGTꢀmediated NNAL glucuronidation could lead to
enhanced NNAL detoxification and thereof may contribute to the reduction in O6ꢀmG.10,31 Since
AhR has been reported to transcriptionally regulate UGTs,14,32 the impact of Ahr status on
NNAL glucuronidation was evaluated herein. The urinary ratio of NNALꢀGluc and free NNAL
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has been used as a convenient parameter to evaluate glucuronidationꢀmediated NNAL
detoxification.33 Higher urinary ratio of NNALꢀGluc to free NNAL would indicate better
detoxification and such a ratio has been used to examine NNK detoxifying capability among
smokers.34 Although both NNALꢀNꢀGluc and NNALꢀOꢀGluc have been detected in human
smokers, only NNALꢀOꢀGluc has been detected in A/J mice.10 In this study we therefore
directly quantified NNALꢀOꢀGluc and free NNAL in the mouse urine,10 and the ratio of NNALꢀ
OꢀGluc to free NNAL was calculated. As shown in Figure 2, DHM at a dose of 1 mg/g of diet
significantly increased the urinary ratio of NNALꢀOꢀGluc to NNAL while DHK had no effect,
consistent with our results in A/J mice,10 supporting that DHM may exert its chemopreventive
effects through increasing NNAL detoxification. In agreement with the results of O6ꢀmG
reduction, the detoxification of NNAL by DHM also seemed to be independent of the AhR
pathway. Although the extent of increase by DHM in AhR+/ꢀ mice seemed to be higher than that
in Ahrꢀ/ꢀ mice, the basal level was slightly higher in AhR+/ꢀ mice as well that further investigation
is needed. While DHM was able to reduce the formation of O6ꢀmG at the dose of 0.05 and 0.2
mg/g of diet, such treatments did not increase NNALꢀOꢀGluc/NNAL ratio (data not shown). A
simple explanation could be glucuronidation of NNAL is not the major mechanism responsible
for the reduction of NNK/NNALꢀinduced O6ꢀmG by DHM treatment. Alternatively, the urinary
ratio of NNALꢀOꢀGluc/NNAL may not accurately reflect the target lung tissue since liver is the
major metabolizing tissue. Our earlier work showed that dietary DHM treatment only
preferentially reduced NNK/NNALꢀinduced DNA damage in the target lung tissue not the liver
tissue.8 It is possible that lower dosages of DHM may be sufficient to enhance NNAL
glucuronidation in the target lung tissue while higher dosages of DHM may be needed to
enhance NNAL glucuronidation in the liver, which requires further investigation.
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The Effect of Ahr Status on CYP1A1/2 Activity by DHM in Mouse Liver Microsome In
addition to its chemopreventive potential, kava usage has been linked to hepatotoxicity in
humans.35 Here we characterized the impact of dietary DHM on CYP1A1/2 to further explore its
safety. We focused on CYP1A1/2 in this study mainly because these two CYP isozymes are
dominantly regulated by AhR36 and Li et al. has demonstrated CYP1A1 activation by DHM in
an AhR dependent manner in vitro.15 In addition, CYP1A1 has been reported to be upꢀregulated
in vivo upon high dosages of kava treatment.22 Moreover, as discussed before, CYP1A1/2 may
activate other carcinogens to potentially promote carcinogenesis.20,37
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Since DHM was given to mice in diet and its impact on Cyp1a1 and Cyp1a2 mRNA was
expected to be temporal, we attempted to quantify CYP1A1 and CYP1A2 proteins in the liver
tissues via Western blotting. Due to the lack of wellꢀvalidated isoform specific antibodies for
CYP1A1 and CYP1A2, Western blotting results were inconclusive (data not shown). In the next
step, enzymatic activity of CYP1A1/2 was evaluated through 7ꢀethoxyꢀresorufinꢀOꢀdeethylation
(EROD) assay (Figure 3).30 In spite of previous in vitro evidence showing CYP1A1 induction by
DHM via the AhR pathway,15 DHM’s effect on the liver microsomal CYP1A1/2 activity (EROD)
was independent of Ahr status in this study. Such a discrepancy can be due to many reasons,
including in vitro and in vivo models, tissue differences, dosage, metabolism, temporal and
spatial distribution of DHM. In comparison to the NNK control groups, DHM at the highest
dose (1 mg/g) significantly increased EROD activity; while the extent of increase induced by
DHK was much weaker and statistically nonꢀsignificant. With lower doses of DHM (0.05 mg/g
or 0.2 mg/g), liver microsomal CYP1A1/2 activities did not show significant difference when
compared to NNK control groups in both Ahr+/ꢀ and Ahrꢀ/ꢀ mice.
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It appeared that the basal level of liver microsomal EROD activity was lower in the Ahrꢀ/ꢀ
mice relative to that in the Ahr+/ꢀ mice, although the differences were not statistically significant,
suggesting that even in the liver, AhR may contribute to the basal levels of CYP1A1/2.
CONCLUSION
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Since the effect of DHM on pulmonary NNK/NNALꢀinduced O6ꢀmG, urinary ratio of
NNALꢀOꢀGluc/NNAL, and liver microsomal EROD were similar in both Ahr+/ꢀ and Ahrꢀ/ꢀ
C57BL/6 mice, AhR clearly is not the upꢀstream target of DHM. Because of the low sensitivity
of C57BL/6 mice to NNK/NNALꢀinduced lung tumorigenesis in comparison to the A/J mice and
the lack of Ahrꢀ/ꢀ A/J mice, we were unable to evaluate the effect of Ahr status on DHM’s tumor
reduction potential. It is clear that O6ꢀmG formation is unlikely the only molecular basis
responsible for NNKꢀinduced lung tumorigenesis, particularly in C57BL/6 mice.38 Nevertheless,
a strong positive correlation between O6ꢀmG levels and lung tumor multiplicity in A/J mice has
been observed.9 It is reasonable to speculate that DHM may block NNK/NNALꢀinduced lung
tumorigenesis in A/J mice independent of the AhR pathway, which needs further validaton.
It was also observed that DHM at 0.2 or 0.05 mg/g of diet, while retaining complete
chemopreventive effect, did not cause any induction of CYP1A1/2 activity in the liver
microsome. DHM therefore has a decent therapeutic window to effectively block lung
tumorigenesis without enhancing CYP1A1/2 activity. In addition, flavokawains A and B, not
DHM, have been recently identified by us and others as the compounds potentially responsible
for kava’s hepatotoxic risk.39 Nevertheless, comprehensive studies are needed to systematically
assess the physiological and pathological impacts of DHM on CYP1A1/2 and other drug
metabolizing enzymes.
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FUNDING INFORMATION
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This work was funded by the grant R01 CA 193278 (C. Xing) from the National Institutes of
Health National Cancer Institute, in part by the National Cancer Institute Cancer Center Support
Grant CA 077598 and by a Canadian Institutes of Health Research (CIHR) grant 123467 (CJB).
CJB was supported by a salary award from the Fonds de recherche du QuebecꢀSante (FRQꢀS).
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ACKNOWLEDGEMENTS
We thank the Masonic Cancer Center’s Analytical Biochemistry Core Facility for their
assistance with LC/MS analyses.
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ABBREVIATIONS LIST
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DHM, dihydromethysticin; NNK, 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone; AhR, aryl
hydrocarbon receptor; NNAL, 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanol; O6ꢀmG, O6ꢀ
methylguanine; DHK, dihydrokavain; NNALꢀOꢀGluc, 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀ
butanol glucuronide; UGT, UDPꢀglucuronosyltransferase; PXR, pregnane X receptor; CAR,
constitutive androstane receptor; XRE, xenobioticꢀresponse element; BaP, benzo[a]pyrene; PhIP,
2ꢀAminoꢀ1ꢀmethylꢀ6ꢀphenylimidazo(4,5ꢀb)pyridine; GAPDH, glyceraldehyde 3ꢀphosphate
dehydrogenase; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; EROD,
7ꢀethoxyꢀresorufinꢀOꢀdeethylation; qRTꢀPCR, quantitative reverse transcription polymerase
chain reaction; and ANOVA, analysis of variance.
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SUPPORTING INFORMATION
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Confirmation of the Ahr status of lung and liver tissues from representative mice. This material
is available free of charge via the Internet at http://pubs.acs.org
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FIGURE LEGEND
Figure 1. Effect of Ahr status on DHM and DHK on NNK/NNALꢀinduced O6ꢀmG in the target
lung tissues. Data represents mean ± standard deviation of 3 mice per treatment group.
Statistical comparison was made with the NNKꢀonly treatment group in each strain of mice via
oneꢀway ANOVA.
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Figure 2. Effect of Ahr status on DHMꢀinduced NNAL glucuronidation in urine. Ratio of
NNALꢀOꢀGluc : free NNAL was used as a measurement of NNAL glucuronidation. Both DHM
and DHK were at a dose of 1 mg/g diet. Data represents mean ± standard deviation of 3 mice
per treatment group. Statistical comparison was made with the NNKꢀonly treatment group in
each strain of mice via oneꢀway ANOVA.
Figure 3. Effect of Ahr status on CYP1A1/2 activity (EROD) in the liver microsome. Data
represents mean ± standard deviation of 3 mice per treatment group. Statistical comparison was
made with the NNKꢀonly treatment group in each strain of mice via oneꢀway ANOVA.
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FIGURES
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Figure 3
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Scheme 1. Putative mechanism of action of DHM via activating the AhR pathway to enhance
NNAL glucuronidation, which reduces NNK and NNALꢀinduced DNA damage and blocks lung
carcinogenesis.
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NNK/NNAL
O6-mG DNA
adduct
formation
DHM
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DHM
CYP1A1/2
Enzyme
activity
AhR
NNAL
detoxification
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