1521-009X/44/3/422–427$25.00 http://dx.doi.org/10.1124/dmd.115.068387 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:422–427, March 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Dietary Dihydromethysticin Increases Glucuronidation of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in A/J Mice, Potentially Enhancing Its Detoxification

Sreekanth C. Narayanapillai, Linda B. von Weymarn, Steven G. Carmella, Pablo Leitzman, Jordan Paladino, Pramod Upadhyaya, Stephen S. Hecht, Sharon E. Murphy, and Chengguo Xing

Department of Medicinal Chemistry, College of Pharmacy (S.C.N., P.L., J.P., C.X.), Masonic Cancer Center (L.B.W., S.G.C., P.U., S.S.H., S.E.M.), and Department of Biochemistry, Molecular Biology and Biophysics (L.B.W., S.E.M.), University of Minnesota, Minneapolis, Minnesota

Received November 16, 2015; accepted January 6, 2016

ABSTRACT Downloaded from Effective chemopreventive agents are needed against lung cancer, P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation the leading cause of cancer death. Results from our previous work in A/J mouse lung), suggesting that it does not inhibit NNAL showed that dietary dihydromethysticin (DHM) effectively blocked bioactivation. Dietary DHM significantly increased O-glucuronidated initiation of lung tumorigenesis by 4-(methylnitrosamino)-1- NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes (3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced from dietary DHM-treated mice showed enhanced activities for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA NNAL O-glucuronidation. These results overall support the notion dmd.aspetjournals.org adducts in lung. This study explored the mechanism(s) respon- that dietary DHM treatment increases NNAL detoxification, sible for DHM’s differential effects on NNK/NNAL-derived DNA potentially accounting for its chemopreventive efficacy against damage by quantifying their metabolites in A/J mice. The results NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary showed that dietary DHM had no effect on NNK or NNAL NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate abundance in vivo, indicating that DHM does not affect NNAL the clinical evaluation of DHM-based lung cancer chemopreventive formation from NNK. DHM had a minimal effect on cytochrome agents. at ASPET Journals on October 3, 2021

Introduction We recently demonstrated that dihydromethysticin (DHM) (Fig. 1) Lung cancer kills ;160,000 people in the United States each year effectively blocked NNK-induced lung adenoma formation in A/J mice (Siegel et al., 2013). Although cigarette smoking cessation is the ideal when administered during the initiation phase (Narayanapillai et al., strategy to reduce lung cancer risk, quitting is difficult because of the 2014), positioning DHM as a promising candidate for lung cancer addictive nature of nicotine in tobacco products. An alternative strategy prevention. Mechanistically, DHM dose-dependently reduced pulmo- is to develop effective lung cancer chemopreventive agents that would nary DNA adducts derived from 4-(methylnitrosamino)-1-(3-pyridyl)- be complementary to tobacco cessation to help prevent this deadly 1-butanol (NNAL), one major metabolite of NNK (Carmella et al., disease. Tobacco smoke contains various classes of carcinogens, 1993) and a potent pulmonary carcinogen (Upadhyaya et al., 1999). including N-nitrosamines, polycyclic aromatic hydrocarbons, hetero- Interestingly DHM had minimal effects on DNA adducts derived cyclic aromatic amines, and others. NNK [4-(methylnitrosamino)-1- directly from NNK bioactivation. Dihydrokavain (DHK) (Fig. 1), (3-pyridyl)-1-butanone] is a tobacco-specific N-nitrosamine with potent despite its close structural similarity to DHM, was completely void pulmonary carcinogenicity, inducing the formation of lung adenoma of these properties and thus can serve as a control compound for and adenocarcinoma in multiple species (Hecht, 1998). Substantial mechanistic investigation of DHM. evidence suggests that NNK in cigarettes contributes to lung adeno- Based on the known pathways of NNK and NNAL metabolism carcinoma incidence among U.S. smokers (Hecht, 2014), which makes (Hecht, 1998), we hypothesized that the preferential reduction in blocking NNK-induced lung tumorigenesis a plausible approach to NNAL-derived DNA damage by DHM in the lung may be mediated decrease tobacco smoke-induced lung cancer. via inhibiting NNAL formation from NNK, inhibiting cytochrome P450 enzyme-mediated NNAL bioactivation, or enhancing UDP- glucuronosyltransferase (UGT)–mediated NNAL detoxification (Fig. 2). We therefore quantified NNK, NNAL, and glucuronidated NNAL This work was supported by a grant from the National Institutes of Health (NNAL-gluc) in serum and urine samples from mice exposed to dietary National Cancer Institute [Grant R01 CA193278]. DHM in comparison with those from control mice or DHK-treated dx.doi.org/10.1124/dmd.115.068387. mice. We also evaluated the effect of DHM on the activity of

ABBREVIATIONS: AHR, aryl hydrocarbon receptor; CE, collision energy; CYP2A5, cytochrome P450 2A5; DHK, dihydrokavain; DHM, dihydromethysticin; LC-MS/MS, liquid chromatography with tandem mass spectrometry; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanol; NNAL-gluc, glucuronidated NNAL; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; O6-mG, O6-methylguanine; RT, retention time; UGT, UDP-glucuronosyltransferase.

422 Dihydromethysticin Increases NNAL-O-Glucuronidation In Vivo 423

were prepared following our reported procedures (Narayanapillai et al., 2014). After 1 week of consuming the various diets, each mouse was treated with NNK via i.p. injection (100 mg/kg of body weight in 100 ml saline). One group of mice from each treatment regimen was euthanized at 0.5, 1.0, 2.0, or 4.0 hours after NNK exposure, and serum and urine samples were collected. Serum collection and storage followed established procedures (Narayanapillai et al., 2014). For urine collection, each individual mouse was placed on a piece of aluminum foil, and urine

was passively released upon CO2 euthanasia. Typically 25 to 150 mlofurinewas Fig. 1. Dihydromethysticin (DHM) and dihydrokavain (DHK). collected from each mouse. The urine samples were stored at 280C until analysis. Study 2. Fifteen female mice were randomized into three groups of five. One group was maintained on AIN-G powdered diet, one group on AIN-G powdered cytochrome P450 2A5 (CYP2A5) in liver microsomes. The results diet supplemented with DHM at a dose of 1 mg/g of diet, and the other group on AIN-G powdered diet supplemented with DHK at a dose of 1 mg/g of diet. One indicate that dietary DHM treatment increases NNAL glucuronidation, week thereafter, the mice were euthanized with CO2; the lung and liver tissues potentially leading to enhanced detoxification and accounting for the were collected and stored at 280C until NNAL glucuronidation analysis. preferential reduction in NNAL-derived DNA damage. CYP2A5 Enzymatic Assays Materials and Methods Liver tissues used herein were from previous studies (Narayanapillai et al., 2014). Mouse liver microsomes from control mice (n = 4) and mice treated with

Chemicals Downloaded from NNK, NNK + DHM (1 mg/g of diet), or NNK + DHK (1 mg/g of diet) (n =3 NNK, [13C ]NNK, [13C ]NNAL, [CD ]NNAL-N-gluc, and [4-CD ,CD] 6 6 3 2 3 each) were prepared as previously described elsewhere (von Weymarn and NNAL-O-gluc were purchased from Toronto Research Chemicals (Toronto, Murphy, 2003). Ontario, Canada). [Caution: NNK and NNAL are carcinogenic. They should be The effect of DHM and DHK on CYP2A5 enzymatic activity was evaluated handled in a well-ventilated hood with extreme care, and with proper personal using liver microsomes pooled from the four control mice. Briefly, aliquots of the protective equipment.] AIN-93 G powdered diet was purchased from Harlan m b pooled liver microsomes from untreated mice (75 g protein) were added to Teklad (Madison, WI). -Glucuronidase (recombinant, expressed in an Escher- m reaction mixtures containing coumarin (2 or 20 M), DHM, or DHK dissolved in dmd.aspetjournals.org ichia coli–overproducing strain, cat. no. G8295) was obtained from Sigma- methanol (10 or 100 mM) or methanol control and an NADPH-generating system Aldrich (St. Louis, MO). Glass vials (2 ml of silane-treated), with or without in 50 mM Tris buffer (pH 7.4). The final reaction volume was 200 ml. After 15 fused inserts, were obtained from Chromtech (Apple Valley, MN). Kava was minutes of incubation at 37C, the reaction was terminated with the addition of acquired from Gaia Herbs (Brevard, NC) as an ethanol extract of the wild crafted 20 ml of trifluoroacetic acid (15%). The formation of 7-hydroxycoumarin from lateral root from Vanuatu, which was standardized to 150 mg/ml total coumarin was measured by high-pressure liquid chromatography with fluorescence kavalactones. DHM and DHK were isolated from kava following an established detection as previously described elsewhere (Bock and Bock-Hennig, 2010). procedure (Narayanapillai et al., 2014). All other chemicals and solvents were All experiments were performed in duplicate on at least three separate acquired from Sigma-Aldrich or Fisher Scientific (Fairlawn, NJ) and used

occasions. The enzymatic activity of CYP2A5 in each liver microsome at ASPET Journals on October 3, 2021 without further purification. preparation (20–80 mg protein) from the mice treated with NNK, NNK + DHM, or NNK + DHK (n = 3 each) was individually evaluated. Animal Studies These studies were approved by the University of Minnesota Institutional Urine Processing to Convert NNAL-gluc into NNAL and Liquid Animal Care and Use Committee and performed in accordance with National Chromatography with Tandem Mass Spectrometry Quantification Institutes of Health guidelines. Female A/J mice, 6 weeks of age, were purchased Urine samples were diluted 105 times with saline. The diluted samples were from Jackson Laboratories (Bar Harbor, ME) and housed in the specific divided into two or three portions (0.25 ml each for estimation of free NNAL, free pathogen-free animal facilities of the Research Animal Resources, University NNAL + NNAL-N-gluc, or total NNAL [the sum of free NNAL, NNAL-N-gluc, of Minnesota. Food intake was monitored twice a week, and body weight was and NNAL-O-gluc]). Each sample was mixed with [13C ]NNAL as an internal standard measured once a week. None of the treatment caused detectable changes to the 6 (4 ng/ml), processed, and analyzed by liquid chromatography with tandem mass mice based on food consumption and body weight changes. spectrometry (LC-MS/MS) as previously described elsewhere (Carmella et al., 2013). Study 1. After 1 week of acclimation, 48 female mice were randomized into 16 groups of three. Four groups were maintained on AIN-G powdered diet, four Direct LC-MS/MS Detection and Quantification of NNAL-O-gluc, NNK, groups on AIN-G powdered diet supplemented with kava at a dose of 5 mg/g of and NNAL in Urine and Serum Samples diet, four groups on AIN-G powdered diet supplemented with DHM at a dose of 1 mg/g of diet and the other four groups AIN-G powdered diet supplemented Urine samples were diluted 105 times with saline. The diluted samples (0.1 ml with DHK at a dose of 1 mg/g of diet. Kava, DHM, or DHK supplemented diets each) were mixed with [4-CD2,CD3]NNAL-O-gluc and/or [CD3]NNAL-N-gluc

Fig. 2. Potential mechanisms of DHM on NNAL metabolism, inhibiting NNAL formation, inhibiting NNAL bioactivation, or enhancing NNAL detoxification, which could account for its preferential reduction in NNAL- derived DNA damage. 424 Narayanapillai et al.

Fig. 3. The effect of dietary DHM, DHK, or kava on the abundance of NNK and NNAL in A/J mouse urine (A) and serum (B). Mice maintained on the specified diet were given NNK and euthanized at different time points thereafter (n = 3 per group except for the urine samples with DHM treatment at 0.5 hours; n = 2 because one mouse urinated upon NNK administration, so urine collection at euthana- sia was not successful). Statistical analysis was performed with one-way analysis of variance for urine samples and with two-tailed Student’s t test for serum samples. None of the differ- ences were statistically significant. Cont: con- trol diet.

13 at a final concentration of 5 ng/ml. For NNK and NNAL analysis, [ C6]NNK concentrations of 5 mM and 2 mM, respectively, in a total reaction volume of 100 13 and [ C6]NNAL at a final concentration of 10 ng/ml each were added as the ml. The reaction mixture was incubated for 1 hour at 37C. The samples were 4 internal standards. The serum samples were diluted 10 times with saline, and then mixed with [4-CD2,CD3]NNAL-O-gluc (5 pmol). The reactions were each of the diluted samples (0.1 ml) was mixed with [4-CD2,CD3]NNAL- terminated by the addition of 0.1 volumes of 0.3 M Ba(OH)2 and ZnSO4 each,

13 13 Downloaded from O-gluc, [ C6]NNK, and [ C6]NNAL at a final concentration of 10 ng/ml each. while being cooled on ice. The precipitate was removed by centrifugation, and LC-MS/MS analysis was performed using an Agilent 1100 series capillary NNAL-O-gluc in the supernatant was purified using a Resprep C18 SPE column high-pressure liquid chromatography system (Agilent Technologies, Palo Alto, (cat. no. 26030; Restek, Bellefonte, PA), which was preactivated with methanol

CA) interfaced to a TSQ Quantum Discovery Max triple quadrupole mass and equilibrated with H2O. The sample retained on the column was washed with spectrometer (Thermo Electron, San Jose, CA) operated in selective reaction doubly distilled H2O (1 ml) and eluted with 5% methanol (0.5 ml). The eluted monitoring mode and tuned to NNAL-N-gluc. samples were dried using a SpeedVac (Thermo Fisher Scientific, Waltham, MA),

NNK, NNAL, NNAL-N-gluc, and NNAL-O-gluc were analyzed simulta- reconstituted in 100 mlofH2O, and analyzed for NNAL-O-gluc by LC-MS/MS neously on a Phenomenex Luna C18 (150 Â 0.5 mm, 3 microns) capillary by the method described earlier. dmd.aspetjournals.org column, eluted under the following conditions: 95% A (10 mM ammonium acetate): 5% B (acetonitrile) held for 5 minutes, followed by a linear gradient to Results 30% B in 2 minutes, a 3-minute hold at 30% B, a linear gradient to 50% B in 1 minute, a 2-minute hold at 50% B, a linear gradient to 5% B in 1 minute, and a Effects of DHM, DHK, or Kava on NNK and NNAL Abundance 11-minute hold at 5% B. The flow rate was 10 ml/min. in A/J Mouse Urines and Sera. We first determined the ratio of NNK For NNAL-N-gluc, the mass transitions monitored were as follows: NNAL-N- and total NNAL in the urine samples from A/J mice with different gluc (m/z 386 → 210, collision energy (CE) = 14; and m/z 386 → 180, CE = 25) dietary treatments, including control, DHM, DHK, and kava. Ratios → → at ASPET Journals on October 3, 2021 and [CD3]NNAL-N-gluc (m/z 389 213, CE = 14; and m/z 389 183, CE = instead of absolute quantities were reported because we did not control 25). For NNAL-O-gluc, the mass transitions monitored were as follows: NNAL- for urine excretion during the experimental period, and some mice O-gluc (m/z 386 → 210, CE = 14; and m/z 386 → 162, CE = 20) and [4-CD , 2 might have urinated between NNK treatment and euthanasia. In ad- CD3]NNAL-O-gluc (m/z 391 → 215, CE = 14; and m/z 391 → 167, CE = 20). For 12 dition, the relative amounts of urine loss during collection might be NNK, the mass transitions monitored were as follows: [ C6]NNK (m/z 208 → 13 122, CE = 12) and [ C6] NNK (m/z 214 → 128, CE = 12). For NNAL, the mass different among the mice. However, these variables were not critical to 12 transitions monitored were as follows: [ C6]NNAL (m/z 210 → 92, CE = 12) the ratio. As shown in Fig. 3A, there were no significant differences in 13 and [ C6]NNAL (m/z 216 → 98, CE = 12). the ratios of NNK to total NNAL among different treatment regimens, NNAL Glucuronidation Activity of Mouse Lung and Liver Microsomes. suggesting that DHM and kava treatment had no effect on NNAL Lung and liver microsomes were prepared from the respective tissues (100 mg) formation from NNK. To confirm this, the absolute amount of NNK in study 2 as described previously elsewhere (Wong et al., 2003). The and NNAL in the serum samples from control and DHM-treated mice microsomes were resuspended in 10 mM Tris buffer (pH 7.4, 1 mM EDTA, were quantified (Fig. 3B). The results also showed no significant and 20% glycerol, 0.2 ml) and stored at 280C. Protein concentrations were differences between DHM-treated mice and controls. determined by the standard methods. NNAL glucuronidating activity was evaluated via a reported procedure Effect of Dietary DHM on CYP2A5 Activity. CYP2A5 is the (Crampsie et al., 2011). Briefly, liver or lung microsomes (0.1 mg of protein) major catalyst of NNK and NNAL bioactivation in A/J mice (Hollander were preincubated with 1.1 mg/mL alamethicin, 8.5 mM saccharolactone, et al., 2011; Zhou et al., 2012; Megaraj et al., 2014). Mouse liver

10 mM MgCl2, and 50 mM KH2PO4 on ice for 10 minutes and then at 37C for microsomes were prepared and analyzed for coumarin 7-hydroxylation 3 minutes. After preincubation, NNAL and UDPGA were added to achieve final activity [a CYP2A5-specific reaction (von Weymarn and Murphy,

TABLE 2 TABLE 1 Effect of dietary DHM and DHK on coumarin 7-hydroxylation (CYP2A5) activity Effect of DHM and DHK on coumarin 7-hydroxylation (CYP2A5) activity in mouse in mouse liver microsomes (n = 3) liver microsomes – m m m Mouse liver microsomes (20 80 g protein) were incubated with 20 M coumarin and Mouse liver microsomes were incubated with 2 M coumarin and NADPH for 15 minutes at NADPH for 15 minutes at 37C. 37C in the presence or absence of DHM or DHK, which were dissolved in methanol. Hence, 0.4% methanol was added to the control reactions. 7-Hydroxycoumarin Formationa Concentration Percentage of Inhibitiona pmol/min/mg (mM) Control 12 6 3 DHK 10 62 6 6 DHK (1 mg/g of diet) 16 6 4 DHM 10 19 6 9 DHM (1 mg/g of diet) 17 6 4 DHM 100 71 6 10 a Statistical analysis was performed with one-way analysis of variance. None of the differences a Average of three independent experiments performed in duplicate. were statistically significant. Dihydromethysticin Increases NNAL-O-Glucuronidation In Vivo 425

have been detected in smokers’ urine (Carmella et al., 2002). The ratio of urinary NNAL-gluc (the sum of NNAL-N-gluc and NNAL-O-gluc) and free NNAL is a convenient parameter to evaluate glucuronidation- mediated NNAL detoxification (Carmella et al., 2013). The urinary ratios of NNAL-gluc to free NNAL for DHM or kava-treated mice were about twice those of NNK control mice at all four times of urine collection (Fig. 4). The differences were significantly different at 0.5, 2, and 4-hour time points (P , 0.05). DHK, the inactive analog, had no effect. Although both NNAL-N-gluc and NNAL-O-gluc have been detected in human urine, such information is not available for mice. We therefore analyzed their relative abundance in A/J mouse urine. NNAL-N-gluc and NNAL-O-gluc were first quantified as the released aglycons upon NaOH or b-glucuronidase treatment (Carmella Fig. 4. Effects of dietary DHM, DHK, and kava on the ratio of NNAL-gluc and free et al., 2013). No NNAL-N-gluc was detected in mouse urine, NNAL in A/J mouse urine. Mice maintained on the specified diet were given NNK irrespective of treatment regimens (data not shown). To confirm the and euthanized at different time points thereafter (n = 3 per group except for the absence of NNAL-N-gluc in mouse urine, representative mouse urine urine samples with DHM treatment at 0.5 hours; n = 2). Statistical analysis was performed with one-way analysis of variance followed by Dunnett’s test relative to samples (two from NNK-treated mice, two from NNK + DHM-treated , , the corresponding NNK treatment group; *P 0.05; **P 0.01. Cont: control diet. mice, and two from NNK + DHK-treated mice, all at the 2-hour time Downloaded from point) were directly analyzed for NNAL-N-gluc and NNAL-O-gluc via 2003)] in the presence of DHM or DHK. Coumarin 7-hydroxylation was a modified LC-MS/MS method with the corresponding deuterium- inhibited by 62% with 10 mM DHK whereas DHM exhibited minimal labeled compounds as internal standards. NNAL-N-gluc (RT = 4.9 inhibition at 10 mM and a concentration of 100 mM was required to minutes) and NNAL-O-gluc (RT = 12.8 minutes) were readily achieve similar inhibition as DHK at 10 mM (Table 1). The concentration separated (Fig. 5). No appreciable amount of NNAL-N-gluc was m , O O of DHM and DHK (10 M) was based on our preliminary pharmaco- detected ( 0.1% of NNAL- -gluc), but NNAL- -gluc was readily dmd.aspetjournals.org kinetic quantification of DHM in the mouse lung tissues (data not shown). detectable in micromolar abundance (Panel A). Similar results were The CYP2A5 activity in liver microsomes from mice consuming dietary observed in the other samples. DHM or DHK also did not differ from that in control mice (Table 2). A These data demonstrated that the majority of NNAL-gluc in A/J mouse DHM-enrichedkavafractionalsoshowednoinhibitiononCYP2A13 urine is NNAL-O-gluc. Using this method, the urine samples were (data not shown), the major human pulmonary CYP responsible for analyzed again to directly quantify NNAL-O-gluc. The ratios of NNAL- NNK/NNAL bioactivation (Megaraj et al., 2014). Overall these results O-gluc and free NNAL from this direct quantification were in good suggest that inhibiting CYP2A5-mediated NNAL bioactivation is un- agreement with the ratios from the indirect quantification (data not shown). likely to be the primary mode of action of DHM. To confirm the increase in NNAL-O-gluc abundance upon DHM at ASPET Journals on October 3, 2021 Effect of Dietary DHM and Kava on the Urinary Ratio of treatment, we quantified NNAL-O-gluc in serum samples from A/J NNAL-O-gluc to Free NNAL in A/J Mice. The best characterized mice with and without dietary DHM treatment via the direct LC-MS/ pathway for NNAL detoxification is UGT-catalyzed glucuronidation. Both MS method. Higher amounts of NNAL-O-gluc were detected in all N- and O-glucuronides of NNAL (NNAL-N-gluc and NNAL-O-gluc) samples from DHM-treated mice in comparison with those from control

Fig. 5. LC-MS/MS chromatograms of urinary NNAL-N-gluc and NNAL-O-gluc from a mouse exposed to NNK and dietary DHM with urine collection 2 hours after NNK. (A) Mass transition monitoring m/z 386 → 210 of NNAL-N-gluc (RT = 4.9 minutes) and NNAL-O-gluc (RT = 12.8 minutes). (B) Mass transition monitoring of the internal standards m/z 389 → 213 of [CD3] NNAL-N-gluc and m/z 391 → 215 of [4-CD2,CD3]NNAL-O-gluc. 426 Narayanapillai et al.

Fig. 6. Quantities of NNAL-O-gluc (A) and ratio of NNAL-O-gluc to free NNAL (B) in serum samples from NNK-exposed A/J mice upon dietary DHM in comparison with NNK-exposed mice on control diet (n = 3 per group). Statistical analysis was performed with two-tailed Student’s t test between DHM treatment and control group; *P , 0.05 and **P , 0.01. Cont: control diet.

mice (Fig. 6A), which was also reflected in the ratio of NNAL-O-gluc to 2004; Chen et al., 2008; Balliet et al., 2010). Although both NNAL-N-gluc free NNAL (Fig. 6B). and NNAL-O-gluc have been detected in humans (Carmella et al., 2002), Effect of Dietary DHM on UGT Enzymatic Activity in A/J we only detected NNAL-O-gluc in A/J mice, indicating a species dif- Mouse Lungs and Livers. Upon establishing the increase in NNAL- ference between mice and human in NNAL detoxification. We ob- O-gluc with DHM treatment, we evaluated whether such an increase served a clear increase in NNAL-O-gluc formation in A/J mice exposed to may be mediated via enhanced NNAL glucuronidation activity in lung dietary DHM but not DHK. In addition, both lung and liver microsomes Downloaded from and liver tissues upon dietary DHM treatment. In both tissues the extent from DHM-treated mice had enhanced NNAL glucuronidating activity. of NNAL glucuronidation increased with dietary DHM treatment (Fig. This again was not observed in DHK-treated mice. These results overall 7); the increase was significant in liver whereas DHK treatment had no support the mechanism that dietary DHM treatment enhances NNAL such effects. We next explored whether DHM could biochemically glucuronidation both in the lung and liver tissues, which may lead to enhance NNAL glucuronidation; we incubated DHM (10 mM) with increased NNAL detoxification and reduction in NNAL-derived DNA liver microsomes from the control mice and quantified the NNAL damage in A/J mice. Dietary DHM may up-regulate UGT enzyme(s) via dmd.aspetjournals.org glucuronidation. DHK was evaluated as well. Under such conditions, transcriptional activation of AHR as DHM is a potential AHR agonist (Li DHM and DHK had no significant effect on NNAL glucuronidation et al., 2011) and AHR can transcriptionally activate UGTs (Bock and (data not shown). Bock-Hennig, 2010). Interestingly the concentration of free NNAL was not reduced in A/J Discussion mouse sera upon dietary DHM treatment despite the significant increase in the level of NNAL-O-gluc. The concentration of free NNAL in the Dietary DHM has demonstrated potent efficacy in blocking NNK- lung tissues also did not appear to be reduced upon dietary DHM — at ASPET Journals on October 3, 2021 induced lung tumorigenesis in A/J mice by a unique mechanism treatment (data not shown). The lack of reduction in free NNAL in the preferential reduction in DNA damage derived from NNAL in the target sera and lung tissues raises the question whether the increase in NNAL — lung tissues while having minimal effect on DNA damage derived glucuronidation by DHM is critical for its reduction in NNAL-induced directly from NNK (Narayanapillai et al., 2014). Based on the well- DNA damage. characterized metabolism of NNK and NNAL (Hecht, 1998), we in- It should be pointed out that the amount of free NNAL we quantified vestigated the impact of dietary DHM treatment on NNAL formation from was for the whole lung tissues, which contain different type of cells, NNK, on CYP2A5 activity (the enzyme likely responsible for NNAL including type II cells, Clara cells, and small cells. As demonstrated by bioactivation), and on NNAL glucuronidation, one detoxification pathway Devereux et al., different cells in the lung have varied sensitivity to for NNAL (Fig. 2) that could contribute to DHM-induced preferential NNK/NNAL-induced DNA damage; the amount of O6-mG adduct was reduction in NNAL-derived DNA damage. 2 to 8 times higher in Clara cells and type II cells [potentially the target We found that dietary DHM treatment had no effect on the cells for tumorigenesis (Belinsky et al., 1991)] in comparison with abundance of NNK and NNAL in serum or urine samples (Fig. 3), small cells or the whole lung tissues (Devereux et al., 1993). These demonstrating that DHM does not inhibit the reductive formation of different types of cells also respond differently at the molecular level NNAL from NNK. Results from numerous studies suggest that upon NNK treatment (Belinsky et al., 1988, 1996). It remains to be kavalactones can directly inhibit cytochrome 450 enzymes or indirectly regulate cytochrome 450 enzymes via transcriptional induction (Ma et al., 2004; Zou et al., 2004; Lim et al., 2007; Guo et al., 2010; Li et al., 2011). DHM, one of the major kavalactones, had minimal effect on CYP2A5 enzymatic activity in the mouse liver microsomes at 10 mM. Although it inhibited CYP2A5 by 71% at 100 mM, such a high concentration is likely physiologically irrelevant. DHK indeed was a better CYP2A5 inhibitor in this assay, but it failed to block NNK- induced lung tumorigenesis (Narayanapillai et al., 2014). CYP2A5 activity in liver microsomes from mice exposed to dietary DHM treatment was similar to that from control mice, suggesting that DHM did not affect CYP2A5 activity in vivo. These data overall suggest that it is unlikely that dietary DHM treatment inhibits the bioactivation of NNAL. Fig. 7. NNAL-O-gluc formation by (A) lung microsomes and (B) liver microsomes NNAL can be detoxified via several mechanisms, such as N-oxidation from A/J mice exposed to dietary DHM or DHK in comparison with control (n = 5). We incubated 5 mM NNAL for 30 minutes with NNAL-O-gluc quantified by LC- and glucuronidation. NNAL glucuronidation has been characterized in MS/MS. Statistical analysis was performed with one-way analysis of variance several species, including humans (Carmella et al., 2002; Wiener et al., followed by Dunnett’s test relative to the control group; *P , 0.05. Dihydromethysticin Increases NNAL-O-Glucuronidation In Vivo 427 determined whether DHM treatment may have different effects on these Carmella SG, Akerkar SA, Richie JP, Jr, and Hecht SS (1995) Intraindividual and interindividual differences in metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1- cells. It is possible that DHM may selectively enhance the UGT activity (3-pyridyl)-1-butanone (NNK) in smokers’ urine. Cancer Epidemiol Biomarkers Prev 4:635–642. in a subpopulation of the lung cells, which significantly reduces free Carmella SG, Le Ka KA, Upadhyaya P, and Hecht SS (2002) Analysis of N- and O-glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine. Chem Res Toxicol NNAL in the target cells and prevents DNA adduct formation and 15:545–550. tumorigenesis, even though the levels of free NNAL in the whole lung Carmella SG, Ming X, Olvera N, Brookmeyer C, Yoder A, and Hecht SS (2013) High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitro- and sera are not affected. Work is ongoing to test these hypotheses. samine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in Because DHM-induced increase in NNAL-O-gluc formation may smokers. Chem Res Toxicol 26:1209–1217. Chen G, Dellinger RW, Sun D, Spratt TE, and Lazarus P (2008) Glucuronidation of tobacco- account for its lung cancer chemopreventive activity, the urinary ratio of specific nitrosamines by UGT2B10. Drug Metab Dispos 36:824–830. NNAL-gluc to free NNAL has the potential to facilitate the translational Crampsie MA, Jones N, Das A, Aliaga C, Desai D, Lazarus P, Amin S, and Sharma AK (2011) development of DHM. Results from several human studies demonstrate Phenylbutyl isoselenocyanate modulates phase I and II enzymes and inhibits 4- (methylnitrosamino)-1-(3-pyridyl)- 1-butanone-induced DNA adducts in mice. Cancer Prev that such a ratio varies among individuals (Carmella et al., 1995; Park Res (Phila) 4:1884–1894. et al., 2015). It is possible that individuals with a low basal urinary ratio of Dellinger RW, Chen G, Blevins-Primeau AS, Krzeminski J, Amin S, and Lazarus P (2007) Glucuronidation of PhIP and N-OH-PhIP by UDP-glucuronosyltransferase 1A10. Carcino- NNAL-gluc to free NNAL may more likely benefit from DHM in genesis 28:2412–2418. reducing their lung cancer risk via enhancing/restoring their detoxifi- Devereux TR, Belinsky SA, Maronpot RR, White CM, Hegi ME, Patel AC, Foley JF, Greenwell A, and Anderson MW (1993) Comparison of pulmonary O6-methylguanine DNA adduct levels cation capacity. Such a biomarker could also be used to monitor the and Ki-ras activation in lung tumors from resistant and susceptible mouse strains. Mol Car- chemopreventive effect of DHM; if DHM increases the urinary ratio of cinog 8:177–185. Guo L, Shi Q, Dial S, Xia Q, Mei N, Li QZ, Chan PC, and Fu P (2010) Gene expression profiling NNAL-gluc to free NNAL in a specific individual, DHM may likely in male B6C3F1 mouse livers exposed to kava identifies—changes in drug metabolizing genes – reduce the lung cancer risk via enhancing NNAL detoxification. and potential mechanisms linked to kava toxicity. Food Chem Toxicol 48:686 696. Downloaded from Hecht SS (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Glucuronidation is also involved in the detoxification of other carcino- Chem Res Toxicol 11:559–603. gens (Bock, 1991; Dellinger et al., 2007). DHM may induce UGT(s) to Hecht SS (2014) It is time to regulate carcinogenic tobacco-specific nitrosamines in cigarette tobacco. Cancer Prev Res (Phila) 7:639–647. help detoxify carcinogens besides NNAL, which would have a broader Hollander MC, Zhou X, Maier CR, Patterson AD, Ding X, and Dennis PA (2011) A Cyp2a impact to reduce human cancer risk. On the other hand, glucuronidation is polymorphism predicts susceptibility to NNK-induced lung tumorigenesis in mice. Carcino- genesis 32:1279–1284. critical in the metabolism of various therapeutic drugs (Burchell, 2003) Li Y, Mei H, Wu Q, Zhang S, Fang JL, Shi L, and Guo L (2011) Methysticin and 7,8-dihydromethysticin are two major kavalactones in kava extract to induce CYP1A1. 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