Oral dosing of dihydromethysticin ahead of tobacco carcinogen NNK effectively prevents lung tumorigenesis in A/J mice

Abstract

Our early studies demonstrated an impressive chemopreventive efficacy of dihydromethysticin (DHM), unique in kava, against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, in which DHM was supplemented in the diet. The current work was carried out to validate the efficacy, optimize the dosing schedule and further elucidate the mechanisms using oral bolus dosing of DHM. The results demonstrated a dose-dependent chemopreventive efficacy of DHM (orally administered 1 h before each of the two NNK intraperitoneal injections, 1 week apart) against NNK-induced lung adenoma formation. Temporally, DHM at 0.8 mg per dose (~32 mg per kg body weight) exhibited 100% lung adenoma inhibition when given 3 and 8 h before each NNK injection and attained >93% inhibition when dosed at either 1 h or 16 h before each NNK injection. The simultaneous treatment (0h) or 40 h pretreatment (−40h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1 h to 8 h after each NNK injection) was ineffective against lung tumor formation. In short-term experiments for mechanistic exploration, DHM treatment reduced the formation of NNK-induced O⁶-methylguanine (O⁶-mG, a carcinogenic DNA adduct in A/J mice) in the target lung tissue and increased the urinary excretion of NNK detoxification metabolites as judged by the ratio of urinary NNAL-O-gluc to free NNAL, generally in synchrony with the tumor prevention efficacy outcomes in the dose scheduling time-course experiment. Overall, these results suggest DHM as a potential chemopreventive agent against lung tumorigenesis in smokers, with O⁶-mG and NNAL detoxification as possible surrogate biomarkers.

Graphical Abstract

Introduction

Despite the impressive advancements of checkpoint inhibitor immunotherapies for a sizable fraction of non-small cell lung cancer patients, lung cancer remains the top cause of malignancy-related mortality in the United States,¹ accounting for more cancer-related deaths than the next three most deadliest cancers combined,² Each year, there are over 200,000 newly diagnosed cases and over 140,000 deaths from lung cancer in the United States.² Likewise, the annual global lung cancer related mortality is expected to be over 1.4 million.³ Tobacco smoking is the most important risk factor for lung cancer, many of which could be prevented by smoking cessation ⁴. However, due to the addictive nature of tobacco products^5,6 and other factors that slow the progress in reducing tobacco exposure,^7,8 there is a need for alternative strategies such as chemoprevention to inhibit lung cancer initiation and/or to slow down its development.⁹

Among thousands of chemicals in tobacco smoke, the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is highly potent in selectively inducing adenomas and adenocarcinomas in the lungs of various experimental animal species.¹⁰ NNK-induced lung adenoma model in A/J mice has been widely used for lung cancer chemoprevention studies.¹¹ The A/J mice are prone to lung tumorigenesis because they have the predisposed pulmonary adenoma susceptibility 1 (Pas1) gene, tightly linked to the K-ras protooncogene.¹² With appropriate tobacco carcinogen exposure, such as NNK, A/J mice readily develop multiple lung nodules¹¹ that morphologically, histologically, and molecularly resemble human lung adenocarcinomas.¹³ Mechanistically, NNK can be metabolically activated via α-hydroxylation to generate two reactive species, leading to methyl and pyridyl-oxo-butyl (POB) DNA adducts.^14,15 NNK is also metabolically converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which can be activated via α-hydroxylation resulting in methyl and pyridyl-hydroxo-butyl (PHB) DNA adducts. DNA adduct formation by NNK and NNAL has been proposed as a major underlying mechanism for NNK-induced lung tumorigenesis.¹⁰ Therefore, reducing such DNA damage is a reasonable strategy for lung cancer chemoprevention. Among various DNA adducts induced by NNK, O⁶-methyl guanine (O⁶-mG) is believed essential to initiate lung tumorigenesis in A/J mice because of its high miscoding property^16,17 and O⁶-mG levels have a strong and positive correlation with lung tumor multiplicity.¹⁸

We recently reported the lung cancer chemopreventive efficacy of a herbal natural product prepared from Piper methysticum., commonly known as kava,¹⁹ and identified dihydromethysticin (DHM) as the most active compound using the NNK-induced lung tumorigenesis model in A/J mice.²⁰ These studies showed that continuous dietary supplementation of kava or DHM for 7 days prior to NNK exposure completely blocked lung adenoma formation with a minimum efficacious dose of 0.05 mg DHM per gram of diet. Its chemopreventive efficacy is closely associated with a reduction in methyl and PHB DNA adducts and enhanced glucuronidation-mediated urinary detoxification of NNAL.^20,21 We also demonstrated that dietary DHM was well tolerated in the A/J mice without signs of pathological toxicity in the liver when given at a concentration of 0.5 mg/g diet (500 ppm) for a period of 17-weeks of continuous feeding; this represents at least ten times its minimum efficacious dose.²⁰ Given the purported hepatotoxic risk associated with kava (a mixture of a range of chemicals), DHM as a single compound may have more defined safety profile.

To further validate the chemoprevention efficacy of DHM, dissect its carcinogenesis-stage specificity, and probe its chemopreventive mechanistic connection with DNA adducts and carcinogen detoxification, we investigated the efficacy of orally administered DHM bolus dosing against NNK-induced lung adenoma in A/J mice through dose titration and schedule optimization. We also characterized the DNA adducts and NNK urinary metabolites with time-course experiments to explore their potential contribution to the adenoma prevention efficacy. The data have conclusively established that prophylactic administration of DHM prior to NNK exposure in A/J mice is crucial to prevent lung adenoma formation, and is mechanistically associated with reduced DNA adduct burden and increased NNK detoxification.

Materials and Methods

Caution: NNK is a potential human carcinogen and should be handled carefully in well-ventilated fume hoods with proper protective clothing.

Chemicals, reagents and animal diets

The AIN-93G and AIN-93M powdered mouse diets were purchased from Harlan Teklad (Cambridgeshire, UK). Natural DHM was isolated from a kava product supplied by Gaia Herbs, Inc. (Brevard, NC), following reported protocols.²⁰ NNK, [¹³C₆]NNK, [¹³C₆]NNAL, [CD₃]O⁶-mG and [4-CD₂, CD₃]NNAL-O-gluc were purchased from Toronto Research Chemicals (Toronto, ON, Canada).²² O⁶-methylguanine (O⁶-mG) was purchased from Midwest Research Institute (Kansas City, MO). 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-pobG), O²-[4-(3-pyridyl)-4-oxobut-1yl]thymidine (O²-pobdT), O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O⁶-pobdG), 7-[4-(3-pyridyl)-4-hydroxobut-1-yl]guanine (7-phbG), O²-[4–(3-pyridyl)-4-hydroxobut-1yl]thymidine (O²-phbdT), O⁶-[4-(3-pyridyl)-4-hydroxobut-1-yl]-2′-deoxyguanosine (O⁶-phbdG) and their corresponding deuterated analogs were synthetized following reported procedures.^23,24 Micrococcal nuclease and phosphodiesterase II were purchased from Worthington Biochemical Corporation (Lakewood Township, NJ). Alkaline phosphatase was from Roche Molecular Biochemicals (Pleasanton, CA).

Evaluation of the dose-response efficacy of prophylactic DHM gavage on NNK-induced lung adenoma in A/J mice

Female A/J mice (5–6 weeks of age) were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the specific pathogen-free facilities, according to animal welfare protocols approved by Institutional Animal Care and Use Committee at the University of Minnesota and the University of Florida. After 1-week acclimation, mice were weighed, randomized into different groups and switched to AIN-93G powdered diet, defined as Day 1. The number of mice in each group is specified in the “Results” section.

On Day 7 and Day 14, mice in the non-carcinogen negative control and NNK groups received PEG-400 (200 µL) orally, whereas mice in the other groups received DHM dissolved in PEG-400 (200 µL) to deliver 0.2, 0.4, 0.8 and 2.0 mg per mouse at 1 h before each of the two NNK intraperitoneal (i.p.) injection. Mice in the negative control group received 0.1 mL physiologic saline solution, whereas mice in the other groups received NNK (100 and 67 mg/kg respectively on Day 7 and Day 14 in 0.1 mL of physiologic saline solution) via i.p. injection 1 h after DHM gavage. At the end of Day 21, mice were switched to the AIN-93M powdered diet until the end of the study (Day 119). Diet consumption was measured twice weekly and body weight was monitored weekly. All mice were euthanized on Day 119. The lungs were collected and tumors on the surface of the lungs were counted by a board-certified veterinary pathologist (M.G.O՛S) using a dissecting microscope without knowledge of the treatment regimens.

Evaluation of the effect of DHM on NNK-induced DNA adducts in the lung or liver tissues and urinary NNAL metabolites in A/J mice

Female A/J mice (5–6 weeks of age), after 1-week acclimation, were weighed and randomized to different groups (n=3–5), as specified in the “Results” section and switched to AIN-93G powdered diet with the date being defined as Day 1. On Day 7, mice in the respective groups were given a single dose of 0.8 mg DHM in PEG-400 (200 µL) via oral gavage followed by a single i.p. injection of NNK in saline (100 μL, 100 mg/kg of body weight) at different time intervals as indicated in the “Results” section. Mice in the negative control group were given PEG-400 and saline respectively. Depending on the experimental design, mice were euthanized at different times (0.5 h up to 24 h) after NNK exposure. The lungs were harvested, snap-frozen in liquid N₂ and stored at −80°C until DNA isolation. The urine samples were collected at the time of euthanasia and stored at −80°C until analyses.

Isolation of DNA adducts in the lung or liver tissues and quantification by liquid chromatography-electrospray ionization/tandem mass spectrometry

DNA was isolated from 25 – 50 mg lung or liver tissue of each individual mouse, following Puregene DNA isolation protocol from Qiagen Corp.²⁵ 7-PobG, O²-pobdT, O⁶-pobdG, 7-phbG, O²-phbdT, O⁶-phbdG, O⁶-mG and 7-mG were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS), following reported protocols.^18,25,26

Quantification of urinary NNAL-O-gluc and free NNAL

Urine samples collected from mice upon euthanasia were diluted 10⁵ times with LC-MS grade water. The diluted samples (0.1 ml each) were mixed with [4-CD₂, CD₃]NNAL-O-gluc at a final concentration of 5 ng/ml and [¹³C₆]NNAL at a final concentration of 10 ng/ml. LC-MS/MS analysis was performed following the method published earlier.²¹

The efficacy of time-course DHM gavage with respect to NNK exposure to suppress carcinogen-induced lung adenoma in A/J mice (dosing schedule optimization)

Female A/J mice (5 to 6 weeks of age) were maintained the same as in the Efficacy Dose-Response experiment above. After 1-week acclimation, mice were weighed, randomized into different groups and switched to AIN-93G powdered diet, defined as Day 1. On Day 7 and Day 14, mice in the non-carcinogen negative control and NNK groups received PEG-400 (200 µL) 1h before NNK injection whereas mice in the other groups received oral dose of 0.8 mg DHM dissolved in PEG-400 (200 µL) various hours before (−40h, −16h, −8h, −3h, −1h), simultaneously (0h), or after (1h, 4h, 8h) each of the two NNK i.p. injections (100 and 67 mg/kg respectively in 0.1 mL physiologic saline solution). Mice in the negative control group received 0.1 mL physiologic saline solution. At the end of Day 21, mice were switched to AIN-93M powdered diet until the end of the study (Day 119). Diet consumption was measured twice weekly and body weight was monitored weekly. All mice were euthanized on Day 119. The lungs were collected and tumors on the surface of the lungs were counted by a board-certified veterinarian pathologist without knowledge of the treatment regimens.

Statistical analysis

Data on lung adenoma multiplicity, body weight, liver weight relative to body weight, DNA adducts, and urinary ratio of NNAL-O-gluc to free NNAL were reported as mean ± standard deviation (SD). One-way ANOVA was used to compare means among NNK and NNK + treatment groups for the above parameters. The Dunnett test was used for comparisons of the quantity between NNK control and NNK + treatment groups. P value ≤0.05 was considered statistically significant. All analyses were conducted using GraphPad Prism 4 (GraphPad Software, Inc.).

Results

The prophylactic efficacy of orally administrated bolus DHM doses on NNK-induced lung adenoma burden in A/J mice

In the first experiment, we evaluated the dose-response efficacy of DHM administration at 0.2, 0.4, 0.8 and 2.0 mg/mouse. These doses correspond to the daily intake range of DHM per mouse when it was supplemented in diet at 0.05 – 0.5 mg/g diet, assuming daily food intake of four grams of diet per mouse. The DHM dose was given 1 h before each of the two NNK i.p. injections. As shown in Fig. 1A, the orally dosed DHM demonstrated a clear dose-response relationship in blocking NNK-induced lung tumorigenesis in A/J mice. Specifically DHM at a dose of 2.0 mg/mouse decreased NNK-induced adenoma multiplicity by 98.9%. DHM at 0.8 mg/mouse reduced adenoma multiplicity by 93.3%. Even at 0.4 and 0.2 mg per dose, DHM demonstrated significant effects in reducing adenoma formation (49.8% and 42.0% reduction respectively). The oral bolus administration of DHM (2 doses, one week apart, 1 h before each of the two NNK injections) appeared safe up to the dose of 2 mg/mouse on the basis of mouse body weight (Fig. 1B), liver weight (Fig. 1C) and liver weight relative to body weight (Fig. 1D), which showed no significant differences from the NNK control group.

Fig. 1. The efficacy dose-response of orally administrated DHM 1 h before each of two NNK injections on carcinogen-induced lung adenoma formation in A/J mice.

Female A/J mice in all groups except control group (NC) were treated with NNK (100 and 67 mg/kg body weight on day 7 and day 14 respectively) in 0.1mL saline via intraperitoneal injection (n=14 for NNK group and n=5 for all other groups). DHM was given via gavage in 0.2 mL PEG-400 1 h before each NNK injection. The mice were maintained on AIN-93G diet until day 21 and then on AIN-93M diet for the duration of the experiment. (A) Dose-response DHM dosing on NNK-induced lung adenoma multiplicity. The effects of such treatments on mouse body weight (B), liver weight (C), and liver weight relative to body weight (D). One-way ANOVA was used to compare means among NNK and NNK + treatment groups for lung adenoma multiplicity, body weight and liver weight relative to body weight. The Dunnett test was used for comparisons of the quantity between NNK control and NNK + treatment groups. P value < 0.05 was considered statistically significant. ***: p <0.001; ****: p <0.0001. The numbers above each column in 1A are the percentage of reduction in tumor multiplicity relative to NNK control group.

Profiling the effect of a single prophylactic oral dose DHM on NNK-induced DNA adducts in A/J mouse lung or liver tissues

Our previous studies observed that DHM significantly reduced NNK-induced DNA adducts in the lung tissues when DHM was supplemented in diet, which likely contributes to its chemopreventive activity on adenoma multiplicity.²⁰ We therefore investigated the effect of a single dose of DHM before NNK injection on the profiles of DNA adducts in the lung and liver. Mice were given 0.8 mg DHM via oral gavage at 1 h or 3 h before the i.p. injection of NNK (100 mg/kg dose), and euthanized after 24 h. DNA isolated from the lung and liver tissues were used to estimate the levels of methyl, POB and PHB adducts as described in “Methods” section. Consistent with the previous results, DHM at a dose of 0.8 mg/mouse significantly decreased NNK-induced DNA adducts in the target lung tissues (Fig. 2A) and the non-target liver tissues (Fig. 2B), including methyl, PHB and POB adducts. In the target lung tissue, the extent of reduction in methyl adducts was the most significant while there appeared a slight preferential reduction in PHB adducts relative to POB adducts, consistent with the results when DHM was supplemented in the diet ²⁰. There were no obvious preferences in DNA adduct reduction in the liver.

Fig. 2. Effects of orally administered DHM on NNK-induced DNA adducts in A/J mouse lung or liver tissue.

Mice in groups (n = 3 – 5) were maintained on AIN93-G diet and gavaged with 0.8 mg DHM at different time points relative to NNK i.p. injection (100 mg/kg body weight on day 7) and euthanized at indicated time points after NNK treatment as specified. DNA isolated from (A) lung and (B) liver and analyzed for methyl, POB and PHB adducts as described in “Materials and Methods” with DHM (0.8 mg) given 1 or 3 h before NNK injection and the mice euthanized at 24 h after NNK. (C) Time-course of lung O⁶-mG adduct abundance with DHM (0.8 mg) administered 1 h before NNK and the mice were euthanized at 0.5, 1, 2, 4, and 8 h after NNK treatment. (D) Lung O⁶-mG adduct abundance from mice with DHM (0.8 mg) administered 40, 16, 8, 3, 1 h before or simultaneously (0 h) with NNK and the mice were euthanized at 2 h after NNK treatment. One-way ANOVA was used to compare means among NNK and NNK + treatment groups for A, B and D. The Dunnett test was used for comparisons of the quantity between NNK control and NNK + treatment groups. P value ≤0.05 was considered statistically significant. Two-tailed student t-test was used for comparisons of the quantity between NNK control and NNK + treatment groups in C. *: p < 0.05; **: p < 0.01; ***: p <0.001; ****: p <0.0001.

Since the DNA adducts in the above experiment were profiled 24 h after the NNK injection, we next characterized the kinetics of lung DNA adduct formation (focusing on O⁶-mG) with respect to the speed for DHM’s action to occur. Mice were given 0.8 mg DHM via oral gavage 1 h before the NNK i.p. injection with lungs collected at 0.5, 1, 2, 4 and 8 h after NNK exposure. DHM treatment significantly reduced O⁶-mG in the target lung tissues from as early as 0.5 h after the NNK injection, which was 1.5 h after DHM gavage (Fig. 2C). The reduction persisted through the collection duration.

Given the rapid onset of DHM’s effects on DNA adducts (Fig. 2C), we then characterized the time course of prophylactic DHM dosing duration from 40 h before (−40h) to simultaneously (0h) with the NNK exposure on lung O⁶-mG abundance at 2 h after the NNK injection. The data (Fig. 2D) showed an inverse bell-shaped time-course response curve in reducing NNK-induced O⁶-mG in the target lung tissues with DHM given 8 h before NNK exposure resulting in the peak reduction (88.2%). Whereas 40 h prior dosing (−40h) of DHM resulted in a marginal reduction in O⁶-mG, the −16h prophylactic dose still exerted a substantial reduction (74.8%). Even simultaneous dosing of DHM with the NNK injection resulted in a remarkable reduction (63.3%). Since DNA adduct burden reduction is one of the major mechanisms of DHM to decrease NNK-induction of lung tumorigenesis, we hypothesize that the full-course adenoma efficacy outcome will tightly correlate with the O⁶-mG metrics, which was tested in our final efficacy experiment detailed later.

Profiling the effects of a single prophylactic oral dose DHM on NNK to NNAL conversion or glucuronidation-mediated NNAL detoxification

Since a prophylactic oral DHM dose exerted significant and rapid reductions in O⁶-mG adduct (Fig. 2C and 2D), we investigated whether DHM had any effect on NNK/NNAL conversion by quantifying the ratio of NNK and total NNAL in the urine samples from A/J mice as described earlier.²¹ When examined at 0.5, 1 and 2 h after the NNK injection, there were no significant changes in the ratios of NNK to total NNAL with 1 h pre-administration of 0.8 mg DHM dose (Fig. 3A). The ratio was used herein because the urine volume of the mice varied and could not be rigorously controlled (some mice may have urinated right before euthanasia). These data presented herein are consistent with our previous study, which demonstrated that dietary DHM does not affect the conversion of NNK to NNAL.²¹ The analysis could not be extended beyond 2 h because NNK was not detectable in mouse urine samples collected at the later time points. These data suggest that DHM administered via oral gavage does not perturb the conversion of NNK to NNAL.

Fig. 3. Effect of orally administered DHM on levels of urinary NNK metabolites in A/J mice.

(A) Mice in different groups (n=3) were maintained on AIN93-G diet and gavaged with DHM (0.8 mg) 1h before NNK i.p. injection (100 mg/kg body weight on day 7). Mice were euthanized 0.5, 1, 2, 4, and 8h after NNK treatment. Urine samples collected upon euthanasia were analyzed to estimate the ratios of NNK:total NNAL. (B) Mice in different groups (n=4–5) were maintained on AIN93-G diet and gavaged with DHM (0.8 mg) 40, 16, 8, 3, 1 h before or simultaneously (0 h) with NNK (100 mg/kg body weight i.p. on day 7). Mice were euthanized after 2 h of NNK treatment. Urine samples collected upon euthanasia were analyzed to estimate the ratios of NNAL-O-gluc:free NNAL. One-way ANOVA was used to compare means among NNK and NNK + treatment groups for B. The Dunnett test was used for comparisons of the quantity between NNK control and NNK + treatment groups. P value < 0.05 was considered statistically significant. *: p < 0.05; **: p < 0.01; ***: p <0.001; ****: p <0.0001.

Our early studies suggest that enhancing NNAL O-glucuronidation (NNAL-O-gluc, NNAL-N-gluc is not detectable in mice) and urinary detoxification may contribute towards the chemopreventive activity of dietary DHM as kavalactones that did not enhance NNAL-O-gluc had no chemopreventive activity.²¹ We therefore tested whether a prophylactic oral DHM dose would modulate the ratio of urinary NNAL-O-gluc and free NNAL, which is a convenient and promising parameter to evaluate glucuronidation-mediated NNAL detoxification.²⁷ In a pilot experiment, a 1-h prophylactic DHM dose before NNK exposure had no significant effect (data not shown). Because changes in the expression and activity of phase II detoxification enzymes might require longer time periods than the short prophylactic DHM dose duration (1 h) examined, we next measured the ratio of urinary NNAL-O-gluc and free NNAL using the urine samples from the detailed pretreatment time course experiment (Fig. 2D). The ratio of NNAL-O-gluc to free NNAL demonstrated a bell-shaped time-course response curve, peaking at 8 h prophylactic DHM dosing (−8h) (Fig. 3B). The bell shape direction is opposite to the curve of lung O⁶-mG adduct abundance (Fig. 2D).

Optimizing lung adenoma efficacy of prophylactic DHM dosing schedule with respect to NNK exposure

The short-term “mechanistic” assessments on lung DNA adduct burden and NNK detoxification prompted us to evaluate the time-course effect of DHM oral dosing (0.8 mg/mouse) relative to the NNK exposure on blocking lung tumorigenesis in the long-term adenoma efficacy bioassay. The DHM gavage was given between 40 h (−40h) and 1 h (−1h) before, concurrently (0 h), or up to 8h after each NNK exposure. The results demonstrated a bell-shaped time course effect of DHM in decreasing NNK-induced lung adenoma formation (Fig. 4A). Specifically, DHM effectively blocked lung adenoma formation when it was given 3 – 8 h before the NNK exposure (100%). The efficacy was still remarkable when given as short as 1 h (93.3% reduction) or as long as 16 h (97.8% reduction) before the NNK exposure. Surprisingly, DHM orally administered 40 h before the NNK exposure (−40h) still resulted in a 52.1% reduction in tumor multiplicity. On the other hand, DHM given simultaneously with the NNK exposure (0h) showed a comparable 49.8% reduction in lung adenoma burden. However, DHM dosed after the NNK exposure (1 h to 8 h) showed no reduction in lung adenoma multiplicity. The DHM doses were well tolerated without any adverse impact on body weight or liver weight relative to body weight (data not shown). These data clearly indicate that DHM treatment ahead of the NNK exposure is essential to impact the mouse physiology and tissue biochemistry to maximize its tumor initiation-blocking efficacy. The lack of efficacy of DHM in the post-NNK delivery suggested its lack of impact on the repair of preformed NNK-induced DNA adducts, most of which have been formed by 0.5 h after NNK injection (Fig. 2C).

Fig. 4. Dosing schedule optimization for orally administrated DHM before, concurrent or after NNK injection on NNK-induced lung adenoma formation in A/J mice.

(A) Time-response DHM dosing on NNK-induced lung adenoma multiplicity. Female A/J mice in all groups except the control group were treated with NNK (100 and 67 mg/kg body weight on day 7 and day 14 respectively) in 0.1mL saline via i.p. injection (n=14 for NNK alone group and n=5 for all other groups). DHM (0.8 mg) was given via gavage in 0.2 mL PEG-400 at 40, 16, 8, 3, 1 h before (−40h to −1h), simultaneously (0 h), or 1, 4 or 8 h after NNK injection. The mice were maintained on AIN-93G diet until day 21 and then given AIN-93M diet for the duration of the adenoma bioassay. One-way ANOVA was used to compare means among NNK and NNK + treatment groups. The Dunnett test was used for comparisons of the quantity between NNK control and NNK + treatment groups. P value < 0.05 was considered statistically significant. *: p < 0.05; **: p < 0.01; ***: p <0.001; ****: p <0.0001. (B) Relationship of tumor reduction efficacy (% reduction from the NNK group), O⁶-mG reduction (% reduction from the NNK group), and increase in NNAL-O-gluc detoxification (% over the NNK group) vs. DHM pretreatment time.

Discussion

The results of this study clearly demonstrated that bolus dosing of DHM by oral gavage (two DHM doses one week apart and 1 h ahead of each of the two NNK injections) dose-dependently inhibited lung carcinogenesis in A/J mice (Fig. 1). The time course design allowed us to define the optimal window of prophylactic DHM dosing in relation to NNK exposure to maximize its adenoma blocking efficacy (Fig. 4A). The efficacious dose of 0.8 mg DHM per mouse, given by gavage between 8 and 3 h before NNK injection, completely blocked lung adenoma formation (Fig. 4A). The efficacy was retained for 16 h (97.8% reduction) and remained impressive with ~50% reduction of adenoma burden when administered 40 h prior to or concurrently with the NNK exposure (Fig. 4A). Although DHM was not effective when given after the NNK exposure, the long-retained action of orally administered DHM before NNK exposure strongly supports its chemoprevention potential for active smokers since it can provide current smokers with up to 40 hours of protection from NNK-induced DNA damage. These findings validated and extended our previous observations with dietary delivery, which firmly establish that DHM effectively prevents NNK-induced lung tumorigenesis initiation in the A/J mouse model. Per allometric conversion, the efficacious dose of 0.8 mg per mouse (weighing ~25 g) yields a predicted equivalent human dose of 200 mg DHM (75 kg body weight). Such a low dose of DHM effectively blocks DNA damage produced by a high dose of NNK in A/J mice. This suggests that the dose requirement of DHM should be feasible for oral delivery as a neat compound. Given the substantially lower dose of NNK exposure among smokers in comparison to the injection dose in the A/J mouse model, a smaller daily dose of DHM is expected to offer significant protection for current smokers against NNK induced tumorigenesis.

Our earlier work had established a strong association of reduction in NNK-induced lung O⁶-mG and lung tumor chemoprevention efficacy in A/J mice with the dietary delivery of DHM.^19,20,28 That relationship was further illuminated with the more detailed time-course evaluation of the impacts of dosing schedule between DHM gavage and NNK injection on lung O⁶-mG levels (Fig. 2D) and the reduction in lung adenoma multiplicity (Fig. 4A and B). The overall synchrony of these changes within the time frame of DHM 16 h pretreatment (−16h) to concurrent treatment (0h) with respect to the NNK exposure is consistent with the hypothesis that reduction in DNA damage by DHM, particularly O⁶-mG, is one key mechanism for its chemopreventive activity against NNK-induced lung carcinogenesis in A/J mice. On the other hand, DHM when given 40 h before the NNK exposure exerted only a marginal reduction in O⁶-mG while it reduced lung adenoma multiplicity by 52.1% (Fig. 2D, 4A and 4B). These data suggest that there might be DNA adduct/damage-independent mechanisms by which DHM pretreatment could modulate to prevent NNK-induced lung carcinogenesis. Proteomic and transcriptomic approaches are being deployed to interrogate such a possibility.

It should also be noted that the short-term O⁶-mG data and the long-term adenoma multiplicity data were obtained from two separated rounds of animal studies. The potential contribution of mouse-to-mouse and experimental variations could not be excluded. Another limitation is that O⁶-mG quantified herein reflected the whole lung tissues, which included different types of cells in the lung. Earlier work by others suggests that lung adenomas induced by NNK might be derived from specific types of cells in the lung, such as the type II cells²⁹ or lung cells with stem cell properties.³⁰ DHM might differentially reduce NNK-induced DNA damage in certain types of lung cells, which could be of varied importance to NNK-induced lung tumorigenesis. Further studies may be needed to separate different types of cells in the lung tissues to characterize the effects of DHM, which is technically challenging.

Although some reports showed that DHM could inhibit CYP2C9, 2C19, 2D6, and 3A4 in human liver microsomes with an IC₅₀ around 10 µM,³¹ none of these CYPs have been demonstrated to activate NNK or NNAL. There has been no knowledge of DHM’s effects on UGTs and other transporters. Our previous work suggested that continuous dietary DHM increased NNAL glucuronidation with no effect on CYP2A5 inhibition,²¹ leading to enhanced NNAL detoxification and reduced DNA damage. The current work provided further insights into the time frame necessary for DHM to induce an increase in NNAL detoxification (the ratio of urinary NNAL-O-gluc to free NNAL) and its connection with lung tumor chemoprevention efficacy outcomes (Fig. 3B and 4B). The bell-shape time course profile of the urinary ratio of NNAL-O-gluc to free NNAL upon DHM treatment (Fig. 3B) was in reasonable synchrony with the reverse bell-shape time-course profile of lung tissue O⁶-mG (Fig. 2D) and lung adenoma multiplicity (Fig. 4A), except with the longest pretreatment time point (−40 h). Given that NNAL glucuronidation is mediated via UDP-glucuronosyltransferases (UGT),^32,33 the time-course profile suggested that DHM might activate the responsible UGT(s) likely via transcriptional activation to enhance NNAL detoxification, peaking with 8 h DHM pretreatment. Exploration of DHM’s effect on transcription of certain UGTs is ongoing. At the same time, the urinary data reflect the collective activities of all tissues in the body while DHM could have tissue variations in enhancing NNAL detoxification. In another word, DHM may activate UGTs more efficiently in certain tissues to preferentially and efficiently remove NNAL in such tissues. The potential increase in NNAL detoxification by DHM in such tissues may be under-estimated in our analysis of urinary NNAL detoxification. Another complicating factor for the data interpretation is the lack of quantitative knowledge of NNK metabolism in A/J mice under these experimental conditions. Although this A/J mouse model has been widely used to evaluate lung cancer chemopreventive agents ¹¹ and some metabolites of NNK have been quantitatively characterized,^34,35 it is still not known what percentage of NNK would be eliminated via urinary NNAL excretion, what percentage of NNK would be bioactivated, and the dose-response relationship between bioactivated NNK/NNAL and induced DNA damage. If NNAL urinary excretion is a dominant pathway of NNK metabolism under the experimental conditions in A/J mice, a slight increase in NNAL detoxification (the ratio of urinary NNAL-O-gluc to free NNAL) could have a significant reduction in NNK-induced DNA damage. Further quantitative analyses of NNK metabolism, therefore, may be essential to more accurately interpret the connections of NNK-induced DNA damage, NNAL urinary excretion, and the impact of DHM on these processes in association with blocking lung tumor formation.

Overall, these correlative data (Fig. 4B) suggest a causal relationship between DHM enhanced NNAL detoxification via O-glucuronidation, reduction in O⁶-mG and other DNA adducts in the lung tissues, and the eventual blockade of lung adenoma formation. The short-term assay parameters, therefore, have the potential as biomarkers to timely monitor the chemopreventive efficacy of DHM and kava in reducing NNK-induced lung carcinogenesis risk. Indeed, in a short-term pilot human trial, we have documented that kava as a dietary supplement enhances the urinary NNAL clearance and reduces urinary DNA adducts among current smokers,³⁶ indicating that kava dietary supplement, upon proven safe with rigorous composition characterization, may reduce the lung carcinogenesis risk induced by NNK in tobacco users. Reflecting from these translational experiences, the NNK-induced lung tumorigenesis A/J mouse model has several limitations. First, the dose of NNK used in A/J mice is very high relative to its exposure among smokers. Such a high dose may modulate pathways in A/J mice that are physiologically less important among smokers when the dose of NNK is low. Second, tobacco smoke contains many other carcinogens and co-carcinogens, such as polyaromatic hydrocarbons (PAH) and catechols that facilitate lung carcinogenesis among smokers, which are missing in the NNK-induced lung tumorigenesis A/J mouse model. Third, other risk factors in human lung carcinogenesis among smokers, such as air pollution and psychosocial stress, are not captured in the A/J mouse model. The potential impact of DHM and kava on these aspects needs to be evaluated creatively in the future.

In summary, the data from this study support the potential of DHM as a highly efficacious, easily administrable lung cancer initiation blocking agent with durable protective effect against tobacco carcinogen NNK-induced carcinogenesis. Further pre-clinical and clinical evaluation of DHM for its effects on tobacco smoke-induced lung carcinogenesis, ideally better capitulating human pathogenesis, is warranted to eventually translate its benefits to current smokers and second-hand smokers.

Abbreviations

DHM

dihydromethysticin

NNK

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

NNAL

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

POB

pyridyl-oxo-butyl

PHB

pyridyl-hydroxo-butyl

O⁶-mG

O⁶-methylguanine

7-pobG

7-[4–(3-pyridyl)-4-oxobut-1-yl]guanine

O⁶-pobdG

O⁶-[4–(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine

O²-pobdT

O²-[4–(3-pyridyl)-4-oxobut-1-yl]thymidine

7-phbG

7-[4–(3-pyridyl)-4-hydroxobut-1-yl]guanine

O⁶-phbdG

O⁶-[4–(3-pyridyl)-4-hydroxobut-1-yl]-2′-deoxyguanosine

O²-phbdT

O²-[4–(3-pyridyl)-4-hydroxobut-1-yl]thymidine

NNAL-O-gluc

NNAL-O-glucuronide

ANOVA

analysis of variance

SD

standard deviation

UGT

UDP-glucuronosyltransferase