We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kava's hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.
We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice during the initiation and post-initiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by ~ 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in complete inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched Fraction B fully recapitulated kava’s chemopreventive efficacy while kavalactone-free Fractions A and C were much less effective. Mechanistically, kava and Fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O6-methylguanine (O6-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O6-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava.
Effective chemopreventive agents are needed against lung cancer, the leading cause of cancer death. Results from our previous work showed that dietary dihydromethysticin (DHM) effectively blocked initiation of lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA adducts in lung. This study explored the mechanism(s) responsible for DHM's differential effects on NNK/NNAL-derived DNA damage by quantifying their metabolites in A/J mice. The results showed that dietary DHM had no effect on NNK or NNAL abundance in vivo, indicating that DHM does not affect NNAL formation from NNK. DHM had a minimal effect on cytochrome P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation in A/J mouse lung), suggesting that it does not inhibit NNAL bioactivation. Dietary DHM significantly increased O-glucuronidated NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes from dietary DHM-treated mice showed enhanced activities for NNAL O-glucuronidation. These results overall support the notion that dietary DHM treatment increases NNAL detoxification, potentially accounting for its chemopreventive efficacy against NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate the clinical evaluation of DHM-based lung cancer chemopreventive agents.
Anxiolytic kava products have been associated with rare but severe hepatotoxicity in humans. This adverse potential has never been captured in animal models, and the responsible compound(s) remains to be determined. The lack of such knowledge greatly hinders the preparation of a safer kava product and limits its beneficial applications. In this study we evaluated the toxicity of kava as a single entity or in combination with acetaminophen (APAP) in C57BL/6 mice. Kava alone revealed no adverse effects for long-term usage even at a dose of 500 mg/kg bodyweight. On the contrary a three-day kava pretreatment potentiated APAP-induced hepatotoxicity, resulted in an increase in serum ALT and AST, and increased severity of liver lesions. Chalcone-based flavokawains A (FKA) and B (FKB) in kava recapitulated its hepatotoxic synergism with APAP while dihydromethysticin (DHM, a representative kavalactone and a potential lung cancer chemopreventive agent) had no such effect. These results, for the first time, demonstrate the hepatotoxic risk of kava and its chalcone-based FKA and FKB in vivo and suggest that herb–drug interaction may account for the rare hepatotoxicity associated with anxiolytic kava usage in humans.
(+)-Dihydromethysticin was recently identified as a promising lung cancer chemopreventive agent while (+)-dihydrokavain was completely ineffective. A pilot in vivo structure-activity relationship (SAR) was explored, evaluating the efficacy of its analogs in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced short-term O6-methylguanine and long-term adenoma formation in the lung tissues in A/J mice. Both results revealed cohesive SARs, demonstrating that the methylenedioxy functional group in DHM is essential while the lactone functional group tolerates modifications.
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), O-methylguanine (O-mG) is likely the most carcinogen in A/J mice. Results of our previous studies showed that levels of O-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 O-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 O-mG in the target lung tissue, but there were no significant differences in O-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.
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 or 16 h before each NNK injection. The simultaneous treatment (0 h) or 40 h pretreatment (−40 h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1−8 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 O 6 -methylguanine (O 6 -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 O 6 -mG and NNAL detoxification as possible surrogate biomarkers.
Kava (Piper methysticum Forster) extract and its major kavalactones have been shown to block chemically induced lung tumor initiation in mouse models. Here we evaluated the chemopreventive effect of a kavalactone-rich Kava fraction B (KFB), free of flavokavains, on carcinogenesis in a transgenic adenocarcinoma of mouse prostate (TRAMP) model and characterized the prostate gene expression signatures. Male C57BL/6 TRAMP mice were fed AIN93M diet with or without 0.4% KFB from 8 wk of age. Mice were euthanized at 16 or 28 wk. The growth of the dorsolateral prostate (DLP) lobes in KFB-treated TRAMP mice was inhibited by 66% and 58% at the respective endpoint. Anterior and ventral prostate lobes in KFB-treated TRAMP mice were suppressed by 40% and 49% at 28 wk, respectively. KFB consumption decreased cell proliferation biomarker Ki-67 and epithelial lesion severity in TRAMP DLP, without detectable apoptosis enhancement. Real time qRT-PCR detection of mRNA from DLP at 28 wk showed decreased expression of cell cycle regulatory genes congruent with Ki-67 suppression. Microarray profiling of DLP mRNA indicated that "oncogene-like" genes related to angiogenesis and cell proliferation were suppressed by KFB but tumor suppressor, immunity, muscle/neuro, and metabolism-related genes were upregulated by KFB in both TRAMP and WT DLP. TRAMP mice fed KFB diet developed lower incidence of neuroendocrine carcinomas (NECa) (2 out of 14 mice) than those fed the basal diet (8 out of 14 mice, χ = 5.6, P < 0.025). KFB may, therefore, inhibit not only TRAMP DLP epithelial lesions involving multiple molecular pathways, but also NECa. © 2016 Wiley Periodicals, Inc.
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