Nitrosamine Impurities in Herbal Formulations: A Review of Risks and Mitigation Strategies.

Nitrosamines are a class of chemical compounds that have been found to be impurities in a variety of pharmaceutical products. These impurities have raised concerns due to their potential carcinogenic effects. Recent studies have identified nitrosamines as impurities in a number of pharmaceutical products including angiotensin II receptor blockers (ARBs) and proton pump inhibitors (PPIs). The presence of nitrosamines in these products has led to recalls and market withdrawals. In addition to pharmaceuticals, nitrosamines have also been found in some herbal medicines particularly those containing traditional Chinese medicinal ingredients. The presence of nitrosamines in herbal formulations poses a significant risk to public health and highlights the need for quality control and regulations in the herbal drug industry. The present review article aims to discuss nitrosamine impurities (NMI) prominent causes, risks and scientific strategies for preventing NMI in herbal formulations. The primary objective of this study is to examine the origins of nitrosamine contamination in herbal formulations, the risks associated with these contaminants, and the methods for reducing them. The significance of thorough testing and examination before releasing herbal products to the public is also emphasized. In conclusion, the presence of nitrosamines is not limited to pharmaceutical products and poses a significant threat to the safety of herbal drugs as well. Adequate testing and extensive research are crucial for producing and distributing herbal medicines to the general population.

Changes in N-nitrosamines, Residual Nitrites, Lipid Oxidation, Biogenic Amines, and Microbiota in Chinese Sausages Following Treatment with Tea Polyphenols and Their Palmitic Acid-modified Derivatives.

This study aimed to investigate the effects of tea polyphenol (TP), epigallocatechin gallate (EGCG), and their palmitic acid-modified derivatives palmitoyl-TP (pTP) and palmitoyl-EGCG (pEGCG) on the accumulation of N-nitrosamine and biogenic amines (BAs), residual nitrites, and lipid oxidation in Chinese sausages. The microorganisms, color, and texture properties of sausages were evaluated. TP, EGCG, pTP, or pEGCG significantly inhibited the accumulation of N-nitrosodimethylamine (NDMA) and BAs, residual nitrites, and lipid oxidation, but enhanced the redness, hardness, and chewiness of sausages. The concentration of NDMA in sausages was reduced by 58.11%, 63.51%, 36.49%, and 44.59%, respectively, after treatment with TP, EGCG, pTP, and pEGCG. Both EGCG and pEGCG exhibited excellent inhibitory effects on the predominant BAs, including putrescine, tyramine, cadaverine, histamine, and 2-phenylethylamine. Palmitoyl-EGCG was found to be the strongest inhibitor of lipid oxidation. Besides, the four antioxidants weakly affected the population of total aerobic bacteria and lactic acid bacteria but totally suppressed the growth of undesirable Enterobacteriaceae. The principal component and correlation analyses proved that BAs, nitrites, lipid oxidation, and microbiota were responsible for the formation of NDMA. The results indicated that palmitic acid-modified TPs and similar derivatives might serve as potential preservatives to improve the safety and quality of fermented meat products.

Dietary Phytochemicals as Potential Chemopreventive Agents against Tobacco-Induced Lung Carcinogenesis.

Lung cancer is the second most common cancer in the world. Cigarette smoking is strongly connected with lung cancer. Benzo[a]pyrene (BaP) and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-butanone (NNK) are the main carcinogens in cigarette smoking. Evidence has supported the correlation between these two carcinogens and lung cancer. Epidemiology analysis suggests that lung cancer can be effectively prevented through daily diet adjustments. This review aims to summarize the studies published in the past 20 years exploring dietary phytochemicals using Google Scholar, PubMed, and Web of Science databases. Dietary phytochemicals mainly include medicinal plants, beverages, fruits, vegetables, spices, etc. Moreover, the perspectives on the challenges and future directions of dietary phytochemicals for lung cancer chemoprevention will be provided. Taken together, treatment based on the consumption of dietary phytochemicals for lung cancer chemoprevention will produce more positive outcomes in the future and offer the possibility of reducing cancer risk in society.

Nitroso Impurities in Drug Products: An Overview of Risk Assessment, Regulatory Milieu, and Control Strategy.

Many nitrosamines have been recognized to be carcinogenic for many decades. Despite the fact that several nitrosamine precursors are frequently used in the manufacturing of pharmaceutical products, their potential presence in pharmaceutical products has previously been overlooked due to a lack of understanding on how they form during the manufacturing process. From the risk assessment, it is clear that nitrosamines or their precursors may be present in any component of the finished dosage form. As a risk mitigation strategy, components with a high potential to form nitrosamine should be avoided. In the absence of suitable alternatives, sufficient measures to maintain nitrosamines below acceptable intake levels must be applied. Excipient manufacturing pathways must be extensively studied in order to identify probable excipient components that may contribute to nitrosamine formation. The manufacturers must not solely rely on pharmacopeial specifications for APIs and excipients, rather, they should also develop and implement additional strategies to control nitrosamine impurities. The formulation can be supplemented with nitrosating inhibitors, such as vitamin C, to stop the generation of nitrosamine. The purpose of this review is to identify key risk factors with regard to nitrosamine formation in pharmaceutical dosage forms and provide an effective control strategy to contain them below acceptable daily intake limits.

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies.

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% - 117%; repeatability was estimated in the range of 1.5% - 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.

Lipid metabolism disorders contribute to hepatotoxicity of ICR mice induced by nitrosamines exposure.

Health risks caused by crucial environmental carcinogens N-nitrosamines triggered ubiquitous attention. As the liver exerted vital function through metabolic process, lipid metabolism disorders have been confirmed as potential drivers for toxicological effects, and the mechanisms of lipid regulation related to hepatotoxicity induced by N-nitrosamines remained largely unclear. In this study, we comprehensively explored the disturbance of hepatic lipid homeostasis in mice induced by nitrosamines. The results implied that nitrosamines exposure induced hepatotoxicity accompanied by liver injury, inflammatory infiltration, and hepatic edema. Lipidomics profiling analysis indicated the decreased levels of phosphatidic acids (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), lyso-phosphatidylcholines (LPC), lyso-phosphatidylethanolamines (LPE), diacylglycerols (DAG) and triacylglycerols (TAG), the elevation of ceramides (Cer) and decomposition of free fatty acids (FFA) in high-dose nitrosamines exposure group. Importantly, nitrosamines exposure promoted fatty acid oxidation (FAO) by facilitating fatty acid uptake and decomposition, together with the upregulation of genes associated with FAO accompanied by the activation of inflammatory cytokines TNF-α, IL-1ß and NLRP3. Furthermore, fatty acid translocase CD36-mediated fatty acid oxidation was correlated with the enhancement of oxidative stress in the liver caused by nitrosamines exposure. Overall, our results contributed to the new strategies to interpret the early toxic effects of nitrosamines exposure.

Evaluation of coloration, nitrite residue and antioxidant capacity of theaflavins, tea polyphenols in cured sausage.

This study evaluated the effects of theaflavins (TFs), tea polyphenols (TP) and vitamin C (VC) on the nitrite residue amount, color, antioxidant capacity and N-nitrosamines inhibition in cured sausage. The addition of TFs, TP and VC combined with NaNO2 respectively could significantly increase the a* value, nitroso pigment content and DPPH free radical scavenging rate, and effectively reduced the content of residual nitrite, metmyoglobin (MetMb) and total N-nitrosamines in cured sausages than treated only with NaNO2 (P < 0.05), of which TFs group was the most significant (P < 0.05). It was indicated that the addition of TFs, TP could better inhibit the oxidation of cured sausages. UV-vis spectroscopy also showed pentacoordinate nitrosyl ferrohemochrome was the main pigment component in the samples. The results demonstrated that TFs and TP could contribute to the desired color and safety of sausage.

Safety and efficacy of a digital therapeutic for substance use disorder: Secondary analysis of data from a NIDA clinical trials network study.

Background: Traditional treatments for substance use disorders (SUDs) rely heavily on face-to-face interactions, which pose substantial limitations for patients. A clinical trial of a digital therapeutic (DT), delivering behavioral therapy demonstrated safety and efficacy in a population including patients with opioid use disorder (OUD) not treated with buprenorphine, which is not a guideline-recommended approach. This study re-analyzed the data excluding patients with OUD to more closely approximate real-world patient populations. Methods: Secondary analysis of patients with substance use disorders related to alcohol, cannabis, cocaine, or other stimulants (n = 399, patients with OUD excluded) from a previously-published randomized controlled trial. Patients received 12-weeks of outpatient treatment-as-usual (TAU; n = 193) or TAU with reduced counseling plus a DT (n = 206) providing computerized cognitive behavioral therapy and contingency management. Primary outcomes were abstinence in weeks 9-12 and retention in treatment. Results: The 399 patients in this analysis (206 in the DT group and 193 in the TAU group) reported substance use disorders related to alcohol, cannabis, cocaine, or other stimulants (e.g., methamphetamines). Demographic and baseline characteristics including age, sex, race, education, and reported primary substance use disorder were balanced between treatment groups. Abstinence was significantly higher in the DT group compared to TAU (40.3 vs. 17.6%; p < 0.001) as was retention in therapy (76.2 vs. 63.2%, p = 0.004). Intergroup adverse event rates were not significantly different (p = 0.68). Conclusions: The results demonstrate that use of a DT safely increased abstinence (reduced substance use) and retention in treatment among patients with substance use disorders related to alcohol, cannabis, cocaine, or other stimulants (including methamphetamines).

Metabolites profiling reveals gut microbiome-mediated biotransformation of green tea polyphenols in the presence of N-nitrosamine as pro-oxidant.

The gut microbiome contributes to host physiology and nutrition metabolism. The interaction between nutrition components and the gut microbiota results in thousands of metabolites that can contribute to various health and disease outcomes. In parallel, the interactions between foods and their toxicants have captured increasing interest due to their impact on human health.  Taken together, investigating dietary interactions with endogenous and exogenous factors and detecting interaction biomarkers in a specific and sensitive manner is an important task. The present study sought  to identify for the first time the metabolites produced during the interaction of diet-derived toxicants e.g., N-nitrosamines with green tea polyphenols, using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). In addition, the metabolic products resulting from the incubation of green tea with a complex gut microbiome in the presence of N-nitrosamine were assessed in the same manner. The quinone products of (epi)catechin, quercetin, and kaempferol were identified when green tea was incubated with N-nitrosamine only; whereas, incubation of green tea with N-nitrosamine and a complex gut microbiome prevented the formation of these metabolites. This study provides a new perspective on the role of gut microbiome in protecting against potential negative interactions between food-derived toxicants and dietary polyphenols.

Orchestrating Nitric Oxide and Carbon Monoxide Signaling Molecules for Synergistic Treatment of MRSA Infections.

The local delivery of gaseous signaling molecules (GSMs) has shown promising therapeutic potential. However, although GSMs have a subtle interplay in physiological and pathological conditions, the co-delivery of different GSMs for therapeutic purposes remains unexplored. Herein, we covalently graft a nitric oxide (NO)-releasing N-nitrosamine moiety onto the carbon monoxide (CO)-releasing 3-hydroxyflavone (3-HF) antenna, resulting in the first NO/CO-releasing donor. Under visible light irradiation, photo-mediated co-release of NO and CO reveals a superior antimicrobial effect toward Gram-positive bacteria with a combination index of 0.053. The synergy of NO and CO hyperpolarizes and permeabilizes bacterial membranes, which, however, shows negligible hemolysis and no evident toxicity toward normal mammalian cells. Moreover, the co-release of NO and CO can efficiently treat MRSA infection in a murine skin wound model, showing a better therapeutic capacity than vancomycin.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats.

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.

Garlic oil blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis by inducing phase II drug-metabolizing enzymes.

Lung cancer caused one-quarter of all cancer deaths that was more than other cancers. Chemoprevention is a potential strategy to reducing lung cancer incidence and death, and the effective chemopreventive agents are needed. We investigated the efficacy and mechanism of garlic oil (GO), the garlic product, in the chemoprevention of tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer in A/J mice and MRC-5 cell models in the present study. As a result, it was demonstrated that GO significantly inhibited the NNK-induced lung cancer in vivo and protected MRC-5 cells from NNK-induced cell damage. GO could induce the expressions of the phase II drug-metabolizing enzymes, including NAD(P)H: quinone oxidoreductase 1 (NQO-1), glutathione S-transferase alpha 1 (GSTA1), and antioxidative enzymes heme oxygenase-1 (HO-1). These results supported the potential of GO as a novel candidate agent for the chemoprevention of tobacco carcinogens induced lung cancer.

Oral Dosing of Dihydromethysticin Ahead of Tobacco Carcinogen NNK Effectively Prevents Lung Tumorigenesis in A/J Mice.

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 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.

Anthocyanin-rich haskap (Lonicera caerulea L.) berry extracts reduce nitrosamine-induced DNA damage in human normal lung epithelial cells in vitro.

Diets rich in polyphenols are known to reduce cancer among high-risk populations. Haskap (Lonicera caerulea L.) berry has abundant phenolic acids and flavonoids, especially anthocyanins. Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) present in cigarette smoke, is a major lung carcinogenic factor. We analyzed the efficacy of anthocyanin-rich haskap berry extracts in preventing DNA damage induced by 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc), a precursor of NKK, in human lung epithelial BEAS-2B cells in vitro. A cocktail of monomeric polyphenols from haskap berries was extracted separately in ethanol and water and profiled. Sub-lethal concentrations of NNKOAc were used to induce DNA damage in BEAS-2B cells, and a cell viability assay was performed to confirm that the tested concentrations of haskap extracts were not cytotoxic to BEAS-2B cells. Cells were pre-treated with the haskap extracts prior to NNKOAc exposure. Dose-dependent DNA damage was observed with carcinogenic NNKOAc, but did not occur in the presence of the haskap extracts. Pre-treatment of the cells with the haskap extracts significantly reduced NNKOAc-induced DNA damage, DNA fragmentation, and intracellular reactive oxygen species and upregulated the ATM-dependent DNA damage repair cascade compared to non-treated BEAS-2B cells. The protective effect of haskap extracts could be related to their polyphenol content and high antioxidant capacity.

A Nitrate-Rich Vegetable Intervention Elevates Plasma Nitrate and Nitrite Concentrations and Reduces Blood Pressure in Healthy Young Adults.

BACKGROUND: Emerging evidence suggests that increasing dietary nitrate intake may be an effective approach to reduce blood pressure. Beetroot juice is often used to supplement dietary nitrate, whereas nitrate intake levels from habitual diet are low. An increase in the habitual intake of nitrate-rich vegetables may represent an alternative to nitrate supplementation. However, the effectiveness and acceptability of a nitrate-rich-vegetables diet remain to be established. OBJECTIVE: The aim was to investigate the effect and feasibility of two different intervention strategies to increase dietary nitrate intake, on plasma nitrate/nitrite concentrations and blood pressure. DESIGN: A randomized, crossover trial was used. PARTICIPANTS: Participants were healthy men and women (both n=15; age: 24±6 years) from the Netherlands. INTERVENTION: Participants were instructed to consume ∼400 mg nitrate at lunch, provided through nitrate-rich vegetables and dietary counseling, or beetroot juice supplementation. Both interventions lasted 1 week, with 1-week washout (January to April 2017). MAIN OUTCOME: Plasma nitrate and nitrite concentrations and resting systolic and diastolic blood pressure were measured in an overnight fasted state (before and after intervention) and ∼2.5 hours after lunch (before and throughout intervention on day 1, 4, and 7). STATISTICAL ANALYSIS: Two-factor (time × treatment) repeated-measures analyses of variance were performed. RESULTS: Mean plasma nitrate concentrations increased with both interventions, with a larger increase in beetroot juice vs nitrate-rich vegetables, both in a fasted state and ∼2.5 hours after lunch (day 1, beetroot juice: 2.31±0.56 mg/dL [373±90 µmol/L] vs nitrate-rich vegetables: 1.71±0.83 mg/dL [277±134 µmol/L]; P<0.001). Likewise, mean plasma nitrite concentrations increased with both interventions, but were higher after lunch in beetroot juice than in nitrate-rich vegetables (day 1: 2.58±1.52 µg/dL [560±331 nmol/L] vs 2.15±1.21 µg/dL [468±263 nmol/L]; P=0.020). Fasting mean systolic and diastolic blood pressure did not change, but mean systolic and diastolic blood pressure assessed ∼2.5 hours after lunch were significantly reduced throughout both intervention periods (P<0.05), with no differences between beetroot juice and nitrate-rich vegetables (day 1, systolic blood pressure: -5.1±9.5 mm Hg and diastolic blood pressure: -5.3±8.9 mm Hg). CONCLUSION: Short-term consumption of dietary nitrate in the form of nitrate-rich vegetables represents an effective means to increase plasma nitrate and nitrite concentrations, and reduces blood pressure to the same extent as beetroot juice supplementation.

Abrogation of esophageal carcinoma development in miR-31 knockout rats.

MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10-6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB-controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31-associated EGLN3/NF-κB-controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31-/- rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.

The Impact of One-week Dietary Supplementation with Kava on Biomarkers of Tobacco Use and Nitrosamine-based Carcinogenesis Risk among Active Smokers.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Dose-Related Modulatory Effects of Polymeric Black Tea Polyphenols (PBPs) on Initiation and Promotion Events in B(a)P and NNK-Induced Lung Carcinogenesis.

Our understanding of dose-related effects of polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, is limited. In the present study, the effect of various doses of black tea (0.75, 1.5, and 3%)-derived PBP-rich extract on biochemical parameters and lung carcinogenicity in A/J mice was investigated. Pretreatment with PBPs showed the dose-related decrease in B(a)P-induced expression and activity of CYP1A1 in the liver while CYP1A2 expression and activity in the lung. Dose-dependent significant increase in PBP-mediated over-expression and activity of GSTs (alpha in the liver while pi in the lung) were observed in polyphenol-treated groups. Significant dose-related decrease in number and intensity of BPDE-DNA adducts were observed in liver and lung. Black tea (1.5%, 3%)-derived PBPs showed dose-mediated decrease in lung tumor incidence and multiplicity which was further correlated with different molecular markers like cell proliferation and apoptosis in B(a)P and NNK model. In conclusion, dose-dependent chemopreventive effects of PBPs, both anti-initiating (induction of phase II and inhibition of carcinogen-induced phase-I enzymes leading to decrease in BPDE-DNA adducts) and anti-promoting (decreased cell proliferation and increased apoptosis lowering incidence and/or multiplicity of lung lesions), were observed in A/J mice without significant toxicity.

Video-based kinetic analysis of calcification in live osteogenic human embryonic stem cell cultures reveals the developmentally toxic effect of Snus tobacco extract.

Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of congenital anomalies, including low bone mass and increased fracture risk. Despite intensive public health initiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10-20% of women in the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco. These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose users to fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not reduced overall health risk to consumers and virtually nothing is known about whether skeletal problems from intrauterine exposure arise in the embryo. Utilizing a newly developed video-based calcification assay we determined that extracts from Snus tobacco hindered calcification of osteoblasts derived from pluripotent stem cells early on in their differentiation. Nicotine, a major component of tobacco products, had no measurable effect in the tested concentration range. However, through the extraction of video data, we determined that the tobacco-specific nitrosamine N'-nitrosonornicotine caused a reduction in calcification with similar kinetics as the complete Snus extract. From measurements of actual nitrosamine concentrations in Snus tobacco extract we furthermore conclude that N'-nitrosonornicotine has the potential to be a major trigger of developmental osteotoxicity caused by Snus tobacco.

Flaxseed Consumption Inhibits Chemically Induced Lung Tumorigenesis and Modulates Expression of Phase II Enzymes and Inflammatory Cytokines in A/J Mice.

Flaxseed consumption is associated with reduced oxidative stress and inflammation in lung injury models and has shown anticancer effects for breast and prostate tissues. However, the chemopreventive potential of flaxseed remains unexplored for lung cancer. In this study, we investigated the effect of flaxseed on tobacco smoke carcinogen (NNK)-induced lung tumorigenesis in an A/J mouse model. Mice exposed to NNK were fed a control diet or a 10% flaxseed-supplemented diet for 26 weeks. Flaxseed-fed mice showed reduced lung tumor incidence (78%) and multiplicity, with an average of 2.7 ± 2.3 surface lung tumor nodules and 1.0 ± 0.9 H&E cross-section nodules per lung compared with the control group, which had 100% tumor incidence and an average of 10.2 ± 5.7 surface lung tumor nodules and 3.9 ± 2.6 H&E cross-section nodules per lung. Furthermore, flaxseed-fed mice had a lower incidence of adenocarcinomas compared with control-fed mice. Western blotting performed on normal lung tissues showed flaxseed suppressed phosphorylation (activation) of p-AKT, p-ERK, and p-JNK kinases. RNA-Seq data obtained from normal lung and lung tumors of control and flaxseed-fed mice suggested that flaxseed intake resulted in differential expression of genes involved in inflammation-mediated cytokine signaling (IL1, 6, 8, 9, and 12α), xenobiotic metabolism (several CYPs, GSTs, and UGTs), and signaling pathways (AKT and MAPK) involved in tumor cell proliferation. Together, our results indicate that dietary flaxseed supplementation may be an effective chemoprevention strategy for chemically induced lung carcinogenesis by altering signaling pathways, inflammation, and oxidative stress. Cancer Prev Res; 11(1); 27-37. ©2017 AACR.