Short-Term Exposure to Waterpipe/Hookah Smoke Triggers a Hyperactive Platelet Activation State and Increases the Risk of Thrombogenesis.

OBJECTIVE: Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbß3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. CONCLUSIONS: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.

Structural binding perspectives of a major tobacco alkaloid, nicotine, and its metabolite cotinine with sex-steroid nuclear receptors.

Globally, more than a billion people smoke tobacco making it one of the biggest public health problems and a leading risk factor for global deaths. Nicotine, the main alkaloid in tobacco, has been shown to be associated with fertility problems in men and women. The adverse effects of tobacco/nicotine on reproduction have been attributed to deleterious effects on gametes, steroidogenic imbalance, and competitive inhibition of steroid receptors. The present study reports the sex-steroid receptor disrupting potential of nicotine and its major metabolite cotinine against the estrogen receptor-α (ERα), ERß, androgen receptor (AR), and progesterone receptor (PR). Both ligands bound in the ligand-binding pockets of ERα, ERß, AR and PR and formed important hydrophobic interactions with different amino-acid residues of receptors. Most of the residues of ERα, ERß, AR and PR interacting with nicotine and cotinine were common with those of native/bound ligands of the receptors. Interacting amino acids most important for binding of nicotine and cotinine with each receptor were identified by loss in accessible surface area. Amino acids Leucine-346, Leucine-384 and Phenylalanine-404 for ERα; Methionine-336, Phenylalanine-356 and Leucine-298 for ERß; and Leucine-704 and Leucine-718, respectively for AR and PR, were the most important residues for binding with nicotine and cotinine. Among the four receptors, based on the number of interactions, nicotine and cotinine had greater potential to interfere in the signaling of ERß. In conclusion, the results suggested that nicotine and cotinine bind and interact with sex-steroid nuclear receptors and have potential to interfere in the steroid hormone signaling resulting in reproductive dysfunction.

Age-dependent sensitivity of the mouse kidney to chronic nicotine exposure.

BackgroundMany adolescents are exposed to nicotine via smoking, e-cigarette use, or second-hand smoke. Nicotine-induced renal oxidative stress and its long-term consequences may be higher in adolescents than in adults because of intrinsic factors in the adolescent kidney.MethodsAdolescent and adult male C57Bl/6J mice were subjected to 2 or 200 µg/ml nicotine, which closely emulates passive or active smoking, respectively, for 4 weeks. Extent of nicotine exposure (cotinine content), oxidative stress (HNE), renal function (creatinine), tubular injury (KIM-1), and pretreatment renal levels of select pro-oxidant (p66shc) and antioxidant (Nrf2/MnSOD) genes were determined. Impact of p66shc overexpression or Nrf2/MnSOD knockdown on low-/high-dose nicotine-induced oxidative stress was determined in cultured renal proximal tubule cells.ResultsDespite similar plasma/renal cotinine levels, renal HNE and KIM-1 contents were higher in adolescents compared with those in adults, whereas renal function was unaltered after passive or active smoking-equivalent nicotine exposure. Pretreatment levels of p66shc were higher, whereas Nrf2/MnSOD levels were lower in the adolescent kidney. In agreement with this, overexpression of p66shc or knockdown of Nrf2/MnSOD augmented nicotine-induced ROS production in renal proximal tubule cells.ConclusionChronic nicotine exposure incites higher oxidative stress in the adolescent than in adult kidney because of a pre-existent pro-oxidant milieu.

Efficacy of vitamins E and C for reversing the cytotoxic effects of nicotine and cotinine.

Nicotine has adverse cellular and molecular effects on oral mucosa, bone, and teeth. Vitamin E (α-tocopherol) and vitamin C (ascorbic acid) are biological antioxidants with positive effects on wound healing and bone formation. This in vitro study sought to assess the cytotoxic effects of different concentrations of nicotine and cotinine (a metabolite of nicotine) on MG-63 osteoblast-like cells and human gingival fibroblasts (HGFs) in the presence and absence of antioxidant vitamins E and C (separately and combined). Cell viability and proliferation were assessed using the methyl thiazol tetrazolium (MTT) assay. Cell migration was assessed using the scratch test, and expression of apoptosis-related genes was quantitatively analyzed using real-time PCR. Dose-dependent negative effects of nicotine on the morphology, viability, proliferation, and migration of MG-63 and HGF cells were statistically significantly greater than those of cotinine. Vitamin E (separately and combined with vitamin C) was statistically significantly more effective than vitamin C (at the concentration used in this study) at improving cell viability, proliferation, and migration, and at reducing apoptosis of cells exposed to nicotine or cotinine. Based on the positive results of this study, vitamin C and especially vitamin E (systemically and/or locally) may be useful in the repair and regeneration of oral hard and soft tissues in smokers.

Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells.

The liver is a dynamic organ which is both multifunctional and highly regenerative. A major role of the liver is to process both endo and xenobiotics. Cigarettes are an example of a legal and widely used drug which can cause major health problems for adults and constitute a particular risk to the foetus, if the mother smokes during pregnancy. Cigarette smoke contains a complex mixture of thousands of different xenobiotics, including nicotine and polycyclic aromatic hydrocarbons. These affect foetal development in a sex-specific manner, inducing sex-dependant molecular responses in different organs. To date, the effect of maternal smoking on the foetal liver has been studied in vitro using cell lines, primary tissue and animal models. While these models have proven to be useful, poor cell phenotype, tissue scarcity, batch-to-batch variation and species differences have led to difficulties in data extrapolation toward human development. Therefore, in this study we have employed hepatoblasts, derived from pluripotent stem cells, to model the effects of xenobiotics from cigarette smoke on human hepatocyte development. Highly pure hepatocyte populations (>90%) were produced in vitro and exposed to factors present in cigarette smoke. Analysis of ATP levels revealed that, independent of the sex, the majority of smoking derivatives tested individually did not deplete ATP levels below 50%. However, following exposure to a cocktail of smoking derivatives, ATP production fell below 50% in a sex-dependent manner. This was paralleled by a loss metabolic activity and secretory ability in both female and male hepatocytes. Interestingly, cell depletion was less pronounced in female hepatocytes, whereas caspase activation was ~twofold greater, indicating sex differences in cell death upon exposure to the smoking derivatives tested.

Nicotine and Cotinine Levels With Electronic Cigarette: A Review.

BACKGROUND: Since their introduction in 2004, electronic cigarettes (e-cigarettes) have gained popularity worldwide. E-cigarettes are marketed as nicotine delivery devices. Commonly reported reasons for use include to quit smoking, to reduce urge to smoke, or the perceived lower risk alternative to smoking. But what are the actual amounts of nicotine delivered? AIM: This review summarizes all the published studies concerning nicotine or cotinine levels following e-cigarette use. METHODS: A literature search was conducted from the PubMed database, from 1985 to January 2014, using the following terms: electronic cigarette(s), e-cigarette(s), electronic nicotine delivery system, cotinine, and nicotine. Articles were excluded if they were not pertinent according to our criteria. References of all relevant articles were also evaluated. RESULTS: Eight studies were included in this review. The following information was extracted from the articles: population size, age of participants, recruitment, inclusion and exclusion criteria, concentration of nicotine in refills liquids, study sample design, and observed concentrations. Following design of studies, plasma nicotine Cmax was observed between 0 and 5 ng/mL (no significant changes) or between 13.9 and 16.3 ng/mL (similar to a tobacco cigarette) with a Tmax between 70 and 75 minutes. Cotinine levels after "vaping" an e-cigarette are similar to a tobacco cigarette. CONCLUSION: This review summarizes e-cigarette studies that contain information on nicotine or cotinine levels. The peak concentration of nicotine appears to be dependent on the use and dose level of e-cigarette cartridge. The value of this peak concentration is similar to the value found with a tobacco cigarette. However, the time corresponding to the peak concentration is delayed compared to a tobacco cigarette.

Influence of nicotine and cotinine impregnation on the first step of periodontal regeneration: clot stabilization.

This study analyzes the clot stabilization on root surfaces of teeth impregnated with cotinine and nicotine and the influence of the scaling in the adhesion of blood components, observing the influence of new exposition to nicotine and/or cotinine after scaling. Fifteen human teeth extracted due to periodontal disease of non-smokers patients were selected and manually scaled. Four dentin blocks were obtained from each tooth (n = 60). Samples received blood application or reimpregnation with nicotine and/or cotinine, depending on the groups. Group 1: PBS immersion + root scaling + blood; group 2: nicotine + root scaling + blood; group 3: nicotine + root scaling + nicotine reapplication + blood; group 4: cotinine + root scaling + blood; group 5: cotinine + root scaling + cotinine reapplication+ blood; group 6: nicotine and cotinine + root scaling + nicotine and cotinine + blood. Samples were kept in 2 ml of each substance for 24 hours. Each group received a blood drop and was analyzed by SEM. The higher amount of blood components was present in teeth exposed to cotinine and the groups submitted to scaling and blood application in comparison with groups that received reapplication of toxic substances after scaling. The greater toxic effect on root dentin surface was after the exposure to nicotine and cotinine. Results suggest that periodontal healing may be delayed in smokers due to the direct inhibition of clot stabilization on the root surface when nicotine and cotinine are present concomitantly.

Early-life nicotine or cotinine exposure produces long-lasting sleep alterations and downregulation of hippocampal corticosteroid receptors in adult mice.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Blood BTEXS and heavy metal levels are associated with liver injury and systemic inflammation in Gulf states residents.

INTRODUCTION: Exposures to volatile organic compounds and metals have previously been associated with liver diseases including steatohepatitis, although more data are needed. Benzene, toluene, ethylbenzene, xylenes, styrene (BTEXS) and metals were measured in blood samples collected between May 2012-July 2013 from volunteers participating in home visits for the Gulf Long-term Follow-up (GuLF) Study. This cross-sectional analysis evaluates associations of exposure biomarkers with serum liver injury and adipocytokine biomarkers in a sample of 214 men. METHODS: Adult nonsmoking men without a history of liver disease or heavy alcohol consumption were included. The serologic disease biomarkers evaluated were the hepatocellular injury biomarker, cytokeratin 18 [whole (CK18 M65) and caspase-cleaved fragment (CK18 M30)]; and adipocytokines. Confounder-adjusted beta coefficients were determined using linear regression models for the overall sample (primary endpoints) and for obesity-classified sub-groups (secondary endpoints). A product interaction term between the exposure of interest and a dichotomized indicator of obesity was included to determine the disease modifying effects of obesity on the biomarker associations. RESULTS: The study sample was 57% white and 51% obese. In the overall sample, lead was positively associated with CK18 M30 (ß = 21.7 ± 6.0 (SE), p = 0.0004); IL-1ß (ß = 32.8 ± 5.2, p < 0.0001); IL-6 (ß = 72.8 ± 18.3, p = 0.0001); and IL-8 (ß = 140.8 ± 42.2, p = 0.001). Cadmium exposures were associated with increased IL-1ß (ß = 77.8 ± 26.3, p = 0.003) and IL-8 (ß = 419.5 ± 201.2, p = 0.04). There were multiple significant interactions between obesity and exposure to lead, cadmium, benzene and toluene in relation to outcome biomarkers. Among obese participants (n = 108), benzene, lead, and cadmium were each positively associated with CK18 M30, IL-1ß, IL-6, and IL-8. In obese subjects, lead was also inversely associated with leptin, and toluene was positively associated with IL-1ß. CONCLUSION: For the overall sample, heavy metal exposures were associated with liver injury (lead only) and/or systemic inflammation (lead and cadmium). Obesity modified the associations between BTEXS and heavy metal exposures on several of the outcome variables. In the obesity subgroup, liver injury was positively associated with lead, cadmium and benzene exposures; systemic inflammation was increased with lead, cadmium, benzene, and toluene exposures; and leptin was inversely associated with lead exposures. The cross-sectional design of this study makes it difficult to determine causality, and all results should be interpreted cautiously. Nonetheless, the potential impact of exposures to lead, cadmium, benzene and toluene in steatohepatitis, an obesity-associated inflammatory liver disease, warrants further investigation.

Cotinine, a major nicotine metabolite, induces cell proliferation on urothelium in vitro and in vivo.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3'-hydroxy cotinine and N'-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 µM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.

The effect of cotinine on telomerase activity in human vascular smooth muscle cells.

AIM: Cotinine, the main stable metabolite of nicotine, has been shown to have a biological half-life approximately 10 times longer than nicotine. It has also been demonstrated to have a powerful effect on vascular smooth muscle cell (VSMC) proliferation. Telomerase activation is known to play an important role in cell viability and proliferation. The purpose of our experiment was to evaluate the effect of cotinine on proliferative potential of vascular smooth muscle cells via its effects on telomerase activity. METHODS: Primary cultures of human VSMC obtained from greater saphenous veins were used in this experiment from 3(rd) to 5(th) passage. Cotinine was added in doses equivalent to plasma levels of cotinine in an active smoker by dissolving, 0.0, 2.88x10(-6), 5.76x10(-6), and 1.44x10(-5) mol/L of cotinine in the media. The number of viable cells was assessed by trypan blue exclusion. The Telomeric Repeat Amplification Protocol (TRAP) was used to detect telomerase activity. TRAP products were detected by ELISA. RESULTS: The mitogenic effect of cotinine in VSMC was observed at 48 hours after treatment. The viable cell numbers were significantly increased (4.0x10(7)) at lower doses of cotinine exposure as compared to untreated cultures (2.5x10(5)). At the concentration of 1.44x10(-5) mol/L, cotinine was cytotoxic to VSMCs. Telomerase activity was detected in all sets of VSMC cultures treated with cotinine (P<0.01). CONCLUSIONS: Cotinine causes abnormal cell proliferation as demonstrated by increased cell numbers and reactivation of telomerase in a dose dependent manner. This study demonstrated cotinine's stimulatory effect on human SMC proliferation in vitro at low doses while high doses of cotinine had a toxic effect. These data correlate with the results of other studies concerning the mitogenic effect of cotinine and telomerase activation during cellular proliferative response.

High dose cotinine may induce neural tube defects in a chick embryo model.

AIM: Nicotine is a well-known agent among 4000 chemicals in cigarettes. About 70 to 80% of nicotine is converted to cotinine, a major metabolite. The aim of the present study is to investigate the effect of cotinine on neural tube development in a chick embryo model. MATERIAL AND METHODS: Sixty fertile, specific pathogen free eggs were divided into 6 groups for this study. In the first group, a fixed cotinine concentration for each egg was calculated just to simulate the concentration of a smoker's blood level. A second experimental group was designed at a higher cotinine concentration. Embryos that succeeded to reach Hamburger-Hamilton stage 12 from each group were then embedded into paraffin for permanent sections. These two groups were compared with eggs subjected to vehicle (standard alcohol and ten times more alcohol concentration) and control groups (saline and sham groups). RESULTS: Embryos of the cotinine (regular dose), vehicle and control groups were normal, but embryos subjected to higher cotinine concentrations were malformed at the cranial part of the thoracic neural tube. CONCLUSION: Association of cotinine with neural tube defects was demonstrated in the present study. Cigarette smoking may induce hazardous effects on neural tube development.

Preliminary evidence from planarians that cotinine establishes a conditioned place preference.

While the psychoactive stimulant nicotine has been the subject of extensive research, considerably less attention has focused on other compounds found in either tobacco smoke or that are nicotine metabolites. Recent papers have suggested that some of the compounds in question may either alter nicotine's effects or have reinforcing properties themselves, although they would only be experienced after consumption of tobacco. The potential for these compounds to function as reinforcers or to potentiate the reinforcing properties of nicotine merits investigation. To pursue this line of inquiry, we examined cotinine in a planarian model of environmental place preference. In the present study, planarians demonstrated that the compound cotinine, which is present in tobacco smoke, and is also the principal nicotine metabolite, establishes a conditioned place preference. These data represent the first ever demonstration that cotinine will establish a conditioned place preference in planarians and possibly contribute to the addictive properties of nicotine.

Polycyclic aromatic hydrocarbon exposure, urinary mutagenicity, and DNA adducts in rubber manufacturing workers.

OBJECTIVES: Several studies have suggested that genotoxic risks might still be present in the contemporary rubber manufacturing industry. Previously, we observed elevated levels of urinary mutagenicity and bladder DNA adducts in rubber workers. Presently, we investigated whether DNA adducts in peripheral blood mononuclear cells (PBMC) and/or urothelial cells may be caused by polycyclic aromatic hydrocarbons or other genotoxic compounds. METHODS: Spot urine samples from 116 rubber manufacturing workers were collected on Sunday and during the workweek (post-shift) to determine 1-hydroxypyrene and mutagenicity levels. For 52 nonsmokers, urothelial cell DNA adducts and PBMC DNA adducts were measured additionally. RESULTS: Urinary 1-hydroxypyrene levels were significantly higher in workweek samples compared with Sunday (P = 0.0001). This increase was not uniform across tasks and only reached statistical significance for the curing department (+99%; P = 0.003). Weekday urinary mutagenicity was significantly increased for mixing (+56%) and curing (+21%) workers when compared with that for Sunday. Total urothelial cell DNA adducts were related to urinary 1-hydroxypyrene (P = 0.021) and mutagenicity (P = 0.027). No significant relationship was found between the adduct levels in PBMC and urothelial cells or between the former and urinary 1-hydroxypyrene or mutagenicity. CONCLUSIONS: Workers in the compounding, mixing, and curing departments were at highest genotoxic risk among rubber manufacturing workers. Increased levels of urinary 1-hydroxypyrene, mutagenicity, and urothelial cell DNA adducts were found in these workers. Urothelial cell and PBMC DNA adducts were not related, hinting possibly to the presence of specific bladder carcinogens in the rubber manufacturing industry.

Cell proliferation kinetics and genotoxicity in lymphocytes of smokers living in Mexico City.

Genotoxicity caused by tobacco smoke was assessed in peripheral blood lymphocytes of smokers living in Mexico City by determining sister chromatid exchange (SCE), cell proliferation kinetics (CPK), replication index (RI) and mitotic index (MI). Nicotine levels, and its major metabolite cotinine, were also estimated in urine samples using gas-chromatography-mass spectrometry to quantify smoking intensity. The outcome of the analysis and the comparison of the 77-smoker group with a non-smoking control group showed that moderate and heavy smokers exhibited significant differences (P < 0.001 and P < 0.05, respectively) in CPK, with an underlying delay in the cellular cycle; similarly, RI was significantly different in these groups (P < 0.001 and P < 0.0001, respectively). There were significant correlations (P < 0.05) between age and number of years the subject had been smoking, as well as between RI and nicotine and cotinine levels and between CPK (M1, M2 and M3) and nicotine and cotinine levels. Smokers were classified for the analysis according to the nicotine levels (it is in relation to number of cigarettes smoked per day) found in urine (ng/mL) as: light (10-250), moderate (251-850) and heavy (851-4110). Significant differences in CPK were found (P < 0.05) between moderate and heavy smokers and non-smokers. Significant differences in RI were found between moderate (P < 0.001) and heavy smokers (P < 0.0001) and non-smokers, but not for the light smoking group. MI was determined in 57 of the smokers, whereas SCE frequency was only recorded in 34 smokers. Both parameters yielded no significant differences, nor correlations with any of the assessed variables. In conclusion, cytokinetic and cytostatic effects were mainly detected in heavy and moderate smokers. Cell cycle delay and RI decrease were found in all ;healthy' smokers. The nicotine and cotinine exposure (causing oxidative damage to DNA) may have implications in the decrease in cell replication due to direct damage to DNA and/or a decrease in the DNA repair mechanisms. Alternatively, nicotine and cotinine may possibly induce apoptosis.

Evaluation of the effects of cotinine and nicotine-N'-oxides on the development of tumors in rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.

The effects of cotinine and nicotine-N'-oxides on tumor development in F344 rats initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide [(FANFT) CAS: 24554-26-5] were evaluated. When rats were 6 weeks old, FANFT in an agar diet was administered for a 6-week period. Subsequently, cotinine, trans-nicotine-N'-oxide, and a mixture of cis-nicotine-N'-oxide and trans-nicotine-N'-oxide in drinking water were given as promoters in concentrations of 0.1, 0.02, and 0.02%, respectively. These nicotine metabolites were offered ad libitum for 78 weeks. Control groups consisted of rats that received tap water with or without prior administration of FANFT. Cotinine, trans-nicotine-N'-oxide, and the mixture of cis- and trans-nicotine-N'-oxides were neither carcinogens nor promoters of urinary bladder tumors in rats initiated with FANFT. A reduced incidence of urinary bladder tumors was observed in FANFT-pretreated animals that also received a mixture of cis- and trans-nicotine-N'-oxides. FANFT administration increased the incidences of mesothelioma of the peritoneum and thyroid tumors. Tumor formation in the tongue and palate observed in FANFT-treated rats was not affected by administration of these nicotine metabolites. There was, however, a significant increase in the incidence of forestomach tumors in rats that were initiated with FANFT and subsequently received either trans-nicotine-N'-oxide or a mixture of cis- and trans-nicotine-N'-oxides.

[Determination of nicotine and cotinine in human biological materials and their significance in toxicological studies]. / Problematyka oznaczania nikotyny i kotyniny w ludzkim materiale biologicznym w aspekcie badan toksykologicznych.

The aim of this study was the preparation of reliable procedure of the determination of nicotine and cotinine both in classic (serum, urine) and alternative biological materials (hair, saliva) and evaluation of their significance for clinical and forensic toxicology. Biological material samples (blood, urine, saliva) were taken from patients after Percutaneous Trans-luminal Coronary Angioplasty (PTCA). The determination of cotinine and nicotine concentration in the biological material should be optimized depending on the aim of analysis. Liquid-liquid extraction procedure and high performance liquid chromatography HPLC/UV-DAD are reliable, specific and relatively cheap. Serum and saliva are valuable biological materials which allow to determine temporary nicotine and cotinine content on the similar level of concentrations. In the near future it will be able to replace blood with saliva sample because of an easy and non-invasive way of sampling. Evaluation of cotinine concentration in urine allows to distinguish the passive from the active tobacco smokers. Hair analysis allows to control a nicotine abstinence as well as a long-term evaluation of the history of smoking. However usage of hair is limited because of difficulty with sampling. Interpretation of results in analysis of alternative materials (hair, saliva) pose a problem because of lack of sampling standardization and lack of standardization of final analysis method.

Influence of nicotine and cotinine on epithelial colonization by periodontopathogens.

BACKGROUND: Since smoking is an established risk factor for the development of periodontitis, the present study investigated whether nicotine and cotinine can make epithelial cells more prone to colonization by periodontopathogens. METHODS: Primary epithelial cell mono-layers were inoculated with nicotine and cotinine prior to adhesion experiments with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The number of bacteria associated with cells inoculated or not with nicotine or cotinine were assessed by an indirect culture viability assay. The same experimental set-up was used for assessing HeLa cells exposed to cigarette smoke extract (CSE). RESULTS: Primary epithelial cells inoculated with concentrations of nicotine and cotinine, found in smokers and non smokers, did not show significant differences (P>0.05) in colonization susceptibility to A. actinomycetemcomitans. When these concentrations were increased to 1 mg/ml, a significant (P<0.05) and species-specific effect of the colonization susceptibility of epithelial cells was observed: It increased for A. actinomycetemcomitans, while it decreased for P. gingivalis. For both species the effects were more pronounced for nicotine, although this was not statistically significant. The change in colonization susceptibility did not result from alterations of the bacterial viability due to nicotine or cotinine. Treatment of HeLa cells with CSE also led to a species-specific variation in colonization tendency; i.e., increased for A. actinomycetemcomitans (P<0.05), but not for P. gingivalis. CONCLUSIONS: The susceptibility of epithelial cells to become colonized by either A. actinomycetemcomitans or P. gingivalis could be altered by nicotine, cotinine, or CSE in a time-dependent, species-specific manner. Whether these findings that support the hypothesis of an increased patient susceptibility for bacterial adhesion to epithelial cells in smokers are clinically relevant remains to be proven.

Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells.

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an "epimutagen combination" that is effective at low concentrations as detected in maternal peripheral and cord blood.

The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (i.p.) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.