Antibody-catalyzed oxidative degradation of nicotine using riboflavin

Tobacco abuse remains a major cause of death worldwide despite ample evidence linking nicotine to various disease states. Consequently, immunopharmacotherapeutic approaches for the treatment of nicotine abuse have received increasing attention. Although a number of nicotine-binding antibodies have been disclosed, no antibody catalysts exist which efficiently degrade nicotine into pharmacologically inactive substances. Herein, we report the first catalytic antibodies which can oxidatively degrade nicotine. These biocatalysts use the micronutrient riboflavin and visible light as a source of singlet oxygen for the production of reactive oxygen species. Along with various known nicotine metabolites, antibody-catalyzed nicotine oxidations produce two novel nicotine oxidation products that were also detected in control ozonation reactions of nicotine. The reaction is efficient, with multiple turnovers of catalyst observed and total consumption of nicotine attained. These results demonstrate the potential of harnessing riboflavin as an endogenous sensitizer for antibody-catalyzed oxidations and demonstrate a new approach for the development of an active vaccine for the treatment of nicotine addiction using in vivo catalytically active antibodies.     

Nicotine administration to rats: methodological considerations

The effects of nicotine on normal physiological function are of increasing concern. Preliminary to studies on the effects of prenatal exposure to nicotine, we examined methods of administering nicotine to rats. Drinking water containing nicotine was not palatable to rats and was an unsatisfactory method in our hands, producing weight loss and large decreases in fluid intake. Administration of nicotine in a complete liquid diet produced better results but the data suggest that oral administration of nicotine may interfere with absorption of some nutrients. Osmotic mini-pumps were found to be the best mechanism of nicotine delivery of those tried. There were no significant effects on food or water intake nor on weight gain, particularly when using a short term anesthetic for pump implantation. Plasma nicotine and cotinine levels were directly correlated to dose of nicotine delivered. Plasma nicotine levels similar to levels reported in humans were obtained.     

Dose-response curves of central and peripheral airways to nicotine injections in dogs

The dose-response curves of the central and peripheral airways to intravenously injected nicotine were studied in 55 anesthetized dogs. With intact vagi, nicotine caused a dose-dependent increase in central airway resistance (Rc) similar to the increase in peripheral airway resistance (Rp) at concentrations ranging from 4 to 64 micrograms/kg. However, the responses of both Rc and Rp fell progressively when sequential doses of nicotine greater than 256 micrograms/kg were administered. With intact vagi and the administration of propranolol, there was a greater increase in Rp than in Rc at a nicotine dose of 64 micrograms/kg (P less than 0.05). With vagotomy, the responsiveness of both central and peripheral airways to nicotine decreased with doses of nicotine less than 64 micrograms/kg, but with doses of nicotine greater than 256 micrograms/kg the suppressive effect of nicotine on both Rc and Rp was less than that seen with intact vagi. Under conditions in which the vagi were cut and atropine administered, the responsiveness of nicotine was even further depressed. Combinations either of atropine and chlorpheniramine or atropine and phenoxybenzamine also completely blocked reactions to nicotine. Additionally reactions to nicotine were completely blocked by hexamethonium. These results suggest that nicotine increases both Rc and Rp mainly through a vagal reflex and stimulation of the parasympathetic ganglia.     

Nicotine moderates the effects of macronutrient balance on nutrient intake by parasitized <Emphasis Type="Italic">Manduca sexta</Emphasis> L

Effects of dietary nicotine and macronutrient ratio on M. sexta larvae were examined. Larvae were fed a carbohydrate-biased, protein-biased or diet having equal amounts of casein and sucrose, with and without nicotine. Without nicotine, larvae displayed compensatory feeding on the low protein diet, but despite consuming more, grew least on this diet. Nicotine at 0.5% had no effect on nutrient consumption. Nicotine at 1.0 and 2.0% reduced overall consumption and thereby also reduced nicotine consumption. Larvae parasitized by C congregata displayed reduced nutrient intake and growth on all diets. Parasitized larvae responded to 1% nicotine similarly to unparasitized larvae. At 0.5% nicotine, they displayed reduced consumption on all diets, possibly due to altered chemoreceptor sensitivity to nicotine. When offered a choice of two diets having different macronutrient ratios, one with and the other without 0.1% nicotine, all larvae preferred the diet lacking nicotine and failed to regulate nutrient intake such that the nutrient intake target, a ratio of nutrients supporting optimal growth, was achieved. Parasitized larvae consumed less nicotine on a fresh weight basis than unparasitized insects, suggesting that the feeding response of parasitized larvae to nicotine minimizes the exposure of nicotine to developing parasites.     

Nicotine dependence in rats

Health hazards associated with nicotine and tobacco use are well known. A contributing factor, the dependence producing potential of this drug, has become widely accepted. However, there are only a few human and animal studies that provide objective measures of the behavioral consequences of nicotine abstinence. The purpose of the present experiment was to use sensitive measures to examine behavioral disruptions that resulted when nicotine administration was terminated. Six rats were administered 96 daily intravenous infusions of nicotine (0.125 mg/kg/infusion) for at least 10 days. They were trained to respond on a tongue-operated solenoid-driven drinking device that delivered 0.005 ml of a glucose and saccharin solution (G + S) per lick. When nicotine access was terminated for six days, there was a marked suppression in behavior reinforced by the sweetened solution, and this disruption was immediately reversed when nicotine was reinstated. In contrast, nicotine removal also resulted in a decrease in food intake on the first day, but on subsequent days food intake was significantly higher than when nicotine was administered. When cotinine (0.25 mg/kg/infusion), a metabolite of nicotine was substituted for nicotine for six days, similar disruptions resulted in responding maintained by G + S, but food intake was not significantly decreased on the first day of nicotine abstinence. These findings illustrate the utility of sensitive behavioral tests to reveal effects of nicotine abstinence.     

The Influence of Stress on the Affective Modulation of the Startle Response to Nicotine Cues

Recent research suggesting that nicotine cues are appetitive in nature promotes the affective modulation of the startle reflex (AMSR) paradigm as a potentially valuable psychophysiological tool for elucidating mechanisms involved in nicotine addiction. Despite numerous studies indicating stress as a key factor in nicotine dependence, specific behavioral mechanisms linking stress and smoking have yet to be explicated. The current study aimed to determine the effects of stress, a negative affective state intimately linked with nicotine use, on the psychophysiological responding of nicotine dependent individuals during smoking cues. Twenty-nine nicotine dependent individuals were randomly assigned to the trier social stress test or control condition directly prior to administration of the AMSR paradigm, which examined their physiological responses to appetitive, neutral, aversive, and nicotine cue images. Both groups evinced significantly decreased startle magnitudes in response to nicotine cues as compared to aversive images. However, exposure to stress did not significantly modulate the startle reflex while viewing nicotine cues. Stress induction does not appear to modulate the AMSR paradigm when evaluating responses to nicotine images. These findings suggest that AMSR is robust to effects of acute stress induction in nicotine dependent individuals which may increase its viability as a clinical tool for assessing success in smoking cessation interventions.     

Enantiomeric composition of nicotine in tobacco leaf,cigarette, smokeless tobacco,and e‐liquid by normal phase high‐performance liquid chromatography

Evaluating the source of nicotine in e‐liquid is a problem. Tobacco‐derived nicotine contains predominantly (S)‐(?)‐nicotine, whereas tobacco‐free nicotine products may not. Thus, we developed a new normal phase high‐performance liquid chromatography method to determinate the enantiomeric composition of nicotine in 10 kinds of flue‐cured tobacco, 3 kinds of burley, 1 kind of cigar tobacco, 2 kinds of oriental tobacco, 5 kinds of Virginia cigarette, 5 kinds of blend cigarette, 10 kinds of e‐liquid, and 4 kinds of smokeless tobacco. The amount of (R)‐(+)‐nicotine ranged from ~0.02% to ~0.76% of total nicotine. An e‐liquid sample had the highest level of (R)‐(+)‐nicotine. The extraction and purification processes used to obtain commercial (S)‐(?)‐nicotine from the tobacco do not decrease the amount of (R)‐(+)‐nicotine in tobacco. So the amount of (R)‐(+)‐nicotine in samples in our work were the same as tobacco samples.     

The secretory response of hypothalamic β-endorphin neurons to acute and chronic nicotine treatments and following nicotine withdrawal

In the present study, we determined the effect of acute and chronic nicotine treatments on the secretion of immunoreactive β-endorphin (IR-β-EP) and cell viability of cultured hypothalamic neurons. Also, we determined the secretory response of IR-β-EP following withdrawal from a long-term nicotine treatment. Fetal hypothalamic cells were dissociated and maintained in cultures for 9 days and were treated with various doses of nicotine (1, 6,12 and 18 μM) for 6 h (acute treatment) or treated with nicotine at 12 h intervals for 96 h (chronic treatment). Determination of IR-β-EP concentrations in the media revealed that 6–18 μM doses of nicotine increased IR-β-EP secretion from these cultures for a period of 24 h; after this period, the cultured cells did not respond to these doses of nicotine. The desensitization of β-EP neurons 24 h after treatment with nicotine did not appear to be related to the loss of viable cells. Determination of withdrawal response after 72 h of constant nicotine (6 μM) treatments revealed that the hypothalamic neurons secrete elevated amounts of IR-β-EP for a period of 72 h after nicotine withdrawal. These results suggest that: 1) acute treatment with nicotine stimulates hypothalamic IR-β-EP release; 2) chronic nicotine treatment desensitizes β-EP-secreting neurons and, 3) β-EP neurons in primary culture show withdrawal response to nicotine.     

Pathways and Networks-Based Analysis of Candidate Genes Associated with Nicotine Addiction

Nicotine is the addictive substance in tobacco and it has a broad impact on both the central and peripheral nervous systems. Over the past decades, an increasing number of genes potentially involved in nicotine addiction have been identified by different technical approaches. However, the molecular mechanisms underlying nicotine addiction remain largely unclear. Under such situation, a comprehensive analysis focusing on the overall functional characteristics of these genes, as well as how they interact with each other will provide us valuable information to understand nicotine addiction. In this study, we presented a systematic analysis on nicotine addiction-related genes to identify the major underlying biological themes. Functional analysis revealed that biological processes and biochemical pathways related to neurodevelopment, immune system and metabolism were significantly enriched in the nicotine addiction-related genes. By extracting the nicotine addiction-specific subnetwork, a number of novel genes associated with addiction were identified. Moreover, we constructed a schematic molecular network for nicotine addiction via integrating the pathways and network, providing an intuitional view to understand the development of nicotine addiction. Pathway and network analysis indicated that the biological processes related to nicotine addiction were complex. Results from our work may have important implications for understanding the molecular mechanism underlying nicotine addiction.     

Propensity for social interaction predicts nicotine‐reinforced behaviors in outbred rats

Social and genetic factors can influence smoking behavior. Using olfactogustatory stimuli as the sensory cue for intravenous nicotine self‐administration (SA), we previously showed that social learning of nicotine contingent odor cue prevented rats from developing conditioned taste aversion and allowed them to instead establish stable nicotine SA. We hypothesized that genetic factors influenced socially acquired nicotine SA. A heterogeneous stock (HS; N/NIH) of outbred rats was trained to self‐administer nicotine using the social learning protocol. Both male and female HS rats acquired nicotine SA, but females self‐administered more nicotine than males. After extinction, the context previously paired with nicotine SA, in conjunction with socially transmitted drug cues, was sufficient to cause reinstatement of drug‐seeking behavior. Wide variation in both nicotine intake and reinstatement was observed. Using multiple regression analysis, we found that measures of social interaction were significant predictors of nicotine intake and reinstatement of drug seeking in both males and females. Furthermore, measures of depression were predictors of nicotine intake in both males and females, anxiety was a predictor only in males and response to novelty was a predictor only in females. In males, measures of both depression and anxiety predicted nicotine reinstatement. Together, these data supported the ideas that genetically determined propensities for emotional and social phenotypes are significant determinants for nicotine‐reinforced behavior, and that the HS rat is a suitable tool for dissecting genetic mechanisms that may underlie the interaction between social behavior, anxiety, depression and smoking .     

High reinforcing efficacy of nicotine in non-human primates

Although tobacco appears highly addictive in humans, there has been persistent controversy about the ability of its psychoactive ingredient nicotine to induce self-administration behavior in laboratory animals, bringing into question nicotine's role in reinforcing tobacco smoking. Because of ethical difficulties in inducing nicotine dependence in na?ve human subjects, we explored reinforcing effects of nicotine in experimentally-naive non-human primates given access to nicotine for periods of time up to two years. Five squirrel monkeys with no experimental history were allowed to intravenously self-administer nicotine by pressing one of two levers. The number of presses on the active lever needed to obtain each injection was fixed (fixed-ratio schedule) or increased progressively with successive injections during the session (progressive-ratio schedule), allowing evaluation of both reinforcing and motivational effects of nicotine under conditions of increasing response cost. Over time, a progressive shift toward high rates of responding on the active lever, but not the inactive lever, developed. The monkeys' behavior was clearly directed toward nicotine self-administration, rather than presentation of environmental stimuli associated with nicotine injection. Both schedules of reinforcement revealed a high motivation to self-administer nicotine, with monkeys continuing to press the lever when up to 600 lever-presses were needed for each injection of nicotine. Thus, nicotine, by itself, in the absence of behavioral or drug-exposure history, is a robust and highly effective reinforcer of drug-taking behavior in a non-human primate model predictive of human behavior. This supports the use of nicotinic ligands for the treatment of smokers, and this novel preclinical model offers opportunities to test future medications for the treatment of nicotine dependence.     

Enhancement of haloperidol-induced catalepsy by nicotine: an investigation of possible mechanisms

Nicotine has been reported to potentiate the cataleptic effect of the dopamine receptor antagonist haloperidol in rats. This effect is paradoxical, since nicotine alone tends to increase nigrostriatal dopamine release. In the present experiments, a pro-cataleptic effect of nicotine was confirmed statistically but was small and variable. Three potential mechanisms underlying this effect were investigated. (i) Desensitization of brain nicotinic receptors appears to make little if any contribution to the pro-cataleptic effect of nicotine, insofar as the latter was not mimicked by two centrally active nicotinic antagonists (mecamylamine and chlorisondamine). (ii) Depolarization inactivation resulting from combined treatment with haloperidol and nicotine does not appear to be critical, since the pro-cataleptic effect of nicotine was not enhanced by chronic haloperidol administration, a treatment designed to enhance depolarization inactivation. (iii) The slow emergence and persistence of the acute pro-cataleptic effect of nicotine suggested possible mediation by a nicotine metabolite. However, neither cotinine nor nornicotine, the principal pharmacologically-active metabolites of nicotine, exerted a significant pro-cataleptic effect. In conclusion, the pro-cataleptic effect of nicotine was weak and variable in the present study, and its mechanism remains obscure.     

A Two-Day Continuous Nicotine Infusion Is Sufficient to Demonstrate Nicotine Withdrawal in Rats as Measured Using Intracranial Self-Stimulation

Avoidance of the negative affective (emotional) symptoms of nicotine withdrawal (e.g., anhedonia, anxiety) contributes to tobacco addiction. Establishing the minimal nicotine exposure conditions required to demonstrate negative affective withdrawal signs in animals, as well as understanding moderators of these conditions, could inform tobacco addiction-related research, treatment, and policy. The goal of this study was to determine the minimal duration of continuous nicotine infusion required to demonstrate nicotine withdrawal in rats as measured by elevations in intracranial self-stimulation (ICSS) thresholds (anhedonia-like behavior). Administration of the nicotinic acetylcholine receptor antagonist mecamylamine (3.0 mg/kg, s.c.) on alternate test days throughout the course of a 2-week continuous nicotine infusion (3.2 mg/kg/day via osmotic minipump) elicited elevations in ICSS thresholds beginning on the second day of infusion. Magnitude of antagonist-precipitated withdrawal did not change with further nicotine exposure and mecamylamine injections, and was similar to that observed in a positive control group receiving mecamylamine following a 14-day nicotine infusion. Expression of a significant withdrawal effect was delayed in nicotine-infused rats receiving mecamylamine on all test days rather than on alternate test days. In a separate study, rats exhibited a transient increase in ICSS thresholds following cessation of a 2-day continuous nicotine infusion (3.2 mg/kg/day). Magnitude of this spontaneous withdrawal effect was similar to that observed in rats receiving a 9-day nicotine infusion. Our findings demonstrate that rats exhibit antagonist-precipitated and spontaneous nicotine withdrawal following a 2-day continuous nicotine infusion, at least under the experimental conditions studied here. Magnitude of these effects were similar to those observed in traditional models involving more prolonged nicotine exposure. Further development of these models, including evaluation of more clinically relevant nicotine dosing regimens and other measures of nicotine withdrawal (e.g., anxiety-like behavior, somatic signs), may be useful for understanding the development of the nicotine withdrawal syndrome.     

Actions of nicotine on renal function in dogs

Renal excretory and circulatory responses to nicotine were investigated in anesthetized dogs under three sets of conditions: (a) infusion of nicotine into the left renal artery (ia) at a dose of 0.5 microgram X min-1 X kg body wt-1 X 15 min; (b) ia nicotine after 1.0 mg/kg ia propranolol; and (c) ia nicotine after bilateral adrenalectomy. Measured and calculated left and right renal excretory variables included sodium, potassium, and chloride excretion rates (UNaV, UKV, and UClV, respectively), total solute excretion (UOsV), glomerular filtration rate (GFR), fractional sodium excretion (FENa), and urine flow rate. Systemic arterial pressure and left renal artery blood flow (RBF) were also measured. In seven intact dogs administered nicotine alone, there were significant increases in UNaV, UClV, UOsV, GFR, and urine flow rates from both kidneys. However, nicotine did not significantly affect UKV, FENa, arterial pressure, or RBF. The lack of circulatory effects of nicotine was also observed after either propranolol or adrenalectomy. However, when nicotine was administered after propranolol, the drug evoked significant decreases in UOsV, UNaV, UClV, and GFR, compared with prenicotine values. When nicotine was administered after bilateral adrenalectomy, the drug evoked decreases in the excretory parameters similar to those observed after propranolol. These findings seem to support several inferences: (a) nicotine stimulates renal excretory functions-the alkaloid is saluretic and diuretic; (b) the action of nicotine on the kidney is mediated mainly by the release of catecholamines from the adrenal medulla; (c) catecholamines released by nicotine act mainly on beta-adrenergic receptors; and (d) the saluresis prompted by the release of catecholamines in response to nicotine is due to a subsequent increase in GFR.     

Underground herbivory and the costs of constitutive defense in tobacco

Nicotine is both a constitutive and induced defense in cultivated tobacco (Nicotiana tabacum). Nicotine is thought primarily to defend against above-ground herbivory; however, below-ground herbivores like the nematode Meloidogyne incognita can also damage plants. We evaluated the costs and benefits of constitutive nicotine production in four near-isogenic lines of N. tabacum differing in nicotine content. We exposed the four lines to levels of nematode infection below that found to induce nicotine synthesis, and measured nematode density and each line's response to nematode presence. Nematode density did not differ among lines and was not related to leaf nicotine content in any of the lines, suggesting that constitutive nicotine content did not affect nematode survival or reproduction. Most measures of plant performance were unaffected by nematodes; however, nematode infection decreased flowering in the high nicotine line relative to the other lines. Lines with less constitutive nicotine did not incur similar costs, suggesting a tradeoff between nicotine production and tolerance of low levels of herbivory. A cost of nicotine production is also suggested by the fact that flowering was inversely correlated with leaf nicotine content in all four lines. Although nicotine conferred no resistance to nematodes, high nicotine content reduced the plant's tolerance of low levels of nematode infection and was correlated with reduced flowering. In examining the costs and benefits of a constitutive plant defense, this work complements and extends previous research addressing the relationship between plant tolerance and induced defenses.     

Formation of nicotine addiction in mongrel white rats

A study was made of the possibility of forming nicotine addiction in laboratory rats and using it as the basis for the design of experimental nicotine toxicomania. Experiments were carried out on 56 rats placed in individual cages with a possibility of free choice between water and 0.005% nicotine solution for 2 to 4 months. It was established that the population of intact laboratory rats with 8- and 16-week contact with nicotine solution could be divided into groups demonstrating 3 main types of attitude toward nicotine: aversion (68% of all the animals), moderate addiction (4%), and pronounced addiction (28%). These quantitative relationships remained unchanged whatever the time of contact with nicotine. Thus, the possibility has been shown of designing experimental nicotine toxicomania with marked elements of physical dependence in rats consuming nicotine on a voluntary basis.     

Interactive effects of nicotine and MPTP on striatal tetrahydrobiopterin in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin known to cause dopamine (DA) neuron degeneration, while the psychoactive compound nicotine is known to excite DA neurons. Tetrahydrobiopterin is the cofactor for tyrosine hydroxylase (TOH) in the regulation of DA biosynthesis. The present study investigated the interactions between nicotine and MPTP on striatal biopterin, DA and TOH activity in BALB/c mice. The results indicated that both acute and chronic nicotine administrations at various concentrations significantly increased biopterin and DA levels in the striatum, while MPTP markedly decreased these measures. Pretreatment with nicotine at a dose having no significant effect alone, partially protected against MPTP's toxicity on biopterin and DA. Increasing the dose of nicotine did not have a further protective action. The toxicity of MPTP on TOH was also prevented by nicotine. Further, the above effects of nicotine were probably mediated through the cholinergic nicotinic receptors since mecamylamine reversed the effects of nicotine. These results suggest that nicotine interacts with the dopaminergic system probably at the level of DA biosynthesis through activating TOH and its coenzyme tetrahydrobiopterin.     

Radioimmunoassay of nicotine.

A sensitive and specific radioimmunoassay of nicotine was developed using antisera raised against 6-(p-aminobenzamido) nicotine coupled to bovine serum albumin. Inhibition studies with various nicotine analogues revealed that the antisera are highly specific for both the N-methylpyrrolidine ring and the pyridine ring of nicotine, and especially for the structural changes of the former. The use of these antisera in an assay of nicotine in biological fluids is desirable, since the pyrrolidine ring of nicotine is first metabolized in vivo and antibodies must, therefore, discriminate nicotine from other nicotine metabolites.     

Should intake of carbon monoxide be used as a guide to intake of other smoke constituents?

The relation between blood carboxyhaemoglobin (COHb) and plasma nicotine concentrations was studied in a group of 12 smokers smoking cigarettes of three levels of standard delivery. While the intake of carbon monoxide from a single cigarette was unrelated to the intake of nicotine, presmoking "trough" concentrations of the two substances (reflecting longer-term exposure) were highly correlated. Various other measures of nicotine exposure were at best only moderately correlated with blood nicotine concentrations. Thus trough COHb concentrations might be used to provide a reliable indication of the exposure to nicotine of individual smokers smoking the same type of cigarette, and of the relative exposure to nicotine of populations smoking cigarettes of different standard deliveries.