Nicotine:
Tolerance and its Effects on the Cardiovascular System, The Lungs and The Fetus

        Although there have been some experiment that disprove nicotine's adverse effects, most studies point to nicotine as a very toxic agent. Nicotine is not essential to tobacco products. It is a naturally occurring slightly basic alkaloid with a pyridine and a pyrrolidine ring. It is estimated that the average smoker inhales with each puff, a dose of nicotine equivalent to .1mg nicotine given intravenously. Nicotines pKa is 9. Its half-life is two hours. Nicotine is converted to its metabolites by the P450 enzyme system and by aldehyde oxidases. Nicotine and its metabolites, such as cotinine, N-nitrosomornicotinine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK) are toxic to humans. As is shown in P4, many people in the United States smoke. The average mg of nicotine per cigarette has declined in the United States in the1980s.(P5) However, it is still a substantial amount. The effects of nicotine are dose-related. Therefore, if a smoker smokes more low-nicotine cigarettes and inhales more to obtain the same phychopharmacological effects, the amount of nicotine would be equal to that if cigarettes with a higher level of nicotine had been smoked.(F1)
        N-nitrosamines formed form nicotine during curing and processing by the nitrosation of the tertiary amine nicotine by nitrate, found in tobacco leaf stems and ribs.(P2,3,4) Cleavage of the N-CH3 bond and the loss of formaldehyde yields NNN. Cleavage of the 2 N or 5 bond yields NNK. These nitrosamines are among the most toxic and are present in both mainstream and sidestream smoke. (F2) They are converted to unstable electrophiles that react with DNA to form mutations which can lead to cancer. More than 300 nitrosamines have been shown to be carcinogenic in one or more of 40 animal species. (F3) It has been shown that a smoker has a greater chance of endogenously forming nitrosamines than a nonsmoker. (F4)

ADDICTIVE EFFECTS OF NICOTINE AND TOLERANCE TO NICOTINE

       There is much experimental evidence that nicotine is an addictive drug and that it causes a level of tolerance in lab animals and humans, through its effect on the CNS. For a drug to produce dependence, it must rapidly enter in to the blood steam, must be psychoactive, must readily cross the blood brain barrier and the psychosomatic effects must be related to levels of drug in the brain. All of the above is true for nicotine. Tolerance is when, after repeated doses, a given dose of a drug produces less effect. Smoking a first cigarette as a teenager may produce nausea, and dizziness, effects to which the smoker rapidly becomes tolerant. When nicotinic receptors in the brain are continuously exposed to nicotine, tolerance develops. In 1979 Hirschhorn et al. showed that stimulatory effects of nicotine were due to nicotinic, not muscarinic cholinergic receptors. Intravenously administered nicotine increased the heart rate and the motor effects of rats (tested by lever pushing). Only nicotinic receptor blocker mecamylamine reversed nicotine's effects. Cholinergic receptor blockers did not.

       When people are asked why they smoke, they often point to the psychosomatic effects such as relaxation and increased awareness. There are many nicotinic receptors in the brain. Nicotinic receptor protein has been mapped with [3H] nicotine (Clarke et al., 1987). Lab animals work hard to stimulate dopaminergic neurotransmission in the mesolimbic dopaminergic system which provides the entry point by which psychomotor stimulatory drugs such as nicotine gain access to the reward centers of the brain. Drugs such as cocaine produce rewarding effects by increasing levels of dopamine and it has been postulated that nicotine does the same. Evidence linking dopamine and the reward system of drug (Pettit and Justice,1989) is: 1. Dopamine receptor antagonists reduce the rewarding effects of the drug; 2. Depletion of dopamine decreases the rewarding effects of the drug; 3. injection of d-amphetamine is most rewarding when injecting directly into the nucleus accumbens, a major terminal area of the mesolimbic dopamine system.

       In a study by Porcket et al., 1988, subjects received paired intravenous infusions of nicotine, separated by different time intervals. Despite higher blood concentrations of nicotine, heart rate acceleration was less when a second infusion was given at 60 or 120 minutes after the first infusion. (P6)
       In 1988 Collins and Romm et al. measured the time course of the development and loss of tolerance to nicotine in female rats injected sub- cutaneously twice daily with 1.6 mg/kg of nicotine. Tolerance to nicotine-induced increases in locomotor activity and body temperature were observed within two-four day post treatment test period. In addition, the binding of [3H]l-nicotine was measured in the cortex, midbrain, hindbrain, hippocampus, striatum and hypothalamus and corresponded with the development of tolerance. These results suggest that changes in receptor binding relate to the development of tolerance.

       Nicotinic receptors are present in moderate to high density in the brain areas containing dopamine cell bodies - the ventral tegmental area, the nucleus accumbens and the olfactory tubercle. Destruction of the mesolimbic and nigrostriatal dopamine neurons by 6-OHDA, reduced [3H]nicotine binding in these areas (Clarke & Pert, 1985.) Clarke et al., 1988, showed that nicotine increased dopamine levels in the nucleus accumbens in a dose-dependent and stereoselective manner. Wonnacott & Drasdo in 1989 (F5) showed that nicotine acts in a dose dependent manner(EC50=4 m) to elicit Ca2+ dependent dopamine release. Nicotine acts through presynaptic acetylcholine receptors. Neuronal bungarotoxin inhibits nicotine-evoked dopamine release by 50%. Dopamine mediates nicotines stimulatory effects.

       Fuxe et al., 1990(F6), studied the effects of chronic nicotine treatment via minipumps on retrograde and anterograde degenerative processes in the nigrostriatal dopamine neurons following a partial hemitransection. They showed that nicotine protect against the degeneration of nigrostriatal dopamine neurons in the male rat. The hemitransection produced a decrease of dopamine nerve terminals in the neostriatum and in the anterior parts of the nucleus accumbens and tuberculum olfactorium. Rats received four intraperitoneal injections of nicotine tartrate in a dose of .5 mg/kg body weight with thirty minute intervals. Controls were: hemitransection and saline, sham operation and nicotine and sham operation and saline. As shown in P5, the nicotine produced a protection against the disappearance of nerve cell bodies, dendrites, and terminals in the nigrostriatal dopamine neurons following hemitransection. This experiment was another link between nicotine and the dopamine reward system.
       In 1991 Janson et al.(F7) showed that (-)nicotine treatment protects against the neurotoxic effects of 1-mehtyl-4-phenyl-1,2,3,6-tetrahydropyrridine (MPTP) on dopamine neurons.(P6) MPTP was administered subcutaneously using minipumps to male C57BL/6 mice. (-)Nicotine was given after injection of MPTP. Controls were mice given MPTP and saline. Different amount of nicotine were injected into the mice. A dosedependent enhancement by chronic (-) nicotine of the MPTP-induced depletion of dopamine stores in the neostriatum and of the disappearance of THIR nerve cells in the substantia nigra was observed. The saline control group showed no change in the dopamine store levels. Pauly et al., in 1992 (F8) injected nicotine intraperitaneously into C57BL/6 mice and showed that animals receiving chronic nicotine were less sensitive to nicotine than animals that received chronic saline. Two weeks following cessation of nicotine treatment, nicotine-treated animals were still tolerant to acute nicotine challenges. (P7) Nicotine sensitivity was measured by locomotor, heart rate, and body temperature tests. With increasing doses of nicotine, only a slight change in heart rate and body temperature were observed in the nicotine tolerant mice.
        Several other experiments have been performed linking tolerance to nicotines effect on the CNS. Marks et al., in 1985 (F9) administered nicotine at 4mg/kg/hr intravenously in the right jugular veins of DBA mice and showed that tolerance to y-maze activity ( a locomotor test), body temperature increases and accelerated heart rate developed. The time for tolerance to be lost after cessation of nicotine administration was: eight days for the locomotor test, twelve days for the body temperature test and twenty days for the heart rate test. In this experiment, tolerance persisted after brain nicotine levels had returned to their normal level after an increase caused by nicotine administration. Also, nicotine binding had returned to normal levels before the effects of tolerance had ceased. In an experiment in 1983, Marks had reported that nicotinic receptor levels in the brain increase after nicotine administration. It is not yet clear what role the amount of nicotine receptors plays in tolerance. In 1994 Grady et al.(F10) demonstrated that nicotine-stimulated dopamine release from mouse striatal synaptosomes was concentration-dependent and that [3H]-nicotine binding site could be the presynaptic receptor involved in [3H]dopamine release in mouse striatal synaptosomes.

        NICOTINE AND ITS EFFECTS ON THE CARDIOVASCULAR SYSTEM
In many studies, nicotine has been shown to adversely affect the cardiovascular system , chiefly through inducing vasoconstriction, thrombosis and increasing blood pressure and heart rate.(P8) Causal relationships have linked nicotine to myocardial infarction, unstable angina pectoris, sudden cardiac death, peripheral artery occlusive disease, arteriosclerosis, aortic aneurysm and stroke. Several studies indicate that nicotine leads to arteriosclerosis by lipid deposition in macrophages on the inner surface of arterial walls, calcified lesions that may ulcerate or hemorrhage and by thrombosis. Nicotine has been shown to increase levels of LDL and to cause injury to vascular endothelial cells. There is also platelet aggregation and release of thromboxane A2 which leads to vasoconstriction. Nicotine releases catecholamines and induces lipolysis and increases plasma free fatty acid concentrations leading to an increase in LDLs.
Cryer et al., 1976 (F11) in a study using ten patients, showed that nicotine increased levels of norepinephrine and epinephrine in the blood in proportion to increases in pulse rate, blood pressure, blood glycerol and blood lactate/pyruvate. Norepinephrine and epinephrine stimulate platelet aggregation and thrombosis by stimulating the second messenger adenylate cyclase system. This may be one way in which nicotine leads to arteriosclerosis. In this experiment, after adrenergic receptor blockage with phentolamine and propanodol, cardiovascular effects were not noted. It was noted that nicotine induces hemodynamic changes through adrenergic mechanisms, such as increasing the secretion of norepinephrine.
In 1978 Booyse et al. (F12), administered nicotine to rabbits and observed significant adverse changes in their aortic endothelial cells. Controls were rabbits fed the same diet but no nicotine. Endothelial focal cells similar to those seen in early stages of arteriosclerosis were seen. Gnasso et al.(1982), showed that cigarette smoking produced a reduction in levels of HDL while Stubbe et al.(1986), observed increasing levels of HDL in patients after two weeks of smoking cessation. Cluette-Brown et al.(1986), fed nicotine to mice and observed increasing levels of LDLs with increasing doses of nicotine.
       In 1986 Burgher et al. showed that there is an increased platelet activity in smokers of high nicotine content cigarettes. In 1987 Grodzdjak et al. showed that nicotine lessens the ability of the heart to convert oxygen to ATP. Activity of cytochrome oxidase fell by 25% in rabbit hearts after eight weeks of nicotine treatment. This happened in a dose-related manner.
       Bounameaux et al. (1987)(F13) compared the haemodynamic changes in six healthy volunteers who smoked one low (.1mg) or one high (1.2mg) nicotine cigarette and those who chewed nicotine gum (4mg) and a placebo gum. Systemic blood pressure rose 9% for those who smoked the high nicotine content cigarette, 4% for those who smoked the low nicotine cigarette and 7% for those who chewed the nicotine gum. Heart rate rose by 25% for those who smoked the high nicotine content cigarette, by 18% for those who smoked the low content nicotine cigarette, and 25% for those who chewed the nicotine gum. Changes were seen five minutes after smoking. No changes were seen for those who chewed the placebo gum. Results can be seen in P9.
        Laustiola et al, (1990) (F14) showed that cessation of smoking decreased norepinephrine levels and increased adrenergic receptor levels on mononuclear leukocyte cells. Impaired vasodilation in chronic smokers is thought to be due to decreased number of b-adrenergic receptors. Also, decreased levels of the vasodilator prostacyclin have also been reported(Wennmalm et al., 1980). Vasoconstriction leads to coronary artery disease. There is a lower risk for this disease after a person quits smoking.
        In 1992 Moreyra et al. (F14) (P10)found a correlation between blood nicotine levels and arterial and venal vasoconstriction and increased heart rate. They studied the effects of smoking two low nicotine cigarettes in twelve patients. Coronary arterial levels of nicotine rose form a baseline of 5+1ng/ml to 37+7 ng/ml after the first cigarette to 45+8 ng/ml after the second cigarette. Venous levels of nicotine rose from 8+2 ng/ml to 15+3 ng/ml after the first cigarette to 20+3ng/ml after the second cigarette. The left anterior descending artery diameter changed from 2.31+ .2 nm before smoking to 2.03 + .2nm after the first cigarette to 2.10 +.2 nm after the second cigarette. Circumflex artery
diameter changed from baseline 2.49+.2nm to 2.19+ .1nm after the first cigarette to 2.21 + .2 nm after the second cigarette. Heart rate rose 8+2 beats per minute after the first cigarette and 9+1beats per minute after the second cigarette. Therefore even low nicotine cigarettes produce substantial blood nicotine levels and lead to vasoconstriction. This, combined with the fact that nicotine may be addictive and smokers may increase the amount of low nicotine cigarettes they smoke of inhale deeper, shows that low nicotine cigarettes pose a significant danger to health.

       In 1993 Khoala et al. showed that nicotine causes vasoconstriction, release of vasopressin, increased muscle blood flow and increased platelet aggregation. Adrenergic receptor blockage stops the cardiovascular effects of nicotine. This was yet another study indicating that nicotine stimulates the CNS.
Nicotine has also been shown to increase the amounts of oxygen free radicals in heart muscle. (Churchet et al., 1994) Free radicals decrease the ability of mitochondria to convert oxygen to ATP. This leads to cardiac ischemia. Nicotine has been shown to sensitize lung neutrophils. Inappropriatel y activated neutrophils release oxygen radicals and play a role in cardiovascular tissue damage. It has been postulated that nonsmokers exposed to passive smoke are more likely to suffer form its adverse effect becauase they do not have tolerance to nicotine or acquired immune defenses against it and are more susceptible to its effects. For example, in the latter experiment, nonsmokers had an increased activation of neutrophils than smokers when smoking cigarettes with the same nicotine levels.

NICOTINE AND ITS EFFECTS ON THE LUNG
        Several studies have shown that nicotine has adverse effects on the lung such as lung cancer, bronchoconstriction, and alveolar damage.
Pulmonary neuroendocrine cells are thought to be the origin of many lung tumors. They proliferate rapidly during hyperoxia or hypoxia and exposure to nitrosamines. PNE secrete bombesin, a mitogenic growth factor. It stimulates the release of cytokines such as granulocyte/macrophage stimulating factor and interleukin 1 from alveolar macrophages and periphenol from monocuclear cells. These cytokines mediate inflammation and recruit and activate neutrophils and eosinophils causing pulmonary obstruction. PNE also secrete calcitonin which increases Ca2+ levels. Ca2+ can activate proteins that lead to the activation of oncogenes. Acetylcholine stimulates PNE proliferation and secretion. Nicotine increases acetylcholine secretion.
        Tabassian et al. (1989) showed that nicotine binds to nicotinic cholinergic receptors to cause hyperplasia of PNE and increased calcitonin levels. Reznik et al. (1976) had previously shown that N-nitrosoethylamine (DEN) and
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) causes similar effects.
Schuller et al. (1989) (F15) conducted in vitro experiments of human lung Clara cells, alveolar type II cells, and PNE. Nicotine, NNK and DEN caused proliferation of these cells. Proliferation was blocked by nicotinic receptor antagonists. Schuller et al. (1990) (F16) exposed Syrian golden hamsters to hyperoxia and gave them subcutaneous injections of DEN or NNK. A significant number of animals developed neuroendocrine lung tumors which secrete calcitonin and bombesin. However, some studies ( Nyelin et al. 1988) have shown that nicotine or nitrosamines alone without abnormal oxygen conditions do not cause an increase in bombesin levels. Clearly, more studies need to be made. P11 shows two proposed mechanisms of nitrosamine initiation of cell proliferation. The nicotine-receptor complex may induce a second messenger pathway leading to cell proliferation or may cause the cell proliferation themselves by interacting with DNA. It has also been shown that NNK causes proliferation of Clara cells. Antagonists of adrenergic receptors block this effect. As before stated, nicotine is thought to increase secretion of norepinephrine and epinephrine which bind to adrenergic receptors. This provides a link between nicotine and the proliferative effects of norepinephrine and epinephrine.
        Hartiala et al. in 1985 (F17) demonstrated that nicotine induced bronchoconstriction and airway smooth muscle tension in dogs. Cigarette smoke of increasing amount of nicotine was introduced into the lungs of donor dogs and injected into the arterial blood of recipient dogs. Blockage of nicotinic receptors in the CNS and airway parasympathetic ganglia inhibited the effects of nicotine on bronchomotor tone. Bronchoconstriction may be a defense mechanism of lungs to reduce airborne pollutants those in cigarette smoke. However, long-term bronchoconstriction may lead to chronic obstructive pulmonary disease and other respiratory complication. Results from this experiment are shown in P12. Nicotine activated nicotinic receptors that stimulated the central respiratory pattern generators which increase breathing and airway smooth muscle tone and airway parasympathetic ganglia.

       Cattaneo et al. (1993) (F18) showed that (-) nicotine induced dose-dependent tumor cell proliferation in small cell lung carcinoma cells with nicotinic receptors. (-) Nicotine increased levels of serotonin, believed to have proliferative effects on lung cells. Mecamylamine, a ganglionic nicottagonist, blocked nicotines effects.

       Leader et al.(1994) (F19), monitored the symptoms of chronic obstructive pulmonary disease (COPD) in eight patients that ceased smoking for 28 weeks. Symptoms of COPD - cough, sputum production, dyspnea, air flow limitation, and impaired gas exchange - decreased with decreasing levels of cotinine as the weeks after smoking cessation passed by, as shown in P13.
Many studies have linked smoking, although not nicotine per se, to asthma, emphysema and other disorders. Kondo et al. (1994) showed that cigarette smoke decreased antioxidants in the alveolar macrophages of elderly men. Bonham et al. (1995) showed that sidestream smoke decreases rapidly adapting receptor responsiveness in the lungs of guinea pig, leaving the lung more susceptible to noxious agents.
Although there is overwhelming evidence that nicotine and its metabolites are involved in cancer, especially lung cancer , some experiments do not support this hypothesis. Doolittle et al. (1995) performed Ames assays and Sister Chromatid Exchange tests on CHO with or without S9 liver homogenate. Nicotine and its metabolites did not cause a significant number of mutations in any of the tests. Yim et al.(1995) (F20) found nicotine not to be genotoxic in the bacterial luminescence test. Cotinine was found to be genotoxic. There are, however, enough studies to that nicotine has a role in cancer. More studies are needed to exactly pinpoint the mechanisms on how nicotine causes or is a promoter in cancer.

NICOTINE AND ITS EFFECT ON THE FETUS
        Nicotine has been linked to causing adverse effects such as lung problems and low birth weights in the fetus and in newborns. Martin et al. (1986) (F22) studied exposing nonsmoking mothers to sidestream smoke before and after pregnancy. They reported that passive smoke was significantly related to low birth weights of newborns. Smoke from high nicotine content cigarettes produced lower birth weight babies. On average, mothers exposed to cigarette smoke delivered babies 24g lighter than mothers not exposed to cigarette smoke.
Nicotine has been shown to cause hypoxia in the fetus, resulting in growth retardation. Maritz et al. (1994) (F23)(P14) showed that nicotine caused swelling of Type II alveolar cells and interstilial cell mitosis, decreased the ratio of Type I:II alveolar cells, and lowered the number of capillaries per unit length of septum in fetal lungs. Blood-air barriers were ruptured, making the lungs more susceptible to airborne toxins.
       Formation of alveolar septa is an important process in pulmonary development. The majority of the lungs functional gas exchange units develop in postnatal life. Type II cells of the septa differentiate into Type I cells. High degree of Type II cell proliferation as opposed to differentiation may compromise lung function and lead to cancer. Nicotine may impair metabolism, also leading to a lower Type I:II ratio.
       Rubin et al.(1986) and Martin et al. (1986) also reported lower fetal weight in sidestream exposed rats. Rajini et al.,(1993) (F25) reported an increase in the proliferation of epithelium in neonatal mice of the A/J strain but not the C57BL/6 strain after exposure to sidestream smoke with varying levels of nicotine. Thus, interspecies differences and genetics may play a role in nicotines effects.
        Maritz et al. in 1992(F24) exposed pregnant rats to nicotine and control rats to saline. Nicotine was found to interfere with ATP production in mitochondria. It disrupted mitochondrial cristae. Decreases in glycolysis due to destruction of cristae and to hypoxia interfered with Type I:II cell ratio. Based on studies such as these it is not only conceivable that nicotine harms the fetus, but also that nicotine causes adverse effects on the lungs of smokers.(Dodge et al..1982, Ekwo et al. 1985 and Wright et al., 1991).
        In 1995 Joad et al. (F25)exposed pregnant Sprague-Dawley rats to: in utero filtered air(FA) followed by postnatal FA; in utero FA followed by postnatal SS with 344+.85 g nicotine; in utero SS followed by postnatal FA; and in utero SS followed by post natal SS. Only lungs exposed to in utero and postnatal SS showed less compliance and an increased number of neuroendocrine cells.(P15)

CONCLUSIONS
       In this paper, several of the adverse effects of nicotine - tolerance, cardiovascular and lung changes, fetal problems - were explored. Nicotine has other adverse effects such as changing insulin levels and altering carbohydrate metabolism. Also, each of its metabolites would have to be studied for their effects on the human body. Problems with animal studies include that it is sometimes not precise to extrapolate their results to humans. Problems with epidemiological studies include that if a long-time smoker dies, it cannot be said for sure that they died from smoking and not drinking, genetics, etc.. Also, tobacco contains 2500 compounds and tobacco smoke over 3800.(F2) It would be hard to say that nicotine was the only or most harmful chemical. Although there are reports showing inconclusive evidence as to nicotines toxic effects, there is enough evidence to conclude that nicotine is addictive and that its overall effects on human health are definitely not good. Past studies on nicotine and other cigarette smoke components prompted the United States Surgeon General in 1986 to conclude that cigarette smoking is causally associated with cancer of the lung, larynx, oral cavity, and esophagus and that it is correlated with cancer of the pancreas, kidney, bladder and cervix.(F28) Ten years later, the FDA is conducting more studies to show a closer link between nicotine and addiction, cancer and other health problems.

FOOTNOTES

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