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6-Hydroxy-l -Nicotine and Memory Impairment
C H A P T E R
21 6-Hydroxy-L-Nicotine and Memory Impairment Lucian Hritcu, Marius Mihasan Department of Biology, Alexandru Ioan Cuza University of Iasi, Iasi, Romania
Abbreviations 2,3,6-THP 2,6-DHP 2,6-DHPON 6HDNO 6HLN 6HLNO 6-HMM 6-HPON AD CHL DHPH DHPONH KDH MGABA nAChBP nAChR NB NDH
2,3,6-trihydroxypyridine 2,6-dihydroxypyridine 2,6-dihydroxypseudooxynicotine 6-hydroxy-D-nicotine oxidase 6-hydroxy-L-nicotine 6-hydroxy-L-nicotine oxidase 6-hydroxy-methylmyosmine 6-hydroxy-pseudooxynicotine Alzheimer’s disease chlorisondamine 2,6-dihydroxypyridine-3-hydroxylase 2,6-dihydroxypseudooxynicotine hydrolase ketone dehydrogenase γ-N-methylaminobutyrate nicotinic receptor binding domain homolog nicotinic acetylcholine receptors nicotine blue nicotine dehydrogenase
21.1 INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with a gradual decline in cognition. It is considered to be the most common pathological cause of dementia (Sase, Yamamoto, Kawashima, Tan, & Sawa, 2017). It has been reported that in 2016, about 2 million people who have AD are at the age of 85 or older, accounting for 37% of all people with the AD (Hebert, Weuve, Scherr, & Evans, 2013). By 2050, as many as 7 million people at the age of 85 and older may have AD, accounting for half (51%) of all people at the age of 65 and older with the AD (Hebert et al., 2013). It has been reported that nicotinic receptors are therapeutic targets in the nervous system (Taly, Corringer, Guedin, Lestage, & Changeux, 2009). Also, the involvement of the nicotinic acetylcholine receptor (nAChR) subtypes α7 and α4β2 in AD pathology was evidenced (Parri, Hernandez, & Dineley, 2011). The AD is characterized by substantial decreasing in both nicotinic and
Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00021-6
muscarinic acetylcholine receptor expression in the basal forebrain, cerebral cortex, and hippocampus (Kása, Rakonczay, & Gulya, 1997). Consequently, in the cerebral cortex, a significant loss of α4β2 nAChR subtypes (Aubert et al., 1992) was evidenced, while in the hippocampus, a loss of α7 nAChRs was reported being correlated with the progressive alteration of the memory function (Nordberg, 2001). Some epidemiological studies have shown data suggesting an inverse relationship between tobacco consumption and the development of AD (Echeverria Moran, 2013). The putative beneficial effect of tobacco has been mainly attributed to nicotine, which has been reported to improve cognitive abilities and reduce plaques in a mouse model of the AD (Nordberg et al., 2002). Numerous studies suggested that acute nicotine administration significantly improved the performance of patients with AD in cognitive tasks (Taly et al., 2009). Since nAChRs play an important role in attention and learning and memory, the positive effects of nicotine on memory have been mostly credited to the activation of these receptors (Sabbagh, Lukas, Sparks, & Reid, 2002). Moreover, the postmortem levels of the beta-amyloid peptide that considered the neurotoxic agents in the AD brains were significantly decreased in the brains of smoking AD patients compared to nonsmokers with the disease (Boopathi & Kolandaivel, 2014). The high potency of nicotine as a cognition-enhancing agent and AD therapeutic strategy (Murray & Abeles, 2002) can be explained not only by its ability to bind and modulate nAChRs but also through its antioxidant effects at low concentrations (Newman et al., 2002) and through its ability to interact with beta-amyloid peptide. However, nicotine has not demonstrated in clinical studies to be a useful treatment for the AD (LópezArrieta & Sanz, 2001). Because of its short halftime (about 2 h), its proved negative effects on various other organs such as the lungs (Hecht, Hochalter, Villalta, &
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Murphy, 2000), its linkage to cigarettes, and the negative publicity associated with smoking (Buccafusco, 2004), nicotine did not impose itself as a feasible therapeutic agent for AD. Numerous studies have focused primarily on the effects of nicotine and nicotine metabolites found in mammalian cells, because of their potential contribution to the neuropharmacological effects resulting from nicotine exposure (Pogocki et al., 2007; Riveles, Huang, & Quik, 2008). In mammalian cells, nicotine is extensively metabolized in the liver to six primary metabolites (nicotine glucuronide, nicotine N-oxide, nornicotine, isomethonium ion, cotinine, and 2-hydroxynicotine). The predominant pathway during the first-pass metabolism yields cotinine (70%–80% of nicotine is metabolized to cotinine in humans) that may have some relevance in the diverse neurobiological effects of smoking as a ligand of nAChRs (Dome, Lazary, Kalapos, & Rihmer, 2010). Furthermore, there are several other indications that cotinine is a pharmacologically active substance. It has neuroprotective and/or cell-protective effects in the cell-culture model, attention-enhancing effects, antipsychotic-like effects (in prepulse inhibition), dopamine-releasing effects in striatal tissue samples, etc. (O’Leary, Parameswaran, McIntosh, & Quik, 2008). All of these results indicate that the impact of nicotine and nicotine metabolites on brain functions is not negligible. Thus, it has become increasingly important to obtain more information on the biological effects of nicotine metabolites derived from various sources, in order to possibly manage neurological abnormalities in some neurodegenerative afflictions.
21.2 IMPLICATION TO TREATMENTS There are many treatments available for subjects who have an addiction to nicotine/tobacco products such as nicotine replacement therapy (the patch and the nicotine gum), nicotine sprays and inhalers, and medication (varenicline, Chantix, and bupropion, Wellbutrin). Additionally, there are mentioned different psychological and behavioral treatments such as hypnotherapy, cognitivebehavioral therapy, and neurolinguistic programming that help people to fight against tobacco addiction.
21.3 NICOTINE DERIVATIVES IN BACTERIA The crystal structure determination of the nicotinic receptor binding domain homolog (nAChBP) with bound nicotine (Celie et al., 2004) followed by the structure of α7 nAChRs (Mowrey et al., 2013) has spurred the interest of
several academic and pharmaceutical laboratories in the possibility that new nicotinic drugs could be designed. From this point of view, nicotine derivatives are ideal candidates, offering a wide array of possibilities. The difficulty resides, firstly, in the identification of molecules that have the beneficial effects of nicotine but elude its side effects (Pogocki et al., 2007) and, secondly, in providing simple and reliable methods for production and isolation of the identified compounds. In this context, the nicotine-degrading bacteria with their ability to use this alkaloid as the sole carbon source offer a wide range of nicotine derivatives with unexplored biotechnological potential. A multitude of reports indicate that bacteria including Pseudomonas (Liu et al., 2014; Tang et al., 2011), Bacillus and Pusillimonas (Ma et al., 2015) strains, Ochrobactrum sp. SJY1 (Yu, Tang, Zhu, Li, & Xu, 2015), Agrobacterium tumefaciens S33 (Wang, Liu, & Xu, 2009), Shinella sp. HZN7 (Ma et al., 2014), and Achromobacter nicotinophagum (Hylin, 1958) can degrade nicotine by using three related pathways: the pyridine pathway (Brandsch, 2006), the pyrrolidine pathway (Tang et al., 2013), and a variant of pyridine and pyrrolidine pathways named the VPP pathway (Wang, Huang, Xie, & Xu, 2012). The intermediates and enzymes involved in each pathway have been extensively presented in two reviews as described by Brandsch (2006) and Gurusamy and Natarajan (2013), respectively.
21.4 NICOTINE CATABOLISM IN ARTHROBACTER NICOTINOVORANS By far, the most extensively studied is the nicotine metabolism of A. nicotinovorans pAO1. This bacterium makes use of the pyridine pathway to degrade nicotine. Briefly, nicotine degradation starts with the hydroxylation of the pyridine ring to 6-hydroxy-L-nicotine (6HLN) by nicotine dehydrogenase (NDH) (Andreesen & Fetzner, 2002). Next, the pyrrolidine ring is oxidized by 6-hydroxy-Lnicotine oxidase (6HLNO) (Schenk & Decker, 1999), and 6-hydroxy-methylmyosmine (6-HMM) is formed. The compound reacts spontaneously with water and turns to 6-hydroxy-pseudooxynicotine (6-HPON; N-methylaminopropyl-(6-hydroxypyridyl-3)-ketone). 6HLNO has a high stereoselectivity and acts only on 6-hydroxy-L-nicotine. As small amounts of D-nicotine can be produced by the tobacco plant, there is a 6-hydroxy-D-nicotine oxidase (6HDNO) made by A. nicotinovorans pAO1 that can transform 6-hydroxy-L-nicotine to 6-hydroxy-methylmyosmine and, consecutively, to 6-HPON (Decker & Bleeg, 1965). A second hydroxylation that takes place at C2 of 6-HPON yields 2,6-dihydroxypseudooxynicotine (2,6-DHPON). This step is encoded by a multimeric ketone dehydrogenase (KDH). A key step in nicotine catabolism is catalyzed by
21.6 6HLN PRODUCED USING AN ARTHROBACTER-BASED BIOTECHNOLOGY
2,6-dihydroxypseudooxynicotine hydrolase (DHPONH) (Sachelaru, Schiltz, Igloi, & Brandsch, 2005) and consists of the cleavage of 2,6-DHPON into two main metabolites: 2,6-dihydroxypyridine (2,6-DHP) and γ-Nmethylaminobutyrate (MGABA). 2,6-DHP is further hydroxylated by a FAD-dependent monooxygenase named 2,6-dihydroxypyridine-3hydroxylase (DHPH) yielding 2,3,6-trihydroxypyridine (2,3,6-THP). In the presence of oxygen, 2,3,6-THP suffers an apparently spontaneous dimerization. The dimerization takes place inside the cell, and the product of the reaction is 4,40 ,5,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 ), which is then excreted and accumulates in the medium forming characteristic nicotine blue (NB) color. Even though the metabolic pathway in A.nicotinovorans was described in detail by the group of Prof. Roderich Brandsch, Albert Ludwigs University, Freiburg, Germany, until now, there are no biotechnological applications of the identified metabolic products.
21.5 6HLN IS ABLE TO INTERACT WITH nAChRs We were interested to see if any of the nicotine intermediates found in the A. nicotinovorans catabolic pathway have any affinity for nAChRs, and we used in silico experiments to evaluate their binding potential (Mihasan, Capatina, Neagu, Stefan, & Hritcu, 2013). For this, the 3-D structures of all of the intermediates found in the nicotine degradation pathway of A.nicotinovorans were downloaded from PubChem (Kim et al., 2016) and converted into a format suitable for docking using FROG v.1.01—free online drug conformation generation (Leite et al., 2007). All steps required for directed docking (molecular surface generation, sphere box, and energy grid generation) were achieved using ADT 1.5.4 (Morris et al., 2009). In silico docking was performed using Autodock 4 (Morris et al., 2009) using the default parameters and the acetylcholine binding protein (AChBP, PDB ID: 1UW6) (Celie et al., 2004) as a receptor. The receptor was kept rigid, and all ligands were flexible. All bonds within the ligand molecules were allowed to rotate. The targeted region for docking was a cube of approx 150 Å centered on Tyr143 from the subunit A of AChBP. First, L-nicotine was docked at the binding site. The computationally determined orientation of L-nicotine in the binding site of AChBP was compared with the experimentally observed orientation described by Celie et al. (2004). A very good fit between the computationally obtained pose of L-nicotine with the experimentally determined one (root-mean-square deviation, RMSD, of 0.2 Å for 12 superimposed atoms of the ligand) was obtained, indicating the reliability of the docking method employed.
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The resulted theoretical binding pose of 6HLN was very similar to the one experimentally observed for nicotine with an RMSD of 0.19 Å for 12 atoms of the ligand. Not only this compound would fit into the binding pocket, but also the increased interaction energy calculated by AutoDockTools for 6HLN is due to an extra H bond between the hydroxyl group of the 6HLN and the Tyr185 residue. Moreover, the antioxidant activity of 6HLN was evaluated using the quantum chemical quantitative structure-activity relationship (QSAR) equation proposed by Rastija and Medic-Šaric (2009). We showed that the antioxidant activity of 6HLN calculated as IC50 ¼ 43.06 is improved compared to the IC50 ¼ 54.54 for nicotine.
21.6 6HLN PRODUCED USING AN ARTHROBACTER-BASED BIOTECHNOLOGY As a result of the promising data produced by this computational pilot study, one might easily see the high biotechnological potential of 6HLN. Currently, the compound can be chemically synthesized using a diazotization reaction on 6-aminonicotine and is available from different suppliers (Santa Cruz Biotechnology or Carbosynth) at a fairly expensive price. Arthrobacter nicotinovorans cells could nevertheless provide an alternative way to produce this compound using waste and by-products from the tobacco industry. HPLC analysis of A. nicotinovorans growth medium showed a small accumulation of 6HLN during the growth of the bacteria in the presence of nicotine (Boiangiu, Guzun, & Mihasan, 2014). Two previously published articles indicate that 6HLN can be found in not only the nicotine growth media of A. nicotinovorans (Hochstein & Rittenberg, 1959) but also a possible export mechanism involving a facilitated transport system (Ganas, Igloi, & Brandsch, 2009). The accumulation is only temporary, and as soon as the amount of nicotine in the medium is depleted, the 6HLN levels drop (Fig. 21.1). Apparently, a metabolic imbalance is involved. NDH, the trimeric enzyme containing a molybdenum pyranopterin dinucleotide cofactor, FAD, and two ironsulfur clusters (Andreesen & Fetzner, 2002) responsible for converting L-nicotine to 6HNLO, has roughly the same affinity (Km of 0.037 mM) for its substrate but a higher reaction rate (specific activity of 29.2 μmol/min/ mg) (Hochstein & Dalton, 1967) compared to downstream enzyme 6-hydroxy-L-nicotine oxidase (6HLNO). The dimeric enzyme 6HLNO contains one FAD per subunit (Schenk & Decker, 1999) and has a Km of 0.02 mM and a much lower specific activity—4.73 μmol/min/mg (Decker, Dai, M€ ohler, & Br€ uhm€ uller, 1972; Kachalova et al., 2010). Basically, as long as nicotine is still in the
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FIG. 21.1 Dynamics of nicotine and 6HLN in the growth medium of A. nicotinovorans. A temporary accumulation of 6HLN can be observed (continuous line). As soon as nicotine is depleted from the medium (dashed line), 6HLN levels drop.
FIG. 21.2 Upstream and downstream reactions of 6HLN in the nicotine catabolic pathway of A. nicotinovorans. NDH, nicotine dehydrogenase; 6HLN, 6-hydroxy-L-nicotine; 6HLNO, 6-hydroxy-L-nicotine oxidase; 6-HMM, 6-hydroxy-methylmyosmine.
medium, NDH converts it to 6HLN at a rather fast pace, 6HLNO cannot keep up, and 6HLN accumulates in the medium. As soon as nicotine is depleted, 6HLNO can quickly recover, and 6HLN is consumed (Fig. 21.2). The observed metabolic imbalance could be used to produce 6HLN. In order to increase yield, a genetically engineered strain of A. nicotinovorans was created. The ndh genes were cloned in the custom pART2 expression vector (Andrei & Mihasan, 2013; Sandu, Chiribau, Sachelaru, & Brandsch, 2005) that allows the overexpression of the NDH in a nicotine-dependent manner (Fig. 21.3). Currently, our lab uses low-density A. nicotinovorans pART2ndh cell cultures cultivated in flasks under constant agitation at 28°C in order to produce 6HLN. By overexpressing NDH and chemically inhibiting 6HLNO using 0.05 mM ZnSO4, we are able to commonly produce approx 50–60 mg of 6HLN per 100 mL medium. 6HLN produced has memory-enhancing effects.
Next, 6HLN was tested in vivo on male naive Wistar rats and proved to have surprisingly positive effects on memory processes and oxidative brain status. Hritcu, Stefan, Brandsch, and Mihasan (2013) continue to demonstrate that in vivo chronic treatment of 6HLN (0.3 mg/kg, seven consecutive days) significantly increased spontaneous alternations in Y-maze task and working memory in radial-arm-maze task, suggesting effects on short-term memory, without affecting longterm memory, explored by reference memory in radialarm-maze task. Also, 6HLN significantly improved locomotor activity, as evidenced by the number of arm entries within Y-maze test (Table 21.1). In addition, 6HLN increased antioxidant enzyme activity (SOD and GPX) and decreased the production of lipid peroxidation (MDA) in the rat temporal lobe homogenates, suggesting antioxidant effects (Table 21.2). We concluded that positive effects of 6HLN on spatial memory may occur by antioxidant actions.
FIG. 21.3 Location of the ndh and 6hlno genes (red) in the nic-gene cluster from the pAO1 megaplasmid of A. nicotinovorans Dark blue, genes experimentally shown to be involved in nicotine metabolism; light blue, genes putatively involved in nicotine metabolism; white, genes with no experimental or putative function; black, transposons and insertion elements; yellow, putative transcription factors.
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TABLE 21.1
Effects of 6HLN in Experimental Models of Memory
Nicotine derivative
Animal species
Behavioral test
Intraperitoneal in naive rats 0.3 mg/kg
Rats
Intraperitoneal in naive rats 0.3 mg/kg
Administration
Observed effects
Reference
Y-maze
Improved short-term memory and locomotor activity
Hritcu et al. (2013)
Rats
Radialarm-maze
Enhanced working memory, without affecting reference memory
Hritcu et al. (2013)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
Y-maze
Sustained short-term memory and locomotor activity
Hritcu, Stefan, Brandsch, and Mihasan (2015)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
Radialarm-maze
Enhanced working memory and reference memory
Hritcu et al. (2015)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
Y-maze
Enhanced spontaneous alternation
Hritcu et al. (2017)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
Radialarm-maze
Improved working memory and reference memory
Hritcu et al. (2017)
Hritcu et al. (2015) demonstrated that 6HLN ameliorated scopolamine-induced spatial memory impairment (Table 21.1). Decreased activities of superoxide dismutase, glutathione peroxidase, and catalase along with a decrease of total content of reduced glutathione were observed in the rat hippocampal homogenates of scopolamine-treated animals as compared with control. Production of malondialdehyde (lipid peroxidation) significantly increased in the rat hippocampal homogenates of scopolamine-treated animals as compared with control, as a consequence of impaired antioxidant enzyme activities. Additionally, in scopolamine-treated rats, 6HLN significantly improved memory formation and TABLE 21.2
decreased oxidative stress (Table 21.2), suggesting memory-enhancing and antioxidant effects. Therefore, our results suggest that the administration of 6HLN ameliorates scopolamine-induced spatial memory impairment by attenuation of the oxidative stress in the rat hippocampus. Recently, Hritcu et al. (2017) reported that 6HLN administration attenuated the cognitive deficits (Table 21.1) and recovered the antioxidant capacity (Table 21.2) in the rat hippocampus of the chlorisondamine (CHL) rat model. Taken together, administration of 6HLN as compared to nicotine improved significantly spatial memory in the Y-maze, and radial-arm-maze
Effects of 6HLN on Oxidative Stress
Nicotine derivative
Animal species
Biochemical markers
Observed effects
References
Intraperitoneal in naive rats 0.3 mg/kg
Rats
SOD, GPX, MDA
Increased the activity of SOD and GPX and decreased MDA level
Hritcu et al. (2013)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
SOD, GPX, CAT, GSH, MDA
Increased the activity of SOD, GPX, CAT, and GSH and decreased MDA level
Hritcu et al. (2015)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
SOD, GPX, CAT, GSH, MDA
Increased the activity of SOD, GPX, CAT, and GSH and decreased MDA level
Hritcu et al. (2017)
Administration
SOD, superoxide dismutase; GPX, glutathione peroxidase; CAT, catalase; GSH, glutathione; MDA, malondialdehyde.
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21. 6-HYDROXY-L-NICOTINE AND MEMORY IMPAIRMENT
FIG. 21.4 Possible mechanism of action of 6HLN with memory-enhancing and antioxidant activity.
tasks decreased lipid peroxidation and enhanced antioxidant status in the hippocampus of the CHL-treated rats. This observed effect could suggest possible neuroprotective effects of both nicotine and 6HLN. However, the effects of 6HLN are significantly increased as compared with nicotine in the CHL-treated rats. From the observation of this study, we can suggest that 6HLN may have a beneficial role in the treatment and/or management of memory disorders such as AD and indicate the important significance of the nicotinic acetylcholine system in brain dysfunction. Additionally, we also reported (Ionita et al., 2017) that both nicotine and 6HLN act as anxiolytic and antidepressant agents in the chlorisondamine rat model. The observed effects could be mediated by the nicotinic acetylcholine receptors. In summary, our data contribute with additional support concerning the role of nicotine and nicotine metabolites in memory processes and brain oxidative status. Moreover, 6HLN should be considered for its beneficial pharmacological properties and for its improved safety profile relative to nicotine. Finally, we summarize in Fig. 21.4 the mechanisms by which 6HLN acts as memory-enhancing and antioxidant agent.
MINI-DICTIONARY OF TERMS Arthrobacter nicotinovorans Gram-positive actinobacteria isolated from soil that has the ability to degrade nicotine. It was previously described as A. oxydans. Brain oxidative stress An imbalance between oxidants and antioxidants, characterized by decreasing ability of the brain to counteract the free-radical production. In silico method A method based on computer programs and algorithms. pAO1 megaplasmid A large bacterial plasmid isolated from A. nicotinovorans and harbors the genes responsible for bacterial degradation of nicotine. Radial-arm-maze test A behavioral test used to evaluate working memory and reference memory in rodents.
Y-maze test A behavioral test used to evaluate spatial short-term memory in rodents.
Key Facts of Memory impairment • Alzheimer’s disease (AD) is a progressive neurodegenerative disorder mainly characterized by the loss of learning and memory. • Loss of cholinergic neurons and a significant fall in acetylcholine level were evidenced during the AD, leading to memory impairment. • Increasing of brain oxidative stress as a consequence of decreasing of the antioxidant enzyme activity may occur in the AD. • The search for new therapeutic options is of high interest and growth. Summary Points • This chapter focuses on 6-hydroxy-L-nicotine (6HLN), which is a nicotine metabolite. • 6HLN is produced by nicotine degradation within A. nicotinovorans pAO1 strain. • In silico and in vivo behavioral approaches were used to assess the target-ligand interactions and the effects on memory processes and brain oxidative stress of 6HLN using animal models of cognitive impairment. • 6HLN is a potent agonist of the nicotine acetylcholine receptors and poses memory-enhancing and antioxidant profile. • However, we argue that 6HLN could represent a valuable therapeutic agent to improve memory impairment in the AD.
Acknowledgment This work was supported by PED-PN-III-P2-2.1-PED-2016-0177.
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Murray, K. N., & Abeles, N. (2002). Nicotine’s effect on neural and cognitive functioning in an aging population. Aging & Mental Health, 6 (2), 129–138. Newman, M. B., Arendash, G. W., Shytle, R. D., Bickford, P. C., Tighe, T., & Sanberg, P. R. (2002). Nicotine’s oxidative and antioxidant properties in CNS. Life Sciences, 71(24), 2807-2820. Doi: https://doi.org/10.1016/S0024-3205(02)02135-5 Nordberg, A. (2001). Nicotinic receptor abnormalities of Alzheimer’s disease: Therapeutic implications. Biological Psychiatry, 49, 200–210. Nordberg, A., Hellstr€ om-Lindahl, E., Lee, M., Johnson, M., Mousavi, M., Hall, R., et al. (2002). Chronic nicotine treatment reduces β-amyloidosis in the brain of a mouse model of Alzheimer’s disease (APPsw). Journal of Neurochemistry, 81(3), 655–658. https://doi.org/ 10.1046/j.1471-4159.2002.00874.x. O’Leary, K., Parameswaran, N., McIntosh, J. M., & Quik, M. (2008). Cotinine selectively activates a subpopulation of alpha3/alpha6beta2 nicotinic receptors in monkey striatum. Journal of Pharmacology and Experimental Therapeutics, 325, 646–654. Parri, H. R., Hernandez, C. M., & Dineley, K. T. (2011). Research update: alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer’s disease. Biochemical Pharmacology, 82(8), 931–942. (2011). https:// doi.org/10.1016/j.bcp.2011.06.039. Pogocki, D., Ruman, T., Danilczuk, M., Danilczuk, M., Celuch, M., & Wałajtys-Rode, E. (2007). Application of nicotine enantiomers, derivatives and analogues in therapy of neurodegenerative disorders. European Journal of Pharmacology, 563, 18–39. Rastija, V., & Medic-Šaric, M. (2009). QSAR study of antioxidant activity of wine polyphenols. European Journal of Medicinal Chemistry, 44(1), 400–408. https://doi.org/10.1016/j.ejmech.2008.03.001. Riveles, K., Huang, L. Z., & Quik, M. (2008). Cigarette smoke, nicotine and cotinine protect against 6-hydroxydopamine-induced toxicity in SH-SY5Y cells. Neurotoxicology, 29, 421–427. Sabbagh, M., Lukas, R., Sparks, D., & Reid, R. (2002). The nicotinic acetylcholine receptor, smoking, and Alzheimer’s disease. Journal of Alzheimer’s disease: JAD, 4, 317–325. Sachelaru, P., Schiltz, E., Igloi, G. L., & Brandsch, R. (2005). An α/β-fold C—C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans. Journal of Bacteriology, 187(24), 8516–8519. https://doi.org/10.1128/JB.187.24.85168519.2005.
Sandu, C., Chiribau, C.-B., Sachelaru, P., & Brandsch, R. (2005). Plasmids for nicotine-dependent and -independent gene expression in Arthrobacter nicotinovorans and other arthrobacter species. Applied and Environmental Microbiology, 71(12), 8920–8924. https://doi.org/ 10.1128/AEM.71.12.8920-8924.2005. Sase, S., Yamamoto, H., Kawashima, E., Tan, X., & Sawa, Y. (2017). Discrimination between patients with Alzheimer disease and healthy subjects using layer analysis of cerebral blood flow and xenon solubility coefficient in xenon-enhanced computed tomography. Journal of Computer Assisted Tomography, 41(3), 477–483. https://doi.org/ 10.1097/rct.0000000000000525. Schenk, S., & Decker, K. (1999). Horizontal gene transfer involved in the convergent evolution of the plasmid-encoded enantioselective 6hydroxynicotine oxidases. Jounal of Molecular Evolution, 48, 178–186. Taly, A., Corringer, P.-J., Guedin, D., Lestage, P., & Changeux, J.P. (2009). Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nature Reviews Drug Discovery, 8, 733. https://doi.org/10.1038/nrd2927. Tang, H., Wang, L., Wang, W., Yu, H., Zhang, K., Yao, Y., et al. (2013). Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genetics. 9(10), e1003923. https://doi.org/ 10.1371/journal.pgen.1003923. Tang, H., Yao, Y., Zhang, D., Meng, X., Wang, L., Yu, H., et al. (2011). A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida. The Journal of Biological Chemistry, 286(45), 39179–39187. https://doi.org/ 10.1074/jbc.M111.283929. Wang, S., Huang, H., Xie, K., & Xu, P. (2012). Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway. Applied Microbiology and Biotechnology, 95(6), 1567–1578. https://doi.org/10.1007/ s00253-012-4007-2. Wang, S. N., Liu, Z., & Xu, P. (2009). Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33. Journal of Applied Microbiology, 107(3), 838–847. https://doi.org/10.1111/j.1365-2672.2009. 04259.x. Yu, H., Tang, H., Zhu, X., Li, Y., & Xu, P. (2015). Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1. Applied and Environmental Microbiology, 81(1), 272–281. https://doi.org/10.1128/AEM.02265-14.
21 6-Hydroxy-L-Nicotine and Memory Impairment Lucian Hritcu, Marius Mihasan Department of Biology, Alexandru Ioan Cuza University of Iasi, Iasi, Romania
Abbreviations 2,3,6-THP 2,6-DHP 2,6-DHPON 6HDNO 6HLN 6HLNO 6-HMM 6-HPON AD CHL DHPH DHPONH KDH MGABA nAChBP nAChR NB NDH
2,3,6-trihydroxypyridine 2,6-dihydroxypyridine 2,6-dihydroxypseudooxynicotine 6-hydroxy-D-nicotine oxidase 6-hydroxy-L-nicotine 6-hydroxy-L-nicotine oxidase 6-hydroxy-methylmyosmine 6-hydroxy-pseudooxynicotine Alzheimer’s disease chlorisondamine 2,6-dihydroxypyridine-3-hydroxylase 2,6-dihydroxypseudooxynicotine hydrolase ketone dehydrogenase γ-N-methylaminobutyrate nicotinic receptor binding domain homolog nicotinic acetylcholine receptors nicotine blue nicotine dehydrogenase
21.1 INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with a gradual decline in cognition. It is considered to be the most common pathological cause of dementia (Sase, Yamamoto, Kawashima, Tan, & Sawa, 2017). It has been reported that in 2016, about 2 million people who have AD are at the age of 85 or older, accounting for 37% of all people with the AD (Hebert, Weuve, Scherr, & Evans, 2013). By 2050, as many as 7 million people at the age of 85 and older may have AD, accounting for half (51%) of all people at the age of 65 and older with the AD (Hebert et al., 2013). It has been reported that nicotinic receptors are therapeutic targets in the nervous system (Taly, Corringer, Guedin, Lestage, & Changeux, 2009). Also, the involvement of the nicotinic acetylcholine receptor (nAChR) subtypes α7 and α4β2 in AD pathology was evidenced (Parri, Hernandez, & Dineley, 2011). The AD is characterized by substantial decreasing in both nicotinic and
Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00021-6
muscarinic acetylcholine receptor expression in the basal forebrain, cerebral cortex, and hippocampus (Kása, Rakonczay, & Gulya, 1997). Consequently, in the cerebral cortex, a significant loss of α4β2 nAChR subtypes (Aubert et al., 1992) was evidenced, while in the hippocampus, a loss of α7 nAChRs was reported being correlated with the progressive alteration of the memory function (Nordberg, 2001). Some epidemiological studies have shown data suggesting an inverse relationship between tobacco consumption and the development of AD (Echeverria Moran, 2013). The putative beneficial effect of tobacco has been mainly attributed to nicotine, which has been reported to improve cognitive abilities and reduce plaques in a mouse model of the AD (Nordberg et al., 2002). Numerous studies suggested that acute nicotine administration significantly improved the performance of patients with AD in cognitive tasks (Taly et al., 2009). Since nAChRs play an important role in attention and learning and memory, the positive effects of nicotine on memory have been mostly credited to the activation of these receptors (Sabbagh, Lukas, Sparks, & Reid, 2002). Moreover, the postmortem levels of the beta-amyloid peptide that considered the neurotoxic agents in the AD brains were significantly decreased in the brains of smoking AD patients compared to nonsmokers with the disease (Boopathi & Kolandaivel, 2014). The high potency of nicotine as a cognition-enhancing agent and AD therapeutic strategy (Murray & Abeles, 2002) can be explained not only by its ability to bind and modulate nAChRs but also through its antioxidant effects at low concentrations (Newman et al., 2002) and through its ability to interact with beta-amyloid peptide. However, nicotine has not demonstrated in clinical studies to be a useful treatment for the AD (LópezArrieta & Sanz, 2001). Because of its short halftime (about 2 h), its proved negative effects on various other organs such as the lungs (Hecht, Hochalter, Villalta, &
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Murphy, 2000), its linkage to cigarettes, and the negative publicity associated with smoking (Buccafusco, 2004), nicotine did not impose itself as a feasible therapeutic agent for AD. Numerous studies have focused primarily on the effects of nicotine and nicotine metabolites found in mammalian cells, because of their potential contribution to the neuropharmacological effects resulting from nicotine exposure (Pogocki et al., 2007; Riveles, Huang, & Quik, 2008). In mammalian cells, nicotine is extensively metabolized in the liver to six primary metabolites (nicotine glucuronide, nicotine N-oxide, nornicotine, isomethonium ion, cotinine, and 2-hydroxynicotine). The predominant pathway during the first-pass metabolism yields cotinine (70%–80% of nicotine is metabolized to cotinine in humans) that may have some relevance in the diverse neurobiological effects of smoking as a ligand of nAChRs (Dome, Lazary, Kalapos, & Rihmer, 2010). Furthermore, there are several other indications that cotinine is a pharmacologically active substance. It has neuroprotective and/or cell-protective effects in the cell-culture model, attention-enhancing effects, antipsychotic-like effects (in prepulse inhibition), dopamine-releasing effects in striatal tissue samples, etc. (O’Leary, Parameswaran, McIntosh, & Quik, 2008). All of these results indicate that the impact of nicotine and nicotine metabolites on brain functions is not negligible. Thus, it has become increasingly important to obtain more information on the biological effects of nicotine metabolites derived from various sources, in order to possibly manage neurological abnormalities in some neurodegenerative afflictions.
21.2 IMPLICATION TO TREATMENTS There are many treatments available for subjects who have an addiction to nicotine/tobacco products such as nicotine replacement therapy (the patch and the nicotine gum), nicotine sprays and inhalers, and medication (varenicline, Chantix, and bupropion, Wellbutrin). Additionally, there are mentioned different psychological and behavioral treatments such as hypnotherapy, cognitivebehavioral therapy, and neurolinguistic programming that help people to fight against tobacco addiction.
21.3 NICOTINE DERIVATIVES IN BACTERIA The crystal structure determination of the nicotinic receptor binding domain homolog (nAChBP) with bound nicotine (Celie et al., 2004) followed by the structure of α7 nAChRs (Mowrey et al., 2013) has spurred the interest of
several academic and pharmaceutical laboratories in the possibility that new nicotinic drugs could be designed. From this point of view, nicotine derivatives are ideal candidates, offering a wide array of possibilities. The difficulty resides, firstly, in the identification of molecules that have the beneficial effects of nicotine but elude its side effects (Pogocki et al., 2007) and, secondly, in providing simple and reliable methods for production and isolation of the identified compounds. In this context, the nicotine-degrading bacteria with their ability to use this alkaloid as the sole carbon source offer a wide range of nicotine derivatives with unexplored biotechnological potential. A multitude of reports indicate that bacteria including Pseudomonas (Liu et al., 2014; Tang et al., 2011), Bacillus and Pusillimonas (Ma et al., 2015) strains, Ochrobactrum sp. SJY1 (Yu, Tang, Zhu, Li, & Xu, 2015), Agrobacterium tumefaciens S33 (Wang, Liu, & Xu, 2009), Shinella sp. HZN7 (Ma et al., 2014), and Achromobacter nicotinophagum (Hylin, 1958) can degrade nicotine by using three related pathways: the pyridine pathway (Brandsch, 2006), the pyrrolidine pathway (Tang et al., 2013), and a variant of pyridine and pyrrolidine pathways named the VPP pathway (Wang, Huang, Xie, & Xu, 2012). The intermediates and enzymes involved in each pathway have been extensively presented in two reviews as described by Brandsch (2006) and Gurusamy and Natarajan (2013), respectively.
21.4 NICOTINE CATABOLISM IN ARTHROBACTER NICOTINOVORANS By far, the most extensively studied is the nicotine metabolism of A. nicotinovorans pAO1. This bacterium makes use of the pyridine pathway to degrade nicotine. Briefly, nicotine degradation starts with the hydroxylation of the pyridine ring to 6-hydroxy-L-nicotine (6HLN) by nicotine dehydrogenase (NDH) (Andreesen & Fetzner, 2002). Next, the pyrrolidine ring is oxidized by 6-hydroxy-Lnicotine oxidase (6HLNO) (Schenk & Decker, 1999), and 6-hydroxy-methylmyosmine (6-HMM) is formed. The compound reacts spontaneously with water and turns to 6-hydroxy-pseudooxynicotine (6-HPON; N-methylaminopropyl-(6-hydroxypyridyl-3)-ketone). 6HLNO has a high stereoselectivity and acts only on 6-hydroxy-L-nicotine. As small amounts of D-nicotine can be produced by the tobacco plant, there is a 6-hydroxy-D-nicotine oxidase (6HDNO) made by A. nicotinovorans pAO1 that can transform 6-hydroxy-L-nicotine to 6-hydroxy-methylmyosmine and, consecutively, to 6-HPON (Decker & Bleeg, 1965). A second hydroxylation that takes place at C2 of 6-HPON yields 2,6-dihydroxypseudooxynicotine (2,6-DHPON). This step is encoded by a multimeric ketone dehydrogenase (KDH). A key step in nicotine catabolism is catalyzed by
21.6 6HLN PRODUCED USING AN ARTHROBACTER-BASED BIOTECHNOLOGY
2,6-dihydroxypseudooxynicotine hydrolase (DHPONH) (Sachelaru, Schiltz, Igloi, & Brandsch, 2005) and consists of the cleavage of 2,6-DHPON into two main metabolites: 2,6-dihydroxypyridine (2,6-DHP) and γ-Nmethylaminobutyrate (MGABA). 2,6-DHP is further hydroxylated by a FAD-dependent monooxygenase named 2,6-dihydroxypyridine-3hydroxylase (DHPH) yielding 2,3,6-trihydroxypyridine (2,3,6-THP). In the presence of oxygen, 2,3,6-THP suffers an apparently spontaneous dimerization. The dimerization takes place inside the cell, and the product of the reaction is 4,40 ,5,50 -tetrahydroxy-3,30 -diazadiphenoquinone-(2,20 ), which is then excreted and accumulates in the medium forming characteristic nicotine blue (NB) color. Even though the metabolic pathway in A.nicotinovorans was described in detail by the group of Prof. Roderich Brandsch, Albert Ludwigs University, Freiburg, Germany, until now, there are no biotechnological applications of the identified metabolic products.
21.5 6HLN IS ABLE TO INTERACT WITH nAChRs We were interested to see if any of the nicotine intermediates found in the A. nicotinovorans catabolic pathway have any affinity for nAChRs, and we used in silico experiments to evaluate their binding potential (Mihasan, Capatina, Neagu, Stefan, & Hritcu, 2013). For this, the 3-D structures of all of the intermediates found in the nicotine degradation pathway of A.nicotinovorans were downloaded from PubChem (Kim et al., 2016) and converted into a format suitable for docking using FROG v.1.01—free online drug conformation generation (Leite et al., 2007). All steps required for directed docking (molecular surface generation, sphere box, and energy grid generation) were achieved using ADT 1.5.4 (Morris et al., 2009). In silico docking was performed using Autodock 4 (Morris et al., 2009) using the default parameters and the acetylcholine binding protein (AChBP, PDB ID: 1UW6) (Celie et al., 2004) as a receptor. The receptor was kept rigid, and all ligands were flexible. All bonds within the ligand molecules were allowed to rotate. The targeted region for docking was a cube of approx 150 Å centered on Tyr143 from the subunit A of AChBP. First, L-nicotine was docked at the binding site. The computationally determined orientation of L-nicotine in the binding site of AChBP was compared with the experimentally observed orientation described by Celie et al. (2004). A very good fit between the computationally obtained pose of L-nicotine with the experimentally determined one (root-mean-square deviation, RMSD, of 0.2 Å for 12 superimposed atoms of the ligand) was obtained, indicating the reliability of the docking method employed.
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The resulted theoretical binding pose of 6HLN was very similar to the one experimentally observed for nicotine with an RMSD of 0.19 Å for 12 atoms of the ligand. Not only this compound would fit into the binding pocket, but also the increased interaction energy calculated by AutoDockTools for 6HLN is due to an extra H bond between the hydroxyl group of the 6HLN and the Tyr185 residue. Moreover, the antioxidant activity of 6HLN was evaluated using the quantum chemical quantitative structure-activity relationship (QSAR) equation proposed by Rastija and Medic-Šaric (2009). We showed that the antioxidant activity of 6HLN calculated as IC50 ¼ 43.06 is improved compared to the IC50 ¼ 54.54 for nicotine.
21.6 6HLN PRODUCED USING AN ARTHROBACTER-BASED BIOTECHNOLOGY As a result of the promising data produced by this computational pilot study, one might easily see the high biotechnological potential of 6HLN. Currently, the compound can be chemically synthesized using a diazotization reaction on 6-aminonicotine and is available from different suppliers (Santa Cruz Biotechnology or Carbosynth) at a fairly expensive price. Arthrobacter nicotinovorans cells could nevertheless provide an alternative way to produce this compound using waste and by-products from the tobacco industry. HPLC analysis of A. nicotinovorans growth medium showed a small accumulation of 6HLN during the growth of the bacteria in the presence of nicotine (Boiangiu, Guzun, & Mihasan, 2014). Two previously published articles indicate that 6HLN can be found in not only the nicotine growth media of A. nicotinovorans (Hochstein & Rittenberg, 1959) but also a possible export mechanism involving a facilitated transport system (Ganas, Igloi, & Brandsch, 2009). The accumulation is only temporary, and as soon as the amount of nicotine in the medium is depleted, the 6HLN levels drop (Fig. 21.1). Apparently, a metabolic imbalance is involved. NDH, the trimeric enzyme containing a molybdenum pyranopterin dinucleotide cofactor, FAD, and two ironsulfur clusters (Andreesen & Fetzner, 2002) responsible for converting L-nicotine to 6HNLO, has roughly the same affinity (Km of 0.037 mM) for its substrate but a higher reaction rate (specific activity of 29.2 μmol/min/ mg) (Hochstein & Dalton, 1967) compared to downstream enzyme 6-hydroxy-L-nicotine oxidase (6HLNO). The dimeric enzyme 6HLNO contains one FAD per subunit (Schenk & Decker, 1999) and has a Km of 0.02 mM and a much lower specific activity—4.73 μmol/min/mg (Decker, Dai, M€ ohler, & Br€ uhm€ uller, 1972; Kachalova et al., 2010). Basically, as long as nicotine is still in the
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FIG. 21.1 Dynamics of nicotine and 6HLN in the growth medium of A. nicotinovorans. A temporary accumulation of 6HLN can be observed (continuous line). As soon as nicotine is depleted from the medium (dashed line), 6HLN levels drop.
FIG. 21.2 Upstream and downstream reactions of 6HLN in the nicotine catabolic pathway of A. nicotinovorans. NDH, nicotine dehydrogenase; 6HLN, 6-hydroxy-L-nicotine; 6HLNO, 6-hydroxy-L-nicotine oxidase; 6-HMM, 6-hydroxy-methylmyosmine.
medium, NDH converts it to 6HLN at a rather fast pace, 6HLNO cannot keep up, and 6HLN accumulates in the medium. As soon as nicotine is depleted, 6HLNO can quickly recover, and 6HLN is consumed (Fig. 21.2). The observed metabolic imbalance could be used to produce 6HLN. In order to increase yield, a genetically engineered strain of A. nicotinovorans was created. The ndh genes were cloned in the custom pART2 expression vector (Andrei & Mihasan, 2013; Sandu, Chiribau, Sachelaru, & Brandsch, 2005) that allows the overexpression of the NDH in a nicotine-dependent manner (Fig. 21.3). Currently, our lab uses low-density A. nicotinovorans pART2ndh cell cultures cultivated in flasks under constant agitation at 28°C in order to produce 6HLN. By overexpressing NDH and chemically inhibiting 6HLNO using 0.05 mM ZnSO4, we are able to commonly produce approx 50–60 mg of 6HLN per 100 mL medium. 6HLN produced has memory-enhancing effects.
Next, 6HLN was tested in vivo on male naive Wistar rats and proved to have surprisingly positive effects on memory processes and oxidative brain status. Hritcu, Stefan, Brandsch, and Mihasan (2013) continue to demonstrate that in vivo chronic treatment of 6HLN (0.3 mg/kg, seven consecutive days) significantly increased spontaneous alternations in Y-maze task and working memory in radial-arm-maze task, suggesting effects on short-term memory, without affecting longterm memory, explored by reference memory in radialarm-maze task. Also, 6HLN significantly improved locomotor activity, as evidenced by the number of arm entries within Y-maze test (Table 21.1). In addition, 6HLN increased antioxidant enzyme activity (SOD and GPX) and decreased the production of lipid peroxidation (MDA) in the rat temporal lobe homogenates, suggesting antioxidant effects (Table 21.2). We concluded that positive effects of 6HLN on spatial memory may occur by antioxidant actions.
FIG. 21.3 Location of the ndh and 6hlno genes (red) in the nic-gene cluster from the pAO1 megaplasmid of A. nicotinovorans Dark blue, genes experimentally shown to be involved in nicotine metabolism; light blue, genes putatively involved in nicotine metabolism; white, genes with no experimental or putative function; black, transposons and insertion elements; yellow, putative transcription factors.
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TABLE 21.1
Effects of 6HLN in Experimental Models of Memory
Nicotine derivative
Animal species
Behavioral test
Intraperitoneal in naive rats 0.3 mg/kg
Rats
Intraperitoneal in naive rats 0.3 mg/kg
Administration
Observed effects
Reference
Y-maze
Improved short-term memory and locomotor activity
Hritcu et al. (2013)
Rats
Radialarm-maze
Enhanced working memory, without affecting reference memory
Hritcu et al. (2013)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
Y-maze
Sustained short-term memory and locomotor activity
Hritcu, Stefan, Brandsch, and Mihasan (2015)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
Radialarm-maze
Enhanced working memory and reference memory
Hritcu et al. (2015)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
Y-maze
Enhanced spontaneous alternation
Hritcu et al. (2017)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
Radialarm-maze
Improved working memory and reference memory
Hritcu et al. (2017)
Hritcu et al. (2015) demonstrated that 6HLN ameliorated scopolamine-induced spatial memory impairment (Table 21.1). Decreased activities of superoxide dismutase, glutathione peroxidase, and catalase along with a decrease of total content of reduced glutathione were observed in the rat hippocampal homogenates of scopolamine-treated animals as compared with control. Production of malondialdehyde (lipid peroxidation) significantly increased in the rat hippocampal homogenates of scopolamine-treated animals as compared with control, as a consequence of impaired antioxidant enzyme activities. Additionally, in scopolamine-treated rats, 6HLN significantly improved memory formation and TABLE 21.2
decreased oxidative stress (Table 21.2), suggesting memory-enhancing and antioxidant effects. Therefore, our results suggest that the administration of 6HLN ameliorates scopolamine-induced spatial memory impairment by attenuation of the oxidative stress in the rat hippocampus. Recently, Hritcu et al. (2017) reported that 6HLN administration attenuated the cognitive deficits (Table 21.1) and recovered the antioxidant capacity (Table 21.2) in the rat hippocampus of the chlorisondamine (CHL) rat model. Taken together, administration of 6HLN as compared to nicotine improved significantly spatial memory in the Y-maze, and radial-arm-maze
Effects of 6HLN on Oxidative Stress
Nicotine derivative
Animal species
Biochemical markers
Observed effects
References
Intraperitoneal in naive rats 0.3 mg/kg
Rats
SOD, GPX, MDA
Increased the activity of SOD and GPX and decreased MDA level
Hritcu et al. (2013)
Intraperitoneal in scopolamine (0.7 mg/kg)-treated rats 0.3 mg/kg
Rats
SOD, GPX, CAT, GSH, MDA
Increased the activity of SOD, GPX, CAT, and GSH and decreased MDA level
Hritcu et al. (2015)
Intraperitoneal in chlorisondamine (10 mg/kg)-treated rats 0.3 mg/kg
Rats
SOD, GPX, CAT, GSH, MDA
Increased the activity of SOD, GPX, CAT, and GSH and decreased MDA level
Hritcu et al. (2017)
Administration
SOD, superoxide dismutase; GPX, glutathione peroxidase; CAT, catalase; GSH, glutathione; MDA, malondialdehyde.
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21. 6-HYDROXY-L-NICOTINE AND MEMORY IMPAIRMENT
FIG. 21.4 Possible mechanism of action of 6HLN with memory-enhancing and antioxidant activity.
tasks decreased lipid peroxidation and enhanced antioxidant status in the hippocampus of the CHL-treated rats. This observed effect could suggest possible neuroprotective effects of both nicotine and 6HLN. However, the effects of 6HLN are significantly increased as compared with nicotine in the CHL-treated rats. From the observation of this study, we can suggest that 6HLN may have a beneficial role in the treatment and/or management of memory disorders such as AD and indicate the important significance of the nicotinic acetylcholine system in brain dysfunction. Additionally, we also reported (Ionita et al., 2017) that both nicotine and 6HLN act as anxiolytic and antidepressant agents in the chlorisondamine rat model. The observed effects could be mediated by the nicotinic acetylcholine receptors. In summary, our data contribute with additional support concerning the role of nicotine and nicotine metabolites in memory processes and brain oxidative status. Moreover, 6HLN should be considered for its beneficial pharmacological properties and for its improved safety profile relative to nicotine. Finally, we summarize in Fig. 21.4 the mechanisms by which 6HLN acts as memory-enhancing and antioxidant agent.
MINI-DICTIONARY OF TERMS Arthrobacter nicotinovorans Gram-positive actinobacteria isolated from soil that has the ability to degrade nicotine. It was previously described as A. oxydans. Brain oxidative stress An imbalance between oxidants and antioxidants, characterized by decreasing ability of the brain to counteract the free-radical production. In silico method A method based on computer programs and algorithms. pAO1 megaplasmid A large bacterial plasmid isolated from A. nicotinovorans and harbors the genes responsible for bacterial degradation of nicotine. Radial-arm-maze test A behavioral test used to evaluate working memory and reference memory in rodents.
Y-maze test A behavioral test used to evaluate spatial short-term memory in rodents.
Key Facts of Memory impairment • Alzheimer’s disease (AD) is a progressive neurodegenerative disorder mainly characterized by the loss of learning and memory. • Loss of cholinergic neurons and a significant fall in acetylcholine level were evidenced during the AD, leading to memory impairment. • Increasing of brain oxidative stress as a consequence of decreasing of the antioxidant enzyme activity may occur in the AD. • The search for new therapeutic options is of high interest and growth. Summary Points • This chapter focuses on 6-hydroxy-L-nicotine (6HLN), which is a nicotine metabolite. • 6HLN is produced by nicotine degradation within A. nicotinovorans pAO1 strain. • In silico and in vivo behavioral approaches were used to assess the target-ligand interactions and the effects on memory processes and brain oxidative stress of 6HLN using animal models of cognitive impairment. • 6HLN is a potent agonist of the nicotine acetylcholine receptors and poses memory-enhancing and antioxidant profile. • However, we argue that 6HLN could represent a valuable therapeutic agent to improve memory impairment in the AD.
Acknowledgment This work was supported by PED-PN-III-P2-2.1-PED-2016-0177.
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