Table 1 Relationships between plant neurotoxins commonly used as ...

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R.J. SULLIVAN ET AL.

Table 1 Relationships between plant neurotoxins commonly used as drugs and CNS receptors. Drug Plant Toxin Neurotransmitter Tobacco, Pituri Nicotiana, Duboisia Nicotinea Acetylcholine Betel nut Areca catechu Arecolinea Acetylcholine Coca Erythroxylum Cocainec Norepinephrine, epinephrine Norepinephrine, epinephrine Khat Catha edulis Ephedrinec , cathinonea,c Coca Erythroxylum Cocainec Dopamine Khat Catha edulis Cathinonea,c Dopamine Coffee, Cola nut Coffea, Cola nitida Caffeineb Adenosine Adenosine Tea Camellia sinensis Caffeineb , theophyllineb , theobromineb Chocolate Theobromine cacao Theobromineb Adenosine Endorphins Opium Papaver somniferum Codeinea , morphinea Cannabis Cannabis sativa ∆9-THCa Anandamide a receptor agonist, b receptor antagonist, c reuptake inhibitor

the cytochrome P450 (CYP) haemoproteins. CYPs are ubiquitous in Bacteria and Eukarya, and have been found in many Archaea species, suggesting that the ancestral CYP gene evolved approximately 3.5 billion years ago. Metabolism of endogenous fatty acids and steroidogenesis appear to have been the original (and still central) functions of most CYP genes. With the rise of terrestrial plants and animals about 400 million years ago, the major functions of CYPs expanded to encompass the detoxification of dietary phytochemicals, via a co-evolutionary process involving dozens of gene duplication events (Lewis, 2001; Nelson, 1999; Nelson et al., 2004).There are approximately 76 CYP families known in animals, with 57 of these present in humans (Nelson, n.d.). Although CYPs are found in many tissues, in humans and other mammals they are concentrated in the liver, where they catalyze the oxidation of a wide range of endogenous and exogenous chemicals in Phase I metabolism. CYP oxidases introduce an atom of molecular oxygen into the structure of a lipophilic substrate (such as a toxin/drug) to render it more hydrophilic prior to conjugation to a carrier molecule in Phase II metabolism for export from the body. In mammals, CYPs are responsible for oxidizing over 90% of drugs and other xenobiotics (Lewis, 2001). Several CYP families are highly conserved across species, whereas others are quite variable. The conserved CYP 5 and higher families have endogenous functions like bile acid metabolism and cholesterol and steroid biosynthesis, and show remarkable cross-species similarity. For example, 21 of 22 human and mouse genes in these families are orthologous (Nelson, 1999). In contrast, most of the drug-metabolizing enzymes are in the variable group (Nelson, 1999). The CYP 2 and 3 families, for example, are phylogenetically divergent with sixteen human CYP 2 genes and four CYP 3 genes, as opposed to fifty-

Receptor Nicotinic receptor Muscarinic receptor Adrenergic receptors Adrenergic receptors Dopamine receptor Dopamine receptor Adenosine receptor Adenosine receptor Adenosine receptor Opioid receptor Cannabinoid receptor

one CYP 2 and eight CYP 3 genes in mice (Nelson et al., 2004). There is only one 2D gene (2D6) in humans, whereas there are nine 2D genes in mice (Nelson et al., 2004). A comparison of the human genome with the initial sequence of the chimpanzee genome similarly found rapid evolutionary divergence in xenobiotic metabolizing genes (The Chimpanzee Sequencing and Analysis Consortium, 2005), as well as divergence in genes expressed in the liver (Khaitovich et al., 2005). The latter finding is supported by in vivo pharmacokinetic studies. For instance, systemic clearance of propranolol, verapamil, theophylline and 12 other synthetic drugs in chimpanzees and humans ranged from close to parity to a 10-fold variation, with CYP2D enzyme activity approximately 10 times higher in the chimpanzee, a species that notably subsists primarily on plants (Wong et al., 2004). The emergence of xenobiotic-metabolizing CYP in animals at about the same time as the evolution of terrestrial plants, the localization of cross-species variation in CYP genes within the xenobiotic-metabolizing subset, and the large species differences in drug metabolism suggest speciesspecific adaptation to frequently encountered plant toxins and other environmental chemicals.

Evolution in human xenobiotic-metabolizing cytochrome P450 The mammalian cytochrome P450 phylogenetic data are compelling evidence of a long evolutionary history of exposure to plant toxins. As mammals, humans have phylogenetically “inherited” the cytochrome P450 system for detoxification of environmental chemicals. This fact alone would seem to falsify the hypothesis that human exposure to drugs is evolutionarily novel – that there has been a “mismatch” between contemporary drug profligate environments and ancestral environments that were “drug” free. But humans are taxonomically unique in several respects, particularly in re-

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Table 2 Human cytochrome P450 natural substrates and enzyme kinetics constants. Vmax is the maximum reaction rate per unit enzyme. Km , the Michaelis-Menten constant, is the substrate concentration at which the reaction rate = Vmax /2 (lower values indicate higher enzyme affinity for substrate). Vmax / Km is an index of enzyme activity (higher values indicate higher enzyme activity). Kinetic values can vary widely; values here are representative of one metabolic pathway (substrates are typically metabolized via multiple pathways). Plants listed are often not the exclusive source of the neurotoxin. Enzyme/substrates Xenobiotic and endogenous sources Km (µM) Vmax Vmax / Km CYP1A2 Caffeine Plant neurotoxin (Coffea – coffee) 460 570b 1.24 Theophylline Plant neurotoxin (Camellia sinensis – tea) 310 43.3b 0.14 Theobromine Plant neurotoxin (Theobromine cacao – chocolate) 2580 1720b 0.67 Aflatoxin B1 Fungus neurotoxin 36 0.92a 0.026 PhIP Cooked meat 46 1.79a 0.039 Estradiol Sex hormone 27.5 17.4a 0.63 Melatonin Hormone 25.9 10.6a 0.41 CYP2A6 Nicotine Plant neurotoxin (Nicotiana – tobacco) 95.3 154.1b 1.69 Coumarin Plant neurotoxin (Dipteryx odorata – tonka bean) 0.6 0.6a 1.0 Cotinine Nicotine metabolite 234.5 37.2b 0.16 CYP2B6 Nicotine Plant neurotoxin [induces 2B6 in the brain] Diazepam Synthetic drug; trace amounts in plants 181 8.5a 0.05 CYP2C8 Taxol Plant neurotoxin (Taxus brevifolia) 5.4 30a 5.6 Arachidonic acid Essential omega-6 fatty acid 71 0.078a 0.001 Retinol Vitamin A 50 1.2a 0.024 CYP2C9 ∆9-THC Plant neurotoxin (Cannabis sativa – marijuana) 2.1 6.4a 3.0 CYP2C19 Melatonin Hormone 282.2 Progesterone Hormone 3.6 1.4a 0.39 CYP2D6 Codeine Plant neurotoxin (Papaver somniferum – opium poppy) 190 6.4a 0.034 Harmaline Plant neurotoxin (Peganum harmala) 1.41 39.9a 28.3 Harmine Plant neurotoxin (Peganum harmala) 7.42 29.7a 4.0 Sparteine Plant neurotoxin (Lupinus) 44 Yohimbine Plant neurotoxin (Pausinystalia yohimbe) 2.0 147.4b 75.5 CYP2E1 Theobromine Plant neurotoxin (Theobromine cacao – chocolate) 3400 Ethanol Yeast waste product 23400 23.2a 0.001 CYP3A4 Cocaine Plant neurotoxin (Erythroxylum coca) 2700 3744.4b 1.4 Quinine Plant neurotoxin (Cinchona) 106 1330b 13 Aflatoxin B1 Fungus neurotoxin 139 61a 0.45 Testosterone Hormone 52 5400b 101 Cortisol Hormone 15 6.4b 0.42 a pmol/min/pmol P450; b pmol/min/mg microsomal protein Data from Bland, Haining, Tracy, and Callery (2005); Bu (2006); Gates and Miners (1999); Ladona, Gonzalez, Rane, Peter, and Torre (2000); Le Corre et al. (2004); Lewis (2001, 2003); Osikowska-Evers and Eichelbaum (1986); Projean, Morin, Tu, and Ducharme (2003); Yang et al. (1998); Yu, Kneller, Rettie, and Haining (2002); Yu, Idle, Krausz, Kupfer, and Gonzalez (2003); Ma, Idle, Krausz, and Gonzalez (2005); Usmani, Cho, Rose, and Hodgson (2006); Yamazaki and Shimada (1997); Bloomer, Clarke, and Chenery (1995); Murphy, Raulinaitis, and Brown (2005); Hammons et al. (1997); Nakajima et al. (1996, 1996); Tjia, Colbert, and Back (1996); Ha, Follath, Chen, and Kr¨ahenb¨uhl (1996); Asai, Imaoka, Kuroki, Monna, and Funae (1996); Marill, Cresteil, Lanotte, and Chabot (2000); Rahman, Korzekwa, Grogan, Gonzalez, and Harris (1994); Campbell, Grant, Inaba, and Kalow (1987); Gallagher, Kunze, Stapleton, and Eaton (1996).

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Table 3 Example ethnic population frequencies of CYP2A6 and CYP2D6 alleles with known in vivo enzyme activity. Frequencies compiled from different studies in the same ethnic population are only approximately comparable. Allele Enzyme activity Population frequencies (%) Ghanaian1 Caucasian2,3 Chinese1,2,3 Japanese1,2,3,4 CYP2A6*1A/B Normal 91.9 88.4 61.7 48.3 CYP2A6*2 None 0 2.3 0 0 CYP2A6*4 None 1.9 1.2 15.1 20.1 CYP2A6*5 None 0 0 1 0 CYP2A6*7 Reduced 0 0 2.2 6.5 CYP2A6*9 Reduced 5.7 5.2 15.7 21.3 CYP2A6*10 Reduced 0 0 0.4 1.1 CYP2A6*12 Reduced 3 0 0 Black African5 Caucasian5 Asian5 Ethiopian6 Saudi Arabian7 CYP2D6*2xn Increased 2 1-5 0-2 16.0 10.4 CYP2D6*4 None 2 12-21 1 1.2 3.5 CYP2D6*5 None 4 2-7 6 3.3 1.0 CYP2D6*10 Reduced 6 1-2 51 8.6 3.0 CYP2D6*17 Reduced* 20-35 0 0 9.0 3.0 1: Gyamfi, Fujieda, Kiyotani, Yamazaki, and Kamataki (2005); 2: Nakajima, Kuroiwa, and Yokoi (2002); 3: Haberl et al. (2005); 4: Yoshida et al. (2003); 5: Ingelman-Sundberg (2005); 6: Aklillu et al. (1996); 7: McLellan, Oscarson, Seideg˚ard, Evans, and Ingelman-Sundberg (1997)

zymes, the statistical evidence of stabilizing selection, and the existence of both species- and population-specific polymorphisms as evidence that humans have undergone relatively recent selection by plant toxins frequently encountered in local environments. This hypothesis, if correct, has important implications for reward models of drug seeking and acute drug use.

The paradox of human drug use Are we inherently vulnerable to drugs? The notion that we are inherently vulnerable to drugs is implicit in neurobiological models of the mammalian MDS. The current assumption is that the MDS is easily triggered by a broad range of neurotoxins because it was not exposed to such toxins during its evolution (Nesse & Berridge, 1997). The co-evolution of the xenobiotic-metabolizing CYP families contradicts this view by demonstrating that heterotroph signalling systems have successfully endured a relentless chemical assault by autotrophs for hundreds of millions of years. The long exposure to plant neurotoxins indicated by the CYP data makes it unlikely that humans, or other mammals, are inherently vulnerable to drugs. This conclusion amounts to a rejection of the conventional evolutionary explanation of drug use, dependent as it is on the notion that the MDS evolved in the absence of selection pressures from plant neurotoxins (Nesse & Berridge, 1997) or, in broader theoretical terms, that the reward and/or reinforcement functions of the MDS were somehow exempt from the implications of evolutionary biology’s punishment model.

To reiterate our broader argument here, the phylogenetic evidence for co-evolution of animal CYP and plant toxins reinforces the evolutionary biological perspective that plant neurotoxins evolved because they punished and deterred consumption by herbivores (Karban & Baldwin, 1997; Roberts & Wink, 1998), and is in direct conflict with neurobiology’s reward model which sees drug use as rewarded and reinforced in the MDS. We termed this incompatibility the paradox of drug reward. Before sketching potential resolutions of the paradox of drug reward, we respond to several objections to our argument that emerge directly from the substantial data generated in support of current reward models like the MDS drug-reward pathway.

Does initial drug use elicit hedonic rewards and false-positive fitness signals? Nesse and Berridge (Nesse & Berridge, 1997) and others (Johns, 1999; Smith, 1999; Tooby & Cosmides, 1990; Kelley & Berridge, 2002; Newlin, 2002) propose that positive and negative affective experiences and sensations are related to fitness consequences, and that drugs interfere with affectively-mediated fitness signals. We find this perspective problematic in several ways. First, commonly-used drugs have multiple nervoussystem “targets” and may activate physiological responses that are unpleasant, or physiological systems that do not mediate affective experiences. For example, the widely-used drug arecoline (betel nut) binds to muscarinic receptors in the brain, but also exerts potent effects in the peripheral nervous system (PNS) inducing tremor, face flushing, sweating, changes in heart rate and blood pressure, salivation, nausea and broncoconstriction (Chu, 1993, 1995). The unpleasant

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