家兔血清、尿中甲基苯丙胺及其代谢产物苯丙胺的气相色谱/质谱分析

目的:建立血清、尿中甲基苯丙胺(MA)及其主要活性代谢产物苯丙胺(AP)的气相色谱/质谱(GC/MS)定性定量分析方法,探讨样品稳定性和家兔血、尿中AP和MA的比值关系。方法:样品中加入内标物丙基解痉素(SKF525A)后碱化,乙醚萃取,三氟醋酸酐衍生化,GC/MS法分析甲基苯丙胺和苯丙胺。结果:血清和尿中甲基苯丙胺与苯丙胺的线性范围分别为0.01~5.0mg.L-1和0.1~50.0mg.L-1;最低检出限为0.005mg.L-1(S/N≥3);提取回收率均大于74.3%;方法回收率为95.50%~103.16%;日内及日间RSD均小于10%。18h内苯丙胺与甲基苯丙胺在血、尿中比值分别为0.16~1.5和0.021~0.079。血清样品在室温和冷冻条件下存放24h甲基苯丙胺、苯丙胺的相对误差为5.3%~6.1%。结论:甲基苯丙胺中毒血清样品在室温和冷冻条件下稳定,建立的GC/MS分析方法同时测定血清和尿液中甲基苯丙胺及其主要代谢产物苯丙胺,方法简便、灵敏、重复性好,适用于甲基苯丙胺中毒与滥用案例的快速鉴定。     

Chronic Methamphetamine Induces Structural Changes in Frontal Cortex Neurons and Upregulates Type I Interferons

While methamphetamine-induced changes in brain neurotransmitters, their receptors, and transporters are well studied, the means by which methamphetamine abuse results in cognitive and behavioral abnormalities is unknown. Here, we administered methamphetamine chronically, in doses relevant to recreational usage patterns, to nonhuman primates. Neurostructural analysis revealed decreased dendritic material and loss of spines in frontal lobe neurons. Molecular examination demonstrated that type I interferons (interferon-alpha and interferon-beta) increased in the frontal lobe in response to chronic methamphetamine treatment, in correlation with the neuronal changes. Chronic methamphetamine thus results in significant changes in the primate brain, inducing cytokines and altering neuronal structure, both of which can contribute to functional abnormalities.     

Reduced (+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro

"Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (+/-)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles (*)2, (*)10, and (*)17, all of which have been linked to decreased enzyme activity. With MDMA, intrinsic clearances (V(max)/K(m)) in CYP2D6.2, CYP2D6.17, and CYP2D6.10 were reduced 15-, 13-, and 135-fold, respectively, compared with wild-type CYP2D6.1. With DEX, intrinsic clearances were reduced by 37-, 51-, and 164-fold, respectively. It was evident that CYP2D6.17 displayed substrate-specific changes in drug affinity (K(m)). Compounds potentially used with MDMA [fluoxetine, paroxetine, (-)-cocaine] demonstrated significant inhibition of MDMA metabolism in both human liver and CYP2D6.1-expressing microsomes. These data demonstrate that individuals possessing the CYP2D6(*)2, (*)17, and, particularly, (*)10 alleles may show significantly reduced MDMA metabolism. These individuals, and those taking CYP2D6 inhibitors, may demonstrate altered acute and/or long-term MDMA-related toxicity.     

Drugs of Abuse, Immune Modulation, and AIDS

Illicit drugs such as amphetamines, cocaine, marijuana, and opiates alter immune function and decrease host resistance to microbes in vitro and in experimental animal models. Effects on the immune system may be mediated indirectly as a result of drug interactions in the central nervous system (CNS) or directly through activation of cognate receptors on various immune cell types. For marijuana and opioids, seven-transmembranal G protein-coupled receptors have been identified in the CNS and in the immune system that may play a functionally relevant role in immune modulation. There is accumulating evidence that sigma₁ receptors play a comparable role in cocaine-mediated alteration of immune responses. A mode by which these exogenously introduced substances affects immunity and host resistance may be by perturbing the balance of Th₁ proinflammatory versus Th₂ anti-inflammatory cytokines and lipid bioeffectors. However, while illicit drugs have been documented to alter immune functions in vitro and in animal models, there is a paucity of controlled longitudinal epidemiological studies that definitively correlate immunosuppressive effects with increased incidence of infections or immune disorders in humans, including infection with the human immunodeficiency virus (HIV) or disease progression to AIDS.     

l-THP对大鼠甲基苯丙胺对条件位置偏爱效应的影响

目的：研究左旋四氢巴马汀(l—tetrahydropalmatine，l—THP)对甲基苯丙胺诱导的大鼠条件位置偏爱(conditioned place preference．CPP)模型的干预作用，并初步探讨其神经生化机制。方法：结果：通过条件位置偏爱实验建立大鼠甲基苯丙胺精神依赖模型，用左旋四氢巴马汀和多巴胺受体拮抗剂干预模型的消退、重建过程，比较分析各组实验结果。CPP实验中大鼠对甲基苯丙胺有明显的偏爱效应，但大鼠腹腔注射l—THP未显示明显的偏爱效应．l—THP对实验大鼠不能诱导条件位置偏爱效应。l—THP能不同程度地促进甲基苯丙胺诱导的大鼠条件位置偏爱效应的消退．而且这一作用有可能呈现一定的剂量依赖性，而且可以在一定程度上抑制低剂量甲基苯丙胺引起的大鼠CPP效应的重建。结论：l—THP在大鼠甲基苯丙胺精神依赖CPP实验中能够影响模型的表达、消退、重建过程。而l—THP在CPP实验中却没有显示出依赖性潜力。     

甲基苯丙胺诱导大鼠神经元凋亡与半胱氨酸天冬氨酸特异性蛋白酶-3的表达

目的:探讨甲基苯丙胺对实验大鼠神经元凋亡的诱导以及凋亡相关因子半胱氨酸天冬氨酸特异性蛋白酶-3(Caspase-3)的表达。方法:选择Wistar♂大鼠20只,将动物随机分为4组,每组5只。4组中随机选3组为甲基苯丙胺实验组(B、C、D),1组为对照组(A)。B组给予甲基苯丙胺,20mg·kg-1,单次ip;C组给予甲基苯丙胺,每次20mg·kg-1,每天2次(8:00,20:00)ip,连续2d;D组给予甲基苯丙胺,每次20mg·kg-1,每天2次(8:00,20:00)ip,连续4d;A组给予等体积生理盐水。采用TUNEL法检测神经元凋亡,免疫组织化学方法检测Caspase-3的表达。结果:给予甲基苯丙胺后,大鼠各脑区有凋亡细胞形成,凋亡相关因子Caspase-3有不同程度的表达。结论:甲基苯丙胺可导致神经元的凋亡,并诱导凋亡相关因子Caspase-3的表达。     

Substance initiation sequences among Taiwanese adolescents using methamphetamine

The aim of this study was to examine substance initiation sequences in Taiwanese adolescent methamphetamine (MAMP) users. Three hundred adolescent MAMP users were questioned about their use of cigarettes, alcohol, areca quid, benzodiazepine, glue, marijuana, MAMP and heroin. The sequences of substance use before MAMP use were determined in every subject according to their age at substance initiation. The sequences of substance initiation were compared between genders and between early-onset and late-onset MAMP users. The results revealed that the adolescents had used cigarettes, alcohol and areca quid before MAMP, and very few (4%) adolescents had started using MAMP before using other substances. Female MAMP users were more likely to use MAMP as their first substance and were less likely to use areca quid than males. Male early-onset MAMP users were more likely to drink alcohol and chew areca quid before MAMP use than were late-onset users. This study found that the sequences of substance initiation were different between genders and between the early-onset and late-onset MAMP users. There are several findings that are different from those in Western studies on the sequence of substance initiation in the adolescent population.     

Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.     

Focused motor stereotypies do not require enhanced activation of neurons in striosomes

Stereotypic motor behavior is a widespread phenomenon of many neurologic and psychiatric disorders. Studies on the mechanisms controlling motor stereotypies have focused on the role of dopamine in modulating the activity of basal ganglia neuronal circuits, and recent results demonstrated that stereotypic motor responses characteristic of psychomotor stimulant sensitization correlate with an enhanced activation of neurons located in striatal striosomes that substantially exceeds that of the surrounding matrix. The present study tested whether predominant striosomal activation is a general predictor for stereotypy. Wild-type and dopamine D(2) and D(3) receptor knockout mice were treated either three times with methamphetamine (METH; 3 x 5 mg/kg every 2 hours) or once with a full D(1) agonist. Depending on the genotype, both treatments elicit the same focused stereotypy (taffy pulling). Repeated METH-treatment elicits intense stereotypy in wild-type and D(3) mutants but not in D(2) single and D(2)/D(3) double mutants. The stereotypic response of wild-type and D(3) mutants correlates with a predominant activation of neurons located in striosomes. No striosomal predominance is detected in METH-treated D(2) single and D(2)/D(3) double mutants. In contrast, D(2) single and D(2)/D(3) double mutants exhibited the most severe stereotypic response to D(1)-agonist treatment. However, this treatment did not result in enhanced striosomal activation. Thus, whereas the expression of stereotypy in response to repeated METH treatment requires D(2) receptor expression, D(2) receptor expression diminishes stereotypic responses to an acute dose of a D(1) agonist. Enhanced striosomal activation, however, is a reliable indicator of D(1)- and D(2)-receptor coactivation but not a predictor for repetitive motor behavior in general.