Chemical and physical variations of cannabis smoke from a variety of cannabis samples in New Zealand.

Studies have compared the chemical properties of tobacco smoke to those of cannabis smoke, with the objective of identifying the chemical attributes responsible for the mutagenicity and carcinogenicity of cannabis smoke. Comparative studies have included small sample sizes and produced conflicting results. The aim of this study was to assess the major chemical and physical variations of cannabis smoke across a range of cannabis samples of different potencies and origins, sourced from the illegal market in New Zealand. Twelve cannabis samples were studied ranging from 1.0% to 13.4% delta-9-tetrahydrocannabinol (Δ9THC) content. A smoking machine was used to smoke "joints" (cannabis cigarettes) and the chemical/physical properties of the smoke assessed. The chemical constituents of the smoke extracts were analysed by gas chromatography/mass spectrometry. A range of different chemical constituents (in addition to Δ9THC) were identified and their concentrations estimated. Terpenoids were identified as the major variable in cannabis smoke, showing a 40-fold range in total terpenoid content. Analysis of the total particulate matter showed that significantly different levels of particulate matter were produced between the different cannabis samples, ranging from 14.6 to 66.3 mg/g of cannabis smoked. The Δ9THC delivery efficiency during smoking was also investigated and produced consistent results showing a mean and median of 12.6% and 10.8%, respectively, of the theoretically available Δ9THC (ranging from 7.2% to 28.0%).

Normal glutathione levels in autopsied brain of chronic users of heroin and of cocaine.

BACKGROUND: Animal studies suggest that exposure to either of the two widely used drugs of abuse, heroin or cocaine, causes depletion of the antioxidant, reduced glutathione, a hallmark of oxidative stress, in the brain. However, the relevance of the animal findings to the human is uncertain and clinical trials with the antioxidant GSH precursor n-acetylcysteine have produced mixed results in cocaine dependence. METHODS: Our major objective was to compare glutathione levels, determined by an HPLC-coulometric procedure, in autopsied brain of chronic heroin (n = 11) and cocaine users (n = 9), who were positive for the drugs in the brain, to those of matched controls (n = 16). Six brain regions were examined, including caudate, hippocampus, thalamus and frontal, temporal and insular cortices. RESULTS: In contrast to experimental animal findings, we found no statistically significant difference between mean levels of reduced or oxidized glutathione in the drug user vs. control groups. Moreover, no correlation was found between levels of drugs in the brain and those of glutathione. CONCLUSIONS: Acknowledging the many generic limitations of an autopsied human brain study and the preliminary nature of the findings, our data nevertheless suggest that any oxidative stress caused by heroin or cocaine in chronic users of the drugs might not be sufficient to cause substantial loss of stores of glutathione in the human brain, at least during early withdrawal. These findings, requiring replication, might also have some relevance to future clinical trials employing glutathione supplement therapy as an anti-oxidative strategy in chronic users of the two abused drugs.

Do glutathione levels decline in aging human brain?

For the past 60 years a major theory of "aging" is that age-related damage is largely caused by excessive uncompensated oxidative stress. The ubiquitous tripeptide glutathione is a major antioxidant defense mechanism against reactive free radicals and has also served as a marker of changes in oxidative stress. Some (albeit conflicting) animal data suggest a loss of glutathione in brain senescence, which might compromise the ability of the aging brain to meet the demands of oxidative stress. Our objective was to establish whether advancing age is associated with glutathione deficiency in human brain. We measured reduced glutathione (GSH) levels in multiple regions of autopsied brain of normal subjects (n=74) aged one day to 99 years. Brain GSH levels during the infancy/teenage years were generally similar to those in the oldest examined adult group (76-99 years). During adulthood (23-99 years) GSH levels remained either stable (occipital cortex) or increased (caudate nucleus, frontal and cerebellar cortices). To the extent that GSH levels represent glutathione antioxidant capacity, our postmortem data suggest that human brain aging is not associated with declining glutathione status. We suggest that aged healthy human brains can maintain antioxidant capacity related to glutathione and that an age-related increase in GSH levels in some brain regions might possibly be a compensatory response to increased oxidative stress. Since our findings, although suggestive, suffer from the generic limitations of all postmortem brain studies, we also suggest the need for "replication" investigations employing the new (1)H MRS imaging procedures in living human brain.

Low levels of astroglial markers in Parkinson's disease: relationship to α-synuclein accumulation.

Although gliosis is a normal response to brain injury, reports on the extent of astrogliosis in the degenerating substantia nigra in Parkinson's disease (PD) are conflicting. It has also been recently suggested that accumulation of nigral α-synuclein in this disorder might suppress astrocyte activation which in turn could exacerbate the degenerative process. This study examined brain protein levels (intact protein, fragments, and aggregates, if any) of astroglial markers and their relationship to α-synuclein in PD and in the positive control parkinson-plus conditions multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Autopsied brain homogenates of patients with PD (n=10), MSA (n=11), PSP (n=11) and matched controls (n=10) were examined for the astroglial markers glial fibrillary acidic protein (GFAP), vimentin, and heat shock protein-27 (Hsp27) by quantitative immunoblotting. As expected, both MSA (putamen>substantia nigra>caudate>frontal cortex) and PSP (substantia nigra>caudate>putamen, frontal cortex) showed widespread but regionally specific pattern of increased immunoreactivity of the markers, in particular for the partially proteolyzed fragments (all three) and aggregates (GFAP). In contrast, immunoreactivity of the three markers was largely normal in PD in brain regions examined with the exception of trends for variably increased levels of cleaved vimentin in substantia nigra and frontal cortex. In patients with PD, GFAP levels in the substantia nigra correlated inversely with α-synuclein accumulation whereas the opposite was true for MSA. Our biochemical findings of generally normal protein levels of astroglial markers in substantia nigra of PD, and negative correlation with α-synuclein concentration, are consistent with some recent neuropathology reports of mild astroglial response and with the speculation that astrogliosis might be suppressed in this disorder by excessive α-synuclein accumulation. Should astrogliosis protect, to some extent, the degenerating substantia nigra from damage, therapeutics aimed at normalization of astrocyte reaction in PD could be helpful.

Is brain gliosis a characteristic of chronic methamphetamine use in the human?

Animal data show that high doses of the stimulant drug methamphetamine can damage brain dopamine neurones; however, it is still uncertain whether methamphetamine, at any dose, is neurotoxic to human brain. Since gliosis is typically associated with brain damage and is observed in animal models of methamphetamine exposure, we measured protein levels (intact protein and fragments, if any) of markers of microgliosis (glucose transporter-5, human leukocyte antigens HLA-DRα [TAL.1B5] and HLA-DR/DQ/DPß [CR3/43]) and astrogliosis (glial fibrillary acidic protein, vimentin, and heat shock protein-27) in homogenates of autopsied brain of chronic methamphetamine users (n=20) and matched controls (n=23). Intact protein levels of all markers were, as expected, elevated (+28%-1270%, P<0.05) in putamen of patients with the neurodegenerative disorder multiple system atrophy (as a positive control) as were concentrations of fragments of glial fibrillary acidic protein, vimentin and heat shock protein-27 (+170%-4700%, P<0.005). In contrast, intact protein concentrations of the markers were normal in dopamine-rich striatum (caudate, putamen) and in the frontal cortex of the drug users. However, striatal levels of cleaved vimentin and heat shock protein-27 were increased (by 98%-211%, P<0.05), with positive correlations (r=0.41-0.60) observed between concentrations of truncated heat shock protein-27 and extent of dopamine loss (P=0.006) and levels of lipid peroxidation products 4-hydroxynonenal (P=0.046) and malondialdehyde (P=0.11). Our failure to detect increased intact protein levels of commonly used markers of microgliosis and astrogliosis could be explained by exposure to methamphetamine insufficient to cause a toxic process associated with overt gliosis; however, about half of the subjects had died of drug intoxication suggesting that "high" drug doses might have been used. Alternatively, drug tolerance to toxic effects might have occurred in the subjects, who were all chronic methamphetamine users. Nevertheless, the finding of above-normal levels of striatal vimentin and heat shock protein-27 fragments (which constituted 10-28% of the intact protein), for which changes in the latter correlated with those of several markers possibly suggestive of damage, does suggest that some astrocytic "disturbance" had occurred, which might in principle be related to methamphetamine neurotoxicity or to a neuroplastic remodeling process. Taken together, our neurochemical findings do not provide strong evidence for either marked microgliosis or astrogliosis in at least a subgroup of human recreational methamphetamine users who used the drug chronically and shortly before death. However, a logistically more difficult quantitative histopathological study is needed to confirm whether glial changes occur or do not occur in brain of human methamphetamine (and amphetamine) users.

Methamphetamine self-administration and the effect of contingency on monoamine and metabolite tissue levels in the rat.

A number of studies have shown that exposure to high doses of methamphetamine (MA) is toxic to central dopamine (DA) and serotonin (5-HT) neurons. In most of those studies, however, high doses of MA were experimenter-administered during a short exposure time. Because contingency is a determinant for many effects of drug exposure, the present objective was to investigate the effects of self-administered MA on tissue monoamine levels following a short (24 hours) or longer (7 days) withdrawal period. As previously reported, a noncontingent "binge" high-dose treatment regimen (4 injections of 10 mg/kg MA administered every 2 hours) produced persistent depletion of cortical 5-HT and striatal DA. Effects of self-administered MA (0.1 mg/kg/infusion) were then determined following a 20-day duration where a yoked design was employed such that some rats received MA contingent on an operant lever press and others received either MA or saline dependent on the responses of the contingent rat. Self-administered MA produced a transient striatal DA depletion with a more persistent increase in DA turnover, indicating the presence of some lasting adaptations. Furthermore, the yoked design revealed that there was no effect of contingency on these parameters.

Effect of D1-like and D2-like receptor antagonists on methamphetamine and 3,4-methylenedioxymethamphetamine self-administration in rats.

It has been suggested that activation of dopamine D1-like and D2-like receptors contribute equally to the maintenance of drug self-administration. This study compared the contribution of these receptor subtypes to 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MA) self-administration. Effects of pretreatment with the D2-like receptor antagonist, eticlopride (0.0, 0.0125, 0.025 or 0.05 mg/kg, intraperitoneal), on responding maintained by several doses of MDMA (0.5, 1.0 and 2.0 mg/kg/infusion) and MA (0.05, 0.1 and 0.2 mg/kg/infusion) were determined. As we have published data showing the effects of the D1-like receptor antagonist, SCH23390 (0.0, 0.01 or 0.02 mg/kg, subcutaneous), on MDMA self-administration, effects of this dose range on the MA dose-response curve were determined. In our previous study, 0.02 mg/kg SCH23390 produced a rightward shift in the MDMA dose response curve, whereas in the present results, this dose decreased responding maintained by most doses of MA. Eticlopride increased the responding maintained by most doses of MDMA but failed to alter MA self-administration. The present results suggest that both D1-like and D2-like receptors contribute to the maintenance of MDMA self-administration, whereas MA self-administration was more sensitive to D1-like receptor blockade.

Brain serotonin transporter in human methamphetamine users.

RATIONALE: Research on methamphetamine (MA) toxicity primarily focuses on the possibility that some of the behavioural problems in human MA users might be caused by damage to brain dopamine neurones. However, animal data also indicate that MA can damage brain serotonin neurones, and it has been suggested that cognitive problems and aggression in MA users might be explained by serotonergic damage. As information on the brain serotonin system in human MA users is fragmentary, our objective was to determine whether protein levels of serotonin transporter (SERT), a key marker of serotonin neurones, are decreased in brain of chronic MA users. METHODS: SERT immunoreactivity was measured using an immunoblotting procedure in autopsied brain of 16 chronic MA users testing positive for the drug in blood and brain and matched controls. RESULTS: SERT levels were non-significantly decreased (-14% to -33%) in caudate, putamen and thalamus (normal in hippocampus), and, unlike the robust striatal dopamine reduction, there was marked overlap between control and MA user ranges. Concentrations of SERT were significantly decreased (-23% to -39%) in orbitofrontal and occipital cortices (normal in frontopolar and temporal cortices). CONCLUSIONS: Our data suggest that MA might modestly damage brain serotonin neurones and/or inhibit SERT protein expression, with cerebral cortex being more affected than sub-cortical regions. The SERT reduction in orbitofrontal cortex complements other data suggesting involvement of this area in MA-related behaviour. Decreased brain SERT could also be related to the clinical finding that treatment with a selective serotonin re-uptake inhibitor might increase relapse to MA.

N-benzylpiperazine has characteristics of a drug of abuse.

The ability of benzylpiperazine (BZP) to substitute for cocaine and to initiate self-administration in drug-naive subjects was assessed to determine whether BZP has abuse liability. Further, the effects of a pretreatment with dopamine D1-like receptor antagonist (SCH23390) were examined to elucidate the mechanisms associated with BZP reward. First, the ability for BZP (0.125, 0.25 and 0.5 mg/kg/infusion) to substitute for cocaine self-administration was assessed, and the acquisition of BZP (0.5 mg/kg/infusion) self-administration by drug-naive and untrained rats was determined during a 15-day period. Subsequently, dose-effect curves for cocaine (0.06, 0.125, 0.25 or 0.5 mg/kg/infusion) and BZP self-administration (0.125, 0.25, 0.5 or 1.0 mg/kg/infusion) and the effect of SCH23390 (0.00 or 0.02 mg/kg) on BZP and cocaine self-administration were examined. BZP substituted for cocaine, and drug-naive rats rapidly acquired BZP self-administration. BZP self-administration was maintained by a more restricted range of doses than was cocaine self-administration, and responding maintained by BZP was sensitive to dopamine antagonism. The present findings indicate that BZP self-administration, like cocaine self-administration, is readily acquired and mediated by dopaminergic mechanisms.

Benzylpiperazine: a drug of abuse?

N-benzylpiperazine (BZP) is the active ingredient in recreational 'party' or 'p.e.p.' pills, which are used to provide a stimulant, euphoric effect akin to that of methylenedioxymethamphetamine (MDMA, 'ecstasy'). BZP predominantly affects dopamine neurotransmission in a similar fashion to known 'drugs of abuse', such as methamphetamine and cocaine, which strongly suggests BZP has abuse liability. BZP is illegal in many countries including the United States of America and Australia, yet it remains legal in the United Kingdom, Canada and New Zealand. There has been little research, to date, on the neurological consequences of high dose or chronic exposure of BZP. Here we provide a comprehensive review of the information currently available on BZP and suggest a need for further research into the mechanisms of action, long-term effects and potentially addictive properties of BZP.

Levels of 4-hydroxynonenal and malondialdehyde are increased in brain of human chronic users of methamphetamine.

Animal studies suggest that the widely used psychostimulant drug methamphetamine (MA) can harm brain dopamine neurones, possibly by causing oxidative damage. However, evidence of oxidative damage in brain of human MA users is lacking. We tested the hypothesis that levels of two "gold standard" products generated from lipid peroxidation, 4-hydroxynonenal (one of the most reactive lipid peroxidation aldehyde products) and malondialdehyde, would be elevated in post mortem brain of 16 dopamine-deficient chronic MA users compared with those in 21 matched control subjects. Derivatized aldehyde concentrations were determined by gas chromatography-mass spectrometry. In the MA group, we found significantly increased levels of 4-hydroxynonenal and malondialdehyde in the dopamine-rich caudate nucleus (by 67 and 75%, respectively) and to a lesser extent in frontal cortex (48 and 36%, respectively) but not in the cerebellar cortex. Approximately half of the MA users had levels of 4-hydroxynonenal falling above the upper limit of the control range in caudate and frontal cortex. A subgroup of MA users with high brain drug levels had higher concentrations of the aldehydes. Our data suggest that MA exposure in human causes, as in experimental animals, above-normal formation of potentially toxic lipid peroxidation products in brain. This provides evidence for involvement of oxygen-based free radicals in the action of MA in both dopamine-rich (caudate) and -poor (cerebral cortex) areas of human brain.

Brain antioxidant systems in human methamphetamine users.

Animal data suggest that the widely abused psychostimulant methamphetamine can damage brain dopamine neurones by causing dopamine-dependent oxidative stress; however, the relevance to human methamphetamine users is unclear. We measured levels of key antioxidant defences [reduced (GSH) and oxidized (GSSG) glutathione, six major GSH system enzymes, copper-zinc superoxide dismutase (CuZnSOD), uric acid] that are often altered after exposure to oxidative stress, in autopsied brain of human methamphetamine users and matched controls. Changes in the total (n = 20) methamphetamine group were limited to the dopamine-rich caudate (the striatal subdivision with the most severe dopamine loss) in which only activity of CuZnSOD (+ 14%) and GSSG levels (+ 58%) were changed. In the six methamphetamine users with severe (- 72 to - 97%) caudate dopamine loss, caudate CuZnSOD activity (+ 20%) and uric acid levels (+ 63%) were increased with a trend for decreased (- 35%) GSH concentration. Our data suggest that brain levels of many antioxidant systems are preserved in methamphetamine users and that GSH depletion, commonly observed during severe oxidative stress, might occur only with severe dopamine loss. Increased CuZnSOD and uric acid might reflect compensatory responses to oxidative stress. Future studies are necessary to establish whether these changes are associated with oxidative brain damage in human methamphetamine users.

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans.

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.

Brain dopamine-stimulated adenylyl cyclase activity in Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy.

The dopamine D(1) receptor is considered to participate in levodopa's antiparkinsonian action and levodopa-induced dyskinesias. We examined the functional status of the D(1) receptor in brain of patients with Parkinson's disease (PD), multiple system atrophy (MSA), and progressive supranuclear palsy (PSP). Dopamine-stimulated adenylyl cyclase activity was significantly increased in putamen (+43%) and frontal cortex (+52%) in PD, normal in PSP, but decreased by 47% in putamen in MSA. The supersensitive dopamine D(1) receptors in both striatum and cerebral cortex in PD might compensate for dopamine deficiency, but could also contribute to long-term complications of levodopa therapy.

Why is parkinsonism not a feature of human methamphetamine users?

For more than 50 years, methamphetamine has been a widely used stimulant drug taken to maintain wakefulness and performance and, in high doses, to cause intense euphoria. Animal studies show that methamphetamine can cause short-term and even persistent depletion of brain levels of the neurotransmitter dopamine. However, the clinical features of Parkinson's disease, a dopamine deficiency disorder of the brain, do not appear to be characteristic of human methamphetamine users. We compared dopamine levels in autopsied brain tissue of chronic methamphetamine users with those in patients with Parkinson's disease and in a control group. Mean dopamine levels in the methamphetamine users were reduced more in the caudate (-61%) than in the putamen (-50%), a pattern opposite to that of Parkinson's disease. Some methamphetamine users had severely decreased dopamine levels, within the parkinsonian range, in the caudate (up to 97% dopamine loss) but not in the putamen. As the putamen and caudate subserve aspects of motor and cognitive function, respectively, our data suggest that methamphetamine users are not parkinsonian because dopamine levels are not sufficiently decreased in the motor component of the striatum. However, the near-total reduction in the caudate could explain reports of cognitive disturbances, sometimes disabling, in some drug users, and suggests that treatment with dopamine substitution medication (e.g. levodopa) during drug rehabilitation might be helpful.

Nigral glutathione deficiency is not specific for idiopathic Parkinson's disease.

The consistent findings of decreased levels of the major antioxidant glutathione in substantia nigra of patients with idiopathic Parkinson's disease (PD) has provided most of the basis for the oxidative stress hypothesis of the etiology of PD. To establish whether a nigral glutathione deficiency is unique to PD, as is generally assumed, or is present in other Parkinsonian conditions associated with nigral damage, we compared levels of reduced glutathione (GSH) in postmortem brain of patients with PD to those with progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). As compared with the controls, nigral GSH levels were decreased in the PD and PSP patient groups (P < 0.05 for PD [-30%], PSP [-21%]), whereas a similar decrease in the MSA patient group did not reach statistical significance (P = 0.078, MSA [-20%]). GSH levels were normal in all examined normal and degenerating extra-nigral brain areas in PSP and MSA. A trend for decreased levels of uric acid (antioxidant and product of purine catabolism) also was observed in nigra of all patient groups (-19 to -30%). These data suggest that glutathione depletion, possibly consequent to overutilisation in oxidative stress reactions, could play a causal role in nigral degeneration in all nigrostriatal dopamine deficiency disorders, and that antioxidant therapeutic approaches should not be restricted to PD.