Assessing the relationship between biomarkers of exposure and biomarkers of potential harm: PATH Study Wave 1 (2013-2014).

BACKGROUND: The adequacy of biomarkers of potential harm (BOPH) for assessing tobacco products was explored based on their ability to distinguish tobacco use from non-use, change with cessation, and to show biological gradient. METHODS: The sample included individuals with biomarker data in Wave 1 of the Population Assessment of Tobacco Health (PATH) Study who never used tobacco, currently smoke cigarettes exclusively, used to smoke cigarettes exclusively (quit in past 12 months), currently use smokeless tobacco exclusively, and currently use e-cigarettes exclusively. We compared BOPH levels between groups and assessed the relationships between log-transformed biomarkers of exposure (BOE) [Total Nicotine Equivalents including seven nicotine metabolites (TNE-7), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonol (NNAL), N-acetyl-S-(2-cyanoethyl)-L-cysteine (CYMA), 1-Hydroxypyrene (1-OHP), cadmium, and serum cotinine (SCOT)], and BOPH [high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), fibrinogen, soluble intercellular adhesion molecule-1 (sICAM-1) and 8-isoprostane]. RESULTS: Among people who smoke, both sICAM-1 and 8-isoprostane distinguished smoking from non-use and were associated with all six BOE. Among people who use smokeless tobacco, 8-isoprostane was associated with TNE-7 and NNAL whereas hs-CRP was associated with SCOT. Among people who use e-cigarettes, no associations between BOPH and BOE were observed. CONCLUSIONS: Both sICAM-1 and 8-isoprostane may be useful for assessing the use or changes in use of some tobacco products. Studies examining their predictive validity could further strengthen our understanding of these two biomarkers. IMPACT: We found that two BOPH, sICAM-1 and 8-isoprostane, may have utility in studies assessing the potential harm of tobacco use in absence of long-term epidemiological studies.

Acute ethanol reduces reversal cost in discrimination learning by reducing perseverance in adolescent rhesus macaques.

BACKGROUND: Acute alcohol exposure produces cognitive deficits in adults but less is known about the acute cognitive effects of alcohol in adolescents. The cognitive impact of acute alcohol exposure includes deficits in discrimination and reversal learning, but traditional experimental approaches make it difficult to distinguish the effect of alcohol on discrimination learning from the effect of alcohol on reversal learning. Young rhesus macaques can be used to model some aspects of human adolescence because of their anatomical, neurophysiological, and cognitive similarities with humans. METHODS: Adolescent male rhesus monkeys (n = 10) were trained to respond to visual stimuli on touch-sensitive LCD panels controlled by the nonhuman primate version of CANTAB software. Discrimination and reversal learning tasks were subsequently assessed after monkeys were allowed to consume varying amounts of ethanol (EtOH) in a flavored vehicle (vehicle only, up to 0.5 g/kg EtOH, up to 1.0 g/kg EtOH, and up to 1.5 g/kg EtOH). RESULTS: Acute exposure to EtOH reduced perseverance, increased response accuracy, and reduced errors during reversal learning when the task was completed within 90 minutes of EtOH consumption. No reduction in reversal errors was observed when EtOH was consumed 3 or 24 hours prior to reversal learning. EtOH only impaired discrimination learning when monkeys had very little previous EtOH exposure. CONCLUSIONS: The temporal relationship between EtOH consumption and reversal learning was consistent with selective EtOH-induced impairment of retrieval, but not storage, processes. This was evidenced by diminished perseverance on the previously correct stimulus leading to decreased errors to criterion.

Mephedrone (4-methylmethcathinone) supports intravenous self-administration in Sprague-Dawley and Wistar rats.

Recreational use of the drug 4-methylmethcathinone (mephedrone; 4-MMC) became increasingly popular in the United Kingdom in recent years, spurred in part by the fact that it was not criminalized until April 2010. Although several fatalities have been associated with consumption of 4-MMC and cautions for recreational users about its addictive potential have appeared on Internet forums, very little information about abuse liability for this drug is available. This study was conducted to determine if 4-MMC serves as a reinforcer in a traditional intravenous self-administration model. Groups of male Wistar and Sprague-Dawley rats were prepared with intravenous catheters and trained to self-administer 4-MMC in 1-hour sessions. Per-infusion doses of 0.5 and 1.0 mg/kg were consistently self-administered, resulting in greater than 80% discrimination for the drug-paired lever and mean intakes of about 2-3 mg/kg/hour. Dose-substitution studies after acquisition demonstrated that the number of responses and/or the total amount of drug self-administered varied as a function of dose. In addition, radiotelemetry devices were used to show that self-administered 4-MMC was capable of increasing locomotor activity (Wistar) and decreasing body temperature (Sprague-Dawley). Pharmacokinetic studies found that the T1/2 of 4-MMC was about 1 hour in vivo in rat plasma and 90 minutes using in vitro liver microsomal assays. This study provides evidence of stimulant-typical abuse liability for 4-MMC in the traditional pre-clinical self-administration model.

Decreased sensitivity in adolescent vs. adult rats to the locomotor activating effects of toluene.

Volatile organic solvent (inhalant) abuse continues to be a major health concern throughout the world. Of particular concern is the abuse of inhalants by adolescents because of its toxicity and link to illicit drug use. Toluene, which is found in many products such as glues and household cleaners, is among the most commonly abused organic solvents. While studies have assessed outcomes of exposure to inhalants in adult male animals, there is little research on the neurobehavioral effects of inhalants in female or younger animals. In attempt to address these shortcomings, we exposed male and female Long-Evans rats to 20 min of 0, 2000, 4000, or 8000 parts per million (ppm) inhaled toluene for 10 days in rats aged postnatal (PN) day 28-39 (adolescent), PN44-PN55, or >PN70 (adult). Animals were observed individually in 29-l transparent glass cylindrical jars equipped with standard photocells that were used to measure locomotor activity. Toluene significantly increased activity as compared to air exposure in all groups of male and female rats with the magnitude of locomotor stimulation produced by 4000 ppm toluene being significantly greater for female adults than during any age of adolescence. The results demonstrate that exposure to abuse patterns of high concentrations of toluene through inhalation can alter spontaneous locomotor behavior in rats and that the expression of these effects appears to depend upon the postnatal age of testing and sex of the animal.

Comparison of Atipamezole with Yohimbine for Antagonism of Xylazine in Mice Anesthetized with Ketamine and Xylazine.

The α2 adrenergic agonist xylazine produces a sedative effect and is typically combined with ketamine and used for anesthesia or chemical restraint of laboratory mice. Xylazine's sedative effect-and its undesirable side effects of bradycardia, hypotension, and poor tissue perfusion-can be reversed by administration of α2 antagonists, such as atipamezole or yohimbine. Although atipamezole and yohimbine dosing guidelines are available for mice, no controlled comparison has been performed to guide the lab animal community in the selection of one over the other. This study is a single-dose crossover comparison of these 2 antagonist drugs, given intraperitoneally at clinically recommended doses, to determine which results in more rapid recovery of mice from xylazine-ketamine anesthesia. Time to return of righting reflex was used as the primary outcome measure. Mice were anesthetized with xylazine (10 mg/kg IP) and ketamine (80 mg/kg IP), followed 15 min later by injection of an α2 antagonist or saline (control). Time to return of righting reflex differed significantly among groups, with mice recovering in an average of 10.3 min after administration of atipamezole (1 mg/kg IP) as compared with 21.3 min after yohimbine (1.5 mg/kg IP) and 38.2 min after saline. When rapid recovery of mice after xylazine-ketamine anesthesia is desirable, administration of an antagonist to reverse the effects of the xylazine is indicated. When injection of the antagonist by the technically simple intraperitoneal route is desirable, our data indicate that (at the doses evaluated) atipamezole is more effective than yohimbine.

Comparative effects of dextromethorphan and dextrorphan on nicotine discrimination in rats.

While the role of dextrorphan and dextromethorphan as N-methyl-d-aspartate (NMDA) receptor antagonists has received considerable research attention, their effects on nicotinic acetylcholine receptors (nAChR) has been less well characterized. Recent in vitro and in vivo research has suggested that these drugs noncompetitively block alpha3beta4*, alpha4beta2, and alpha7 nAChR subtypes and antagonize nicotine's antinociceptive and reinforcing effects. Both drugs were most potent at blocking alpha3beta4* AChR. This study investigated the effects of dextrorphan and dextromethorphan on nicotine's discriminative stimulus effects. Three groups of rats were trained in a two-lever drug discrimination procedure to discriminate 0.4 mg/kg s.c. nicotine from saline. Nicotine dose-dependently substituted for itself in all three groups. In contrast, when dextrorphan (group 1) or dextromethorphan (group 2) were injected i.p., neither substitution for nor antagonism of nicotine was observed for either drug. Since i.p. administration allows substantial metabolism of dextromethorphan to its parent compound dextrorphan, the two drugs were also tested following s.c. administration (group 3). Discrimination results were similar across both routes of administration, in that neither substitution nor antagonism occurred, however, s.c. administration reduced response rates to a much greater extent than did i.p. administration. Previous work suggests that beta2 subunits are crucial for mediation of nicotine's discriminative stimulus effects and may play a role in its reinforcing effects, albeit other research suggests a role for alpha3beta4* nicotinic receptors in the latter. Our results suggest that alpha3beta4* nicotinic receptors do not play a major role in nicotine's discriminative stimulus effects. Further, they suggest that the role of cholinergic mediation of the behavioral effects of dextrorphan and dextromethorphan related to the abuse properties of nicotine may be minimal.

Advances in Behavioral Laboratory Methods that Inform Tobacco Regulatory Science: A TCORS Working Group Special Issue.

OBJECTIVE: The 2009 Family Smoking Prevention and Tobacco Control Act (TCA) created unprecented enabling conditions for establishing national regulatory policy that reduces the burden of public health and societal problems associated with tobacco product use. The Center for Tobacco Products (CTP), created by the FDA to implement the TCA, developed a first-of-its-kind FDA/National Institutes of Health (NIH) collaborative program to fund Tobacco Centers of Regulatory Science (TCORS). METHODS: To assist the TCORS with addressing research priorites, working groups (WGs) comprised of FDA-CTP liasions and TCORS investigators were formed. Under the direction of the Center for Evaluation and Coordination of Trainin and Research (CECTR), the TCORS WGs seek to develop tangible work products in their respective areas of focus. RESULTS: The focus of the behavioral pharmacology WG evolved from publishing a narrow paper on behavioral methods in electronic cigarette research to a collection of papers on advances in behavioral laboratory methods that may inform tobacco regulatory science. CONCLUSION: This Special Issue contains articles that address all of the CTP research priorities and demonstrates how advances in behavioral laboratory methods made by TCORS investigators can inform FDA efforst to regulate tobacco products.

Chronic periadolescent alcohol consumption produces persistent cognitive deficits in rhesus macaques.

Although human alcoholics exhibit lasting cognitive deficits, it can be difficult to definitively rule out pre-alcohol performance differences. For example, individuals with a family history of alcoholism are at increased risk for alcoholism and are also behaviorally impaired. Animal models of controlled alcohol exposure permit balanced group assignment, thereby ruling out the effects of pre-existing differences. Periadolescent male rhesus macaques (N = 5) consumed alcohol during 200 drinking sessions (M-F) across a 10-month period (mean daily alcohol consumption: 1.38 g/kg/day). A control group (N = 5) consumed a fruit-flavored vehicle during the same period. Spatial working memory, visual discrimination learning and retention and response time behavioral domains were assessed with subtests of the Monkey CANTAB (CAmbridge Neuropsychological Test Automated Battery). Spatial working memory performance was impaired in the alcohol group after 120 drinking sessions (6 mo) in a manner that depended on retention interval. The chronic alcohol animals were also impaired in retaining a visual discrimination over 24 hrs when assessed 6-8 weeks after cessation of alcohol drinking. Finally, the presentation of distractors in the response time task impaired the response time and accuracy of the chronic alcohol group more than controls after 6 months of alcohol cessation. Chronic alcohol consumption over as little as 6 months produces cognitive deficits, with some domains still affected after acute (6-8 wks) and lasting (6 mo) discontinuation from drinking. Animals were matched on alcohol preference and behavioral performance prior to exposure, thus providing strong evidence for the causal role of chronic alcohol in these deficits.

Endocannabinoid contribution to Δ9-tetrahydrocannabinol discrimination in rodents.

The mechanism through which marijuana produces its psychoactive effects is Δ(9)-tetrahydrocannabinol (THC)-induced activation of cannabinoid CB1 receptors. These receptors are normally activated by endogenous lipids, including anandamide and 2-arachidonoyl glycerol (2-AG). A logical "first step" in determination of the role of these endocannabinoids in THC׳s psychoactive effects is to investigate the degree to which pharmacologically induced increases in anandamide and/or 2-AG concentrations through exogenous administration and/or systemic administration of inhibitors of their metabolism, fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), respectively, share THC׳s discriminative stimulus effects. To this end, adult male mice and rats were trained to discriminate THC (5.6 and 3mg/kg, respectively). In Experiment 1, exogenous administration of anandamide or 2-AG did not substitute for THC in mice nor was substitution enhanced by co-administration of the FAAH or MAGL inhibitors, URB597 and N-arachidonyl maleimide (NAM), respectively. Significant decreases in responding may have prevented assessment of adequate endocannabinoid doses. In mice trained at higher baseline response rates (Experiment 2), the FAAH inhibitor PF3845 (10mg/kg) enhanced anandamide substitution for THC without producing effects of its own. The MAGL inhibitor JZL184 increased brain levels of 2-AG in vitro and in vivo, increased THC-like responding without co-administration of 2-AG. In rats, neither URB597 nor JZL184 engendered significant THC-appropriate responding, but co-administration of these two enzyme inhibitors approached full substitution. The present results highlight the complex interplay between anandamide and 2-AG and suggest that endogenous increases of both endocannabinoids are most effective in elicitation of THC-like discriminative stimulus effects.

The influence of acute and chronic alcohol consumption on response time distribution in adolescent rhesus macaques.

BACKGROUND: Analysis of the distribution of reaction times (RTs) in behavioral tasks can illustrate differences attributable to changes in attention, even when no change in mean RT is observed. Detrimental attentional effects of both acute and chronic exposure to alcohol may therefore be revealed by fitting RT data to an ex-Gaussian probability density function which identifies the proportion of long-RT responses. METHODS: Adolescent male rhesus macaques completed a 5-choice serial reaction time task (5CSRT) after acute alcohol consumption (up to 0.0, 1.0 and 1.5 g/kg). Monkeys were next divided into chronic alcohol (N = 5) and control groups (N = 5); the experimental group consumed 1.5-3.0 g/kg alcohol for 200 drinking sessions. Unintoxicated performance in the 5CSRT task was determined systematically across the study period and the effect of acute alcohol was redetermined after the 180th drinking session. The effect of extended abstinence from chronic alcohol was determined across 90 days. RESULTS: Acute alcohol exposure dose-dependently reduced the probability of longer RT responses without changing the mean or the standard deviation of the RT distribution. The RT distribution of control monkeys tightened across 10 months whereas that of the chronic alcohol group was unchanged. Discontinuation from chronic alcohol increased the probability of long RT responses with a difference from control animals observed after 30 days of discontinuation. CONCLUSIONS: Alcohol consumption selectively affected attention as reflected in the probability of long RT responses. Acute alcohol consumption focused attention, chronic alcohol consumption impaired the maturation of attention across the study period and alcohol discontinuation impaired attention.

Cannabidiol attenuates deficits of visuospatial associative memory induced by Δ(9) tetrahydrocannabinol.

BACKGROUND AND PURPOSE: Recent human studies suggest that recreational cannabis strains that are relatively high in cannabidiol (CBD) content produce less cognitive impairment than do strains with negligible CBD and similar Δ(9) tetrahydrocannabinol (THC) content. Self-selection in such studies means it is impossible to rule out additional variables which may determine both cannabis strain selection and basal cognitive performance level. Controlled laboratory studies can better determine a direct relationship. EXPERIMENTAL APPROACH: In this study, adult male rhesus monkeys were assessed on visuospatial Paired Associates Learning and Self-Ordered Spatial Search memory tasks, as well as additional tests of motivation and manual dexterity. Subjects were challenged with THC (0.2, 0.5 mg·kg(-1) , i.m.) in randomized order and evaluated in the presence or absence of 0.5 mg·kg(-1) CBD. KEY RESULTS: CBD attenuated the effects of THC on paired associates learning and a bimanual motor task without affecting the detrimental effects of THC on a Self-Ordered Spatial Search task of working memory. CBD did not significantly reverse THC-induced impairment of a progressive ratio or a rotating turntable task. CONCLUSIONS AND IMPLICATIONS: This study provides direct evidence that CBD can oppose the cognitive-impairing effects of THC and that it does so in a task-selective manner when administered simultaneously in a 1:1 ratio with THC. The addition of CBD to THC-containing therapeutic products may therefore help to ameliorate unwanted cognitive side-effects. LINKED ARTICLE: This article is commented on by Mechoulam and Parker, pp 1363-1364 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12400.

A methamphetamine vaccine attenuates methamphetamine-induced disruptions in thermoregulation and activity in rats.

BACKGROUND: There are no approved pharmacotherapies for d-methamphetamine (METH) addiction and existing therapies have limited efficacy. Advances in using immunotherapeutic approaches for cocaine and nicotine addiction have stimulated interest in creating a similar approach for METH addiction. This study investigated whether active vaccination against METH could potentially attenuate responses to METH in vivo. METHODS: Male Sprague Dawley rats (n = 32) received a four-boost series with one of three candidate anti-METH vaccines (MH2[R], MH6, and MH7) or a control keyhole limpet hemocyanin conjugate vaccine. Effects of METH on rectal temperature and wheel activity at 27°C ambient temperature were determined. The most efficacious vaccine, MH6, was then contrasted with keyhole limpet hemocyanin conjugate vaccine in a subsequent experiment (n = 16), wherein radiotelemetry determined home cage locomotor activity and body temperature at 23°C ambient temperature. RESULTS: The MH6 vaccine produced high antibody titers with nanomolar affinity for METH and sequestered METH in the periphery of rats. In experiment 1, the thermoregulatory and psychomotor responses produced by METH at 27°C were blocked in the MH6 group. In experiment 2, METH-induced decreases in body temperature and locomotor activity at 23°C were also attenuated in the MH6 group. A pharmacokinetic study in experiment 2 showed that MH6-vaccinated rats had higher METH serum concentrations, yet lower brain METH concentrations, than control rats, and METH concentrations correlated with individual antibody titer. CONCLUSIONS: These data demonstrate that active immunopharmacotherapy provides functional protection against physiological and behavioral disruptions induced by METH.

Effect of ambient temperature on the thermoregulatory and locomotor stimulant effects of 4-methylmethcathinone in Wistar and Sprague-Dawley rats.

The drug 4-methylmethcathinone (4-MMC; aka, mephedrone, MMCAT, "plant food", "bath salts") is a recent addition to the list of popular recreational psychomotor-stimulant compounds. Relatively little information about this drug is available in the scientific literature, but popular media reports have driven recent drug control actions in the UK and several US States. Online user reports of subjective similarity to 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") prompted the current investigation of the thermoregulatory and locomotor effects of 4-MMC. Male Wistar and Sprague-Dawley rats were monitored after subcutaneous administration of 4-MMC (1-10 mg/kg ) using an implantable radiotelemetry system under conditions of low (23°C) and high (27°C) ambient temperature. A reliable reduction of body temperature was produced by 4-MMC in Wistar rats at 23°C or 27°C with only minimal effect in Sprague-Dawley rats. Increased locomotor activity was observed after 4-MMC administration in both strains with significantly more activity produced in the Sprague-Dawley strain. The 10 mg/kg s.c. dose evoked greater increase in extracellular serotonin, compared with dopamine, in the nucleus accumbens. Follow-up studies confirmed that the degree of locomotor stimulation produced by 10 mg/kg 4-MMC was nearly identical to that produced by 1 mg/kg d-methamphetamine in each strain. Furthermore, hypothermia produced by the serotonin 1(A/7) receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) was similar in each strain. These results show that the cathinone analog 4-MMC exhibits thermoregulatory and locomotor properties that are distinct from those established for methamphetamine or MDMA in prior work, despite recent evidence of neuropharmacological similarity with MDMA.

Exposure to a high-fat diet decreases sensitivity to Δ9-tetrahydrocannabinol-induced motor effects in female rats.

Arachidonic acid, a fatty acid component of neuronal cell membranes, forms the backbone of endogenous ligands of the endocannabinoid system. The lipid nature of this system may make it particularly susceptible to changes in fat content of the diet, which may, in turn, affect endocannabinoid tone and subsequent changes in receptor expression or activity. The latter would also be expected to affect responses to exogenous cannabinoids. The purpose of the present study was to determine the effects of a high-fat diet on sensitivity to the pharmacological effects of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Male and female Long-Evans rats were fed either a diet of standard rodent chow or chow enhanced with corn oil. Subsequently, they were repeatedly assessed for Δ(9)-THC-induced hypomobility, catalepsy and hypothermia. Female rats that received the high-fat diet beginning in adolescence or in adulthood became significantly less sensitive to the effects of Δ(9)-THC on motor behavior, but not its hypothermic effects, with faster development of decreased sensitivity in female rats that began the high-fat diet as adults. In contrast, diet-induced differences either did not occur, or were less pronounced, in male rats of both ages. After acute injection, brain and blood levels of Δ(9)-THC and its two primary metabolites were similar regardless of diet. Combined with the fact that diet differentially affected only some of the measures, these results suggest that pharmacokinetic differences cannot fully account for the effects of the high-fat diet on response to Δ(9)-THC. Further, these results suggest that dietary fat content may represent an important consideration in predicting the effects of marijuana in females.

Pharmacological effects of acute and repeated administration of Delta(9)-tetrahydrocannabinol in adolescent and adult rats.

Adolescents of many mammalian species exhibit rapid physiological change that is accompanied by behaviors such as increased risk taking and social interaction with peers. Marijuana abusers frequently report that their initial use occurred during adolescence. Our goal was to determine whether the in vivo effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) differed in adolescent and adult rats. Following initial testing with Delta(9)-THC in adolescent [postnatal day (PN)29] and adult (>PN60) rats of both sexes, we injected rats twice daily with 10 mg/kg Delta(9)-THC or vehicle for 9.5 days. Subsequently, rats were again injected with their initial dose of Delta(9)-THC and tested. In all rats, Delta(9)-THC produced dose-dependent locomotor suppression, antinociception, hypothermia and catalepsy. Some age-dependent differences in potency and efficacy were noted. Although Delta(9)-THC dose-effect functions were more similar across age after repeated exposure, subchronic dosing produced greater change in the hypothermic and locomotor effects of Delta(9)-THC in adolescents, but less change in its antinociceptive effects. These results suggest that the effects of initial exposure to Delta(9)-THC may not be entirely predictive of the effects of repeated exposure. Despite similarities in pharmacological effects of Delta(9)-THC after repeated use, adolescents and adults may exhibit differences in the pattern of transition from use to abuse.