SAGE is one of the genome-wide association studies funded as part of the Gene Environment Association Studies under GEI. Assistance with phenotype harmonization and genotype cleaning, as well as with general study coordination, was provided by the GENEVA Coordinating Center . Assistance with data cleaning was provided by the National Center for Biotechnology Information. Support for collection of datasets and samples was provided by the Collaborative Study on the Genetics of Alcoholism , the Collaborative Genetic Study of Nicotine Dependence and the Family Study of Cocaine Dependence . Funding support for genotyping, which was performed at the Johns Hopkins University Center for Inherited Disease Research, was provided by the NIH GEI , the National Institute onAlcohol Abuse and Alcoholism, the National Institute on Drug Abuse, and the NIH contract ‘High throughput genotyping for studying the genetic contributions to human disease’ .Although pain perception is thought to be controlled mainly by neurotransmitter systems that operate within the CNS10 , antinociceptive mechanisms also occur in peripheral tissues. For example, endogenous opioid peptides released from activated immune cells during inflammation inhibit pain transmission by interacting with opioid receptors on peripheral sensory nerve endings11 . To determine whether endogenous cannabinoids have an analogous function to that of opioids, we used the formalin test, a behavioural model of injury-induced acute and tonic pain12 . Injection of dilute formalin into the hind paws of freely moving rodents evokes a pain behaviour consisting of two temporally distinct phases of licking and flexing o f t he injected limb12 . An early phase involving acute activation of pain-sensing C fifibres13 begins immediately after formalin administration, reaches a peak within 5 min, and then rapidly declines. After an interval of 10–15 min, a second phase of sustained pain behaviour appears, in which sensory fibre activity is accompanied by inflammation14 and central sensitization15 .In mice, vertical grow rack the early phase of pain behaviour was blocked when anandamide was injected into the paw together with formalin, whereas both phases were blocked by the synthetic cannabinoid agonists WIN-55212-2 and HU-210 .
These analgesic effectswere prevented by systemic administration of the CB1 antagonist SR141716A , but not of the CB2 antagonist SR144528 16 or of the opioid antagonist naloxone . The lack of effect of anandamide on late-phase pain behaviour may be explained by the short lifespan of this compound, which undergoes rapid biological inactivation in tissues17,18 . In support of this suggestion, the inactivation-resistant analogue methanandamide inhibited pain behaviour during the entire test . Local anandamide injections were not accompanied by central signs of cannabimimetic activity, indicating a peripheral site of action. To test this possibility, we measured the antinociceptive potency of anandamide following local , intravenous or intraperitoneal administrations. Anandamide was 100 times more potent in preventing formalin-evoked pain behaviour when injected locally rather than intravenously, with half-maximal inhibitory doses of 0.1 mg per kg and 10 mg per kg, respectively . Anandamide had no significant effect when injected intraperitoneally . As a further test, we determined the bio-distribution of 3H-labelled anandamide 10 min after i.pl. injection in rats. In three experiments, we found that 94% of recovered 3H-labelled anandamide remained associated with the injected paw , whereas little or no radioactivity above background was detected in forebrain, cerebellum and spinal cord . These results indicate that anandamide inhibits nociception after formalin injection by activating CB1-like receptors, which may be located on peripheral endings of sensory neurons involved in pain transmission. Anandamide is the ethanolamide of arachidonic acid and is thought to be produced by phosphodiesterase-mediated cleavage of N-arachidonyl phosphatidylethanolamine9,19 . Enzymatic cleavage of other N-acyl phosphatidylethanolamines may give rise to additional fatty acylethanolamides, the physiological roles of which are still poorly understood20 . PEA, an acylethanolamide found in neural and non-neural tissues, inhibits mast-cell activation and reduces inflammatory r esponses21,22 b y a m echanism t hat may involve binding to CB2-like receptors23 . The molecular identity of these receptors is unknown, although they are likely to be distinct from the CB2 receptors whose encoding genes have been cloned, as PEA shows little or no affinity for these receptors24 . We found that PEA, but not two closely related analogues, inhibited both early and late phases of formalin-evoked pain behaviour after i.pl. injection in mice . This effect may not be explained by the anti-inflammatory properties of PEA; 30 min after injection, the analgesic effects of PEA were not accompanied by a reduction in inflammatory oedema , which became apparent only 1 h after formalin administration.
The analgesia produced by PEA was reversed by administration of the CB2 antagonist SR144528 , whereas the CB1 antagonist SR141716A and the opioid antagonist naloxone were ineffective . In addition, PEA was more potent when administered locally than systemically . Together, these results indicate that PEA exerts antinociceptive effects that are mediated by peripheral CB2-like receptors. The cellular localization of such receptors and their possible structural relationship with the CB2 receptor whose gene has been cloned, which is primarily expressed in immune cells25, are unknown. The fact that anandamide and PEA activate pharmacologically distinct receptors and that these two substances can be produced simultaneously in tissues9 prompted us to examine their possible interactions in vivo. When injected together in equal amounts, anandamide and PEA inhibited the early phase of formalin-evoked pain behaviour with a potency that was approximately 100-fold greater than each of the compounds separately . A similar synergistic potentiation occurred in the late phase, on which anandamide had no effect when given alone . Earlier administration of either CB1 or CB2 antagonists entirely blocked the response . This interaction did not appear to involve pain-processing structures within the brain: injection of PEA in the cerebral ventricles did not affect the behavioural responses to acute thermal stimuli, assessed in the hot-plate test, and did not enhance the inhibitory activity of anandamide administered by the same route . These results indicate that the parallel activation of peripheral CB1- and CB2-like receptors by anandamide and PEA results in a synergistic inhibition of peripheral pain initiation. To test this idea further, we determined the intrinsic effects of CB1 and CB2 antagonists on formalin-evoked pain behaviour . Blockade of CB1 receptors with SR141716A produced significant hyperalgesia. This effectwas particularly pronounced after local injection of the drug, which resulted in a 10-min prolongation of the early nociceptive phase and in a two- to threefold increase in pain behaviour during the entire testing period . In contrast, systemic administration of the CB2 antagonist SR144528 caused a selective enhancement of early-phase, but not of latephase, responses . The selectivity of this effect may not result from a rapid elimination of SR144528 after early phase, as the drug reversed PEA-induced antinociception during both early- and late-phase pain behaviour . SR144528 could not be administered locally because of its limited solubility in the injection vehicle. Although the hyperalgesic effects of CB1 and CB2 antagonists may be accounted for by their inverse agonist properties16,26 , two lines of evidence suggest that these drugs acted by removing an endogenous cannabinoid tone. First, gas-chromatography/massspectrometry analysesshowed that anandamide and PEAare present in at paw skin . By comparison with internal deuterated standards, we measured 49 6 9 pmol of anandamide and 692 6 119 pmol of PEA per g of tissue . These amounts are five- to tenfold higher than those measured by the same method in rat brain and plasma27,28 , and are probably sufficient to activate cannabinoid receptors7,23 . Second, the CB2 antagonist SR144528 enhanced nociception selectively during the early phase of the formalin response , a result inconsistent with an inverse agonist effect. Thus, a parsimonious interpretation of our findings is that endogenous PEA acting at CB2-like receptors may participate in attenuating the early stages of nociception, whereas endogenous anandamide acting at CB1-like receptors may have a more sustained modulatory effect on both acute and tonic pain. Our results show that the tonic activation of local CB1-like and CB2-like receptors may regulate pain initiation in cutaneous tissue. These findings s upport t he p ossibility t hat e ndogenous cannabinoids, in addition to their spinal and supraspinal sites of action8 ,cannabis grow racks may participate in buffering emerging pain signals at sites of tissue injury. The results also indicate that selective agonists for the CB2-like receptor activated by PEA, or peripherally administered CB1-like/CB2-like agonists, may reduce pain without the dysphoric side effects and perceived abuse potential typical of centrally acting cannabimimetic or opiate drugs. Note added in proof: After submission of this paper, Jaggar et al. 29 have observed an analgesic effect of systemically administered PEA in formalin-induced pain.The District of Columbia and 15 US states – Alaska, Arizona, California, Colorado, Illinois, Maine, Massachusetts, Michigan, Montana, Nevada, New Jersey, Oregon, South Dakota, Vermont, and Washington – have legalized recreational marijuana sales, but few research studies have measured exposure to secondhand marijuana smoke in everyday settings.
On January 1, 2018, California legalized the sale of recreational cannabis to adults, and the state currently has 358 state-licensed stores selling recreational cannabis products . Many studies have measured the psychoactive compound delta-9- tetrahydrocannabinol and other related cannabinoids produced by marijuana use . Berthet et al. identified 958 papers on passive exposure to cannabis, and they selected 21 papers for review. These passive exposure studies generally employed biomarkers of exposure such as urine, blood, oral fluid, hair, and sebum to determine for forensic purposes whether an individual had recently used cannabis. For example, Moore et al. asked 10 healthy volunteers who were not marijuana smokers to spend up to 3 h in a Dutch coffee shop with heavy marijuana smoking. THC exceeding 4 ng/ml was detected in the oral fluid of half the volunteers but not the metabolite 11-nor-9-carboxy-THC , so the authors recommended measuring this metabolite as an indicator to avoid falsely concluding a person was an active cannabis smoker. We reviewed 729 papers on exposure to marijuana in the scientific literature, and we found almost no published papers measuring fine particle mass concentrations from secondhand cannabis smoke in homes. Both marijuana and tobacco cigarettes produce fine particle mass concentrations consisting of airborne particles less than 2.5 μm in diameter. Klepeis et al. and Posis et al. reported results from one of the few studies that measured indoor particles, a randomized clinical survey in San Diego of 298 predominantly low-income homes with an adult smoker and a child less than 14 years old. In each participating residence, a Dylos™ DC1700 monitor was set up for a week to measure indoor particle counts. Homes without indoor smoking had 7-day average particle levels lower than homes with only cannabis smoking or homes with both cigarette and cannabis smoking, and 33 homes reported that marijuana smoking took place from 1 to 7 times per week.The Dylos monitor provides an indication of particle levels, but it does not measure particle mass concentrations with the same accuracy as the gravimetric filter-and-pump “gold standard,” or a research-grade air monitor with its calibration factor based on gravimetric filter measurements. The common methods of smoking marijuana include a pre-rolled joint, which is similar to a cigarette or a cigar, a pipe or bong containing marijuana buds, and a vaping pen that vaporizes cannabis liquid from a commercially available cartridge. A nationwide survey of 4269 adults in 2014 found that 7.2% had used marijuana over the past 30 days . Among current users, 10.5% reported medicinal use only, 53.4% reported recreational use only, and 36.1% reported both. More than half of current users reported only one method of use ; 22.4% reported two methods; and 18.8% reported three methods. For these users, the two most popular methods of smoking marijuana were the joint and the pipe , with less popular use of the bong, water pipe, and hookah . In 2014, 7.6% of the respondents reported using marijuana vaporizers, but more recently battery-powered pens using liquid cannabis cartridges have become increasingly popular for cannabis vaping. In 2015, 5.3–8.0 million children in the US lived with a parent who was a cannabis user, and both current cannabis use and daily cannabis use have been increasing among parents .