The molecular alterations were consistent with the observed phenotypes, as stress exposure induced anxiety like behavior in offspring with pre-gestational WIN exposure, compared to controls without pre-gestational WIN exposure. The epigenetic changes in the offspring could have been due to direct epigenetic modifications on the sperm or testes, as well as related to disruptions in the paternal endocannabinoid system. This animal model supports a transgenerational epigenetic effect of cannabinoid exposure potentially altering stress response in the offspring. However, global DNA methylation measurements lack gene specificity and therefore provide limited biological insights. Another study in rats examined the association between exposure to THC in male and female rats during adolescence and differentially methylated regions in the NAc of offspring using Enhanced Reduced Representation Bisulfite Sequencing. A total of 1027 DMRs, including 406 hypermethylated and 621 hypomethylated regions, were observed in exposed offspring, compared to unexposed controls with genes enriched for cell membrane function, synaptic organization, and receptor activity. The hypomethylated DMR in the first coding exon of glutamate ionotropic receptor NMDA type subunit 2A was consistent with Grin2a mRNA transcript expression differences observed in another rat study by the same research group. This is in line with the hypothesis that hypomethylation in gene bodies may lead to decreased gene expression. The Grin2a gene is involved in calcium channel activity and ionotropic glutamate receptor activity and mediates synaptic plasticity and transmission, with impacts on addictive behavior. The same authors that observed reduced Grin2a mRNA levels in the NAc also observed differential mRNA gene expression in different areas of the brain, depending on the time of evaluation in rats pregestationally exposed to THC. There was increased Cbr1, Grin2a, and Gria2 mRNA expression in pregestationally exposed rats,cannabis grow tray compared to controls at the adolescent time point, while there was a decrease in mRNA expression of Cbr1, Drd2, Grin1, Grin2a, Gria1, and Gria2 in the dorsal striatum in pre-gestationally exposed rats, compared to controls, at the adult time point.
The shift in mRNA expression from the adolescent to adult time point is consistent with the transition from reward-oriented to habitual, compulsive drug-taking that typifies progression from recreational drug use to addiction disorder. The same study authors further evaluated potential sex-specific effects and observed that sex-specific mRNA expression patterns were present in both the adolescent and adult brains. Overall, the findings contribute to evidence that parental history of germline THC exposure could possibly confer enhanced risk for psychiatric disorders in the subsequent generation, as a result of impaired epigenetic regulatory processes in relevant genes and pathways. One other study observed no changes in Drd2 mRNA levels in the NAc of adult rats exposed pre-gestationally to WIN and postnatally to morphine. The latter study, however, found higher opioid receptor mu 1 mRNA levels in WIN-exposed animals, compared to unexposed animals, following a morphine challenge. On the day of the morphine challenge, animals pre-gestationally exposed to WIN had an enhanced response to morphine, compared to controls. The Oprm1 gene encodes at least three opioid receptors in humans, and it is involved in dependence to drugs such as nicotine, cocaine, and alcohol via its modulation of the dopamine system. The study contributed to evidence that pre-gestational cannabis could induce addiction vulnerability in F1 offspring.In order to evaluate the impact of cannabis exposure during adulthood on the sperm methylome, one study examined DNA methylation in adult male subjects that had either cannabis or no cannabis use. Over 6000 CpG sites differed between cannabis and non-cannabis users. Specifically, prostaglandin I2 receptor methylation was inversely correlated with THC level, while casein kinase 1 epsilon methylation was associated with increased THC. The PTGIR gene is associated with reduced sperm fecundity, while CSNK1E phosphorylates period circadian regulator 2 and is implicated in sensitivity to opioids. DiscsLarge Associated Protein 2 was also hypomethylated in the sperm of cannabis-exposed men, compared to controls. The DGLAP2 gene encodes a membrane-associated protein that is involved in synapse organization and signaling in neuronal cells and is linked to psychological andneurological disorders, such as schizophrenia. It has also been identified as an autism candidate gene. While it is biallelically expressed in the brain, only the paternal allele is expressed in the testes due to imprinting. Another recent study by Schrott et al. 2019 further evaluated DNA methylation and mRNA transcript expression using the same study population as the study discussed above.
They first validated the findings related to DLGAP2 in the study discussed above using quantitative bisulfite pyrosequencing, instead of reduced representation bisulfite sequencing], which showed good agreement. The authors noted that it was one of 46 genes with more than 10 CpG sites showing altered DNA methylation in the sperm of cannabis users, compared to controls. They observed sperm hypomethylation of DLGAP2 at 17 CpGs in exposed adult men, compared to controls, in the RRBS assay. The differential DNA methylation was validated in DLGAP2 for nine CpG sites, plus an additional site, in intron 7 in the pyrosequencing assay. They further determined the functional association between DNA methylation and mRNA transcript expression in human brain tissue from terminated pregnancies, a relevant target tissue for the expression of DLGAP2. In these human brain samples, methylation of DLGAP2 intron 7 was inversely correlated with DLGAP2 mRNA expression and significant only in females.The same two studies in human sperm highlighted in the preceding section also validated their findings using male, sexually mature adult rat sperm from animals exposed to THC, compared to unexposed controls. In the first study, 627 genes had altered DNA methylation associated with THC exposure. There were six overlapping genes among the rat and human-exposed sperm, suggesting that these two pathways may be targets of THC exposure across species. Although the study focused on the F0 generation, some DNA methylation changes of non-imprinted genes in gametes can resist post-fertilization reprogramming and persist in the somatic cells of offspring. Supporting this hypothesis, the authors compared the 627 genes exhibiting DMRs in the rat sperm to the 473 DMR genes identified in the NAc of adult rats exposed to THC pregestationally in a study discussed earlier in this review. They found 55 overlapping DMR genes between these two datasets with significant enrichment, suggesting that THC-induced epigenetic modifications in sperm cells could persist in somatic cells. Important strengths of this study include some validation from similar observations in human sperm and in adult rat brain tissue. The same authors pooled data from a new set of sexually mature rats that were given intravenous THC, with the set of rats from Murphy et al. 2018 given oral THC, and identified a region of Dlgap2 that showed differential methylation in eight CpG sites in sperm between exposed and control groups. Hypomethylation at CpG site 2 was detected in the NAc of pre-gestationally exposed offspring , compared to controls, as well as in the sperm of the THC exposed fathers, compared to controls. The study provided evidence of potential intergenerational inheritance of epigenetic marks in Dlgap2, despite evidence from the same study that it is not an imprint control region. Finally, a recent study examined epigenetic and functional alterations in sperm of sexually mature mice exposed to the synthetic CBR2 agonist JWH-133.
Not only did exposed males have decreased sperm count, but their offspring demonstrated impaired placental development and reduced growth, compared to unexposed controls. This was accompanied by increased DNA methylation at the paternally expressed imprinted genes Peg 10 and Plagl1 in sperm, which was retained in the offspring placenta. Although the study highlights that overactivation of CBR2 can promote altered DNA methylation in sperm, which is retained in embryonic tissue and may cause altered offspring phenotypes, it could not confirm the precise effect that the epigenetic alterations may have on offspring. However, considering that cannabis is made up of numerous cannabinoids that could bind with CBR2, it adds to the body of evidence that pre-gestational cannabis may promote epigenetic changes in sperm cells that are functionally relevant in offspring.With its roots in international treaties signed during the League of Nations Era, the transnational legal order of cannabis prohibition represents one of the most sustained efforts to develop internationally applicable standards for governing illicit markets.The vast majority of United Nations member states are now parties to the three major international drug conventions,vertical grow systems for sale which require criminalizing the production, distribution, and use of cannabis. Over the past decades, the cannabis prohibition TLO has come to encompass an extensive array of legal instruments for monitoring implementation efforts,disseminating information on the activities of drug trafficking networks,and facilitating cooperation among national police forces.However, despite the extensive institutionalization of this TLO, cannabis remains the most widely used illegal drug in the world. The 2018 World Drug Report estimates that at least 192 million people aged 15–64 had used cannabis in the preceding year.With the percentage of adults reporting cannabis use in North American and European countries far exceeding the international average, cannabis use has become integrated into mainstream culture in a large number of countries.In an era that is often characterized as one of a growing isomorphism of the laws and procedures governing criminal activities in different countries,the issuearea of cannabis policy undergoes processes of fragmentation and polarization.Others have sought to “separate the market” for cannabis from that of other drugs by decriminalizing the possession of small amounts of marijuana, authorizing its use for medical purposes, and establishing administrative measures for taxing and regulating the commercial sale of the drug.
These reforms have gained international momentum despite resistance from key actors in the international drug control system, including the International Narcotic Control Board and the US federal government.The proliferation of cannabis liberalization reform is frequently depicted as a historical step toward the collapse not only of this TLO but of the entire edifice of the international narcotic control system of which it forms a part.How deep is the current crisis of the cannabis prohibition TLO? What are its causes and consequences? What does this case study reveal about the conditions under which criminal justice TLOs rise and fall? In this Article, I explore these questions to demonstrate the complex ways in which the cannabis prohibition TLO has served as a battleground between competing conceptions of the role of criminal law in addressing social and medical harms. Drawing on TLO theory,the Article shows that the capacity of the cannabis prohibition TLO to regulate the practices of legal actors at the international, national, and local levels has been eroded as a result of effective contestations of the input and output legitimacy of its governance endeavors. The rapid and widespread diffusion of new models of decriminalization, depenalization, and legalization has relied on the operation of mechanisms of recursive transnational lawmaking. These mechanisms originate from the indeterminacy of drug prohibition norms, the ideological contradictions between competing interpretations of their meaning, the impact of diagnostic struggles over the social issues that the international drug control system should address, and the mismatch between the actors shaping formal prohibition norms at the international level and those implementing these norms in national and local contexts. However, our analysis also shows that the cannabis prohibition TLO creates path-dependent trajectories that constrain the development of non-punitive strategies for regulating cannabis markets. In this context, the Article explains why it is too early to sound the death knell for the prohibitionist agenda of cannabis control. The dense array of UN treaties, transnational and regional monitoring schemes, national laws, and local enforcement arrangements put in place throughout the institutionalization of the cannabis prohibition TLO impede efforts to initiate more progressive regulatory innovations in this field. The Article is organized as follows: Section I briefly introduces the historical formation of the international legal framework governing cannabis regulations. It also identifies the inherent ambiguities giving rise to interpretive disagreements regarding the scope of application of cannabis prohibition norms. Section II examines the debates that evolved during the 1960s–70s regarding the criminological logic of drug prohibition policies and the cannabis liberalization reforms shaped by these debates. It then considers the processes leading to the reversal of these liberalizing trends and the extensive institutionalization of new measures reinforcing strict interpretations of the prohibition norms enshrined in the international treaties. Section III discusses the causes and consequences of the legitimation crisis that the cannabis prohibition TLO has experienced since the mid- 1990s as well as the global wave of depenalization, decriminalization, and legalization reforms precipitated by this crisis. Section IV considers the extent to which this wave of cannabis liberalization reform lessens the impact of the prohibitionist approach on the development of cannabis regulations at the international, national, and local levels.