The legalization of the use of cannabis for medical and recreational purposes has encouraged regular usage to become increasingly popular. It is one of the most frequently used psychoactive substances worldwide [19], and as a result, the number of research studies covering all aspects of the dependency on the substance, including epigenetic modifications, has grown. In the present study, we analyzed 33 CpG sites located in the promoter region of the DAT1 gene in cannabis-dependent subjects and in controls. The results of our analyses showed significant differences between both groups—methylation changes are not similar across all sites. In comparison with the control group, some sites in the dependent subjects were hypermethylated, while some were hypomethylated. Further, the assessment of the ability of transcription factors to bind indicated sites revealed a substantial number of those regulators of gene expression.

Three of the analyzed sites were found to be possible PAX5 transcription factor binding sites (positions 3, 22, and 33), and all of them were hypomethylated in the group of dependent subjects. This is crucial, since PAX5 is a transcription factor associated with numerous processes including the development of the nervous system [34]. Unfortunately, we did not analyze the expression of the DAT1 gene in our study group, so we are unable to fully anticipate what role this finding may have in relation to dopaminergic transmission. Additionally, the transcription’s binding ability revealed that the CpG island covers a sequence that may be bound by ligand-bound GR (position 6). As we demonstrated, this was the site that showed a significant difference in methylation status after the Bonferroni correction. This may be of vital importance since glucocorticoids mediate nervous system functions in substance dependency [35]. In response to this, we concluded that hindering glucocorticoid responses could change drug-induced reactions, including the modulation of drug-related learning and memory. Additionally, we postulated that GR may become a promising target in substance use disorders and dependency therapy [36,37].

As of yet, no studies regarding the DAT1 methylation status have been performed in the context of cannabinoid dependency, and it is difficult to say if any direct association may be revealed. Further studies on animal models with the use of DAT1 knock-out animals or chromatin immunoprecipitation (ChIP) experiments should be performed to reveal which TF or other proteins may bind DNA in the DAT1 promoter region. However, since neural development and functionality is determined throughout one’s lifetime, it could not produce expected results. With this said, among possible TFs, which may bind the DAT1 promoter in selected sites, SP1 can be found. In general, SP1 is a type of low specificity transcription factor with a wide spectrum of target sequences.

There is the possibility, however, that the DAT1 methylation status might either predispose or reflect exposure to substances in dependent individuals. Therefore, we do not know if it is more of a prognostic or diagnostic marker. However, we do know that, functionally, epigenetic alterations affect the expression of genes, as measured by RNA and protein synthesis; this may in turn affect the cellular structure and function and consequently affect the whole body’s metabolism and behavior. With regard to drug-taking and -seeking behavior, the preclinical literature demonstrated that changes in the expression of genes, such as brain-derived neurotrophic factor (BDNF) [38] and OPRM1 (the μ-opioid receptor gene) [39,40], alter the reinforcement properties of drugs including alcohol, cocaine, and heroin. Thus, epigenetic changes and resultant changes in gene expression may also contribute to our mechanistic understanding of substance dependencies.

Peripheral tissues have important limitations with respect to generalizability to other tissues of interest, such as the brain. However, there is a growing amount of evidence that proves that the numerous epigenetic changes found in peripheral leukocytes and transformed lymphoblasts also correspond to alterations in the brain cells [41]. Most importantly, a study performed in the previous year demonstrated that peripheral DAT1 promoter methylation may be a predictive factor of dopamine transporter availability in the striatum [27].

Another important factor that should be considered when analyzing the epigenetic effects of THC exposure is the age of a given exposure. Recent animal studies [42,43] showed that the hippocampus, the amygdala, and the nucleus accumbens are impacted differently by chronic THC consumption, depending on age. Histone modifications in particular regions were also affected differently. The adolescent brain showed changes leading to transcriptional repression, whereas the effect on adults showed transcriptional activation. In adolescent hippocampus and nucleus accumbens, the initial repression was followed by a counterbalancing response. The authors suggested that this may have resulted from the adolescent brain being more vulnerable to the adverse effects of THC [42]. Similarly, adolescent exposure was shown to alter plasticity genes, which are important in the development of cognitive deficits. The process was mediated by histone modifications and the altered prefrontal cortex [43].

Conclusively, we think that DAT1 methylation as well as methylation in general should be more extensively investigated in the context of substance dependency pathogenesis. Future studies should involve women and not only men, as in our case, as well as subjects of non-Caucasian origin. Because genetic and epigenetic markers vary by sex and race, the conclusions that can be drawn from this study are limited. Similarly, expression studies of DAT1 should be performed to fully evaluate the impact of epigenetic modifications and other genes, as well as their role in dependency. There have been some reports showing that blood derived samples reveal a significantly lower expression of DAT1 in substance-dependent patients than in controls [44], but without significant alterations in DAT1 methylation [44,45]. However, both studies included opioid-dependent patients, which may constitute a crucial difference to one’s cannabinoid dependency. Findings for DAT1 in other substance use disorders suggested broader implications for this subset of psychiatric disorder. This is crucial given the high rates of comorbidity among psychiatric disorders and the potential for comorbidities as confounding factors [46,47]. Therefore, it seems that DAT1 gene promoter methylation studies may provide important insights into the mechanism of the development of substance dependency.

There is currently very little research concerning epigenetic, and specifically, methylation changes in addiction. To the best of our knowledge, there is no study analyzing the methylation of the few genes in the same group of subjects that also incorporates a full phenotypic description of addiction (i.e., substance(s), age of onset, number of relapse, and many others), psychometric testing, and single nucleotide polymorphism (SNP) data, so the question of replicability is still open.