Additionally, longitudinal epigenetic studies will allow for the examination of the impact of interventions on epigenetic changes. For example, longitudinal examination of smoking-induced DNA methylation patterns identified dynamic and stable markers across time and also observed reversal of smoking induced methylation changes after smoking cessation. Using repeated measures of DNA methylation and marijuana use, we crosssectionally identified numerous marijuana associated epigenetic markers associated at one time point but not the other , including 6 and 10 loci associated with both recent and cumulative marijuana use at Y15 and Y20, respectively. Additionally, one stable epigenetic marker, cg05575921, was associated with both marijuana variables across the examination years with consistent effect estimates . We also performed longitudinal analyses to investigate changes in methylation and marijuana use across the examinations and identified 12 CpGs that varied with change in marijuana use, including markers in AHRR, COL11A2, and TFEB. Together, these results suggest a majority of the observed marijuana associated epigenetic associations are dynamic, although stable epigenetic patterns maybe observed with marijuana use. Furthermore, the identification of dynamic markers across time suggests both recent and cumulative marijuana use may modulate epigenetic changes differently during the aging process. A possible explanation for the observation of different CpGs, as well as biological pathways and diseases, across the time points may relate to the pharmacokinetic properties influenced by age. For example,cannabis drying rack reductions in hepatic and renal clearance can increase the bio-availability of marijuana metabolites with prolongation of its half-life and subsequently, may impact molecular and cellular processes differently by age.

Consistent with our findings, dynamic epigenetic markers are more likely to be identified compared to stable markers during longitudinal analyses. However, further studies investigating the modulatory effects of marijuana on the epigenome on different age groups may provide additional insight. Moreover, changes in marijuana use may alter DNA methylation signatures, which may serve as biomarkers to evaluate continued or ceased marijuana use. Although additional studies are needed to evaluate these markers, our findings demonstrate marijuana may induce dynamic and stable epigenetic signatures that may have utilityas biomarkers for recent and cumulative marijuana use across time. The impact of lifestyle factors and behaviors on health is complex and often involves an integrative approach to elucidate the underlying biological processes. By investigating genetic contributions to methylation markers associated with marijuana use, we identified 650 cis-meQTLs, including 56 cis-meQTLs that mapped to traits in the NHGRI-EBI GWAS Catalog. Among the mapped traits, we observed consistent terms related to immunological factors, cardiovascular traits, and brain measurements. Marijuana use has been associated with alterations in white blood cell counts, blood pressure, and brain structures. Caffeine is the most consumed psychoactive substance in the world and induces dopamine release in the nucleus accumbens, a brain structure mediating pleasure and reward processing. Analogously, marijuana exerts similar effects on the nucleus accumbens via the endocannabinoid system, suggesting the pleasure and reward of caffeine and marijuana use share the same reward center. Additionally, we identified 198 DMRs associated with recent and cumulative use of marijuana at Y15 and Y20. Among the top DMRs, several regions have previously been associated with cognitive function, psychiatric disorders, and immune function. RNF39 was the most significant DMR in two of the four analyses and has previously been associated with general cognitive function and bipolar and major depressive disorders. TRIOBP is the most significant DMR associated with recent marijuana use at Y20 and has been associated with general cognitive function, schizophrenia, and basophil count.

Similarly, SH3RF3 has been associated with general cognitive ability, schizophrenia, and eosinophilia. Lastly ZFP57 has been associated with general cognitive ability, schizophrenia, autism, and rheumatoid arthritis. In sum, these findings suggest marijuana use shares common genetic and epigenetic pathways associated with immunological factors, cognitive function, and brain structures and may regulate similar molecular mechanisms and biological processes. These insights could help lead to the development of new preventive and predictive tools for marijuana-associated health outcomes. As a psychoactive substance, marijuana may modulate pathways and diseases associated with homeostasis and health outcomes. Our pathway analysis revealed differentially methylated markers over represented in pathways associated with cellular proliferation, hormone signaling, and infection. The MAPK signaling cascades are signaling pathways that regulate cellular proliferation, differentiation, and apoptosis; studies have suggested potential therapeutic benefits of CBD on cancer treatment via these pathways. With regard to hormones, the endocannabinoid system modulates dopaminergic neurons and acute use of tetrahydrocannabinol increases dopamine release and neuron activity, whereas long-term use has been associated with diminishing of the dopamine system. THC has also been shown to modulate oxytocin and areas of the brain associated with reward and addiction behaviors. Moreover, cannabinoids have been reported to promote progression of human papillomavirus positive head and neck squamous cell carcinoma, primarily through MAPK activation. Notably, a previous genome-wide DNA methylation study of marijuana identified the latter two pathways during pathway analysis. In addition to these biological pathways, differentially methylated genes associated with marijuana use were over represented in psychiatric diseases and spasticity. Marijuana use has been associated with several psychotic disorders including schizophrenia, bipolar disorder, autism, and psychosis , as well as substance-related disorders. Additionally THC and smoked marijuana have been shown to reduce spasticity among patients with multiple sclerosis and spinal cord injuries. Additionally, connections between the top marijuana associated pathways and diseases have been previously reported. For example, abnormalities in the MAPK signaling and dopamine pathways have been associated with schizophrenia, as well as the use of oxytocin for treatment of substance related disorders.

Collectively, we identified pathways and diseases over represented with marijuana-associated methylation markers, suggesting common epigenetic regulations which could serve as potential diagnostic and therapeutic targets for these related traits. The current CARDIA study leveraged repeated methylation levels and marijuana data to examine the association of marijuana use on DNA methylation. The availability of genetic data enabled the examination of potential genetic modulation of methylation markers associated with marijuana via meQTL analyses. Moreover, compared to other countries where residents use a mixture of marijuana and tobacco, CARDIA is a US-based cohort where mixing of marijuana and tobacco is less prevalent, allowing for a more complete examination of the independent associations of marijuana and tobacco smoking on DNA methylation. This study, however, is not without limitations. Although we identified biologically relevant epigenetic loci and replicated previously reported methylation markers, we were unable to replicate our findings in an independent study, and as such, the findings presented warrant validation. Residual confounding from additional factors, e.g., use of other or co-drug use and social support,vertical grow system may partially explain the observed associations. As marijuana use was considered illegal for most yearly examinations in CARDIA, use may have been under reported. However, at each examination, marijuana use was self-reported , collected at a research site , and participants’ responses were confidential . The route of administration of marijuana can also affect the onset, intensity, and duration of the psychoactive effects, as well as organ systems. Investigations into marijuana use via other routes of administration may provide novel additional insights, including the latter, which was not present during the time points in the current study but is becoming more widely used. Additionally, this study examined acute exposure to marijuana , compared to hyperacute exposure and investigations into DNA methylation changes due to hyperacute exposure may provide further insight into the acuity of exposure on epigenetic factors. And lastly, although CARDIA is a diverse cohort, Black and White participants were sampled from four centers across the US. As such, additional studies from more diverse populations across different geographical locations will enable for better generalizability of the findings presented here.Mas biomarkers for recent and cumulative marijuana use across time. The impact of lifestyle factors and behaviors on health is complex and often involves an integrative approach to elucidate the underlying biological processes. By investigating genetic contributions to methylation markers associated with marijuana use, we identified 650 cis-meQTLs, including 56 cis-meQTLs that mapped to traits in the NHGRI-EBI GWAS Catalog. Among the mapped traits, we observed consistent terms related to immunological factors, cardiovascular traits, and brain measurements. Marijuana use has been associated with alterations in white blood cell counts, blood pressure, and brain structures. We found the most significant mapped cis-meQTL has previously been associated with coffee consumption. Caffeine is the most consumed psychoactive substance in the world and induces dopamine release in the nucleus accumbens, a brain structure mediating pleasure and reward processing. Analogously, marijuana exerts similar effects on the nucleus accumbens via the endocannabinoid system, suggesting the pleasure and reward of caffeine and marijuana use share the same reward center.

Additionally, we identified 198 DMRs associated with recent and cumulative use of marijuana at Y15 and Y20. Among the top DMRs, several regions have previously been associated with cognitive function, psychiatric disorders, and immune function. RNF39 was the most significant DMR in two of the four analyses and has previously been associated with general cognitive function and bipolar and major depressive disorder. TRIOBP is the most significant DMR associated with recent marijuana use at Y20 and has been associated with general cognitive function, schizophrenia,and basophil count. Similarly, SH3RF3 has been associated with general cognitive ability, schizophrenia, and eosinophilia. Lastly ZFP57 has been associated with general cognitive ability , schizophrenia, autism, and rheumatoid arthritis. In sum, these findings suggest marijuana use shares common genetic and epigenetic pathways associated with immunological factors, cognitive function, and brain structures and may regulate similar molecular mechanisms and biological processes. These insights could help lead to the development of new preventive and predictive tools for marijuana-associated health outcomes. As a psychoactive substance, marijuana may modulate pathways and diseases associated with homeostasis and health outcomes. Our pathway analysis revealed differentially methylated markers over represented in pathways associated with cellular proliferation, hormone signaling, and infection. The MAPK signaling cascades are signaling pathways that regulate cellular proliferation, differentiation, and apoptosis; studies have suggested potential therapeutic benefits of CBD on cancer treatment via these pathways. With regard to hormones, the endocannabinoid system modulates dopaminergic neurons and acute use of tetrahydrocannabinol increases dopamine release and neuron activity, whereas long-term use has been associated with diminishing of the dopamine system. THC has also been shown to modulate oxytocin and areas of the brain associated with reward and addiction behaviors. Moreover, cannabinoids have been reported to promote progression of human papillomavirus positive head and neck squamous cell carcinoma, primarily through MAPK activation. Notably, a previous genome-wide DNA methylation study of marijuana identified the latter two pathways during pathway analysis. In addition to these biological pathways, differentially methylated genes associated with marijuana use were over represented in psychiatric diseases and spasticity. Marijuana use has been associated with several psychotic disorders including schizophrenia, bipolar disorder, autism, and psychosis, as well as substance-related disorders . Additionally THC and smoked marijuana have been shown to reduce spasticity among patients with multiple sclerosis and spinal cord injuries. Additionally, connections between the top marijuana associated pathways and diseases have been previously reported. For example, abnormalities in the MAPK signaling and dopamine pathways have been associated with schizophrenia, as well as the use of oxytocin for treatment of substance related disorders. Collectively, we identified pathways and diseases over represented with marijuana-associated methylation markers, suggesting common epigenetic regulations which could serve as potential diagnostic and therapeutic targets for these related traits. The current CARDIA study leveraged repeated methylation levels and marijuana data to examine the association of marijuana use on DNA methylation. The availability of genetic data enabled the examination of potential genetic modulation of methylation markers associated with marijuana via meQTL analyses. Moreover, compared to other countries where residents use a mixture of marijuana and tobacco, CARDIA is a US-based cohort where mixing of marijuana and tobacco is less prevalent, allowing for a more complete examination of the independent associations of marijuana and tobacco smoking on DNA methylation. This study, however, is not without limitations. Although we identified biologically relevant epigenetic loci and replicated previously reported methylation markers, we were unable to replicate our findings in an independent study, and as such, the findings presented warrant validation. Residual confounding from additional factors, e.g., use of other or co-drug use and social support, may partially explain the observed associations.