The effects of substance use are increasing from both an economic and medical perspective in the United States. Research on SUD is continually evolving, driven by the shift towards precision medicine. The National Institute of Health (NIH) has started the Helping to End Addiction Long-term Initiative (HEAL), which is funding over 1000 projects nationwide focused on novel therapeutics [17]. To develop novel theories, research has transitioned to understanding the biological and genetic factors contributing to SUD [18]. Limited information on the frequency of KP IDO1/2, TDO2, and KMO genetic variants in SUD cohorts is available. Therefore, this present study is carried out to investigate the association of genetic polymorphisms of IDO1/2, TDO2, and KMO as biomarkers for increased risk of substance abuse in the AoU database. Our results indicated that fourteen of these polymorphisms had a significantly different frequency in at least one of the SUD cohorts than in the control cohort.

The metabolites of the KP play pivotal roles in several processes involved in neurodevelopment and mood regulation roles in biochemical processes. For example, KYNA can help regulate dopamine release and is currently being researched as a potential target for therapeutic intervention in SUD [5,19]. The involvement of TRP metabolism through the KP as it relates to the function of the dopamine reward system supports the search for possible associations between specific genetic variants of enzymes that regulate TRP metabolism as a genetic risk factor of SUD. Multiple enzymes catalyze the KP, and identifying genetic alterations in these enzymes can help to understand further imbalances in the metabolites of the KP and how that can impact recovery from SUD.

TDO2 is the enzyme catalyzing the initial rate-limiting step in TRP metabolism (Figure 1). Variants in the corresponding gene can impact the activity of the encoded protein and subsequently alter the KP metabolites [20]. TDO2 variants have previously been associated with various disorders, such as Tourette's syndrome, autism, and hypertryptophanemia [11]. In this study, findings for the alcohol cohort were consistent with previous findings from Soichoit et al., which showed that no significant variation in TDO2 variant frequencies was observed in the alcohol cohort [9]. Alternatively, a significant difference was seen in each of the other SUD cohorts. These SNPs could result in either increased or decreased enzyme activity, leading to altered concentrations of the KP metabolites compared to healthy controls. As an example, it was previously reported by Comings et al. that variants in the TDO2 promoter region resulted in an increase in KYN in patients with Tourette syndrome, which could also lead to an increase in QA [8]. These variants might also be expected to be increased in SUD cohorts, as SUD has been reported to have higher QA concentrations than individuals without SUD [21]. QA, a neurotoxic metabolite of KP, can induce an increase in the release of dopamine, which can increase the risk of SUD based on the enhanced release of dopamine. The alternate is true with KA, the neuroprotective metabolite, which can decrease dopamine release, helping with SUD recovery [5].

The IDO1 and IDO2 enzymes also catalyze the initial step, converting TRP to KYN in the KP. IDO1/2 variants have previously been associated with depression, depression treatment outcomes, Crohn’s disease, and systemic sclerosis [11]. Specifically, the rs9657182 SNP has been shown to increase the chances of depression during treatment for infectious disease [22]. Highlighting the role IDO1/2 variants play in depression and the potential role they could have in SUD. Lee et al. investigated the relationship between Crohn’s disease and IDO1/2 and found that with the rs35059413 SNP, serum samples had a decrease in KYN concentration and decreased KYN/TRP ratio [21]. Both the cannabis and cocaine cohorts had significantly increased percentages of participants with this polymorphism. This study further identified that variants within the genes encoding the enzymes of the KP can cause imbalances in the metabolites, potentially increasing the individual’s chance of multiple diseases, one of which being SUD. Two of the polymorphisms of the IDO1 gene are missense mutations. These have been identified as having diminished enzyme activity [11]. Neither the opioid nor the other cohorts had significant frequency differences in the polymorphisms of these genes.

KMO catalyzes the reaction of kynurenine through the neurotoxic pathway, resulting in QA and NAD+. A polymorphism in the KMO gene, rs1053230, has previously been identified to be associated with bipolar disorder [15]. Lavebratt et al. found that the variant C allele of the rs1053230 polymorphism is associated with lower KMO expression, increasing KA concentration. In this study, the cannabis and cocaine cohorts demonstrated a significantly decreased frequency of the variant, likely resulting in less of the KA metabolite being created and more of the neurotoxic metabolite, QA, forming in these individuals. This finding may further explain the habitual use of addictive substances because of the increase in dopamine release when QA is increased and KA is decreased. Neither the alcohol nor the “other” SUD cohort had a significant difference in the KMO gene. Studies have previously found imbalances in KP metabolites, and an increase in QA, a neurotoxic compound, has been seen in serum samples of individuals with SUD [5,21]. One explanation for the elevated QA is the significant difference in gene variance found in SUD cohorts. This increased knowledge about the genetic variants in the KP more frequently associated with SUD and their resulting impact on the metabolites of the KP pathway can aid in identifying therapeutic interventions designed to reestablish balance in the KP metabolites. Research in SUD has recently been focused on evaluation if targeting the KP for therapeutic intervention in SUD is an option [21]. Determining genetic variants of enzymes in the KP in SUD could lead to personalized pharmacotherapy, where genetic testing could be performed, and treatments are tailored to an individual’s metabolic. Targeted therapies could include modulators of specific enzymes to restore balance in the pathway and reduce neuroinflammation, potentially decreasing relapse and withdrawal symptoms. Additionally, neuroprotective agents or anti-inflammatory treatments could be developed to protect against the neurotoxic effects of metabolites such as quinolinic acid, improving cognitive function and overall treatment outcomes. One example is Ro 61-8048, a KMO inhibitor, which has shown promising results as a therapeutic option to help prevent relapse in cannabis and cocaine use disorder [18,19].

This study used a retrospective cohort from the AoU database, providing a large sample cohort with extensive clinical and genomic data. The strengths include the low cost of the study since the information is readily available, and the genomic frequencies were evaluated using data cohorts, which is lower in cost compared to extracting the data using Python or R. A study weakness is that haplotypes were not assessed within the cohorts and should be considered in follow-up research to understand penetrance better. Additionally, future research could determine if further correlations exist with the age of onset, the pattern of use habit, polysubstance abuse, and the timeframe of use to see if these cohort subsets strongly correlate to the genetic variants. Additionally, environmental factors that impact SUD, such as stress, trauma, peer influence, family environment, and socioeconomic status, were not evaluated in this study and could be a confounding factor. Future research could benefit from a prospective study where screening for potential confounding factors and personal surveys designed specifically for identifying potential environmental factors would eliminate the chance of these variables impacting the results. Further research could also exclude participants who have bipolar disorder, schizophrenia, depression, or other diagnoses that could confound the results of the study. These variables could be a reason why the cohorts had different polymorphisms that were seen at significantly different rates and should be controlled for in future studies. This correlation has been seen in different variants that are prevalent in SUD [19]. This research would further the understanding of this molecular mechanism on SUD.

Another limitation of the study was that KP metabolite concentrations for the participants were unavailable for the dataset. Limited research has been completed to determine the relationship of the genetic variants to the concentration of these metabolites in serum [11,12,14,15,20,21]. Based on these studies, expected changes in these metabolites could be hypothesized based on the presence of different variants but could not be confirmed. Additionally, while there is limited research on the alterations of the kynurenine pathway in these SUD cohorts, further studies can explore how alcohol, cannabis, cocaine, opioids, and other addictive substances influence the kynurenine pathway to gain a more comprehensive understanding of their relationships. Studies combining genetic variation data with observed KP metabolite concentrations could provide a further understanding of the association of these genetic variants. Understanding if these variants significantly differ in the SUD cohorts, alter or disrupt the enzyme regulation and activity, or cause protein instability, will help further understand these variants’ impact. This provides further knowledge on the potential for therapeutic targets and drugs that can alter the pathway to regulate the metabolites.