In our study, the patient group (SUD with predominant A4 use) showed significantly reduced BDNF levels and elevated TOS and OSI levels compared to HCs; in contrast, zonulin and TAS levels did not differ between groups. There was no correlation between craving score and biochemical parameters. Based on the ROC analysis, the cut-off values were found to be ≤2.51 pg/mL for BDNF, ≥14.51 μmol/L for TOS and ≥0.69 for OSI ( Figure 1 ).

Neurotrophins are a family of proteins important for many functions in the nervous system and are involved in cell growth and differentiation. Among neurotrophins, BDNF has been one of the most well-established. It is an extracellular signaling molecule promoting neurons development, plasticity and survival (21). BDNF has been examined in several psychiatric disorders and has been proposed as a putative biomarker in several disorders, including SUD (12). In a meta-analysis of 40 studies including 2238 patients with SUD and 2574 HCs, serum BDNF levels were found to be significantly reduced in active users compared to HCs. However, this divergence appeared to diminish during remission. Subgroup analysis revealed that BDNF levels were particularly lower in patients using alcohol and crack/cocaine, suggesting that BDNF may be a putative biomarker of interest in these populations (13). A further meta-analysis, consisting of 11 studies, revealed that changed blood BDNF levels were not significantly associated with cannabis use, suggesting a requirement of additional studies in order to elucidate the interactions among cannabis use and the neurotrophic factors (22). A study on 45 cannabis-dependent patients and 45 HCs found more elevated BDNF levels in patients with cannabis use disorder. These authors hypothesized that this increase may reflect a compensatory mechanism due to a loss of neuronal plasticity in addiction (14). In our research, patients had lower serum BDNF levels. This finding may suggest an impaired neuroplasticity and neuronal damage in central nervous system in A4 users. The finding of reduced BDNF in patients vs. healthy controls indicates that BDNF may be a useful biomarker in this population. Our findings suggest that a BDNF cut-off value of ≤ 2.51 pg/mL may be a reliable indicator of A4 use. However, these results should be supported by studies with larger samples.

Another important result of our research was the increased TOS, and OSI levels, which are important markers of oxidative stress, in patients with A4 use. In contrast, TAS were similar between patients and HCs. Recent studies have suggested that central and peripheral toxicities of SUD might be partially associated with a imbalance in the production of reactive oxygen species and the antioxidant defense systems. A meta-analysis reported higher oxidant markers and lower antioxidant markers in SUD patients than in HCs (6). In a study comparing 34 patients with cannabis use disorder and 34 HCs, it was found that patients with cannabis use disorder had higher levels of TOS, OSI, interleukin 1β, IL-6, IL-8, and tumor necrosis factor α compared to HCs. TAS, IL-12, and interferon γ levels were similar in both groups (11). The balance between oxidants and antioxidants is crucial for brain aging. Free radicals in the body cause oxidative damage to cellular and genetic material, leading to tissue and organ dysfunction, and ultimately resulting in death (11, 23, 24). All substance use can lead to oxidative stress and may contribute to SUD-related toxicity in the brain and peripheral nervous system. Additionally, the higher levels of oxidant markers compared to antioxidants at different stages of treatment in SUD patients can lead to cell dysfunction and death through the oxidation of DNA, proteins, or phospholipids. This can have significant clinical implications for early morbidity and mortality (25, 26). Our findings also indicate that there is an oxidative imbalance in patients with A4 use that can lead to cell damage and multiple morbidities, including accelerated aging. Indeed, while TAS levels are similar to HCs, TOS and OSI levels are much higher than those of HCs. This indicates that patients using synthetic cannabinoids (A4) are exposed to intense oxidative stress. Although oxidant markers were elevated in A4 users, antioxidant levels remained unchanged. This finding may suggest that increased oxidant production exceeded the buffering capacity of the antioxidant defense system, or that chronic substance use may weaken adaptive antioxidant responses. In addition, inflammatory markers such as CRP, IL-1β, IL-6, IL-8, and TNF-α, and antioxidant enzymes including glutathione peroxidase, catalase, and superoxide dismutase, play an important role in oxidative stress and immune regulation (27–29). Although these markers were not assessed in our study, they should be considered in future research to provide a more comprehensive understanding of the biochemical alterations associated with A4 use.

In our study, A4 users exhibited significantly reduced BDNF levels, together with elevated TOS and OSI, while TAS remained unchanged. This pattern may indicate that chronic A4 exposure impairs neuroplasticity and neuronal survival, reflected by decreased BDNF, whereas increased oxidant production exceeds the buffering capacity of the antioxidant defense system, leading to oxidative imbalance without a measurable rise in total antioxidant status. Unlike Δ9-THC, which is a partial agonist, most synthetic cannabinoids are full agonists of cannabinoid receptors. This results in greater efficacy, with more intense psychoactive effects and more severe side effects. Furthermore, synthetic cannabinoids blends lack cannabidiol, a compound present in cannabis that modulates cannabinoid receptor activity and helps counterbalance the psychoactive effects of Δ9-THC. The chemical structure of synthetic cannabinoids in A4 differs markedly from classical cannabis, generating numerous structurally diverse metabolites through CYP450 metabolism. Several of these metabolites have been associated with mitochondrial toxicity and enhanced reactive oxygen species production (7, 30). Taken together, these findings suggest a potential mechanism in which A4 use leads to excessive oxidative stress and impaired antioxidant responses, ultimately contributing to neurotoxicity and reduced BDNF levels. This mechanistic pathway may help explain the severe clinical consequences observed with A4 use and highlights the need for further translational studies.

In our study, the cut-off values were determined as ≥14.51 μmol/L for TOS and ≥0.69 for OSI. Particularly, BDNF along with TOS and OSI could be potential biomarkers for detecting the use of synthetic cannabinoids, which are extremely difficult to identify and pose significant issues in forensic psychiatry.

In our study, no difference was found in zonulin levels. Zonulin is a significant factor in intestinal permeability. It is a protein that increases intestinal permeability in the small intestine and contributes to the innate immunity of the gut. It is produced by the human small intestinal epithelium under the influence of environmental stimuli (31, 32). Substance use causes damage to the intestinal barrier. The intestinal barrier damage caused by SUD allows harmful substances and bacteria to pass through the intestinal barrier and enter the circulatory system, leading to systemic inflammatory responses and immune imbalances (15). However, there is no literature available that evaluates zonulin levels in SUD patients. In our study, zonulin levels were found to be similar to those of healthy controls. This may suggest that synthetic cannabinoids alone may not be sufficient to alter intestinal permeability or that intestinal permeability may also be influenced by other variables. Ultimately, intestinal permeability can be affected by many factors (diet, medications, lifestyle, infections) (33).