Tobacco induces abnormal metabolism of tryptophan via the kynurenine pathway

Introduction

Smoking is one of the most important causes of preventable disabilities, diseases, and premature deaths worldwide [1]. The World Health Organization revealed that approximately 7 million deaths annually are directly connected with tobacco use, and approximately 1.2 million people die due to exposure to cigarette smoke even though they are nonsmokers [2]. Tobacco smoke contains over 7,000 compounds, more than 50 of which have been identified as carcinogens. Some of these chemicals are carbon monoxide, hydrogen cyanide, benzene, formaldehyde, nicotine, phenol, polycyclic aromatic hydrocarbons (PAHs), cadmium, and nitrosamines [3]. Additionally, the most important reactive oxygen derivatives commonly found in cigarette smoke include hydroxyl radicals, superoxide anion radicals, peroxyl radicals, singlet oxygen radicals, and hydrogen peroxide [4]. Therefore, tobacco chemicals have toxic, carcinogenic, and mutagenic effects, and smoking affects immunity, and smoking compounds play an important role in the pathogenesis of many diseases such as respiratory diseases, cancer, cardiovascular diseases, neuropsychiatric diseases, inflammatory diseases, and allergies [5]. Although it is thought that cigarette compounds may play a role in the pathogenesis of these diseases by inducing oxidative stress, changing the inflammatory response and immune system functions, there is still a need for comprehensive studies on this subject, considering the complex chemical structure of tobacco [6].

Tryptophan is an essential amino acid that participates in protein synthesis and is used as the precursor of many neuroactive compounds. Although tryptophan is widely known as the precursor of serotonin and melatonin synthesis, a less well-known metabolic route of tryptophan degradation, the kynurenine pathway, has recently been the subject of an increasing number of studies [7]. The kynurenine pathway is responsible for the catabolism of more than 95 % of tryptophan, except those used in protein synthesis. Under physiological conditions, the kynurenine pathway provides de novo NAD⁺ synthesis [8]. Firstly, tryptophan is metabolized to kynurenine by indoleamine 2,3-dioxygenase (IDO-1) or tryptophan dioxygenase (TDO) enzymes. This is the first and rate-limiting step of the kynurenine pathway [9]. Kynurenine is further metabolized into a series of compounds known as kynurenines, such as 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid, quinolinic acid, picolinic acid, and xanthurenic acid [10]. Although the importance of the kynurenine pathway was initially attributed to its role in NAD⁺ synthesis, subsequent studies have shown that kynurenines have important neurological effects and play an important role in oxidant/antioxidant status, inflammatory response, and immune system regulation. Recently, increasing evidence has shown that abnormal kynurenine pathways may be related to the pathogenesis of many neurological, autoimmune, inflammatory, cardiovascular, and malignant diseases [11], [12], [13], [14]. Considering the role of tobacco chemicals in these diseases and their effects on the immune system, neurological system, and cardiovascular systems. This study aimed to investigate the influence of smoking on the metabolism of kynurenine and thus contribute to the elucidation of the potential mechanism of action of cigarette smoking.

Materials and methods

Results

Participants were matched for age and gender (p=0.582). Although the systolic (p=0.153) and diastolic blood pressures (p=0.148) of smokers were higher than those of nonsmokers, this difference was not statistically significant. However, the FRS of the smoker group was statistically significantly higher than the control group (p<0.001). The study group was also BMI-matched (p=0.833). When FTND was evaluated in the smoker group, it was determined that 13 (15.9 %) of the participants had low, 26 (31.7 %) had medium, and 43 (52.4 %) had high nicotine addiction. The demographic parameters of participants were expressed in Table 1.

Statistical comparison of routine laboratory parameters showed that MCV, MPV, WBC, NEU, LYM, and TG levels were statistically significantly higher in smokers (p<0.05), while MCHC and HDL-C levels were statistically significantly lower (p<0.05). A comparison of routine laboratory parameters of participants was shown in Table 2.

Statistical analysis of kynurenines revealed that serum tryptophan concentrations (p=0.040) were statistically significantly lower in smokers, and the kynurenine/tryptophan ratio and serum kynurenine, kynurenic acid, quinolinic acid, 3-hydroxyanthranilic acid levels were high (p<0.001). There was no statistically significant difference between 3-hydroxykynurenine levels (p=0.688). Kynurenine pathway metabolite levels of participants were revealed in Table 3.

Correlation analysis was conducted by separating the smoker and nonsmoker groups and evaluating each group within itself. Our correlation analysis in the smoker group showed that kynurenic acid levels positively correlated with FTDN (r=0.294, p=0.009). The serum kynurenine levels were positively correlated with the levels of total cholesterol (r=0.260, p=0.023), TG (r=0.301, p=0.008), LDL-C (r=0.264, p=0.021), and FRS (r=0.268, p=0.019). There was a positive correlation between serum quinolinic acid levels and participants’ systolic (r=0.257, p=0.023) and diastolic blood pressures (r=0.251, p=0.026). The kynurenine/tryptophan ratio was positively correlated with FRS (r=0.265, p=0.022) and total cholesterol levels (r=0.267, p=0.020). In nonsmokers, there was a positive correlation between the kynurenine/tryptophan ratio and MPV (r=0.317, p=0.008), WBC (r=0.314, p=0.012), PLT (r=0.329, p=0.007), and NEU (r=0.302, p=0.039) levels.

Linear regression analysis revealed a statistically significant positive association (B=0.293, p=0.011) between serum kynurenine levels and TG levels in smokers. Linear regression analysis demonstrated significant associations of kynurenine/tryptophan ratio with TC levels (B=0.306, p=0.035). There was a positive association (B=0.293, p=0.009) between kynurenic acid levels and FTDN. In the control group, regression analysis showed positive associations between the kynurenine/tryptophan ratio and MPV (B=0.581, p<0.001) and PLT (B=0.387, p=0.004) levels.

Discussion

According to our findings, serum tryptophan levels were statistically significantly lower in smokers, and the kynurenine/tryptophan ratio and serum kynurenine, kynurenic acid, quinolinic acid, 3-hydroxyanthranilic acid levels were higher.

Firstly, the importance of the kynurenine pathway was attributed to its role in NAD⁺ synthesis and energy production. In addition, early studies revealed kynurenines may be related to neurodegenerative and psychiatric conditions. However, further investigations have shown that kynurenine pathway metabolites may be regulated by the immune system, oxidant/antioxidant balance, and inflammatory response. Thus, the relationship of kynurenines with various autoimmune, malignant, and chronic inflammatory disorders has been revealed. The findings coming from various research have also expressed that kynurenines play a role in blood pressure regulation and endothelial function and that disturbances of kynurenines may be connected with the pathogenesis of cardiovascular diseases [21], 22]. Smoking triggers immunological response and vascular damage and modulates inflammation. Smoking has been shown to play a role in the pathogenesis of various diseases such as pulmonary, chronic inflammatory, autoimmune, cardiovascular, and neurodegenerative diseases [23], [24], [25], [26]. However, related to the complex chemical composition of cigarette tobacco, further investigations are needed to clarify the potential mechanisms between smoking and these pathologies. Considering their role in common pathologies, we aimed to investigate whether changes in kynurenine pathway metabolite levels may play a role in smoking-related comorbidities.

Our findings showed reduced serum tryptophan, elevated serum kynurenine levels, and therefore increased kynurenine/tryptophan ratio in smokers. Additionally, the increase in further metabolite levels of the kynurenine route showed that tryptophan degradation was induced in smokers through the kynurenine pathway. Only a few studies have explored the kynurenine pathway in smokers and these studies provide contradictory results. One of them is the research executed by Pertovaara et al. [27]. The kynurenine-tryptophan ratio was applied in this study to assess IDO activity, and the findings indicated that serum IDO activity in smokers was lower than in nonsmokers. Given that activities were similar in nonsmokers and smokers who had not smoked in the two days before the study, this effect was strong and immediate but short-lived. Among individuals who had smoked, but not within the last two days and as long as 10 years ago, IDO levels were comparable to those of never-smokers. Therefore, according to this study, the influence of smoking on IDO was significant, swift, but temporary, and reversible if smoking was not smoked. However, in this study, researchers hypothesized that this effect of smoking may be related to nicotine, excluding other chemicals in the cigarette composition and conditions such as oxidative stress caused by smoking [27]. Önder et al. measured kynurenine and tryptophan levels in individuals with periodontal inflammation. The findings reveal that smokers had a higher salivary kynurenine/tryptophan ratio than nonsmokers, while the serum ratio was greater in nonsmokers. In this study, decreased kynurenine levels in smokers were attributed to the reduction of interferon-γ (IFN-γ) production and therefore IDO activity via carbon monoxide, while high salivary kynurenine levels in smokers were attributed to the dominant effect of exacerbated periodontal inflammation [28]. When two studies evaluating the effect of smoking on kynurenines are evaluated together, they indicate that the effect of smoking on the kynurenine pathway varies depending on the severity of cigarette addiction including the smoking intensity, and the timing since the last cigarette exposure, inflammatory load and inflammation severity. However, in 2022 Liang et al. obtained findings contrary to these two studies. This study showed that chemicals in cigarette composition induce tryptophan metabolism through the induction of IDO. Contrary to other studies, high IDO activity and plasma kynurenine/tryptophan ratio were detected in non-small-cell lung cancer (NSCLC) patients who smoke. According to the research group, carcinogens in tobacco smoke increase tryptophan catabolism by inducing IDO, thereby suppressing the immune response to promote carcinogenesis. Moreover, while the plasma kynurenine/tryptophan ratio was observed to be considerably higher in current smokers compared to nonsmokers, the plasma kynurenine/tryptophan ratio was found to be slightly higher in current smokers than in former smokers, although it was not statistically significant. In smokers, a lower plasma tryptophan/kynurenine ratio was linked to a poorer prognosis, whereas in nonsmoking patients, a lower plasma tryptophan/kynurenine ratio was associated with slightly shorter survival [29].

Our findings suggest a decline in kynurenine/tryptophan levels in smokers, while kynurenine levels and kynurenine/tryptophan ratio elevated relative to the control group. Furthermore, besides kynurenine, levels of 3-hydroxyanthranilic acid, kynurenic acid, and quinolinic acid were also significantly elevated in smokers. Consequently, our findings revealed an increase in IDO activity in smokers, and tryptophan catabolism was induced via kynurenine. Studies indicating that IDO activity is reduced in smokers have hypothesized that this effect may be due to the reduction of IFN-γ production, especially by nicotine and carbon monoxide, and therefore to the downregulation of all biochemical pathways induced by IFN-γ. However, as Önder et al. showed in smokers’ saliva samples, this situation is related to the severity of inflammation, while as Pertovaara et al. showed, it is related to many conditions such as the duration after tobacco exposure and the severity of addiction [27], 28]. In contrast to these studies, one of the carcinogenic tobacco compounds nicotine-derived nitrosaminoketone has been shown to upregulate IDO1 through induction of the transcription factor c-Jun, known to bind to the IDO1 promoter [29]. ​ In another study, it was reported that polycyclic aromatic hydrocarbons, which are also found in cigarette composition, triggered the aryl hydrocarbon receptor (AhR)-IDO axis, leading to increased tryptophan metabolism and elevated kynurenine levels [30]. As pointed out in these studies, chemicals in tobacco composition can activate the IDO enzyme through different mechanisms. However, studies on the toxic effects of these components are still limited. It was found that systemic inflammation indicators of smokers such as WBC, MCV, MPV, and NEU were higher than in nonsmokers. Although these findings initially suggested that the induced kynurenine pathway in smokers might be associated with increased inflammation, as a result of our correlation analysis, no relationship was detected between kynurenine or the kynurenine/tryptophan ratio and these inflammatory markers in smokers. In the control group, a positive correlation was detected between the kynurenine/tryptophan ratio and MPV, NEU, PLT, and WBC. Our correlation findings suggest that enhanced activity of the kynurenine pathway in smokers may be affected by the special mechanisms we mentioned above, which are independent of inflammation and involve a number of tobacco chemicals. In addition, while no relationship could be detected between kynurenine levels and blood lipid levels in the control group, a positive association was observed between FRS, TC, TG, and LDL-C in the smoker group. Numerous studies have highlighted the role of kynurenine in cardiovascular pathologies. For instance, among individuals with renal artery occlusion, elevated plasma angiotensin II levels were positively associated with plasma kynurenine levels and negatively associated with tryptophan levels [31]. Cason and colleagues investigated the link between tryptophan, indole metabolites, and kynurenine derivatives and the presence of advanced atherosclerosis in 100 individuals who had carotid endarterectomy or limb revascularization surgery. This research demonstrated that tryptophan levels were strongly associated with a reduced risk of advanced atherosclerosis, while the kynurenine/tryptophan ratio was positively related to advanced atherosclerotic plaque. Furthermore, the kynurenine/tryptophan ratio was linked with a higher likelihood of postoperative cardiac complications and major adverse events during follow-up [32]. According to the Hordaland Health Study, an increased kynurenine/tryptophan ratio forecasts greater risk for acute coronary incidents, such as unstable angina, non-fatal or fatal myocardial infarction, and sudden death in patients [33]. Zinellu et al. observed that cholesterol-lowering treatment led to decreased kynurenine levels and a reduced kynurenine/tryptophan ratio [34]. Tan et al. showed that all kynurenine metabolites increased in overweight or obese children and that there was a positive correlation between kynurenine, quinolinic acid, xanthurenic acid, hydroxyanthranilic acid levels, and body fat percentage [35]. Moreover, recent findings suggest that atherosclerosis development is strongly associated with AhR functionality, with kynurenine serving as an AhR agonist [36]. Our findings, when evaluated together with these studies, show that increased kynurenine and decreased tryptophan levels in smokers may be associated with cardiovascular and metabolic comorbidities.

The accelerated metabolism of tryptophan via the kynurenine pathway results in the formation of a number of biologically active metabolites such as 3-hydroxykynurenine, 3-hydroxyanthranilic acid, kynurenic acid, quinolinic acid, in addition to kynurenine. Our findings showed that serum levels of these metabolites increased in smokers compared to nonsmokers.

3-Hydroxykynurenine and 3-hydroxyanthranilic acid are metabolites of this pathway with dual antioxidant/pro-oxidant properties. While these metabolites show free radical scavenger and antioxidant properties under normal physiological conditions, they gain oxidant properties as a result of abnormal increases in their concentrations in inflammatory conditions and in case of increased oxidative stress. They bind to amino groups of proteins under oxidative conditions, causing cross-linking of polypeptide chains, lipid peroxidation, and structural modifications in nucleic acids, thus predisposing to many oxidative stress-related diseases [37].

Kynurenic acid and quinolinic acid are metabolites of the kynurenine pathway with neuromodulatory properties. Kynurenic acid is an antagonist of N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), and kainate receptors as well as α7 nicotinic acetylcholine receptor (α7nAChR) receptors. It has neuroprotective properties due to its effects on these receptors. However, it also shows peripheral effects due to its effect on G-protein-coupled receptor 35 (GPR35) and AhR. Particularly its anti-inflammatory and immunomodulatory effects are attributed to its effect on these receptors [38]. Quinolinic acid, unlike kynurenic acid, has neurotoxic effects with its agonistic activity on NMDA receptors. Quinolinic acid causes oxidative stress by increasing the production of reactive oxygen derivatives, thus showing its excitotoxic effects. In addition, it shows peripheral effects by being involved in bone metabolism through NMDA receptors expressed in osteoblasts and osteoclasts [39], 40]. Our findings showed that both quinolinic acid and kynurenic acid levels are increased in smokers. Considering the oxidant, inflammatory, and neurotoxic effects of quinolinic acid, we think that the increase of this metabolite in smokers may be a risk factor for neurological disorders and disorders related to bone metabolism in smokers. Also, correlation analysis showed a positive correlation between serum quinolinic acid levels and participants’ systolic and diastolic blood pressures. Quinolinic acid has agonistic activity on NMDA receptors. It is known that glutamatergic activity, in which glutamate, an excitatory amino acid, plays a role through NMDA receptors, has an important role in the central nervous system. However, cerebral and aortic endothelial cells and smooth muscle cells also express NMDA receptors. NMDA receptor activation in cerebral or aortic endothelium causes endothelial dysfunction, inflammatory cell infiltration, oxidative stress, and proliferation while triggering proliferation and extracellular matrix restructuring in aortic smooth muscle cells. Therefore, it is thought that quinolinic acid may have an effect on the hemodynamic system due to its effect mediated by endothelial NMDA receptors [41].

​However, the increase in kynurenic acid, which has opposite effects with quinolinic acid, suggests that there may be an increase in the levels of this metabolite, especially as a compensatory [42]. Our correlation analysis in the smoker group showed that there was a positive correlation between serum kynurenic acid levels and FTDN. Considering the anti-inflammatory, anti-exitotoxic, and neuroprotective effects of kynurenic acid, our hypothesis that kynurenic acid levels may have increased compensation to tobacco exposure due to nicotine addiction is strengthened.

Conclusions

Our findings showed that tryptophan metabolism via the kynurenine pathway was induced in smokers. It is thought that this induction may be related to specific mechanisms triggered by chemicals found in cigarette composition. Additionally, our other hypothesis is that metabolites formed by activation of the kynurenine pathway may play a role in the pathogenesis of smoking-related comorbidities. However, no significant relationship was found between the amount of cigarette consumption and kynurenines in our study. For this reason, further studies are needed to measure cigarette compounds, especially cotinine, in a larger population. The limitations of our study are that it was a cross-sectional study, was conducted with a limited number of participants, and cotinine measurements were missing in the participants.