The Mercapturomic Profile of Health and Non-Communicable Diseases

The mercapturate pathway is a unique metabolic circuitry that detoxifies electrophiles upon adducts formation with glutathione. Since its discovery over a century ago, most of the knowledge on the mercapturate pathway has been provided from biomonitoring studies on environmental exposure to toxicants. However, the mercapturate pathway-related metabolites that is formed in humans—the mercapturomic profile—in health and disease is yet to be established. In this paper, we put forward the hypothesis that these metabolites are key pathophysiologic factors behind the onset and development of non-communicable chronic inflammatory diseases. This review goes from the evidence in the formation of endogenous metabolites undergoing the mercapturate pathway to the methodologies for their assessment and their association with cancer and respiratory, neurologic and cardiometabolic diseases.


Brief Overview of the Mercapturate Pathway
The mercapturate pathway is one of the key traits of renal proximal tubular cells, although it is also present in hepatocytes [1]. The main function of this pathway is to detoxify electrophilic species [2]. These electrophiles might arise either from the metabolism of endogenous substances or from exogenous compounds (or their biotransformation products) present in air, food or water [3][4][5][6][7]. Once generated in any cell and upon conjugation with glutathione (GSH) (Figure 1), an electrophile-GSH-S-conjugate is formed [8]. As cells are not able to metabolize these conjugates intracellularly, those conjugates are effluxed into the bloodstream to undergo the mercapturate pathway. Thus GSH-S-conjugates are the precursors that will generate mercapturates, through the three sequential steps that constitute this pathway. The first two steps are extracellular and generate cysteinyl-glycine-S-conjugates (CysGly-S-conjugates) and cysteine-S-conjugates (Cys-S-conjugates) by the membrane-bound-enzymes, gamma-glutamyl-transferase (GGT) and dipeptidase or aminopeptidase-M, respectively [9][10][11]. Despite their presence in tissues such as liver, small intestine, lung, brain, spleen and pancreas, the main local of expression of these enzymes is the kidney tubule [12]. The Cys-S-conjugates enter the renal tubular cells and hepatocytes via various transporters including organic anion transport polypeptides and cystine/cysteine transporters for the last reaction of the mercapturate pathway [12][13][14]. The last CysLTs have also been associated to silica-induced lung fibrogenesis [49]. In fact, increased LTE4 levels were observed in exhaled breath condensate of patients with pneumoconiosis derived from asbestos and silica exposure [39].

Cancer
Cys-S-conjugates have also been described in different types of cancer, namely in melanoma, non-Hodgkin lymphoma, breast, ovarian and thyroid cancer ( Table 2).
Melanoma was linked to the melanin metabolite 5-S-Cys-DOPA (Cys-DOPA). In melanocytes, the amino acid L-DOPA is oxidized into a highly reactive dopaquinone that after binding to a sulfhydryl donor as glutathione is further oxidized to pheomelanin, a yellow to reddish form of melanin. Increases in serum Cys-DOPA have been associated with poor prognosis of malignant melanoma and shorter survival times [50][51][52][53][54]. Additionally, Cys-DOPA also increased in melanoma recurrence after chemotherapy or surgery [50,53,54].
Estrogen metabolism is strongly implicated in the development of hormonal cancers [55][56][57]. Estrogen metabolites, namely 2-and 4-hydroxyestrone and 2-and 4-hydroxyestradiol might generate electrophilic metabolites, and for mercapturate pathway-related metabolites their urinary levels were found to be decreased in patients with breast cancer or non-Hodgkin lymphoma relative to healthy subjects [58][59][60]. Additionally, the ratio of depurinating estrogen deoxyribonucleic acid (DNA) adducts to estrogen metabolites and conjugates (including GSH-S-conjugates, Cys-S-conjugates and mercapturates) was higher in cases of thyroid and ovarian cancer in comparison with healthy individuals [56,57]. Changes in Cys-S-conjugates that are disulfides were also observed in leukemia, lymphoma and colorectal adenoma [61,62].

Neurologic Diseases
Parkinson's disease (PD) is characterized by severe depletion of dopamine (DA) [63]. The role of dopamine related cysteinyl-S-conjugates in PD has been investigated in order to evaluate how the failure of anti-oxidative mechanisms, in the prevention of spontaneous dopamine oxidation, might contribute the degeneration of dopaminergic neurons (Table 3).
Dopamine can be oxidized following non-enzymatic and enzymatic pathways. Dopamine can spontaneously oxidize to dopamine-o-quinone, which forms conjugates GSH-S-conjugates. Dopamine can also be oxidized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde, which is further metabolized by aldehyde dehydrogenase to 3,4-dihydrophenylacetic acid (DOPAC) and then into homovanillic acid upon catechol-O-methyltransferase activity [64,65].
In 1989, Fornstedt and colleagues [66] identified 5-Cys-S-conjugates of DOPA, DA and DOPAC in three brain regions (substantia nigra, putamen and caudate nucleus) of post-mortem brains from patients with and without depigmentation and neuronal loss within the substantia nigra. The levels of DOPA, DA and DOPAC were decreased in the depigmented group. Additionally, while no differences were found for the Cys-S-conjugates, the authors observed an increase in the ratio of Cys-DA/DA and Cys-DOPAC/DOPAC in the substantia nigra and Cys-DOPA/DOPA in the putamen of the depigmented group [66]. Similar results were later obtained with patients with PD and parkinsonism (PD and multiple system atrophy parkinsonism). Importantly, patients were not on DOPA therapy. The levels of Cys-DA were not affected in patients with parkinsonism. Nevertheless, as DOPAC or homovanillic acid were decreased, both Cys-DA/DOPAC or Cys-DA/homovanillic acid ratios were increased in these patients [67,68]. The work of Goldstein and co-authors [67] also showed that Cys-DA and DOPAC have the same source: the cytoplasmic dopamine. Thus, the dopamine denervation associated with parkinsonism would be expected to produce equal proportional decreases in Cys-DA and DOPAC levels and consequently unchanged Cys-DA/DOPAC ratios. The authors were not able to explain the observed decrease in DOPAC without the decrease in Cys-DA [67]. Even though, the authors suggested that this might be due to decreased antioxidant capacity [69] and aldehyde dehydrogenase activity [70]. Interestingly, substantia nigra of PD patients has a 50% reduction of their GSH levels [71,72]. This decrease can be presumably due to the reaction of GSH with DA semiquinones or quinones [73]. At the same time, decreased antioxidant capacity might shift the balance from dopamine to dopamine quinone and finally to Cys-DA, which will explain the absence of decreased levels Cys-DA. In opposition, there is one study reporting increased levels of 5-S-Cys-conjugates of DOPA, DA and DOPAC at substantia nigra of patients with PD. However, all patients were under L-DOPA treatment, which could have influenced the results [74].
Catechol estrogens are also present in the brain and, like dopamine, can be bioactivated to catechol quinones able to form adducts with GSH and undergo the mercapturate pathway for elimination. Urinary estrogen-catechol Cys-S-conjugates were lower and estrogen-DNA adducts were higher in PD patients than in healthy controls [75]. The authors suggested that there is an unbalanced estrogen metabolism in PD and that the protective pathways might be unable to avoid the oxidation of catechol estrogens and further DNA adducts formation.
On the other hand, neuro-inflammation might also play a role in autism [76,77]. The levels of CysLTs have been investigated in autistic children, together with a sensitive indicator of bioactive products of lipid peroxidation and oxidative stress, the 8-isoprostane [78,79]. The authors proposed both CysLTs and 8-isoprostane as markers for early recognition of sensory dysfunction in autistic patients that might facilitate earlier interventions [78].
CysLTs increases at the central nervous system [80][81][82], might also be involved in edema formation in brain tumor patients [83].  Ratio depurinating estrogen-DNA adducts to estrogen metabolites and S-conjugates in thyroid cancer > CTL group

Ovarian cancer
Investigate the role of estrogen metabolites and adducts in ovarian cancer Ref [56] Urine samples from 33 women with ovarian cancer (mean age 58 yo) and 34 CTL women (mean age 58 yo). The ratio of depurinating estrogen DNA adducts to estrogen metabolites and S-conjugates was obtained. The estrogen metabolites included Cys, GSH and NAC conjugates of 4-OHE 1 (E 2 ) and 2-OHE 1 (E 2 ).
Ratio depurinating estrogen DNA adducts to estrogen metabolites and S-conjugates in ovarian cancer > CTL.

Breast cancer
Evaluate the urinary levels of estrogen metabolites and adducts in breast cancer Urine samples from 12 women with high-risk for breast cancer (mean age 52 yo), 17 with breast cancer (mean age 54 yo) and 46 CTLs (mean age 50 yo). The ratio of depurinating estrogen DNA adducts to metabolites was obtained. Estrogen metabolites included Cys, GSH and NAC conjugates of 4-OHE1(E2) and 2-OHE1(E2). Ref [58] Cys, GSH and NAC conjugates of 2-OHE 1 (E 2 ) in CTL > other groups.

Autism
Determine the correlation of 8-isoprostane, LTs, age and autism severity scales Ref [78] Plasma samples from 44 autistic children (mean age 7 yo) and 40 CTLs (mean age 7 yo). Autistic cases were all simple and tested negative for the fragile X gene mutations *LTs measured = LTA4 + LTC4 + LTD4 + LTE4

Cardiometabolic Diseases
There are several works reporting the association of CysLTs in cardiometabolic diseases (Table 4) and different mechanisms might explain this association. For instance, in cardiometabolic diseases, the 5-lipoxygenase pathway that contributes to CysLTs formation is activated, the CysLTs receptors (mainly CysLT2R) are strongly expressed in cardiac, endothelial and vascular smooth muscle cells. CysLTs exert negative inotropic action on the myocardium and mediate coronary vasoconstriction [84]. Moreover, CysLTs may have pro-atherogenic effects; they may stimulate proliferation and migration of arterial smooth muscle cells and platelet activation [36].
Winking and collaborators (1998) measured urinary LTs in patients suffering from spontaneous intracerebral hemorrhage. Urinary LTC4, LTD4 and LTE4 levels were positively associated with hematoma volume and decreased after hematoma removal by surgery [85].
Regarding coronary artery diseases, urinary LTE4 levels were increased in patients admitted in the hospital with acute chest pain derived from acute myocardial infarction and unstable angina compared with controls [86]. Likewise, urinary LTE4 levels were higher in patients with chronic stable angina than controls before surgery [87]. In another study, urine and plasma levels of CysLTs increased during, and after, cardiac surgery with cardiopulmonary bypass. Interestingly, that increment was greater in patients with moderate-to-severe chronic obstructive pulmonary disease than in patients without this condition [88]. The authors hypothesize that these differences may be related to neutrophil activation and higher lung and airway production of CysLTs in patients with chronic obstructive pulmonary disease.
CysTL were also evaluated in individuals with atherosclerosis lesions in the carotid artery concomitantly with or without periodontal disease. This study was motivated by several reports that have been associating periodontal disease with the development of early atherosclerosis and increased risk of myocardial infarction [89][90][91]. The sum of LTC4, LTD4 and LTE4 was increased in gingival crevicular fluid in subjects with higher dental plaque and also in subjects with atherosclerotic plaques in the carotid artery, regardless of periodontal status [92].
CysLTs might play a role in the development of the cardiovascular complications associated with obstructive sleep apnea (OSA). Urinary LTE4 levels were associated with obesity and hypoxia severity in patients diagnosed with OSA. Continuous positive air treatment decreased LTE4 by 22% only in OSA patients with normal body max index (BMI). Additionally, LTE4 levels were higher in non-obese OSA patients vs. matched controls [93]. In another study, Gautier-Veyret and co-authors (2018) found that urinary LTE4 levels were independently associated with age, history of cardiovascular events and severity of hypoxia in patients with OSA with and without previous cardiovascular events. As such, LTE4 levels were higher in OSA patients with no previous cardiovascular events than in controls with no previous cardiovascular events. Urinary LTE4 levels were also associated with intima-media thickness, suggesting the activation of CysLTs pathway as a driver of vascular remodeling in OSA [94].
CysLTs were also evaluated in patients with diabetes. Urinary LTE4 levels were higher in patients with type 1 diabetes than in controls [95] and decreased 32% after intensive insulin treatment [96]. These results suggest that hyperglycemia activates arachidonic acid metabolism and consequent CysLTs formation. Interestingly, glucose can also generate Cys-S-conjugates that are far more stable than glucose-GSH. In specific, higher urinary levels of glucose-Cys were detected in patients with diabetes [4].
Cys-S-conjugates that are disulfides were related with hypertension, diabetes and Framingham risk score in coronary heart disease patient [97,98] as well as impaired microvascular function and greater epicardial necrotic core [97]. Moreover, these conjugates and GSH-Cys-S-conjugates were independent predictors of endothelium-dependent vasodilation [97].  Subjects with atherosclerotic plaques in periodontitis > in CTL CysLT higher in periodontitis with higher dental plaque index CysLTs in subjects with > subjects without atherosclerotic plaques in all subjects independently on periodontitis Patients undergoing cardiac surgery with CPB: nine moderate-to-severe COPD (69 yo; 78% men) + 10 non-smoker no COPD patients (64 yo; 60% men). Urine and plasma at baseline, end of CPB, after CPB and 2 h after admission in ICU. CysLTs = LTC4 + LTD4 + LTE4 ↑ urine CysLTs with time in both groups, but more evident in COPD patients; Plasma Cys LTs baseline < at admission to ICU in patients with COPD Assess LTE4 during and after acute coronary syndromes Ref [86] Urine samples from 16 AMI (mean age 51 yo; 88% men); 14 UA patients (mean age 52 yo; 21% men); eight clinical CTLs (non-ischemic heart pain) (88% mean) and 10 normal CTLs (50% men) CTLs (non-evidence of coronary artery disease). Samples collected upon admission with acute chest pain and 3 days after.
LTE4 in MIA and UA at admission > CTL groups. LTE4 on admission > 3 days after UA.

LC-MS Normalization to urine creatinine
Ref [58][59][60]75] LC-MS Normalization with depurinating estrogen DNA adducts Ref [56,57] Glucose-cysteine   CysSSCys Plasma Blood collected in heparin tubes and immediately placed in preservation buffer containing BPDS. The supernatant was added to ice-cold 10% perchloric acid in 10 µmol gamma-glutamylglutamate before freezing LC-FD Ref [61] Blood collected in sodium heparin tubes and transferred into specially prepared tubes with preservative containing serine, sodium heparin, BPDS, iodoacetic acid and borate. Supernatant transferred into a tube containing 10% ice-cold perchloric acid and 0.2 M boric acid solution LC-FD Ref [97] Blood collected in EDTA tubes. After centrifugation, butylated hydroxytoluene and salicylic acid as lipid and aqueous antioxidants were added before freezing LC-FD Ref [62] Aliquots were preserved in a 5% perchloric acid solution containing iodoacetic acid (6.7 µmol/L) and boric acid (0.1 mol/L) before freezing LC-FD Ref [48] Blood collected into heparin tubes and transferred into a preservative solution before freezing LC-MS Ref [103] 2

Methods in Mercapturates Profiling
Mercapturate pathway-related metabolites and their profile might be useful as biomarkers in characterizing human exposure to electrophilic endogenous substrates and its relation to health and disease. The methodological strategies herein reviewed for the determination of mercapturate pathway-related metabolites are presented in Table 5. These compounds have been measured in different human fluids and tissues requiring pre-treatment of samples. The studies herein reviewed quantify only one type or family of mercapturate pathway-related metabolites (dopamine, estrogens, cysteinyl-leukotrienes and cysteinyl-S-conjugates which are disulfides). Those metabolites were quantified by different methodologies including liquid chromatography with ultraviolet detector or fluorescence detector or mass spectrometry detector, enzyme-linked immunosorbent assay and radioimmunoassay (Table 5).

Trends and Limitations
Herein we review the clinical studies that reported associations between, one of or a family of, mercapturate pathway-related metabolites with a particular disease. In fact, most of available evidence on the association of the mercapturomic profile with health and disease has been obtained by a targeted approach (Figure 2). Future work might focus on a comprehensive qualitative and quantitative analysis of the totality of mercapturate-pathway related metabolites, in similarity to what has been done for protein addutomics [104].
One of the main limitations to assess the global mercapturomic profile is the fact that the mercapturate pathway-related metabolites are often minor metabolites [105]. Despite the enormous technological advances in MS instrumentation, the identification of this minor adducts is still challenging. New approaches are needed for providing accuracy and sensitivity along with quantitative information. The future obstacles will involve not only sample pre-treatment procedures, but also optimization of MS and data analysis strategies.
On the other hand, in vivo models of disease will allow to investigate the origin and metabolism of these compounds as well as their distribution in the body. In fact, these compounds have been described to be found in several matrices, including tissues, urine, plasma, exhaled breath condensate, saliva, polymorphic blood mononuclear cells or gingival crevicular fluid that might require different pre-treatment procedures.

Innovative Potential
Many chronic diseases with an inflammatory component display significantly increased levels of electrophiles. The mercapturomic profile might represent a useful tool to globally characterize both environmental and internal electrophile exposomes and its relation to disease ( Figure 2). This holistic omic-approach is expected to provide unique information that includes the identification of new therapeutic targets and commonalities related to mechanisms of different diseases that might facilitate therapeutics development and define preventive strategies. Additionally, this approach might constitute an effective tool to define the mercapturomic phenotypes of drug resistance and adverse reactions; disease progression, encouraging precision medicine standards. Finally, as many environmental compounds undergo this pathway it will also contribute to a better understanding of the contribution of environment to non-communicable diseases.

Conflicts of Interest:
The authors declare no conflict of interests