Our study showed significant changes in FBG, Crea, urea and ALT in Tac group (Table 1), suggesting that the Tac displayed a drug toxicity in the major target organs. Herein, the authors examined the metabolic profile of major organs (serum, brain, heart, liver, lung, kidney, and intestines) after Tac exposure. The authors observed alterations in 8, 7, 2, 5, 7, and 2 metabolites in the serum, brain, heart, liver, lung and kidney, respectively, between the Tac and control mice. Further examination of the significance of these metabolites in biological processes can supply additional intel into the Tac toxicity pathophysiology. Based on our analysis, these altered metabolites were the primary mediators of lipid, amino acid, and carbohydrate metabolites. Moreover, even though Tac is not known to produce serious adverse effects on the lipid profile, our data revealed that the lipid metabolism was strongly dysregulated in Tac mice. In fact, cholesterol, glycerol, MG (16:0/0:0/0:0), MG (0:0/18:0/0:0) were markedly altered. Interestingly, unlike our results, a prior study revealed that cholesterol and triglyceride were strongly diminished by Tac exposure . Hence, additional investigations are warranted to elucidate the true effect of Tac exposure on the lipid profile. In addition, the authors demonstrated that the MG (16:0/0:0/0:0) and MG (0:0/18:0/0:0) were elevated, which was likely due to enhanced lipolysis, in response to glucose metabolic disorders. Our data corroborates with the reported dyslipidemic effects of toxic dosages of Tac exposure.
Patients with diarrheal illness may demonstrate variability and potential toxicity following Tac exposure . However, in our study, the authors observed no significant metabolite alterations within the intestine. Tac metabolism is regulated by the CYP3A enzyme system, and occurs mostly in the small intestine, liver, and kidney . The upper small intestine is the primary site for CYP3A4-based first-pass metabolism in humans, which may explain the metabolic alternation in the intestine. However, additional studies are warranted to validate our results.
Post transplanted diabetes mellitus (PTDM)
Post transplanted diabetes is among the most severe Tac-mediated adverse effects . It is brought on by β-cell apoptosis, diminished insulin gene expression, and direct toxicity to the islets of Langerhans . Tac-mediated islet toxicity is most frequent during solid organ transplantation, and it has a prevalence of newly developed diabetes following transplant of up to 30% within the first year [21,22,23]. Our study uncovered several carbohydrate metabolite alterations in multiple tissues, namely, D-fructose, D-glucitol in the serum, D-Fructose in the heart, D-glucitol in the liver, and D-glucose in the kidney. These alterations indicate carbohydrate metabolism dysfunction following Tac-induced toxicity. Hence, this study confirmed that Tac exposure increases risk of developing PTDM.
Cardiotoxicity-related metabolic alterations
Although cardiotoxicity is relatively rare with Tac exposure, there are reports of arrhythmia and cardiomyopathy following Tac exposure [24, 25]. Although arteritis of cardiac arteries, hypertension, renal vasoconstriction  and reduced nitric oxide formation  are potential mechanisms of Tac-mediated toxicity, the true underlying mechanism remains undetermined. In this study, no specific metabolite alterations were observed in the heart tissue, although a marked increase in acetone may be suggestive of Tac-induced toxicity. The heart requires massive energy, and a constant supply of glucose, lipids, and amino acids to generate ATP to sustain a healthy heart beat . Markedly elevated concentrations of ketone bodies like acetone points to the impairment of the TCA cycle, and a switch from glucose oxidation to β oxidation of fatty acids indicates dysregulation within the energy metabolism .
Hepatic lesion-based metabolic alterations
Liver is essential for glycogen storage, protein synthesis, and detoxification . Emerging evidences suggest that Tac exposure elicits liver damage, and the hepatocytes form a ground-glass appearance, which is an early indicator of Tac toxicity . In terms of the biochemical indexes, ALT, alkaline phosphatase, and total bilirubin are established markers of hepatic lesion. Tac is reported to accelerate cholestasis by suppressing biliary excretion of glutathione .
Herein, the authors demonstrated marked elevation of L-sorbose, palmitic acid, myo-inositol, and uridine in the hepatic tissue. Among these metabolites, palmitic acid concentration was previously reported to be elevated during hepatotoxicity in drug toxicity-based investigations [32, 33]. This increase indicates a rise in de novo synthesis of fatty acids via an alternate axis involving ß-oxidation [34, 35]. Moreover, being a FFA, palmitic acid elicits an elevated hepatic cytotoxic outcome . Uridine minimizes cytotoxicity and improves neurophysiological activities [37, 38]. However, recently, clinical data suggested a direct association between plasma uridine levels and insulin resistance in humans [39, 40]. Prior investigations reported that short uridine exposure accelerated hepatic insulin resistance in C57BL/6 J mice . Combined with PTDM of Tac, uridine dysregulates glucose metabolism in the liver.
Lung toxicity-related metabolic changes
Currently, there are limited reports on Tac-mediated pulmonary toxicity. Akhtar reported that Tac causes structural distortion of the lungs in rats, characterized by alveolar cells necrosis, bronchiolar wall thickening, and interstitial round cell infiltration, which confirms the toxic potential of Tac in lungs . Additionally, a prior investigation revealed that the oxidative stress parameters and proinflammatory markers were significantly increased in rats with Tac-treatment, compared to controls .
In this study, the authors presented the metabolite alterations following Tac toxicity in lung tissues. Our study assessed the metabolic alterations of Tac-induced lung toxicity, and showed marked elevations in the levels of L-lactic acid, L-5-oxoproline, L-threonine, and phosphorylethanolamine in the Tac mice, relative to controls. Lactic acid, produced via aerobic glycolysis, is a potential early indicator of a reversible state in critically ill patients . L-5-oxoproline is an endogenously formed metabolite that elicits adverse effects at chronically elevated dosages. When present at high levels, L-5-oxoproline and lactic acid serve as metabotoxins, which may present as a toxicity index for Tac. Threonine is an immunostimulant which positively regulates thymus gland development, as well as cell immune defense activity. However, the role of threonine in Tac-induced lung toxicity remains unclear.
Neurotoxicity-related metabolic alterations
Tac-related neurotoxicity, mediated by elevated blood concentrations of Tac, produces headache, tremor, delirium, and peripheral neuropathy . In a large prospective study, significant neurological undesirable events were correlated with high plasma levels in > 50% patients . Metabolic investigations involving the whole brain of Tac-exposed mice are relatively rare. Hence, more research is warranted in this area to supplement what is known about Tac-mediated toxicity and its related mechanisms in major organs.
Herein, Tac-exposed mice showed alterations in aminomalonic acid, scyllo-inositol, dihydromorphine, myo-inositol, and 11-octadecenoic acid. Aminomalonic acid strongly inhibits L-asparagine synthase activity, and is up-regulated in the urine of anxiety and major depressive disorders-diagnosed individuals . Multiple metabolomic investigations also revealed that altered serum aminomalonic acid concentration was closely correlated with neuropsychiatric disorders, ketamine overdose, and aortic aneurysm , thus forming a link between aminomalonic acid and various diseases and toxicities. This study revealed that aminomalonic acid can serve as a potential indicator of Tac-mediated neurotoxicity. Scyllo-inositol improves brain cognitive function, reverses memory deficits, and minimizes amyloid-beta (Aβ) plaque within brains of mice . Thus, the low levels of scyllo-inositol in our study may be another indicator of Tac-mediated neurotoxicity.
Nephrotoxicity-related metabolic alterations
Nephrotoxicity is the most common and clinically significant adverse Tac reaction, and it occurs in almost half of the Tac-treated patients . Multiple reports demonstrated glycosuria to be an effective bioindicator of acute renal toxicity [50, 51]. Herein, D-Glucose was observed to be significantly altered, suggesting that the nephrotoxicity was successfully induced by Tac at the selected concentration. Additionally, the authors also observed marked increases in L-valine in our Tac-treated mice. Alteration in L-valine was also found in other toxic kidney’s injury studies [52, 53]. L-valine is a branched-chain amino acid that links only to carbohydrates (glycogenic) . Valine is intricately linked to insulin resistance, and elevated serum valine concentrations were reported in both diabetic mice and humans [55, 56]. Thus, it is obvious that L-valine participates in the progression of Tac-induced nephrotoxicity.
Herein, the authors systematically assessed Tac-toxicity using GC − MS-based profiling of target tissues. Toxicity is the most common and significant result of elevated blood Tac concentrations. Owing to its narrow therapeutic window, close monitoring of this therapeutic drug is imperative for treatment individualization. Unfortunately, it is still difficult to predict a particular dose that is optimal for a specific patient. Therefore, the effect of different dosages on metabolic profiles needs further investigation.
In conclusion, in this study, the authors reported an extensive metabolic profile following Tac exposure in mice. The authors demonstrated that the altered metabolism involved cellular processes like lipid, amino acid, and carbohydrate metabolism, which may, in turn, provide certain insight into the pathogenesis of Tac-mediated toxicity. Our work will greatly benefit clinicians and researchers, particularly in guiding Tac dosing, and to further understand the toxicological mechanism of Tac.