Mass Spectrometry Imaging of Amino Acids Enabled by Quaternized Pyridinium Salt MALDI Probe.

The distribution of small biomolecules, particularly amino acids (AAs), differs between normal cells and cancer cells. Imaging this distribution is crucial for gaining a deeper understanding of their physiological and pathological significance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) provides accurate in situ visualization information. However, the analysis of AAs remains challenging due to the background interference by conventional MALDI matrices. On tissue chemical derivatization (OTCD) MSI serves as an important approach to resolve this issue. We designed, synthesized, and tested a series of pyridinium salt probes and screened out the 1-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-2,4,6-triphenylpyridin-1-ium (DCT) probe with the highest reaction efficiency and the most effective detection. Moreover, a quantum chemistry calculation was executed to address mechanistic insight into the chemical nature of the novel probes. DCT was found to map 20 common AAs in normal mouse tissues for the first time, which allowed differentiation of AA distribution in normal, normal interstitium, tumor, and tumor interstitium regions and provided potential mechanistic insights for cancer research and other biomedical studies.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Chelerythrine Chloride is an Affinity-Labeling Inactivator of CYP3A4 by Modification of Cysteine239.

Chelerythrine chloride (CHE) is a quaternary benzo[c]phenanthridine alkaloid with an iminium group that was found to cause time- and concentration-dependent inhibition of CYP3A4. The loss of CYP3A4 activity was independent of NADPH. CYP3A4 competitive inhibitor ketoconazole and nucleophile N-acetylcysteine (NAC) slowed the inactivation. No recovery of CYP3A4 activity was observed after dialysis. Dihydrochelerythrine hardly inhibited CYP3A4, suggesting that the iminium group was primarily responsible for the inactivation. UV spectral analysis revealed that the maximal absorbance of CHE produced a significant red-shift after being mixed with NAC, suggesting that 1,2-addition possibly took place between the sulfhydryl group of NAC and iminium group of CHE. Molecular dynamics simulation and site-direct mutagenesis studies demonstrated that modification of Cys239 by the iminium group of CHE attributed to the inactivation. In conclusion, CHE is an affinity-labeling inactivator of CYP3A4. The observed enzyme inactivation resulted from the modification of Cys239 of CYP3A4 by the iminium group of CHE.

Mechanism-Based Inactivation of CYP2D6 by Phellopterin and Related Drug-Drug Interaction with Metoprolol.

Phellopterin (PLP) is a linear furanocoumarin widely found in citrus fruits and herbal medicines. The study aims to comprehensively investigate the mechanism of inhibition of CYP2D6 enzyme activity by PLP and its alteration of metoprolol pharmacokinetics. PLP was found to irreversibly inhibit CYP2D6 in time-, concentration-, and nicotinamide adenine dinucleotide phosphate-dependent manners. Coincubation with quinidine, which is a competitive inhibitor of CYP2D6, attenuated this time-dependent inhibition. Glutathione (GSH) and catalase/superoxide dismutase failed to reverse the PLP-induced CYP2D6 inactivation. GSH trapping experiments provided strong evidence that PLP metabolic activation produces epoxide or γ-ketoaldehyde intermediates. In addition, pretreatment with PLP resulted in significant increases in Cmax and area under curve of plasma metoprolol in rats.

Glutathione conjugation and protein modification resulting from metabolic activation of pesticide metalaxyl in vitro and in vivo.

Metalaxyl (MTL), a germicidal agent, is widely used in agriculture. Due to the biological amplification effect, MTL entering the ecological environment would result in a threat to human health through the food chain. MTL is reportedly accumulated in liver. The objectives of the study included investigating the metabolic activation of MTL in liver and defining the mechanisms participating in the hepatotoxicity of MTL. The corresponding glutathione (GSH), N-acetylcysteine (NAC) conjugate, and cysteine conjugates were observed in liver microsomes, prepared from liver tissues of mice, containing MTL and GSH, NAC or cysteine. These conjugates were also detected in urine and bile of rats receiving MTL. Apparently, MTL was biotransformed to a quinone imine intermediate dose-dependently attacking the thiols and cysteine residues of protein. The bioactivation of MTL required cytochrome P450 enzymes, and CYP3A dominated the bio-activation of MTL.

Metabolic activation and cytotoxicity of metaxalone mediated by cytochrome P450 enzymes and sulfotransferases.

Metaxalone (MTX) is a central nervous system (CNS) depressant used for the treatment of acute skeletal muscle pain. Several cases of fatal overdose deaths in the clinical use of MTX, along with the presence of ischemic hepatitis in deceased patients, have been documented. The present study aimed to investigate the metabolic activation of MTX and to define the possible correlation between the metabolic activation and cytotoxicity of MTX. An oxidative metabolite (M1) and a GSH conjugate (M2) were observed in S9 fraction incubations as well as in rat primary hepatocyte culture after exposure to MTX. M1 and M2 were also observed in bile of MTX-treated rats. CYP2A6 was found to dominate the oxidation of MTX. Both methoxsalen (MTS, a CYP2A6 inhibitor) and 2,6-dichloro-4-nitrophenol (DCNP, a sulfotransferase inhibitor) dramatically decreased the formation of M2. Pre-treatment of primary hepatocytes with DCNP or MTS significantly decreased the susceptibility to the cytotoxicity of MTX.

Metabolic activation and cytotoxicity of 4-methylquinoline mediated by CYP3A4 and sulfotransferases in rats.

4-Methylquinoline (4-MQ) is a quinoline derivative widely present in groundwater and soil and has been reported to be genotoxic. The mechanisms of the toxic action remain unknown. This study aimed to elucidate the metabolic activation of 4-MQ and to determine the possible role of reactive metabolites in 4-MQ-induced liver injury in rats. In the present study, a hydroxylation metabolite (M1), a GSH conjugate (M2) and an NAC conjugate (M3) derived from 4-MQ were detected in vitro and in vivo. The structures of the two conjugates were verified by chemical synthesis, mass spectrometry, and nuclear magnetic resonance. CYP3A4 was found to dominate the hydroxylation of 4-MQ. Sulfotransferases also participated in the metabolic activation of 4-MQ. Pretreatment of primary hepatocytes with ketoconazole (KTC) or 2,6-dichloro-4-nitrophenol (DCNP) not only reduced the production of GSH conjugate M2 but also decreased the susceptibility of hepatocytes to the cytotoxicity of 4-MQ. Urinary NAC conjugate M3 was found in rats given 4-MQ, and M3 may be a potential biomarker for 4-MQ exposure.

Difference in hepatotoxicity of furan-containing components in cortex dictamni correlates the efficiency of their metabolic activation.

BACKGROUND: Cortex Dictamni (CD) has been associated with an increased risk of liver injury, which may be attributable to the metabolic activation of its furan-containing components (FCC). However, the hepatotoxic potencies of these FCCs and the mechanisms behind the differences in their toxicity intensity remain unknown. METHODS: The constituents of CD extract were determined by LC-MS/MS. Potentially toxic FCCs were screened by a previously published method. Hepatotoxicity of potentially toxic FCCs was evaluated in cultured mouse primary hepatocytes and mice. The ability to deplete hepatic glutathione (GSH), along with the formation of the corresponding GSH conjugates, resulting from the metabolic activation was determined ex vivo in mice. Intrinsic clearance rates (CLint,Vmax/Km) were assessed by a microsome-bases assay. RESULTS: A total of 18 FCCs were detected in CD extract. Among them, four FCCs, including rutaevin (RUT), limonin (LIM), obacunone (OBA) and fraxinellone (FRA) were found to be bioactivated in microsomal incubations. Only FRA displayed significant hepatotoxicity in vitro and in vivo. Similarly, FRA caused GSH depletion and GSH conjugation the most in vivo. The order of CLint for the four FCCs was FRA>>OBA>LIM>RUT. CONCLUSION: FRA is the major toxic FCC component of hepatotoxic CD extract. The hepatotoxicity of FCCs is closely related to the efficiency of their metabolic activation.

Reduction of emodin-8-O-ß-D-glucoside content participates in processing-based detoxification of polygoni multiflori radix.

BACKGROUND: The occurrence of severe liver injury by the herbal medicine Polygoni Multiflori Radix (PMR) has drawn significant attention. The fact that processing attenuates PMR-induced hepatotoxicity has been well accepted, but the mechanisms are still ambiguous. PURPOSE: This study aimed to illuminate the mechanism of processing-based attenuation of PMR hepatotoxicity. METHODS: The contents of emodin-8-O-ß-d-glucoside (EG) and emodin (EMD) in raw and processed PMR were quantified. The difference in toxicokinetic behaviors of EG and EMD was determined in vivo, and the disposition properties of EG were investigated in vitro and in vivo. RESULTS: Decreased EG content was found in processed (black bean) PMR. Processed PMR showed reduced adverse effects relative to raw PMR. In addition, less hepatic protein adduction derived from EMD was produced in mice after exposure to processed PMR than that in animals receiving raw PMR. Glucose transporters SGLT1 and GLUT2 participated in the absorption of EG, and effective hydrolysis of EG to EMD took place in the intestinal epithelial cells during the process of absorption. Cytosolic broad-specificity ß-glucosidase and lactase phlorizin hydrolase, as well as intestinal flora, participated in the hydrolysis of EG. The circulated EMD resulting from the deglycosylation of EG executed the hepatotoxic action. CONCLUSION: EG is a pre-toxin and can be metabolically activated to EMD participating in the hepatotoxic event. The reduction of EG content due to processing is a key mechanistic factor that initiates the detoxification of PMR.

In Vitro and In Vivo Metabolic Activation of Tolterodine Mediated by CYP3A.

Tolterodine (TOL) is an antimuscarinic drug used for the treatment of patients with overactive bladder presenting urinary frequency, urgency, and urge incontinence. During the clinical use of TOL, adverse events such as liver injury took place. The present study aimed at the investigation of the metabolic activation of TOL possibly associated with its hepatotoxicity. One GSH conjugate, two NAC conjugates, and two cysteine conjugates were found in both mouse and human liver microsomal incubations supplemented with TOL, GSH/NAC/cysteine, and NADPH. The detected conjugates suggest the production of a quinone methide intermediate. The same GSH conjugate was also observed in mouse primary hepatocytes and in the bile of rats receiving TOL. One of the urinary NAC conjugates was observed in rats administered TOL. One of the cysteine conjugates was found in a digestion mixture containing hepatic proteins from animals administered TOL. The observed protein modification was dose-dependent. CYP3A primarily catalyzes the metabolic activation of TOL. Ketoconazole (KTC) pretreatment reduced the generation of the GSH conjugate in mouse liver and cultured primary hepatocytes after TOL treatment. In addition, KTC reduced the susceptibility of primary hepatocytes to TOL cytotoxicity. The quinone methide metabolite may be involved in TOL-induced hepatotoxicity and cytotoxicity.

Metabolic Activation of Pesticide Isoprocarb Mediated by CYP3A4 and the Possible Correlation with Its Cytotoxicity.

Isoprocarb (IPC), one of the most important carbamate pesticides, is used to control pests, such as rice planthoppers in crops. Studies have found that IPC induced hepatotoxicity in poultry chicken. However, the mechanisms of IPC-induced hepatotoxicity are unclear. The objectives of this study were to characterize reactive metabolites of IPC in vitro and in vivo, to identify cytochrome P450 enzymes for metabolic activation, and to define a possible correlation between the metabolic activation and cytotoxicity of IPC. In GSH- or NAC-supplemented microsomal incubations, one GSH conjugate (M6) and two NAC conjugates (M7 and M8) were detected after exposure to IPC. The corresponding GSH conjugate and NAC conjugates were found in the liver homogenates and urine of mice after IPC administration. IPC was found to be metabolized to a quinone intermediate reactive to GSH in vitro and in vivo. IPC was found to induce marked cytotoxicity in cultured mouse primary hepatocytes. Ketoconazole, a selective CYP3A4/5 enzyme inhibitor, attenuated the susceptibility of hepatocytes to IPC cytotoxicity.

Configurational Alteration Results in Change in Hepatotoxicity of Asarone.

α-Asarone (αA) and ß-asarone (ßA) are often used as flavoring agents for alcoholic beverages and food supplements. They possess a double bond in the side chain with different configurations. Double bonds are a class of alert chemical group, due to their metabolic epoxidation to the corresponding epoxides eliciting liver injury. Little is known about changes of configuration on metabolic activation and related toxicity. Here, we report the insight into the mechanisms of hepatotoxicity of asarone with different configurations. In vitro and in vivo comparative studies demonstrated ßA displayed higher metabolic activation effectiveness. Apparently, the major metabolic pathway of ßA underwent epoxidation at C-1' and C-2', while αA was mainly metabolized to the corresponding alcohol resulting from the hydroxylation of C-3'. CYP1A2 dominated the metabolism of αA and ßA. The molecular simulation studies showed that the orientation of ßA at the active site of CYP1A2 favored the epoxidation of ßA over that of αA. These findings not only remind us that configuration is another important factor for toxicities but also facilitate the understanding of the mechanisms of toxic action of asarone. Additionally, these findings would benefit the risk assessment of αA and ßA exposure from foods.

2,3,5,4'-Tetrahydroxy stilbene-2-Ο-ß-D-glucoside, a mechanism-based inactivator of CYP2C19 and CYP3A4, potentiates hepatic protein adduction and hepatotoxicity induced by emodin in vivo.

2,3,5,4'-Tetrahydroxy stilbene-2-Ο-ß-D-glucoside (TSG) and emodin (EMD) are two main components of Polygonum multiflorum Thunb. (PMT). Its root is widely used as herbal medicine and supplement. However, PMT-induced liver injury has drawn increasing attention. The purpose of this study was to investigate the interaction of TSG with EMD in the aspects of enzymology, pharmacokinetics, and hepatotoxicity. Co-administration with TSG increased internal exposure of EMD, EMD-derived hepatic protein adduction, and EMD-induced liver injury in mice. Mouse and human liver microsomal incubation study demonstrated that co-incubation with TSG decreased the formation of hydroxylation metabolites of EMD. Human recombinant cytochrome P450 enzyme incubation study showed that TSG induced time-, concentration-, NADPH-dependent and irreversible inhibition of CYP2C19 and CYP3A4. An epoxide metabolite derived from TSG was responsible for the observed enzyme inactivations. The findings allow us to better understand the mechanisms by which herbal processing detoxifies raw PMT.

Hepatic RNA adduction derived from metabolic activation of retrorsine in vitro and in vivo.

Pyrrolizidine alkaloids (PAs) are among the most significant hepatotoxins widely distributed in plant species. Incidence of liver injuries caused by PAs has been reported worldwide, and the reactive metabolites of PAs are known to play a critical role in causing the hepatotoxicity. To better understand the toxicity-induction mechanisms, we explored the interactions of PA metabolites with cellular RNA molecules, and examined their effects on the biochemical and metabolic properties of hepatic RNAs. After exposure to retrorsine, adduction on adenosine and guanosine were detected in mouse liver microsomal incubations, cultured mouse primary hepatocytes, and mouse liver tissues. NMR analysis showed that the exocyclic amino group participated in the adduction. We found drastically altered properties and metabolism of the adducted RNA such as reverse-transcriptability, translatability, and RNase-susceptibility. In addition, endogenous modification of N6-methyladenosine (m6A) was remarkably reduced.

Metabolic Activation of Perampanel Mediated by CYP1A2.

Perampanel (PRP), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist with high selectivity, has been used as a new adjuvant for the treatment of fractional seizures with or without primary generalized tonic-clonic seizures and secondary generalized seizures in epilepsy patients over the age of 12. Adverse events such as liver injury have been reported during the clinical application of PRP. The purpose of the study is to explore the in vitro and in vivo metabolic activation of PRP. Two GSH conjugates were detected in rat liver microsomal incubations containing PRP, GSH, and NADPH. The two GSH conjugates were both obtained from the bile of rats and rat primary hepatocytes after exposure to PRP. Similar microsomal incubations complemented with N-acetylcysteine (NAC) in place of GSH offered two NAC conjugates. As expected, the NAC conjugates were detected in the urine of PRP-treated rats. One of the two NAC conjugates was identified as NAC conjugate 12 verified by chemical synthesis. The individual human recombinant P450 enzyme incubation assay demonstrated that CYP1A2 dominated the catalysis for the metabolic activation of PRP. Pretreatment with α-naphthoflavone (NTF) decreased the formation of PRP-derived GSH conjugates in both livers of rats and cultured primary hepatocytes after being treated with PRP. Additionally, NTF was found to decrease the susceptibility of primary hepatocytes to the cytotoxicity of PRP. The findings indicate that PRP was metabolized to the corresponding epoxide, which could participate in PRP-induced cytotoxicity.

Tofacitinib Is a Mechanism-Based Inactivator of Cytochrome P450 3A4.

Tofacitinib (TFT) is an oral JAK inhibitor which has been approved for the treatment of moderately and severely active rheumatoid arthritis. TFT was found to show concentration-, time-, and NADPH-dependent inhibition of CYP3A4, and irreversibility of the inactivation was also observed. Incubation (40 min, 37 °C) of recombinant CYP3A4 with TFT at 200 µM resulted in >70% loss of CYP3A4 activity. Estimated kinact and KI were 0.037 min-1 and 93.2 µM, respectively. GSH and superoxide dismutase/catalase revealed minor or little protection against the CYP3A4 inactivation. Furthermore, ketoconazole attenuated TFT-mediated CYP3A4 inactivation. Epoxide and α-keto-aldehyde intermediates of TFT were trapped and characterized in microsomal incubations, respectively. The aldehyde intermediate is believed to be the key for the enzyme inactivation. Multiple P450 enzymes, including CYPs2C19, 3A4, 2D6, and 1A2, participated in the metabolism of TFT to the epoxide, while the formation of the aldehyde was mainly catalyzed by CYP3A4. In conclusion, TFT was proven to be a mechanism-based inactivator of CYP3A4.

Studies on hepatotoxicity and toxicokinetics of colchicine.

Colchicine (COL) is an alkaloid existing in plants of Liliaceous colchicum. It has widely been used in the treatments of many diseases, such as gout, Familial Mediterranean Fever, and tumor. However, the adverse effects of COL are an obstacle to its safe use. The present studies explored the role of metabolic demethylation in the development of COL-induced hepatotoxicity. We found that inhibition of CYP3A increased the susceptibility of mice to COL hepatotoxicity, and induction of CYP3A decreased the susceptibility of animals to the hepatotoxicity. The toxicokinetic study demonstrated that pretreatment with ketoconazole caused elevated area under the concentration-time curve of COL. Three demethylation metabolites of COL were found to be less hepatotoxic than the parent compound. It appears that the formation of electrophilic demethylation metabolites was not involved in the development of COL-induced liver injury.