Metabolic Activation Pathway Research Articles

Comparison of urinary 3-hydroxybenzo(a)Pyrene (3-OHBaP) and trans-anti-7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)Pyrene (TetraolBaP) as biomarkers of exposure to carcinogenic BaP

IntroductionBiomonitoring of exposure to carcinogenic Benzo(a)Pyrene is generally based on measurement of urinary 3-hydroxybenzo(a)pyrene (3-OHBaP), but its analysis is complex and only reflects the BaP detoxification pathway. TetraolBaP, another BaP metabolite resulting from the metabolic activation pathway, is now available but has not yet been studied in occupational settings or compared with 3-OHBaP. MethodsBiomonitoring was carried out on 118 subjects working in the aluminium smelting industry. 3 urine samples were collected from each subject at the beginning and end of the working week. Pyrene metabolite (1-hydroxypyrene) and the two BaP biomarkers (3-OHBaP and TetraolBaP) were analysed using LC-Fluorescence and GC-NCI-MS-MS. ResultsThe workers studied were found to be highly exposed, with 1-OHP and 3-OHBaP frequently exceeding maximum recommended values in occupational settings. Maximum concentrations were measured at end of shift+16h for all biomarkers, highlighting dermal exposure and/or temporary storage. Correlations were strong between 1-OHP and 3-OHBaP (r = 0.68–0.75) as well as between 3-OHBaP and TetraolBaP (r = 0.67–0.78), and moderate between 1-OHP and TetraolBaP (r = 0.59–0.76). While TetraolBaP levels were higher at low PAH exposures, TetraolBaP increased much more slowly at high exposures, indicating progressive saturation of the bioactivation pathway. The [3-OHBaP]/[TetraolBaP] ratio was found to be significantly lower in chronically exposed workers. Urinary TetraolBaP levels corresponding to 1-OHP (2.5 μg/L or 1 μmol/mol creatinine) or 3-OHBaP (0.4 nmol/mol creatinine) guidance values were found to range between 0.84 and 0.95 nmol/mol creatinine. ConclusionsTetraolBaP, resulting from carcinogenic BaP's metabolic activation pathway, was shown to be a diagnostically specific and sensitive biomarker for determining subjects' toxic internal exposure to PAHs in different contexts (occupational settings, environment) and assessing health risks.

Determining recommended acceptable intake limits for N-nitrosamine impurities in pharmaceuticals: Development and application of the Carcinogenic Potency Categorization Approach (CPCA)

N-Nitrosamine impurities, including nitrosamine drug substance-related impurities (NDSRIs), have challenged pharmaceutical industry and regulators alike and affected the global drug supply over the past 5 years. Nitrosamines are a class of known carcinogens, but NDSRIs have posed additional challenges as many lack empirical data to establish acceptable intake (AI) limits. Read-across analysis from surrogates has been used to identify AI limits in some cases; however, this approach is limited by the availability of robustly-tested surrogates matching the structural features of NDSRIs, which usually contain a diverse array of functional groups. Furthermore, the absence of a surrogate has resulted in conservative AI limits in some cases, posing practical challenges for impurity control. Therefore, a new framework for determining recommended AI limits was urgently needed. Here, the Carcinogenic Potency Categorization Approach (CPCA) and its supporting scientific rationale are presented. The CPCA is a rapidly-applied structure-activity relationship-based method that assigns a nitrosamine to 1 of 5 categories, each with a corresponding AI limit, reflecting predicted carcinogenic potency. The CPCA considers the number and distribution of α-hydrogens at the N-nitroso center and other activating and deactivating structural features of a nitrosamine that affect the α-hydroxylation metabolic activation pathway of carcinogenesis. The CPCA has been adopted internationally by several drug regulatory authorities as a simplified approach and a starting point to determine recommended AI limits for nitrosamines without the need for compound-specific empirical data.

Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15–60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

A Systematic Review of Polycyclic Aromatic Hydrocarbon Derivatives: Occurrences, Levels, Biotransformation, Exposure Biomarkers, and Toxicity.

Polycyclic aromatic hydrocarbon (PAH) derivatives constitute a significant class of emerging contaminants that have been ubiquitously detected in diverse environmental matrixes, with some even exhibiting higher toxicities than their corresponding parent PAHs. To date, compared with parent PAHs, fewer systematic summaries and reanalyses are available for PAH derivatives with great environmental concerns. This review summarizes the current knowledge on the chemical species, levels, biotransformation patterns, chemical analytical methods, internal exposure routes with representative biomarkers, and toxicity of PAH derivatives, primarily focusing on nitrated PAHs (NPAHs), oxygenated PAHs (OPAHs), halogenated PAHs (XPAHs), and alkylated PAHs (APAHs). A collection of 188 compounds from four categories, 44 NPAHs, 36 OPAHs, 56 APAHs, and 52 XPAHs, has been compiled from 114 studies that documented the environmental presence of PAH derivatives. These compounds exhibited weighted average air concentrations that varied from a lower limit of 0.019 pg/m3 to a higher threshold of 4060 pg/m3. Different analytical methods utilizing comprehensive two-dimensional gas chromatography coupled with high-resolution time-of-flight mass spectrometry (GC × GC-TOF-MS), gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS), comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC × GC-QQQ-MS), and Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), that adopted untargeted strategies for the identification of PAH derivatives are also reviewed here. Additionally, an in-depth analysis of biotransformation patterns for each category is provided, including the likelihood of specific biotransformation reaction types. For the toxicity, we primarily summarized key metabolic activation pathways, which could result in the formation of reactive metabolites capable of covalently bonding with DNA and tissue proteins, and potential health outcomes such as carcinogenicity and genotoxicity, oxidative stress, inflammation and immunotoxicity, and developmental toxicity that might be mediated by the aryl hydrocarbon receptor (AhR). Finally, we pinpoint research challenges and emphasize the need for further studies on identifying PAH derivatives, tracking external exposure levels, evaluating internal exposure levels and associated toxicity, clarifying exposure routes, and considering mixture exposure effects. This review aims to provide a broad understanding of PAH derivatives' identification, environmental occurrence, human exposure, biotransformation, and toxicity, offering a valuable reference for guiding future research in this underexplored area.

Mass Spectrometric Quantitation of N'-Nitrosonornicotine-1N-oxide in the Urine of Cigarette Smokers and Smokeless Tobacco Users.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which always occur together and are present exclusively in tobacco products, are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. While 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) serves as an excellent biomarker for NNK exposure, the currently available biomarker for NNN exposure is urinary "total NNN" (free NNN plus its N-glucuronide). Quantitation of urinary NNN requires extensive precautions to prevent artifactual formation of NNN resulting from nitrosation of nornicotine during analysis. NNN itself can also be formed endogenously by the same nitrosation reaction, which may sometimes cause an overestimation of exposure to preformed NNN. It is thus important to develop an alternative biomarker to specifically reflect NNN metabolic fate and facilitate relevant cancer etiology studies. In this study, we report the first detection of N'-nitrosonornicotine-1N-oxide (NNN-N-oxide) in human urine. Using a highly specific and sensitive MS3 transition-based method, NNN-N-oxide was quantified with a mean level of 8.40 ± 6.04 fmol/mL in the urine of 10 out of 32 cigarette smokers. It occurred in a substantially higher level in the urine of 13 out of 14 smokeless tobacco users, amounting to a mean concentration of 85.2 ± 96.3 fmol/mL urine. No NNN-N-oxide was detected in any of the nonsmoker urine samples analyzed (n = 20). The possible artifactual formation of NNN-N-oxide during sample preparation steps was excluded by experiments using added ammonium sulfamate. The low levels of NNN-N-oxide in the urine of tobacco users indicate that the pyridine N-oxidation pathway represents a minor detoxification pathway of NNN, which further supports the importance of the α-hydroxylation pathway of NNN metabolic activation in humans.

Distinct Molecular Signatures of Aging in Healthy and HIV-Infected Individuals.

Virally suppressed chronic HIV-infected individuals on antiretroviral therapy experience similar immune impairments as HIV-uninfected elderly. However, they manifest symptoms of premature immune aging such as suboptimal responses to vaccination at a younger age. Mechanisms underlying premature immune aging are unclear. The study site was University of Miami Miller School of Medicine. In this study, we aimed to identify molecular signatures of aging in HIV-infected (HIV) individuals compared with age-matched healthy control (HC) participants. Transcriptomic profiles of peripheral blood mononuclear cells collected cross-sectionally from study participants were evaluated using RNA sequencing, and genes and pathways associated with age and HIV status were identified and compared between study groups. Generalized linear modeling was used to identify transcriptional signatures associated with age. Despite that fewer differentially expressed genes between young (<40 yrs) and old (>59 yrs) were observed in the HIV group, metabolic and innate immune activation pathways were associated with increasing age in both HIV and HC. Age was also associated with pathways involved with T-cell immune activation in HC and with interferon signaling pathways in HIV. We observed signs of precocious immune aging at the transcriptional level in HIV and defined a transcriptional perturbation associated with innate immunity and glucose metabolism induced by aging in both HC and HIV. In this study, we identified distinct molecular signatures predictive of age in HIV versus HC, which suggest precocious immune aging in HIV. Overall, our results highlight the molecular pathways of immune aging in both HC and HIV that may be targeted for additional mechanistic insights or in a therapeutic setting.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

MetMax Human Hepatocyte/HEK Cytotoxic Reactive Metabolite Assay as a Potential In Vitro Experimental System for the Identification of DILI Drugs

In drug development, it is important to identify drug candidates where drug metabolism plays key roles in their toxicity. Major reasons for the identification of drug candidates with metabolism-dependent toxicity are as follows: 1. Safety profiles determined in preclinical animal species may not accurately predict human effects due to species difference in drug metabolism; and 2. idiosyncratic drug toxicity may occur in individuals due to enhanced metabolic activation or compromised detoxification. We recently developed a novel in vitro hepatocyte system, the MetMax Human Hepatocyte (MMHH) system with permeabilized human hepatocytes supplemented with metabolic cofactors, for the evaluation of drug metabolism. The permeabilization treatment removes the plasma membrane as a barrier for drug diffusion into the hepatocytes, and the diffusion of the metabolites of the hepatocytes. A further advantage of MMHH is that cytotoxicity of the intended substrate would not affect drug metabolism, a challenge often encountered in the evaluation of metabolism of toxic drugs in intact hepatocytes. We report here the development of the MMHH/HEK assay for the evaluation of metabolic activation and detoxification of toxic drugs. In this assay, drug toxicity is quantified in a drug metabolism incompetent target cell line (HEK293) in the presence of absence of MMHH as an exogenous metabolic activating system, and in the presence of selective cofactors for the evaluation of key metabolic activation and detoxification pathways. As a proof-of-concept study, the MMHH/HEK assay was applied in the the evaluation of the model metabolically-activated drugs that are known to be associated with severe drug induced liver injuries (DILI) - acetaminophen, cyclophosphamide, and troglitazone. Our results showed that the cytotoxicity of these model DILI drugs was enhanced in the presence of MMHH supplemented with NADPH, thereby confirming the role of oxidative metabolism in metabolic activation. Furthermore, supplementation of NADPH with reduced glutathione (GSH), a physiologically relevant detoxifying conjugating cofactor for reactive metabolites, led to significant attenuation of cytotoxicity, demonstrating that these model drugs were metabolized by hepatocytes to to reactive, cytotoxic metabolites. Supplementation with UDPGA and PAPS also attenuated the in vitro cytotoxicity of these model drugs in the MMHH/HEK assay, demonstrating the involvement of glucuronidation and sulfation conjugating pathways in toxification. Troglitazone cytotoxicity in the MMHH/HEK assay was found to be higher than that observed for the known less hepatotoxic analogs, rosiglitazone and pioglitazone. Our results suggest that MMHH/HEK assay can be applied towards the evaluation of metabolic activation and detoxification and the identification of drugs with idiosyncratic DILI potential.

Mice with a specific deficiency of Pfkfb3 in myeloid cells are protected from hypoxia-induced pulmonary hypertension.

Macrophage infiltration into the lungs is a characteristic of pulmonary hypertension (PH). Glycolysis is the main metabolic pathway for macrophage activation. However, the effect of macrophage glycolysis on the development of PH remains unknown. We investigated the effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKBF3), a critical enzyme of macrophage glycolysis, on PH development. Lung tissues from PH patients were examined by immunostaining with macrophage markers. PH was induced in Wistar rats with SU5416/hypoxia and in mice with hypoxia. Lungs and macrophages were isolated for analysis by RT-PCR, western blot, flow cytometry, and immunostaining. Expression of glycolytic molecules was increased in circulating peripheral blood mononuclear cells (PBMCs) and lung macrophages of PH patients. These results were also found in lung macrophages of SU5416/hypoxia (Su/Hx)-induced PH rats and hypoxia-induced PH mice. PH was ameliorated in myeloid-specific Pfkfb3-deficient mice (Pfkfb3ΔMϕ ) or mice treated with the PFKFB3 inhibitor 3PO, compared with their controls. Alveolar macrophages of PH Pfkfb3ΔMϕ mice produced lower levels of growth factors and pro-inflammatory cytokines than those of control mice. Circulating myeloid cells and lung myeloid cells were much fewer in PH Pfkfb3ΔMϕ mice than controls. Mechanistically, overexpression of Hif1a or Hif2a in bone marrow-derived macrophages (BMDMs) cultured with bone marrow of Pfkfb3ΔMϕ mice restored the decreased expression of pro-inflammatory cytokines and growth factors. Myeloid Pfkfb3 deficiency protects mice from PH, thereby suggesting that myeloid PFKFB3 is one of the important targets in the therapeutic effect of PFKFB3 inhibition in PH treatment.

Metabolic Activation of Pyrrolizidine Alkaloids – Generation of Multiple Reactive Metabolites Leading to DNA Adduct Formation and Potentially Liver Tumor Initiation

Pyrrolizidine alkaloids (PAs) are toxic and carcinogenic phytochemicals widespread in the world. Metabolic formation of dehydro‐PAs and 6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine (DHP) has long been considered the principle metabolic activation pathway. We determined that a set of DHP‐DNA adducts are generally formed from the metabolism of a series of carcinogenic PAs in vitro, in cells and in vivo. The level of DHP‐DNA adducts well correlated with liver tumor potency for a set of PAs. We have proposed that this set of DHP‐DNA adducts is a potential biomarker of PA exposure and PA‐induced liver tumor initiation. Our present studies have found that, in addition to dehydro‐PAs and DHP, there are another seven reactive pyrrolic metabolites that are capable of binding to cellular DNA to produce the same set of DHP‐DNA adducts. These DNA reactive metabolites are: 7‐glutathione‐DHP, 7‐cysteine‐DHP, 7‐N‐acetylcysteine‐ DHP, 1‐CHO‐DHP, cysteinyl‐[2′‐S‐7]‐1‐CHO‐DHP, cysteinyl‐[3′‐N‐7]‐1‐CHO‐DHP, and 1‐cysteinylimino‐DHP. These pyrrolic metabolites are enzymatically and/or chemically formed from the reaction of the corresponding amino acids (and glutathione) with either dehydro‐PAs or DHP. Also, these pyrrolic metabolites are interconvertible, apparently ascribed to their unique reactive bifunctional alkylating character. These overall results represent the first time that concerning the metabolic activation of PAs: (i) there involve the formation of multiple DNA reactive pyrrolic metabolites; and (ii) metabolism of carcinogenic and potential carcinogenic PAs all generate the identical DHP‐DNA adducts. [This abstract is not an official U.S. Food and Drug Administration (FDA) guidance or policy statement. No official support or endorsement by the U.S. FDA is intended or should be inferred. The authors declare no competing financial interest.]

Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

The metabolic plasticity of neoplastic cholangiocytes: perspective for target therapy of intrahepatic cholangiocarcinoma

Background and Aims: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer arising from biliary epithelial cells (BECs) lining the biliary tree. iCCA is a highly chemoresistant tumor and pharmacological therapies are generally unsuccessful. Furthermore, due to the complexity of the in vivo cellular interactions, metabolic activation pathways are largely unknown. We herein aim to elucidate the metabolic asset of both tumoral and non-tumoral primary BECs by profiling both the extra- and endo- metabolome.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B.thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 invitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

Metabolic activation of TM5441 in vitro and in vivo: Formation of reactive metabolites and human enzymes involved

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5′-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.

The ying and yang of idebenone: Not too little, not too much – cell death in NQO1 deficient cells and the mouse retina

Idebenone has recently been investigated as a drug therapy for Leber's hereditary optic neuropathy (LHON), a rare genetic mitochondrial disease that causes rapid and progressive bilateral vision loss. Although several studies have shown that idebenone can promote vision recovery in patients with LHON, the evidence for the efficacy of idebenone is still limited. Idebenone failed to demonstrate superiority over placebo in the primary end-points of the only published randomised, double-blind, placebo-controlled trial. There appears to be a patient-specific response to idebenone with high variability in therapeutic outcomes. A recent study suggested that the cytosolic enzyme NAD(P)H: quinone acceptor oxidoreductase (NQO1) is the major enzyme involved in the activation of idebenone, and the beneficial effects of idebenone are dependent on the expression of NQO1. Here, we confirm the NQO1-dependent activity of idebenone, but we also show, for the first time, that the cytotoxicity of idebenone is linked to cellular expression of NQO1. Upon idebenone administration, cells deficient in NQO1 show a marked decrease in viability in comparison to NQO1 expressing cells, with idebenone causing ROS production and deleterious effects on ATP levels and cell viability. In addition, our data highlights that only cells expressing NQO1 can significantly activate idebenone, indicating that other proposed metabolic activation pathways, such as complex II and glycerol-3-phosphate dehydrogenase, do not play a significant role in idebenone activation. Furthermore, we provide evidence of idebenone-induced toxicity in the retina ex-vivo, which can be explained by the variation of NQO1 expression between different cell types in the mouse retina. Idebenone mediated cell rescue in the rotenone ex vivo model also indicated that this drug has a narrow therapeutic window. These findings will help to guide the development of future therapies and drug delivery strategies including intra-ocular administration. The specific dependence of idebenone activity on NQO1 may also explain the variation in patient outcomes in clinical trials.

1-Formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP): A Potential Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids.

Pyrrolizidine alkaloids (PAs) are phytochemicals present in more than 6000 plant species worldwide; about half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that PA-induced liver tumor initiation is mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-DNA adducts and proposed that these DHP-DNA adducts are biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP-DNA adducts as biomarkers, it is significant to identify reactive metabolites associated with this metabolic activation pathway. Segall et al. ( Segall et al. ( 1984 ) Drug Metab. Dispos. 12 , 68 - 71 ) previously reported that 1-formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP) is generated from the metabolism of senecionine by mouse liver microsomes. In the present study, we examined the metabolism of seven hepatocarcinogenic PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and senkirkine) and one noncarcinogenic PA (platyphylline) by human, rat, and mouse liver microsomes. 1-CHO-DHP was identified as a common metabolite from the metabolism of these hepatotoxic PAs, but not from platyphylline. Incubation of 1-CHO-DHP with HepG2 and A549 cells produced the same set of DHP-DNA adducts, which were identified by both LC/MS MRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of 1-CHO-DHP treated HepG2 cells, both DHP and 7-cysteine-DHP were formed, which were capable of binding to cellular DNA to produce DHP-DNA adducts. These results suggest that 1-CHO-DHP is a proximate DNA metabolite of genotoxic and carcinogenic PAs.

1‐Formyl‐7‐hydroxy‐6,7‐dihydro‐5H‐pyrrolizine (9‐CHO‐DHP) ‐ A Biologically Proximate Pyrrolic Metabolite of Carcinogenic Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are phytochemical constituents of more than 6000 plant species worldwide; approximately half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that the metabolic activation pathway of PA‐induced liver tumor initiation is mediated by a set of four (±)‐6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine (DHP)‐derived DNA adducts, and proposed that these DHP‐DNA adducts are potential biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP‐DNA adducts as biomarkers, it is important to identify the spectrum of reactive metabolites associated with this metabolic activation pathway. Segall et al. (Drug Metab. Disposit., 12, 68–71, 1984) reported that 1‐formyl‐7‐hydroxy‐6,7‐dihydro‐5H‐pyrrolizine (9‐CHO‐DHP) is a metabolite formed from the mouse liver microsomal metabolism of senecionine. In this present study, we examined the metabolism of six hepatocarcinogenic PAs (riddelliine, senecionine, monocrotaline, senkirkine, intermedine, and heliotrine) and one non‐carcinogenic PA (platphylline) by human, rat, and mouse liver microsomes. 9‐CHO‐DHP was found as a common metabolite from the metabolism of the six hepatotoxic PAs, but not from platphylline. Incubation of 9‐CHO‐DHP in HepG2 cells and A549 cells resulted in the formation of the same set of DHP‐DNA adducts, as assessed by both LC/MS SRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of HepG2 cells, both DHP and 7‐cysteine‐DHP were identified. These metabolites have previously shown to be capable of binding to cellular DNA to produce DHP‐DNA adducts. Thus, when incubated with HepG2 cells, 9‐CHO‐DHP was metabolized to the reactive metabolites DHP and 7‐cysteine‐DHP, metabolites associated with the DHP‐DNA adduct formation. These results indicate that 9‐CHO‐DHP is a DNA proximate metabolite of hepatotoxic PAs and further support the generality of this metabolic activation pathway mediated by the formation of these DHP‐DNA adducts. [This abstract is not an official U.S. Food and Drug Administration (FDA) guidance or policy statement. No offcial support or endorsement by the U.S. FDA is intended or should be inferred. The authors declare no competing financial interest.]Support or Funding InformationThis research was supported in part by appointment (X.H.) to the Postgraduate Research Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potential Metabolic Activation of Representative Alkylated Polycyclic Aromatic Hydrocarbons 1-Methylphenanthrene and 9-Ethylphenanthrene Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells.

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. Alkylated phenanthrenes are the most abundant PPAHs present in the crude oil and could contaminate the food chain. We describe the metabolism of a C1-phenanthrene regioisomer 1-methylphenanthrene (1-MP) and a C2-phenanthrene regioisomer 9-ethylphenanthrene (9-EP) in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. Side chain hydroxylation of 1-MP and 9-EP was observed as the major metabolic pathway. The formation of 1-(hydroxymethyl)-phenanthrene was confirmed by reference to an authentic synthetic standard. However, formation of the bioactivated sulfate was not detected. Tetraols were also identified as signature metabolites of 1-MP and 9-EP, indicating that metabolic activation occurred via the diol-epoxide pathway. O-Monosulfonated-catechols were discovered as signature metabolites of the o-quinone pathway of metabolic activation of 1-MP and 9-EP, respectively. The identification of O-monosulfonated-catechols supports the metabolic activation of 1-MP and 9-EP by P450 and AKR isozymes followed by metabolic detoxification of the o-quinone through interception of redox cycling by phase II isozymes. The signature metabolites identified could be used as biomarkers of human exposure to 1-MP and 9-EP resulting from oil spills.

Investigation of the presence in human urine of mercapturic acids derived from phenanthrene, a representative polycyclic aromatic hydrocarbon

Polycyclic aromatic hydrocarbons (PAH) are environmental carcinogens implicated as causes of cancer in certain industrial settings and in cigarette smokers. PAH require metabolic activation to exert their carcinogenic effects. One widely accepted pathway of metabolic activation proceeds through formation of “bay region” diol epoxides which are highly reactive with DNA and can cause mutations. Phenanthrene (Phe) is the simplest PAH with a bay region and an excellent model for the study of PAH metabolism. In previous studies in which [D10]Phe was administered to smokers, we observed higher levels of [D10]Phe-tetraols derived from [D10]Phe-diol epoxides in subjects who were null for the glutathione-S-transferase M1 (GSTM1) gene. We hypothesized that Phe-epoxides, the primary metabolites of Phe, were detoxified by glutathione conjugate formation, which would result ultimately in the excretion of the corresponding mercapturic acids in urine. We synthesized the four stereoisomeric mercapturic acids that would result from attack of glutathione on Phe-epoxides followed by normal processing of the conjugates. We also synthesized the corresponding dehydrated metabolites and sulfoxides. These 12 standards were used in liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry analysis of urine samples from smokers and creosote workers, the latter exposed to unusually high levels of PAH. Only the sulfoxide derivatives were consistently detected in the urine of creosote workers; none of the compounds was detected in the urine of smokers. These results demonstrate a new pathway of PAH-mercapturic acid formation, but do not provide an explanation for the role of GSTM1 null status on Phe-tetraol formation.

Development of In Silico Protocols to Predict Structural Insights into the Metabolic Activation Pathways of Xenobiotics.

To establish in silico model to predict the structural insight into the metabolic bioactivation pathway of xenobiotics, we considered two specific and one non-specific mammary procarcinogen [e.g., dibenzo[a,l]pyrene (DBP), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (BP)]. The CYP1A1, 1B1, 2C9, 1A2 and 2B6 reported in wet-lab studies to actively metabolize DBP also showed strong binding energies (kcal/mol) of -11.50, -10.67, -10.37, -9.76 and -9.72, respectively, with inhibition constants ranging between 0.01 and 0.08µM. The CYP3A4 depicted minimum binding energy (-9.51kcal/mol) which is in agreement with the wet-lab reports. Further, relatively better affinity of CYP1A1 and CYP1B1 with the dibenzo[a,l]pyrene-11,12-diol (DBPD) might be indicative of their involvement in carcinogenicity of parent compound. Like DBP, BP (-10.13kcal/mol, Ki: 0.04µM) and BP-diols (BPD) (-9.01kcal/mol, Ki: 0.25µM) observed plausible binding with CYP1A1 supporting to the reported data that emphasize the major contribution of CYP1A1 in the activation of similar procarcinogens and mutagens. Likewise, in silico results further highlighted the CYP1A1 as key player in bioactivation of DMBA to its carcinogenic metabolites. In case of PhIP metabolism, strong binding interaction predicted with CYP1A1 (-9.63kcal/mol) rather than CYP1A2 (-8.84kcal/mol). Dissimilarity in the binding affinity of PhIP might be due to its basic scaffold. Further, molecular dynamics (MD) simulation of 10ns has been revealed that docked complexes of CYP1A1 with DBP, DMBA and BP are comparatively more stable than the complex of PhIP. Moreover, the current findings might be valuable as reference model in prediction and elucidation of the approximate metabolic pathway of xenobiotics.