Lung Microsomes Research Articles

In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19

BackgroundRecent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PurposeOur study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. MethodsHPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. ResultsHPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4′-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. ConclusionHT was mainly metabolized into 4′-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

Deficiency of Vitamin E and Selenium Enhances Calcium-Independent Phospholipase A2 Activity in Rat Lung and Liver

Conditions promoting oxidative stress, which is implicated in many diseases, activate phospholipases A2, a family of enzymes central to phospholipid metabolism and signal transduction. Little is known about isozyme specificity with respect to this activation process. Accordingly, a dietary deficiency model known to induce oxidative stress was used to investigate phospholipase A2 isozyme activity in rat tissues. Long-Evans hooded rats were fed purified diets for 6 wk with or without the addition of vitamin E and selenium in a 2 ՠ2 factorial design. Phospholipase A2 activity was assessed in lung, liver, kidney and heart cytosol and microsomes in the presence (5 mmol/L CaCl2) or absence (5 mmol/L EGTA) of calcium with dipalmitoylphosphatidylcholine at pH 6.5. Lung phospholipase A2 activity was also assessed with 1-stearoyl-2-arachidonoylphosphatidylcholine as substrate at pH 8.5. Organ samples from rats deficient in both nutrients showed two- to tenfold higher calcium-independent phospholipase A2 activity in lung cytosol and microsomes, and in liver cytosol compared with samples from control and single nutrient-deficient rats. In contrast, the calcium-dependent activity was affected only slightly. The malondialdehyde concentration of the organs was measured and the pattern obtained mirrored that of enhanced phospholipase A2 activity for lung but not for liver. The enhanced phospholipase A2 activity in the lung cytosol and microsomes from rats deficient in both nutrients was partially blocked by p-bromophenacylbromide, further enhanced by dithiothreitol and unaffected by treatment with diisopropylfluorophosphate. These results suggest that deficiency of both vitamin E and selenium activates and/or induces unique calcium-independent forms of phospholipase A2 markedly in rat lung, and to a lesser extent in liver.

Structure-Dependent Toxicokinetics of Selected Pyrrolizidine Alkaloids In Vitro

Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.

Characterizing and Quantifying Extrahepatic Metabolism of (-)-Δ9-Tetrahydrocannabinol (THC) and Its Psychoactive Metabolite, (±)-11-Hydroxy-Δ9-THC (11-OH-THC).

(-)-Δ9-Tetrahydrocannabinol (THC) is the psychoactive constituent of cannabis, a drug recreationally consumed orally or by inhalation. Physiologically based pharmacokinetic (PBPK) modeling can be used to predict systemic and tissue exposure to THC and its psychoactive metabolite, (±)-11-hydroxy-Δ9-THC (11-OH-THC). To populate a THC/11-OH-THC PBPK model, we previously characterized the depletion clearance of THC (by CYP2C9) and 11-OH-THC (by UDP-glucuronosyltransferase (UGT), CYP3A, and CYP2C9) in adult human liver microsomes. Here we focused on quantifying extrahepatic depletion clearance of THC/11-OH-THC, important after oral (intestine) and inhalational (lung) consumption of THC as well as prenatal THC use (placenta and fetal liver). THC (500 nM) was metabolized in adult human intestinal microsomes (n = 3-5) by CYP2C9 [Vmax: 1.1 ± 0.38 nmol/min/mg; Michaelis-Menten constant (Km): 70 nM; intrinsic clearance (CLint): 15 ± 5.4 ml/min/mg; fraction metabolized (fm): 0.89 ± 0.31 at concentration ≪ 70 nM] and CYP3A (CLint: 2.0 ± 0.86 ml/min/mg; fm: 0.11 ± 0.050). 11-OH-THC (50 nM) was metabolized by CYP3A (CLint: 0.26 ± 0.058 ml/min/mg; fm: 0.51 ± 0.11) and UGT2B7 (CLint: 0.13 ± 0.027 ml/min/mg; fm: 0.25 ± 0.053). THC at 500 nM (CLint: 4.7 ± 0.22 ml/min/mg) and 11-OH-THC at 50 nM (CLint: 2.4 ± 0.13 ml/min/mg) were predominately (fm: 0.99 and 0.80, respectively) metabolized by CYP3A in human fetal liver microsomes (n = 3). However, we did not observe significant depletion of THC/11-OH-THC in adult lung, first trimester, second trimester, or term placentae microsomes. Using PBPK modeling and simulation, these data could be used in the future to predict systemic and tissue THC/11-OH-THC exposure in healthy and special populations. SIGNIFICANCE STATEMENT: This is the first characterization and quantification of (-)-Δ9-tetrahydrocannabinol (THC) and (±)-11-hydroxy-Δ9-THC (11-OH-THC) depletion clearance by cytochrome P450 and UDP-glucuronosyltransferase enzymes in extrahepatic human tissues: intestine, fetal liver, lung, and placenta. These data can be used to predict, through physiologically based pharmacokinetic modeling and simulation, systemic and tissue THC/11-OH-THC exposure after inhalational and oral THC use in both healthy and special populations (e.g., pregnant women).

Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice.

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d-/- mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d-/- mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d-/- mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.

Pharmacokinetics and tissue distribution of remdesivir and its metabolites nucleotide monophosphate, nucleotide triphosphate, and nucleoside in mice.

Remdesivir (RDV) exerts anti-severe acute respiratory coronavirus 2 activity following metabolic activation in the target tissues. However, the pharmacokinetics and tissue distributions of the parent drug and its active metabolites have been poorly characterized to date. Blood and tissue levels were evaluated in the current study. After intravenous administration of 20 mg/kg RDV in mice, the concentrations of the parent drug, nucleotide monophosphate (RMP) and triphosphate (RTP), as well as nucleoside (RN), in the blood, heart, liver, lung, kidney, testis, and small intestine were quantified. In blood, RDV was rapidly and completely metabolized and was barely detected at 0.5 h, similar to RTP, while its metabolites RMP and RN exhibited higher blood levels with increased residence times. The area under the concentration versus time curve up to the last measured point in time (AUC0-t) values of RMP and RN were 4558 and 136,572 h∙nM, respectively. The maximum plasma concentration (Cmax) values of RMP and RN were 2896 nM and 35,819 nM, respectively. Moreover, RDV presented an extensive distribution, and the lung, liver and kidney showed high levels of the parent drug and metabolites. The metabolic stabilities of RDV and RMP were also evaluated using lung, liver, and kidney microsomes. RDV showed higher clearances in the liver and kidney than in the lung, with intrinsic clearance (CLint) values of 1740, 1253, and 127 mL/(min∙g microsomal protein), respectively.

Improved Pharmacokinetic Characteristics of Ursolic Acid in Rats Following Intratracheal Instillation and Nose-Only Inhalation Exposure.

Ursolic acid (UA) is a common pentacyclic triterpene phytochemical with various pharmacological activities. However, UA is classified as a class IV drug in BCS system and its development as an oral drug is limited. Pulmonary delivery is an effective way to improve the bioavailability of drugs with low absorption. In this study, the differences in pharmacokinetic behaviors of UA after pulmonary and oral administration was explored in rats. Compared with oral administration, the plasma concentration of UA increased rapidly after pulmonary administration, and the bioavailability increased about 80 times. UA instantly accumulated in the lungs after pulmonary administration, and the pulmonary AUC0-t/dose increased by 114 times compared to oral dosing. Incubation experiments showed that the metabolism of UA in rat lung microsomes was significantly reduced compared with that in liver microsomes, in which the clearance rate of phase I and phase II metabolism was reduced by 14.7 times and 1.4 times respectively. These results indicated that pulmonary administration could improve the bioavailability of UA and reduce its metabolism. This study not only provides a preferable route of administration for the application of UA but also offers new insights for the development of phytochemical drug candidates with poor pharmacokinetic properties.

Pulmonary Metabolism of Substrates for Key Drug-Metabolizing Enzymes by Human Alveolar Type II Cells, Human and Rat Lung Microsomes, and the Isolated Perfused Rat Lung Model.

Significant pulmonary metabolism of inhaled drugs could have drug safety implications or influence pharmacological effectiveness. To study this in vitro, lung microsomes or S9 are often employed. Here, we have determined if rat and human lung microsomes are fit for purpose or whether it is better to use specific cells where drug-metabolizing enzymes are concentrated, such as alveolar type II (ATII) cells. Activities for major hepatic and pulmonary human drug-metabolizing enzymes are assessed and the data contextualized towards an in vivo setting using an ex vivo isolated perfused rat lung model. Very low rates of metabolism are observed in incubations with human ATII cells when compared to isolated hepatocytes and fewer of the substrates are found to be metabolized when compared to human lung microsomal incubations. Reactions selective for flavin-containing monooxygenases (FMOs), CYP1B1, CYP2C9, CYP2J2, and CYP3A4 all show significant rates in human lung microsomal incubations, but all activities are higher when rat lung microsomes are used. The work also demonstrates that a lung microsomal intrinsic clearance value towards the lower limit of detection for this parameter (3 µL/min/mg protein) results in a very low level of pulmonary metabolic clearance during the absorption period, for a drug dosed into the lung in vivo.

Mass spectrometric characterization of carfentanil metabolism in human, dog, and rat lung microsomes via comparison to chemically synthesized metabolite standards

The metabolism of carfentanil was assessed using human, dog, and rat pulmonary microsomes. Mass spectrometry based analysis allowed for metabolite identification and species differentiation. Participation of different metabolic enzymes in carfentanil biotransformation was also assessed. Metabolite profiling was accomplished by incubating 10 µM carfentanil in human, dog, and rat lung microsomes. The metabolites were separated and analyzed by ultra-high performance liquid chromatography/high-resolution mass spectrometry. In total, 18 metabolites were detected. Nine metabolites were authentically identified through comparison to synthesized reference standards. In human lung microsomes, nine metabolites were identified. In dog lung microsomes, 15 metabolites were identified with three being dog specific. In rat lung microsomes, 15 metabolites were identified and two were rat specific. Proposed metabolic pathways included N-dealkylation, monohydroxylation, dihydroxylation, N-oxidation of piperidine ring nitrogen, and ketone formation. Participation of enzymes CYP2B6, CYP2E1, CYP2J2, and CYP3A4/5 to carfentanil metabolism was suggested by selective enzymatic inhibition. The pulmonary clearance in human lung microsomes was lower than the previously reported hepatic metabolism suggesting organ specific metabolic rates. The contribution of multiple cytochrome P450 enzymes to human, dog, and rat pulmonary microsomal carfentanil biotransformation varied between species. The identified metabolites may provide useful markers for possible forensic and clinical determination of the mode of ingestion but the use of dog and rat animal models was not indicated. To our knowledge, this is the first reported use of chemically synthesized reference standards for the unequivocal identification of lung carfentanil metabolites.

Incorporation of in vitro metabolism data and physiologically based pharmacokinetic modeling in a risk assessment for chloroprene

Objective: To develop a physiologically based pharmacokinetic (PBPK) model for chloroprene in the mouse, rat and human, relying only on in vitro data to estimate tissue metabolism rates and partitioning, and to apply the model to calculate an inhalation unit risk (IUR) for chloroprene.Materials and methods: Female B6C3F1 mice were the most sensitive species/gender for lung tumors in the 2-year bioassay conducted with chloroprene. The PBPK model included tissue metabolism rate constants for chloroprene estimated from results of in vitro gas uptake studies using liver and lung microsomes. To assess the validity of the PBPK model, a 6-hr, nose-only chloroprene inhalation study was conducted with female B6C3F1 mice in which both chloroprene blood concentrations and ventilation rates were measured. The PBPK model was then used to predict dose measures – amounts of chloroprene metabolized in lungs per unit time – in mice and humans.Results: The mouse PBPK model accurately predicted in vivo pharmacokinetic data from the 6-hr, nose-only chloroprene inhalation study. The PBPK model was used to conduct a cancer risk assessment based on metabolism of chloroprene to reactive epoxides in the lung, the target tissue in mice. The IUR was over100-fold lower than the IUR from the EPA Integrated Risk Information System (IRIS), which was based on inhaled chloroprene concentration. The different result from the PBPK model risk assessment arises from use of the more relevant tissue dose metric, amount metabolized, rather than inhaled concentrationDiscussion and conclusions: The revised chloroprene PBPK model is based on the best available science, including new test animal in vivo validation, updated literature review and a Markov-Chain Monte Carlo analysis to assess parameter uncertainty. Relying on both mouse and human metabolism data also provides an important advancement in the use of quantitative in vitro to in vivo extrapolation (QIVIVE). Inclusion of the best available science is especially important when deriving a toxicity value based on species extrapolation for the potential carcinogenicity of a reactive metabolite.

Metabolism of 4-methylimidazole in Fischer 344 rats and B6C3F1 mice

4-Methylimidazole (4-MeI) is a widely used chemical, also identified as a by-product of heating foods. In cancer bioassays, 4-MeI induced lung tumors in mice, but not in rats. To establish if metabolic differences could explain species difference in carcinogenicity, this study investigated metabolism of 4-MeI in rat and mouse lung and liver microsomes and S-9 fractions, and in vivo in rats and mice. No metabolites were detected in rat or mouse lung and liver microsomes, or lung S-9 fractions. Male and female F-344 rats and B6C3F1 mice were administered 50 and 150 mg/kg [14C] 4-MeI by gavage. Excreta, exhaled CO2 and volatiles were collected for 48 h. Elimination was mainly via urine, with 79–89% of the radioactivity in urine in rats and 41–70% in mice. Most of the radioactivity (71–88%) in urine was unchanged 4-MeI. Additional radioactive peaks (the largest metabolite was 8–18%) were characterized by LC-MS/MS as 4-hydroxymethylimidazole, its glucuronide, and other oxidized products, including methylhydantoin. 4-MeI was largely excreted unchanged in rats and mice with limited oxidative metabolism and conjugation. 4-MeI was not oxidized in subcellular fractions from rat and mouse lung and liver. Overall, the metabolism of 4-MeI appeared similar between rats and mice.

Carbonyl reduction of NNK by recombinant human lung enzymes: identification of HSD17β12 as the reductase important in (R)-NNAL formation in human lung.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of this study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant cytosolic reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL-formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels ≥HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knock-down of HSD17β12 resulted in significant decreases in (R)-NNAL-formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.

Functional assessment of rat pulmonary flavin-containing monooxygenase activity

The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model.Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible.The predicted lung clearance (CLlung) estimated from microsomes and slices was 16 ± 0.6 and 2.1 ± 0.3 mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CLlung using rat lung microsomes corresponded to approximately 35% of rat CLliver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine.Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

Assessment of the pulmonary CYP1A1 metabolism of mavoglurant (AFQ056) in rat

1. AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat.2. Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition.3. AFQ056 lung clearance (CLlung) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction.4. While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.

Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator, via phosphorylation and dephosphorylation in humans

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes.2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 μM to 1060 μM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate.3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 μM to 32.1 μM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes.4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

Relationship of Metabolism and Cell Proliferation to the Mode of Action of Fluensulfone-Induced Mouse Lung Tumors. II: Additional Mechanistic Studies.

Fluensulfone is a nematicide for agricultural use. Chronic dietary exposure led to bronchiolo-alveolar hyperplasia and bronchiolo-alveolar adenomas in CD-1 mice but not in rats. Genotoxicity could be excluded as a mode of action (MOA). An earlier publication (Strupp, C., Banas, D. A., Cohen, S. M., Gordon, E. B., Jaeger, M., and Weber, K. (2012). Relationship of metabolism and cell proliferation to the mode of action of fluensulfone-induced mouse lung tumors: analysis of their human relevance using the IPCS framework. Toxicol. Sci. 128, 284-294.) reported MOA studies identifying the following key events: increased metabolism of fluensulfone by CYP2f2 in mouse lung Club cells, followed by local proliferation, finally leading to adenoma formation. Human lung microsomes were found not to metabolize fluensulfone. The Joint FAO/WHO Meeting on Pesticide Residues has reviewed the previous data and concluded that the MOA is plausible however some areas of uncertainty were identified. This publication provides additional data to address these. New cell proliferation studies in mice showed that the MOA is functionally independent of sex. A threshold of cell proliferation in Club cells correlating with the dose response for adenoma formation was shown. CYP2f2 knockout mice did not react to fluensulfone exposure with cell proliferation like wild-type mice, confirming the key role of this enzyme. The collective data for fluensulfone were evaluated according to the International Programme on Chemical Safety (IPCS) Mode of Action Framework which leads to the conclusion that the mouse-specific lung tumors after fluensulfone are not relevant to humans.

Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity.

4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.

Dietary Dihydromethysticin Increases Glucuronidation of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in A/J Mice, Potentially Enhancing Its Detoxification.

Effective chemopreventive agents are needed against lung cancer, the leading cause of cancer death. Results from our previous work showed that dietary dihydromethysticin (DHM) effectively blocked initiation of lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice, and it preferentially reduced 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-derived DNA adducts in lung. This study explored the mechanism(s) responsible for DHM's differential effects on NNK/NNAL-derived DNA damage by quantifying their metabolites in A/J mice. The results showed that dietary DHM had no effect on NNK or NNAL abundance in vivo, indicating that DHM does not affect NNAL formation from NNK. DHM had a minimal effect on cytochrome P450 2A5 (CYP2A5, which catalyzes NNK and NNAL bioactivation in A/J mouse lung), suggesting that it does not inhibit NNAL bioactivation. Dietary DHM significantly increased O-glucuronidated NNAL (NNAL-O-gluc) in A/J mice. Lung and liver microsomes from dietary DHM-treated mice showed enhanced activities for NNAL O-glucuronidation. These results overall support the notion that dietary DHM treatment increases NNAL detoxification, potentially accounting for its chemopreventive efficacy against NNK-induced lung tumorigenesis in A/J mice. The ratio of urinary NNAL-O-gluc and free NNAL may serve as a biomarker to facilitate the clinical evaluation of DHM-based lung cancer chemopreventive agents.

Abstract 2722: Characterization of human lung enzymes involved in the reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the primary tobacco-specific nitrosamine (TSNA) related to cancer. The major metabolic pathway for NNK is carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung and pancreatic carcinogen in rodents, with it estimated that 39-100% of NNK is converted to NNAL in cigarette smokers. NNAL is formed as both (R)- and (S)-NNAL enantiomers; while (S)-NNAL is more readily formed from NNK, it is also more readily glucuronidated and detoxified by the phase II UGTs than is (R)-NNAL in monkeys and probably in humans. Therefore, both (S)- and (R)-NNAL are likely to be important metabolites in the NNK carcinogenic process. In the present study, we demonstrated that while (S)-NNAL is the major form (&amp;gt;90%) produced using lung cytosolic fractions, lung microsomes formed primarily (R)-NNAL (&amp;gt;95%). Consistent with previous studies using placenta-fractionated cytosolic enzymes, we demonstrated that several cytosolic enzymes including the aldo-keto reductases (AKRs) 1C1, 1C2, and 1C4, as well as carbonyl reductase I (CBR1) were active against NNK, with all of them forming primarily (&amp;gt;90%) (S)-NNAL. This pattern is consistent with that observed for lung cytosolic fractions. Using HEK293 cells over-expressing microsomal enzymes HSD11β1, HSD11β2, HSD17β12, SDR5A1, SDR5A3, HSD17β1, HSD17β2 or HSD3β7, only HSD11β1 and HSD17β12, all of which were reported to be expressed in lung, exhibited activity against NNK. Most interestingly, both enzymes formed primarily (R)-NNAL (&amp;gt;80%), which is consistent with that observed in lung microsomes. Real-time PCR showed that HSD17β12 was expressed at a level that, on average, was three-fold higher than that observed for HSD11β1 in a screening of normal lung tissue specimens from five individuals. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD11β1 and HSD17β12 are the active microsomal reductases in human lung microsomes, contributing to the exclusive formation of (R)-NNAL Citation Format: Joseph Ashmore, Ying Liu, Amity Peterson, Sabrina Fechtner, Gang Chen, Philip Lazarus. Characterization of human lung enzymes involved in the reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2722. doi:10.1158/1538-7445.AM2015-2722

Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography–tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.