Hydroxy Metabolites Research Articles

Comprehensive evaluation of cocaine and its hydroxy metabolites in seized cocaine and a large cohort of hair samples.

As cocaine (COC) is not only incorporated into hair via blood following ingestion but also by external contamination, hair samples are commonly tested for COC metabolites to prove ingestion. However, COC metabolites can also be present as degradation products in typical street COC samples. The present study investigates minor hydroxycocaine (OH-COC) metabolites p- and m-OH-COC together with p- and m-hydroxybenzoylecgonine (OH-BE) in seized COC (n = 200) and hair samples from routine case work (n = 2389). Analytical results of hair samples were interpreted using an established decision model for the differentiation between actual use and external contamination using metabolic ratios (metabolite to COC). They were further examined concerning background of request, hair color, body site of sample collection, sex, and metabolic ratios of the main metabolites [benzoylecgonine (BE), norcocaine (NC), and cocaethylene (CE)]. All seized COC samples were positive for p- and m-OH-COC with a maximum percentage of 0.025% and 0.052%, respectively; p- and m-OH-BE were detected in 55% and 56% of samples with a maximum percentage of 0.044% and 0.024%, respectively. Analytical results of 424 hair samples (17.7%) were interpreted as being predominantly from contamination; the majority of these samples were from traffic medicine cases (83.7%). Metabolic ratios of minor OH-COC metabolites were significantly higher in hair samples interpreted as originating from use than in samples interpreted as caused by contamination. Metabolic ratios for OH-COCs were significantly higher in forensic cases compared to abstinence controls and also in black hair compared to blond/gray hair. However, this was not the case for OH-BE metabolic ratios. No statistical difference was observed with regard to the donor's sex. OH-COC metabolic ratios increased significantly with increasing ratios of NC and CE to COC, respectively. The study demonstrates that OH-COC metabolites (including thresholds for their metabolic ratios) must be used for a reliable interpretation of positive COC results in hair samples.

Electrochemical Oxidative Degradation and Trapping of the Mefenamic Acid Drug as a Redox-Active Hydroxy Metabolite on a Carbon Black Surface: Mediated Oxidation and Sensing of Thiol Biomarker

Electrochemical Oxidative Degradation and Trapping of the Mefenamic Acid Drug as a Redox-Active Hydroxy Metabolite on a Carbon Black Surface: Mediated Oxidation and Sensing of Thiol Biomarker

Exploration of vitamin D hydroxy metabolites C3 epimers in cardiovascular patients: comparative cross-sectional study

Abstract Background Research on vitamin D deficiency has recently surged in popularity. This issue has been acknowledged as a widespread problem, often called a "pandemic", and approximately 90% of the Polish population suffers from vitamin D deficiency. The 3-epi-25(OH)D2 and 3-epi-25(OH)D3 metabolites are stereoisomers of 25(OH)D2 and 25(OH)D3 metabolites, and they may be accidentally measured when determining 25(OH)D levels and result in overestimating metabolite concentrations. Purpose Our study aims to identify vitamin D hydroxy metabolites (25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and their epimers 3-epi-25-hydroxyvitamin D2 and 3-epi-25-hydroxyvitamin D3) in patients with cardiovascular disease and healthy volunteers. To our knowledge, no similar studies have been conducted on this topic. Method We included 27 adult patients with venous thrombosis (fourteen males and thirteen females, aged 64.11±14.84 years) and 35 healthy volunteers (thirty-three females and two males, aged 36.37±12.29 years). We used our validated UPLC-MS/MS method to measure the concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D2, and 3-epi-25(OH)D3. Statistica 13.3 with Plus Kit 3.0 was used for the statistical assessment. Normal variables were represented as mean and standard deviation, and non-normal variables as the median and interquartile range. A P-value < 0.05 was considered statistically significant for all tests. Results Patients had significantly lower levels of epi-25(OH)D2 than the healthy control group (0.6 [0.4] ng/mL vs. 2.2 [1.3-4.3] ng/mL, p = 0.004). Similarly, they exhibited significantly lower levels of %epi-25(OH)D2 (8.6 [6-22.3] % vs. 39.4 [18.5] %, p = 0.035). Females had higher levels of 25(OH)D3 than males (26.5 [16.2-37.9] ng/mL vs. 18.8 [7.5] ng/mL, p = 0.04). They also exhibited higher levels of total 25(OH)D3 (30.3 [15.6] ng/mL vs. 19.4 [7.8] ng/mL, p = 0.016). Higher age (OR 1.12, 95% CI 1.07 to 1.18), being male (OR 17.77, 95% CI 3.54 to 89.3), and lower % 3-epi-25(OH)D2 (OR 0.92, 95% CI 0.84 to 0.998) were univariate predictors for patients with venous thrombosis. Conclusion: Demographic factors within a given population may affect the 25(OH)D3 concentration. The results indicate that vitamin D epimeric metabolites, particularly epi-25(OH)D2, and their percentage were different in patients compared to healthy volunteers. Additional studies involving a larger population are necessary to fully comprehend the relationship between these vitamin D epimeric metabolites and cardiovascular health

Step-By-Step Procedure to Identify Previously Unknown Compounds by LC-QTOF-MS Exemplified by an Intoxication With the Methaqualone Analog SL-164.

In September 2019, a 22-year-old man with a history of drug abuse presented to the hospital with altered mental status. Due to a suspected drug overdose, a blood sample taken on admission and a urine sample collected 30 h thereafter were submitted to our laboratory to test for illegal drugs, pharmaceutical substances, and designer drugs. During the routine toxicological analysis of the serum sample, morphine and phenobarbital were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Additionally, two compounds showing identical accurate masses and isotope ratios as the designer benzodiazepine diclazepam and the benzodiazepine lormetazepam were found. However, retention times differed significantly from the expected values, and the acquired MS/MS spectra did not match the library entries of the two compounds, indicating the presence of two previously unknown substances. After further investigation, SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone), a methaqualone analog, which has recently emerged on the research chemical market, and its hydroxy metabolite were tentatively identified by accurate mass, isotope matching, and plausible fragmentation. However, for unequivocal confirmation and quantification, a reference standard is required. As no reference material was available by the end of 2019, SL-164 was obtained from an online shop, and its identity and purity (97.8%) were confirmed by nuclear magnetic resonance spectroscopy. The subsequent quantitative analysis revealed a concentration of 390 ng/mL SL-164 in serum. In the urine sample, the parent compound was not detected, but three suspected monohydroxylated metabolites were found. This example shows that LC-QTOF-MS is a powerful approach for the (tentative) identification of unknown compounds in biological matrices.

In vitro metabolism study of ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA using human hepatocytes, liver microsomes, and in-house synthesized references.

Synthetic cannabinoids (SCs) remain a major public health concern, as they continuously are linked to severe intoxications and drug-related deaths worldwide. As new SCs continue to emerge on the illicit drug market, an understanding of SC metabolism is needed to identify formed metabolites that may serve as biomarkers in forensic toxicology screening and for understanding the pharmacokinetics of the drugs. In this work, the metabolism of ADB-4en-P-5Br-INACA and ADB-P-5Br-INACA ((S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-(pent-4-en-1-yl)-1H-indazole-3-carboxamide, (S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-5-bromo-1-pentyl-1H-indazole-3-carboxamide respectively) were investigated using human hepatocytes in vitro and in-house synthesized references. Both SCs were incubated with pooled human hepatocytes over 3h, with the aim to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total nine metabolites were identified for ADB-4en-P-5Br-INACA and 10 metabolites for ADB-P-5Br-INACA. The observed biotransformations included dihydrodiol formation, terminal amide hydrolysis, hydroxylation, dehydrogenation, carbonyl formation, glucuronidation, and combinations thereof. The major metabolites were confirmed by in-house synthesized references. Recommended biomarkers for ADB-P-5Br-INACA and ADB-4en-P-5Br-INACA are the terminal hydroxy and dihydrodiol metabolite respectively.

Chiral Hydroxy Metabolite of Mebendazole: Analytical and Semi-Preparative High-Performance Liquid Chromatography Resolution and Chiroptical Properties.

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 μm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water-acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase.

Quantitation of propofol metabolites by LC–MS/MS demonstrating long detection window for urine drug monitoring

IntroductionChromatographic methods for analysis of propofol and its metabolites have been widely used in pharmacokinetic studies of propofol distribution, metabolism, and clearance. Application of chromatographic methods is also needed in clinical and forensic laboratories for detecting and monitoring propofol misuse. ObjectiveWe report a method for sensitive analysis of propofol, propofol 1-glucuronide (PG), 4-hydroxypropofol 1-glucuronide (1-QG), 4-hydroxypropofol 4-glucuronide (4-QG) and 4-hydroxypropofol 4-sulfate (4-QS) in urine by LC–MS/MS analysis. The method employs a simple dilute-and-analyze sample preparation with stable isotope internal standardization. ResultsValidation studies demonstrate a linear calibration model (100–10,000 ng/mL), with dilution integrity verified for the extended range of concentrations experienced in propofol use. Criteria-based validation was achieved, including an average coefficient of variation of 6.5 % and a percent bias of −4.2 ng/mL. The method was evaluated in 12 surgical patients, with monitoring periods lasting up to 30 days following intravenous propofol administrations of 100–3000 mg on the day of surgery. While the concentration ratio of PG to 4-hydroxy propofol metabolite decreased significantly in the days following surgery, PG maintained the highest concentration in all specimens. Both PG and 1-QG were detectable throughout the monitoring periods, including in a patient monitored for 30 days. Lower concentrations were determined for 4-QG and 4-QS, with evidence of detection up to 20 days. Propofol was not detectable in any urine specimens, thereby proving ineffective for identifying drug use. ConclusionThe validated method for quantifying propofol metabolites demonstrates its applicability for the sensitive detection of propofol misuse over a long window of drug-use detection.

Elucidating the Mechanism of Metabolism of Cannabichromene by Human Cytochrome P450s.

Cannabichromene (CBC) is a nonpsychoactive phytocannabinoid well-known for its wide-ranging health advantages. However, there is limited knowledge regarding its human metabolism following CBC consumption. This research aimed to explore the metabolic pathways of CBC by various human liver cytochrome P450 (CYP) enzymes and support the outcomes using in vivo data from mice. The results unveiled two principal CBC metabolites generated by CYPs: 8'-hydroxy-CBC and 6',7'-epoxy-CBC, along with a minor quantity of 1″-hydroxy-CBC. Notably, among the examined CYPs, CYP2C9 demonstrated the highest efficiency in producing these metabolites. Moreover, through a molecular dynamics simulation spanning 1 μs, it was observed that CBC attains stability at the active site of CYP2J2 by forming hydrogen bonds with I487 and N379, facilitated by water molecules, which specifically promotes the hydroxy metabolite's formation. Additionally, the presence of cytochrome P450 reductase (CPR) amplified CBC's binding affinity to CYPs, particularly with CYP2C8 and CYP3A4. Furthermore, the metabolites derived from CBC reduced cytokine levels, such as IL6 and NO, by approximately 50% in microglia cells. This investigation offers valuable insights into the biotransformation of CBC, underscoring the physiological importance and the potential significance of these metabolites.

Optimization of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its phase-1 metabolites in human biological fluids

Recently we published in this journal an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 3,4-methylenedioxymethamphetamine (MDMA) and its major phase-1 metabolites, 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid and urine. Since we did not achieve simultaneous enantioseparation of all 4 compounds with a single chiral column, two amylose-based chiral columns were used alternatively. Further optimization of the mobile phase in the present study enabled baseline separation of all four pairs of enantiomers on a single Lux AMP column. In addition, by optimization of the column dimension and applied flow-rate it became possible to complete the separation within 6 minutes. These new methods were applied to the analysis of human plasma, oral fluid and urine. While results on the concentration of MDMA and its metabolites in various biological fluids were reported in our recent publication, in the present study an attempt was made to hydrolyze glucuronides in urine samples by using alternatively, hydrochloric acid or glucuronidase and to evaluate the effect of hydrolysis on the concentration and enantiomeric distribution of hydroxy metabolites of MDMA such as HMA and HMMA.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS)methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometrymethod was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

Analytical toxicology of the semi-synthetic cannabinoid hexahydrocannabinol studied in human samples, pooled human liver S9 fraction, rat samples, and drug product using HPLC-HRMS/MS.

Hexahydrocannabinol (HHC) is an emerging semi-synthetic cannabinoid, that is obtained from cyclization of cannabidiol and subsequent hydrogenation. As a potentially legal alternative of Δ9-tetrahydrocannabinol (Δ9-THC), it is increasingly seized in the United States and Europe. The aims of this study were to investigate the metabolism of HHC in pooled human liver S9 fraction (pHLS9), in rat, and human samples. Additionally, a locally obtained low-THC cannabis product was investigated that was advertised with elevated concentration of HHC. Overall, HHC formed an 11-HO metabolite, as well as a carboxy metabolite. While only the parent compound was detected in rat urine and feces, the hydroxy metabolite was additionally detected in pHLS9 and human plasma. The carboxy metabolite was only detectable in human plasma. The metabolism corresponded well to that of Δ9-THC, although glucuronidation and the formation of an 8-HO metabolite was not observed. Detectability of HHC and its carboxy metabolite in rat urine was investigated using GC-MS, but neither the parent compound, nor the metabolite were detectable. The investigated low-THC cannabis product appeared to be an actual cannabis product, since in addition to HHC, cannabinol, cannabidiol, and Δ9-THC were detected after qualitative analysis. Estimation of its content revealed 30.6 % HHC but also 4 % of Δ9-THC.

Consideration of Nevirapine Analogs To Reduce Metabolically Linked Hepatotoxicity: A Cautionary Tale of the Deuteration Approach.

Idiosyncratic drug reactions (IDRs) in their most deleterious form can lead to serious medical complications and potentially fatal events. Nevirapine (NVP), still widely used in developing countries for combinatorial antiretroviral and prophylactic therapies against HIV infection, represents a prototypical example of IDRs causing severe skin rashes and hepatotoxicity. Complex metabolic pathways accompanied by production of multiple reactive metabolites often complicate our understanding of IDR's origin. While assessment of NVP analogs has helped characterize the pathways involved in IDRs for NVP, which are largely driven by metabolism at the 12-methyl position, it has yet to be investigated if some of these analogs could be valuable replacement drugs with reduced reactive metabolite properties and drug-drug interaction (DDI) risks. Here, we evaluated a set of eight NVP analogs, including the deuterated 12-d3-NVP and two NVP metabolites, for their efficacy and inhibitory potencies against HIV reverse transcriptase (HIV-RT). A subset of three analogs, demonstrating >85% inhibition for HIV-RT, was further assessed for their hepatic CYP induction-driven DDI risks. This led to a closer investigation of the inactivation properties of 12-d3-NVP for hepatic CYP3A4 and a comparison of its propensity in generating reactive metabolite species. The metabolic shift triggered with 12-d3-NVP, increasing formation of the 2-hydroxy and glutathione metabolites, emphasized the importance of the dynamic balance between induction and metabolism-dependent inactivation of CYP3A4 and its impact on clearance of NVP during treatment. Unfortunately, the strategy of incorporating deuterium to reduce NVP metabolism and production of the electrophile species elicited opposite results, illustrating the great challenges involved in tackling IDRs through deuteration.

Controlled Release Matrix Formulation of Paliperidone in Concurrence with Regulatory Requirements

Paliperidone is the 9-hydroxy metabolite (9-hydroxy) of risperidone and is a psychotropic drug of the atypical antipsychotic family. Paliperidone has the racemates (+)- and (-)-paliperidone. It is a dopamine D2 antagonist with serotonergic 5-HT2A antagonistic action that acts centrally. ALZA OROS® osmotic medication release technology is used to create Invega ER tablets. It is a tri-layer longitudinally compressed tablet based on a sophisticated osmotic delivery method that is meant to administer the paliperidone in a defined way over 24 hours. This research aims to create a generic controlled-release single-layer matrix tablet of paliperidone. Different combinations of Polyox and hypromellose in the core were used, followed by coating, to assist/build a stable and strong formulation. All strengths have similar in-vitro dissolution profiles. Freeze formulation was assessed for nitrosamine risk assessment as well as challenge for alcohol dose dumping study. Paliperidone is a basic compound with a pKa1 of 8.2 (piperidine moiety) and a pKa2 of 2.6 (pyrimidine moiety). As a result, a substantial portion of the molecule is ionized at physiological pH. It is relatively insoluble in water (0.003 g/100 mL water at pH 7.4). The solubility decreases at higher pH (0.001 g/100 mL at pH 12.9) and significantly increases at lower pH (3 g/100 mL at pH 5.3). The partition coefficient octanol/water (log P) is 2.39. Hence, discriminating media was identified as pH 2.75 buffer. The Higuchi model was used for expressing the in-vitro release profile through matrix composition. Formulation withstands 0–40% alcoholic conditions under in-vitro release tests. It is easy to formulate, stable and cost-effective. The manufacturing process involves dry blending followed by compression and coating so there will be the least chemical interaction of an active substance with other excipients. Hence, there is a negligible possibility to generate nitrosamine impurity in the formulation. The formulation is classified as rugged against dose dumping.

Investigations on the in vitro and in vivo metabolic fate of the new synthetic opioid desmethylmoramide using HPLC-HRMS/MS for toxicological screening purposes.

New synthetic opioids are an increasing challenge for clinical and forensic toxicologists that developed over the recent years. Desmethylmoramide (DMM), a structural analogue of methadone, is one of the most recent appearances on the drug market. This study investigated its metabolic fate in rat and pooled human liver S9 fraction (pHLS9) to allow the identification of suitable urinary screening targets beyond the parent compound. The analysis of rat urine after the administration of DMM revealed five metabolites, which were the result of pyrrolidine ring or morpholine ring hydroxylation and combinations of them. Additionally, an N',N-bisdesalkyl metabolite was formed. Incubations of DMM using pHLS9 revealed a pyrrolidine hydroxy metabolite, as well as an N-oxide. No Phase II metabolites were detected in either rat urine or incubations using pHLS9. The metabolism of DMM did in part comply with that of its archetype dextromoramide (DXM). Although morpholine ring hydroxylation and N-oxidation were described for DXM and detected for DMM, phenyl ring hydroxylation was not found for DMM but described for DXM. An analysis of 24 h pooled rat urine samples after DMM administration identified the hydroxy and dihydroxy metabolite as the most abundant excretion products, and they may, thus, serve as screening targets, as the parent compound was barely detectable.

Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene.

(E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8months. Thus, isomer-specific HC elimination profiles could be monitored. The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

Effect of Fungicide Treatment on Multi-Mycotoxin Occurrence in French Wheat during a 4-Year Period

Wheat represents one of the most widely consumed cereals worldwide. Cultivated in winter and spring, it is vulnerable to an array of different pathogens, including fungi, which are managed largely through the in-field application of fungicides. During this study, a 4-year field investigation (2018–2021) was performed in France, aiming to assess the efficacy of fungicide treatment to reduce mycotoxin contamination in common and durum wheat. Several different commercially available fungicides were applied via sprayers. Concentrations of mycotoxins and fungal metabolites in wheat were determined using a multi-analyte liquid-chromatography–tandem-mass-spectrometry-based method. The highest contamination levels and strongest effects of fungicides were observed in 2018, followed by 2021. A significant fungicide-mediated reduction was observed for the trichothecenes deoxynivalenol, deoxynivalenol-3-glucoside, nivalenol, and nivalenol-3-glucoside. Furthermore, fungicide treatment also reduced levels of culmorin and its hydroxy metabolites 5- and 15-hydroxy-culmorin, as well as aurofusarin. Interestingly, the Alternaria metabolite infectopyron was increased following fungicide treatment. In conclusion, fungicide treatment was effective in reducing mycotoxin levels in wheat. However, as complete prevention of mycotoxin contamination was not achieved, fungicide treatment should always be combined with other pre- and post-harvest mycotoxin mitigation strategies to improve food and feed safety.

Comparative study on ABCB1-dependent efflux of anthracyclines and their metabolites: consequences for cancer resistance

1. ABCB1 (P-glycoprotein, MDR1) is one of the most important transporter involved in cancer multi-drug resistance. It also plays a significant role in cancer resistance against anthracyclines, an anticancer group of drugs, including doxorubicin and daunorubicin. Several intracellular enzymes metabolise anthracyclines to carbonyl-reduced, hydroxy metabolites, which have impaired cytotoxic properties. However, metabolite efflux by ABCB1 transporter is not well characterised, while it may be the mechanism responsible for the metabolites’ lack of activity. 2. In this study recombinant ABCB1 ATPase transporter assay; anthracyclines accumulation assay in resistant cells overexpressing ABCB1; and molecular modelling were used to investigate anthracyclines: doxorubicin and daunorubicin and their carbonyl-reduced metabolites (doxorubicinol, daunorubicinol) susceptibility for ABCB1-dependent efflux. 3. Based on the kinetics parameters of ATPase activity of ABCB1, it was found that daunorubicinol exerted an exceptionally high potential for being effluxed by the ABCB1 transporter. ABCB1 significantly affected the accumulation pattern of studied chemicals in resistant cancer cells. Doxorubicin and daunorubicinol accumulation were influenced by the activity of ABCB1 modulator – valspodar. 4. Results indicate that ABCB1 activity affects not only anthracyclines but also their metabolites. Therefore crosstalk between the process of anthracyclines metabolism and metabolite efflux may be the mechanism of impairing anticancer properties of anthracyclines metabolites.

Lipid metabolic disturbance induced by triphenyl phosphate and hydroxy metabolite in HepG2 cells

Triphenyl phosphate (TPHP) has been widely used as flame retardants and been detected with increasing frequency in environment. TPHP can transform into mono-hydroxylated phosphate (OH-TPHP) and diester diphenyl phosphate (DPHP) through biotransformation. So far, information on the cytotoxicity and molecular regulatory mechanisms of TPHP metabolites are still limit. This study investigated the adverse effects of TPHP, OH-TPHP, and DPHP in HepG2 cells in terms of cell proliferation, lactate dehydrogenase release, reactive oxygen species generation, and mitochondrial membrane potential. The transcriptomic changes were measured using RNA sequencing, and bioinformatics characteristics including biological functions, signal pathways and protein-protein interaction were analyzed to explore the potential molecular mechanisms. Results displayed that the order of cytotoxicity was OH-TPHP> TPHP> DPHP. The prioritized biological functions changes induced by TPHP and OH-TPHP were correlated with lipid metabolism. Significant lipid accumulation was observed as confirmed by increased total cholesterol and triglycerides contents, and enhanced oil red O staining. Enrichment of PPARα/γ and down-stream genes suggested the participation of PPARs signal pathway in lipid metabolism disorder. In addition, TPHP and OH-TPHP induced endoplasmic reticulum stress (ERS), which was further confirmed by the ERS inhibitor experiment. In general, TPHP and OH-TPHP had obvious cytotoxic effects in HepG2 cells. PPARs signal pathway and endoplasmic reticulum stress may be involved in the lipid metabolism disorder induced by TPHP and OH-TPHP.

71 Metabolomics Study of a Lung Model Exposed to Different Ultrafine Particles (UFP) Aerosols

Abstract Ultrafine particles (UFP), diameter of <0.1 µm, contribute to urban particulate air pollution. There is still not much evidence on their effects on humans. Our study aims to address their possible effects on lung metabolome. Alveolar epithelial cells (A549) together with macrophages (THP-1) were seeded on the apical side of the insert membrane, and fibroblasts (MRC-5) on the downside, respectively. Triple cell cultures were exposed at the air liquid interface to different aerosols generated by a combustion aerosol standard (CAST) soot generator. Applied aerosols consisted of UFP with either high or low content of semi-volatile organic compounds (SVOC). Herein, apical cell lysates were extracted for metabolites. Data acquisition was performed by GC-MS and processed with R and then analyzed by MetaboAnalyst. Statistical analysis showed that top features differentiating the high SVOC from clean air controls were amino acids (L-tryptophan and L-lysine), whereas for low SVOC, 5-methylthioadenosine and 5-methoxyindoleacetate. Tryptophan and its metabolite 5-hydroxy- tryptophan were significantly upregulated in high SVOC. However, in low SVOC, 5-methoxyindoleacetate was substantially upregulated. Pathway analysis revealed that purine metabolism was between the top significant enriched pathways in both high and low SVOC groups. Moreover, tryptophan metabolism which modulates immunity and inflammation was enriched similarly in both groups. These findings suggest that lung cell metabolome is modulated distinctively based on the chemical identity of UFP, which could give more understanding about the lung response mechanism to inhaled UFP. This work was funded by Bayerische Staatsministeriums für Umwelt und Verbraucherschutz (BayUFP).

Kinetics, mechanism, and tautomerism in ametryn acid hydrolysis: From molecular structure to environmental impacts

The excessive use of pesticides and the demand for environmentally friendly compounds have driven the focus to detailed studies of the environmental destination of these compounds. Degradation by hydrolysis of pesticides, when released into the soil, can result in the formation of metabolites with potentially adverse effects on the environment. Moving in this direction, we investigated the mechanism of acid hydrolysis of the herbicide ametryn (AMT) and predicted the toxicities of metabolites through experimental and theoretical approaches. The formation of ionized hydroxyatrazine (HA) occurs with the release of the SCH3- group and the addition of H3O+ to the triazine ring. The tautomerization reactions privileged the conversion of AMT into HA. Furthermore, the ionized HA is stabilized by an intramolecular reaction that provides the molecule in two tautomeric states. Experimentally, the hydrolysis of AMT was obtained under acidic conditions and at room temperature with HA as the main product. HA was isolated in a solid state through its crystallization as organic counterions. The mechanism of conversion of AMT to HA and the experimental investigation of the reaction kinetics allowed us to determine the dissociation of CH3SH as the rate-controlling step in the degradation process that culminates in a half-life between 7 and 24 months under typical acid soil conditions of the Brazilian Midwest – region with strong agricultural and livestock vocation. The keto and hydroxy metabolites showed substantial thermodynamic stability and a decrease in toxicity compared to AMT. We hope that this comprehensive study will support the understanding of the degradation of s-triazine-based pesticides.