Biotransformation of carbamazepine and nicotine in juvenile American alligator (Alligator mississippiensis) in vitro hepatic S9 vs. in situ perfused liver.

American alligators (Alligator mississippiensis) are apex predators and sentinel species in the coastal wetland ecosystem along the Gulf of Mexico. There is concern for alligator exposure and susceptibility to chemical contaminants due to their high trophic level and lower metabolic capability. At present, their hepatic biotransformation capacity to metabolize or detoxify contaminants has not been comprehensively determined. In this study, the hepatic biotransformation capability of juvenile American alligators to metabolize two commonly found environmental pharmaceuticals: carbamazepine (CBZ) or nicotine (NCT) was evaluated. The formation of their respective primary metabolites, i.e., carbamazepine-10,11-epoxide (CBZ-E) and cotinine (CTN), was evaluated at 10 µM (within the human therapeutic range). The in vitro S9 and a novel in situ liver perfusion assays were used to characterize and compare metabolic ability in isolated hepatic enzymes vs. whole organ (liver). For CBZ, the perfused livers exhibited only 30% of intrinsic formation clearance (CLf,int) relative to the S9 assay. The metabolism of NCT was not detectable in the S9 assay and was only observed in the perfused liver assay. Compared to the corresponding rat models (S9 or perfused livers),alligators' CLf,int was 2060% for CBZ and 50% for NCT of rats. Additionally, NCT exposure increased lactate levels in perfused livers indicating metabolic stress. This study provides insight into the hepatic capability of alligators to metabolize CBZ and NCT using an established in vitro (S9) system and a newly developed in situ liver perfusion system.

New insights into the oxidative and cytogenotoxic effects of Tetraglyme on human peripheral blood cells.

The present study investigated the oxidative and cytogenotoxic potential of Tetraethylene glycol dimethyl ether (known as Tetraglyme) on healthy human peripheral blood lymphocytes, widely used as an in vitro model for assessing the human health risk posed by different chemical compounds. In a first step, Nuclear Magnetic Resonance (1H NMR) spectroscopy, and Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) were employed to estimate Tetraglyme's stability under a wide range of pH values (4-12), and thus to identify potential by-products. Thereafter, isolated lymphocytes were treated with different concentrations of Tetraglyme (0.02-20 mg L-1) for assessing its oxidative (using the DCFH-DA staining), and cytogenotoxic potential (using the trypan blue exclusion test for estimating cell viability, Comet assay, as well as the cytokinesis-block micronucleus assay, with or without the addition of S9 metabolic activation system). According to the results, Tetraglyme remains stable at pH 4, but two additional derivatives (i.e. 1-[2-(2-ethoxyethoxy)ethoxy]-2-methoxyethane [C9H20O4] and 1-ethoxy-2-(2-ethoxyethoxy)ethane (Diethylene glycol diethyl ether) [C8H18O3]) were found in traces, under alkaline conditions (pH ≥7). Moreover, although Tetraglyme (and/or its derivatives) showed negligible alterations of cell viability (>92 %) in all cases, the pronounced ROS formation, DNA damage, cell proliferation arrest, and MN frequencies in challenged cells are indicative of its oxidative and cytogenotoxic potential. The significant alterations of Cytokinesis-Block Proliferation Index (CBPI) and Micronucleus (MN) frequencies in S9 challenged cells give further evidence for the potential involvement of Tetraglyme's metabolites in the observed cytogenotoxic mode of action. Although not conclusive, the present findings give rise to further research, utilizing different cell types and biological models, for elucidating Tetraglyme's toxic mode of action, as well as its environmental and human risk.

Differential genotoxicity of Polygoni Multiflori in rat and human: insights from Ames test and S9 metabolic activation system.

The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.

Subcellular effects of imidazolium-based ionic liquids with varying anions on the marine bivalve Mytilus galloprovincialis.

Green Chemistry involves applying a set of principles aimed at minimizing the use of hazardous substances in the design, production, and application of chemical products. In recent decades, Ionic Liquids (ILs) have emerged as more environmentally friendly substitutes for traditional organic solvents. This preference is primarily due to their low vapor pressure, which results in minimal atmospheric pollution and enhanced industrial safety. However, existing literature highlights the toxicity of ILs towards aquatic invertebrates. Consequently, this study points to assess the biochemical effects of a selection of ILs through an in vitro approach. Specifically, digestive gland and gill cellular fractions (S9) of the marine bivalve Mytilus galloprovincialis were exposed to varying concentrations (0.05-2 µM) of three ILs featuring identical cations but different anions. The ILs tested were 1-ethyl-3-methylimidazolium octanoate ([EMIM][Oct]), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), and 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIM][EtSO4]). The results indicate that [EMIM][Oct] induces higher toxicity in both S9 tissues, highlighting a strong effect of the anion. Overall, antioxidant and biotransformation defenses were significantly altered for all three ILs assessed. While acetylcholinesterase activity was significantly inhibited of about half of control activity, indicating neurotoxic damage as part of the toxicity mode of action of these ILs, neither lipid peroxidation nor alterations to DNA integrity were observed (≥100 %). This study supports the use of in vitro techniques as important tools capable of generating reliable ecotoxicological data, which can be further considered as a screening before in vivo testing and used for in silico modeling.

Electrochemical Probing of Human Liver Subcellular S9 Fractions for Drug Metabolite Synthesis.

Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O2-saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s-1, with a formal potential of -0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography-mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms.

Computational analysis of human gut microbial prolyl oligopeptidases (POPs) reveal candidate genes as therapeutics for celiac disease.

Celiac disease (CD) is a common autoimmune disorder in which the patients are unable to digest gluten, which is present in foods made up of wheat, barley and rye. Whilst diagnosis happens late in 80% of the cases, avoidance of such foods appears to be the common solution. Alternative management strategies are required for the patients and their families since CD is also genetically carried over. Probiotic therapeutics and the consumption of appropriate enzymes, such as prolyloligopeptidases (POPs), from gut-friendly bacteria could reduce the disease burden and provide a better lifestyle for CD patients. We have examined around 5000 gut bacterial genomes and identified nearly 4000 non-redundant putative POPs. A select set of 10 gut bacterial POP sequences were subject to three-dimensional modelling, ligand docking and molecular dynamics simulations where stable interactions were observed between the POPs and gluten peptides. Our study provides sequence and structural analysis of potential POP enzymes in gut bacterial genomes, which form a strong basis to offer probiotic solutions to CD patients. In particular, these enzymes could be lead future therapeutics for this disease.

In vitro evaluation of the selective cytotoxicity and genotoxicity of three synthetic ortho-nitrobenzyl derivatives in human cancer cell lines, with and without metabolic activation.

Although the presence of nitro groups in chemicals can be recognized as structural alerts for mutagenicity and carcinogenicity, nitroaromatic compounds have attracted considerable interest as a class of agents that can serve as source of potential new anticancer agents. In the present study, the in vitro cytotoxicity, genotoxicity, and mutagenicity of three synthetic ortho-nitrobenzyl derivatives (named ON-1, ON-2 and ON-3) were evaluated by employing human breast and ovarian cancer cell lines. A series of biological assays was carried out with and without metabolic activation. Complementarily, computational predictions of the pharmacokinetic properties and druglikeness of the compounds were performed in the Swiss ADME platform. The MTT assay showed that the compounds selectively affected selectively the cell viability of cancer cells in comparison with a nontumoral cell line. Additionally, the metabolic activation enhanced cytotoxicity, and the compounds affected cell survival, as demonstrated by the clonogenic assay. The comet assay, the cytokinesis-block micronucleus assay, and the immunofluorescence of the γ-H2AX foci formation assay have that the compounds caused chromosomal damage to the cancer cells, with and without metabolic activation. The results obtained in the present study showed that the compounds assessed were genotoxic and mutagenic, inducing double-strand breaks in the DNA structure. The high selectivity indices observed for the compounds ON-2 and ON-3, especially after metabolic activation with the S9 fraction, must be highlighted. These experimental biological results, as well as the theoretical properties predicted for the compounds have shown that they are promising anticancer candidates to be exploited in additional studies.

Metabolism of alcohol ethoxylates (AEs) in rat, hamster, and human hepatocytes and liver S9: a pilot study for metabolic stability, metabolic pathway, and metabolites identification in vitro and in silico.

Alcohol ethoxylates (AEs) are a well-known class of non-ionic surfactants widely used by the personal care market. The aim of this study was to evaluate and characterize the in vitro metabolism of AEs and identify metabolites. Five selected individual homologue AEs (C8EO4, C10EO5, C12EO4, C16EO8, and C18EO3) were incubated using human, rat, and hamster liver S9 fraction and cryopreserved hepatocytes. LC-MS was used to identify metabolites following the incubation of AEs by liver S9 and hepatocytes of all three species. All AEs were metabolized in these systems with a half-life ranging from 2 to 139 min. In general, incubation of AE with human liver S9 showed a shorter half-life compared to rat liver S9. While rat hepatocytes metabolized AEs faster than human hepatocytes. Both hydrophobic alkyl chain and hydrophilic EO head group groups of AEs were found to be target sites of metabolism. Metabolites were identified that show primary hydroxylation and dehydrogenation, followed by O-dealkylation (shortening of EO head groups) and glucuronidation. Additionally, the detection of whole EO groups indicates the cleavage of the ether bond between the alkyl chain and the EO groups as a minor metabolic pathway in the current testing system. Furthermore, no difference in metabolic patterns of each individual homologue AE investigated was observed, regardless of alkyl chain length or the number of EO groups. Moreover, there is an excellent agreement between the in vitro experimental data and the metabolite profile simulations using in silico approaches (OECD QSAR Toolbox). Altogether, these data indicate fast metabolism of all AEs with a qualitatively similar metabolic pathway with some quantitative differences observed in the metabolite profiles. These metabolic studies using different species can provide important reference values for further safety evaluation.

Application of a generic PBK model for beef cattle: Tissue/fluid correlations of paracetamol and NSAIDs.

Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) and paracetamol can be administered off-label to cattle. Since the use of these veterinary medicines in cattle may pose a public health risk after meat consumption, it is important to translate measured concentrations in urine and tissues into concentrations in meat for human consumption. A generic physiologically-based kinetic (PBK) model for cattle can enable this translation. In this work, a beef cattle PBK model was applied to calculate the relationships between concentrations in different bovine tissues and those were compared to measured concentrations in different matrices. Sixty-seven kidney samples, the corresponding urine and meat samples, and available 19 serum samples were analysed. Overall, 70% of the PBK model predictions are within a 2-fold factor and relationships for kidney/meat, urine/meat, and plasma/meat ratios were established. The conversions of measured kidney concentrations into meat concentrations were mostly within a factor two, while those based on plasma and urine were underpredicted. Based on these ratios, plasma and urine could be used as an appropriate surrogate matrix for a fast, simple in vivo sample screening test under field conditions, such as in local farms and slaughterhouses, to predict a maximum residue level exceedance in meat, reducing the number of test samples.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCR sample solution.

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

The European Union has banned Triton X-100. All reagents containing it should be replaced. Could a new Viral PCR Sample Solution (VPSS) containing Tergitol 15-S-9 be a suitable replacement?

A Comparison of In Vitro Metabolic Clearance of Various Regulatory Fish Species Using Hepatic S9 Fractions.

Bioaccumulation predictions can be substantially improved by combining in vitro metabolic rate measurements derived from rainbow trout hepatocytes and/or hepatic S9 fractions with quantitative structure-activity relationship (QSAR) modeling approaches. Compared with in vivo testing guidelines Organisation for Economic Co-operation and Development (OECD) 305 and Office of Chemical Safety and Pollution Prevention (OCSPP; an office of the US Environmental Protection Agency) 850.1730, the recently adopted OECD test guidelines 319A and 319B are in vitro approaches that have the potential to provide a time- and cost-efficient, humane solution, reducing animal use while addressing uncertainties in bioaccumulation across species. The present study compares the hepatic clearance of the S9 subcellular fraction of rainbow trout, bluegill, common carp, fathead minnow, and largemouth bass, discerning potential differences in metabolism between different warm- and cold-water species. With refinements to the in vitro metabolic S9 assay for high-throughput analysis, we measured in vitro clearance rates of seven chemicals crossing multiple classes of chemistry and modes of action. We confirmed that data from rainbow trout liver S9 fraction metabolic rates can be utilized to predict rainbow trout bioconcentration factors using an in vitro to in vivo extrapolation model, as intended in the OECD 319B applicability domain per the bioaccumulation prediction. Also, we determined that OECD 319B can be applied to other species, modified according to their habitat, adaptations to feeding behavior, and environmental conditions (e.g., temperature). Once toxicokinetics for each species is better understood and appropriate models are developed, this method can be an excellent tool to determine hepatic clearance and potential bioaccumulation across species. The present study could be leveraged prior to or in place of initiating in vivo bioconcentration studies, thus optimizing selection of appropriate fish species. Environ Toxicol Chem 2024;43:1390-1405. © 2024 SETAC.

Smartphone-based device for rapid and single-step detection of piRNA-651 using anti-DNA:RNA hybrid antibody and enzymatic signal amplification.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs/piRs) are a class of small noncoding RNAs that play a crucial role in regulating various biological processes, including carcinogenesis. One specific piRNA, piR-651, has been reported to be overexpressed in both human blood serum and solid cancer tissues, that can be used a viable biomarker in cancer diagnosis. Early diagnosis of cancer can help reduce the burden of the disease and improve survival rates. In the present work, we report for the first time a smartphone-based colorimetric biosensor for highly sensitive and specific detection of piR-651 thanks to an enzymatic signal amplification, which yielded high colorimetric intensities. Indeed, a heteroduplex DNA:RNA was formed in the presence of piR-651 with the capture DNA probe immobilized on the magnetic beads for easy magnetic separation. Then, a HRP tethered to anti-DNA:RNA (S9.6) was used to reveal the DNA-RNA heteroduplex formed by catalyzing the oxidation of TMB substrate into colorimetric TMBox, which absorbs at 630 nm. The absorbance is positively proportional to the piR-651 concentrations. On the other hand, the colorimetric product of the assay can be photographed with a smartphone camera and analyzed using ImageJ software. Using a smartphone and under optimal conditions, the biosensor responded linearly to the logarithm of piRNA-651 from 8 fM to 100 pM with a detection limit of 2.3 fM and discriminates against other piRNAs. It was also successfully applied to the determination of piRNA-651 levels in spiked human serum.

Approaches for Mapping and Analysis of R-loops.

R-loops are nucleic acid structures composed of a DNA:RNA hybrid with a displaced non-template single-stranded DNA. Current approaches to identify and map R-loop formation across the genome employ either an antibody targeted against R-loops (S9.6) or a catalytically inactivated form of RNase H1 (dRNH1), a nuclease that can bind and resolve DNA:RNA hybrids via RNA exonuclease activity. This overview article outlines several ways to map R-loops using either methodology, explaining the differences and similarities among the approaches. Bioinformatic analysis of R-loops involves several layers of quality control and processing before visualizing the data. This article provides resources and tools that can be used to accurately process R-loop mapping data and explains the advantages and disadvantages of the resources as compared to one another. © 2024 Wiley Periodicals LLC.

Metabolic activation of short-chain alkyl N-nitrosamines using Aroclor 1254 or phenobarbital/beta-naphthoflavone-induced rat or hamster S9 - A comparative analysis.

N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to be mutagenic carcinogens and have recently been detected as impurities in several marketed pharmaceuticals. Despite their known mutagenic properties, the suitability of the bacterial reverse mutation (Ames) assay and in particular the use of induced rat liver S9 to detect their mutagenic potential, is often discussed. Recently, it could be demonstrated that induced rat liver S9 is capable of metabolizing small alkyl nitrosamines to exert their mutagenic potential (Bringezu & Simon, 2022). In this project, the mutagenic potential of nitrosamines in vitro under different S9 conditions applying the preincubation protocol and OECD 471-compliant standard Ames test recommendations was investigated. These conditions included various amounts of S9 fraction from hamster and rat, uninduced or induced with Aroclor 1254 or Phenobarbital/beta-Naphthoflavone (PB/NF). The findings indicated that in addition to induced S9, uninduced hamster S9 also demonstrated effectiveness. Moreover, both rat and hamster S9 fractions exhibited suitable responses in terms of mutation frequencies. Increasing the S9 content did not increase the sensitivity of the Ames test. However, above 20% S9, reduced mutation frequency was observed in the higher concentration range suggesting cytotoxicity to the bacteria. Thus, limiting the S9 content to 10% provides reliable results and relates to a lower number of animals required for S9 production which is in concordance with the 3R principles (reduce, refine, replace) for animal testing. In addition, results obtained show that uninduced and induced hamster S9 are similarly effective, doubting the requirement of pretreating animals with enzyme inducers. Further investigations to compare mutagenicity data and rat and hamster S9 proteome analyses are ongoing.

S-9-PAHSA's neuroprotective effect mediated by CAIII suppresses apoptosis and oxidative stress in a mouse model of type 2 diabetes.

BACKGROUND: With the rapidly increasing prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM), neuronal complications associated with these diseases have resulted in significant burdens on healthcare systems. Meanwhile, effective therapies have remained insufficient. A novel fatty acid called S-9-PAHSA has been reported to provide metabolic benefits in T2DM by regulating glucose metabolism. However, whether S-9-PAHSA has a neuroprotective effect in mouse models of T2DM remains unclear. METHODS: This in vivo study in mice fed a high-fat diet (HFD) for 5 months used fasting blood glucose, glucose tolerance, and insulin tolerance tests to examine the effect of S-9-PAHSA on glucose metabolism. The Morris water maze test was also used to assess the impact of S-9-PAHSA on cognition in the mice, while the neuroprotective effect of S-9-PAHSA was evaluated by measuring the expression of proteins related to apoptosis and oxidative stress. In addition, an in vitro study in PC12 cells assessed apoptosis, oxidative stress, and mitochondrial membrane potential with or without CAIII knockdown to determine the role of CAIII in the neuroprotective effect of S-9-PAHSA. RESULTS: S-9-PAHSA reduced fasting blood glucose levels significantly, increased insulin sensitivity in the HFD mice and also suppressed apoptosis and oxidative stress in the cortex of the mice and PC12 cells in a diabetic setting. By suppressing oxidative stress and apoptosis, S-9-PAHSA protected both neuronal cells and microvascular endothelial cells in in vivo and in vitro diabetic environments. Interestingly, this protective effect of S-9-PAHSA was reduced significantly when CAIII was knocked down in the PC12 cells, suggesting that CAIII has a major role in the neuroprotective effect of S-9-PAHSA. However, overexpression of CAIII did not significantly enhance the protective effect of S-9-PAHSA. CONCLUSION: S-9-PAHSA mediated by CAIII has the potential to exert a neuroprotective effect by suppressing apoptosis and oxidative stress in neuronal cells exposed to diabetic conditions. Furthermore, S-9-PAHSA has the capability to reduce fasting blood glucose and LDL levels and enhance insulin sensitivity in mice fed with HFD.

Curcumol metabolized by rat liver S9 fraction and orally administered in mouse suppressed the proliferation of colon cancer in vitro and in vivo.

Following 3R (reduction, refinement, and replacement) principles, we employed the rat liver S9 fraction to mimic liver metabolism of curcumol having high in vitro IC50 on cancer cells. In HCT116 and HT29 colon cancer cells, the metabolites of curcumol by S9 fraction exerted more enhanced activity in inducing cell cycle arrest and apoptosis via regulating the expression of cyclin D1, CDK1, p21, PARP and Bcl-2 than curcumol. In addition, oral administration of curcumol at 4 mg/kg BW significantly suppressed the development of colon tumor induced by azoxymethane/dextran sulfate sodium, and induced cell cycle arrest and apoptosis in tumor tissues. In mass analysis, curcumenol and curzerene were identified as the metabolites of curcumol by S9 fraction metabolism. Taken together, curcumol metabolites showed the enhanced suppressive effect on colon cancer, suggesting that S9 fraction can be considered as simple, fast, and bio-mimicking platform for the screening of chemical libraries on different chronic diseases.

Interspecies comparison of metabolism of two novel prototype PFAS.

As a result of proposed global restrictions and regulations on current-use per-and polyfluoroalkyl substances (PFAS), research on possible alternatives is highly required. In this study, phase I in vitro metabolism of two novel prototype PFAS in human and rat was investigated. These prototype chemicals are intended to be safer-by-design and expected to mineralize completely, and thus be less persistent in the environment compared to the PFAS available on the market. Following incubation with rat liver S9 (RL-S9) fractions, two main metabolites per initial substance were produced, namely an alcohol and a short-chain carboxylic acid. While with human liver S9 (HL-S9) fractions, only the short-chain carboxylic acid was detected. Beyond these major metabolites, two and five additional metabolites were identified at very low levels by non-targeted screening for the ether- and thioether-linked prototype chemicals, respectively. Overall, complete mineralization during the in vitro hepatic metabolism of these novel PFAS by HL-S9 and RL-S9 fractions was not observed. The reaction kinetics of the surfactants was determined by using the metabolite formation, rather than the substrate depletion approach. With rat liver enzymes, the formation rates of primary metabolite alcohols were at least two orders of magnitude higher than those of secondary metabolite carboxylic acids. When incubating with human liver enzymes, the formation rates of single metabolite carboxylic acids, were similar or smaller than those experienced in rat. It also indicates that the overall metabolic rate and clearance of surfactants are significantly higher in rat liver than in human liver. The maximum formation rate of the thioether congener exceeded 10-fold that of the ether in humans but were similar in rats. Overall, the results suggest that metabolism of the prototype chemicals followed a similar trend to those reported in studies of fluorotelomer alcohols.

Metabolite characterisation of the peptide-drug conjugate LN005 in liver S9s by UHPLC-Orbitrap-HRMS.

LN005 is a peptide-drug conjugate (PDC) targeting glucose-regulated protein 78 (GRP78) to treat several types of cancer, such as breast, colon, and prostate cancer.As a new drug modality, understanding its metabolism and elimination pathways will help us to have a whole picture of it. Currently, there are no metabolic studies on LN005; therefore, this study aimed to investigate the metabolism of LN005, clarify its metabolic profile in the liver S9s of different species, and identify the major metabolic pathways and differences between species.The incubation samples were measured by ultra-high performance liquid chromatography combined with orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS).The results showed that LN005 was metabolised by liver S9s, and four metabolites were identified. The main metabolic pathway of LN005 in liver S9s was oxidative deamination to ketone or hydrolysis. Similar metabolic profiles were observed in mouse, rat, dog, monkey, and human liver S9s, indicating no differences between these four animal species and humans.This study provides information for the structural modification and optimisation of LN005 and affords a reference for subsequent animal experiments and human metabolism of other PDCs.

Non-mutagenic impurities - Recent industry experience of using dose durational limits in drug development.

Absence of clear guidance on the qualification threshold for non-mutagenic impurities during clinical development is a source of inconsistency in both sponsor qualification approaches and health authority requests. A survey was conducted in March 2020 with 6 member companies of the European Federation of Pharmaceutical Industries and Associations (EFPIA). Thirteen examples were gathered of where non-International Council for Harmonisation (ICH) limits have been used in regulatory submissions for various indications and stages of development, together with the regulatory outcomes. As expected, few challenges were faced in early clinical development, with health authorities generally commenting that sponsors should work towards ICH Q3A and Q3B guideline specification limits as development progresses. However, inconsistent health authority requests were noted even for early phase clinical trials in late-stage oncology patients. For an optimised use of resources, consistent approaches would have the benefit of supporting faster access of safe medicines to patients while including Replacement, Reduction and Refinement (the 3Rs) considerations with respect to animal testing.