Human Arylamine N-Acetyltransferase 1 (NAT1) Knockout in MDA-MB-231 Breast Cancer Cell Lines Leads to Transcription of NAT2

Many cancers, including breast cancer, have shown differential expression of human arylamine N-acetyltransferase 1 (NAT1). The exact effect this differential expression has on disease risk and progression remains unclear. While NAT1 is classically defined as a xenobiotic metabolizing enzyme, other functions and roles in endogenous metabolism have recently been described providing additional impetus for investigating the effects of varying levels of NAT1 on global gene expression. Our objective is to further evaluate the role of NAT1 in breast cancer by determining the effect of NAT1 overexpression, knockdown, and knockout on global gene expression in MDA-MB-231 cell lines. RNA-seq was utilized to interrogate differential gene expression (genes correlated with NAT1 activity) across three biological replicates of previously constructed and characterized MDA-MB-231 breast cancer cell lines expressing parental (Scrambled), increased (Up), decreased (Down, CRISPR 2–12), or knockout (CRISPR 2–19, CRISPR 5–50) levels of NAT1. 3,889 genes were significantly associated with the NAT1 N-acetylation activity of the cell lines (adjusted p ≤ 0.05); of those 3,889 genes, 1,756 were positively associated with NAT1 N-acetylation activity and 2,133 were negatively associated with NAT1 N-acetylation activity. An enrichment of genes involved in cell adhesion was observed. Additionally, human arylamine N-acetyltransferase 2 (NAT2) transcripts were observed in the complete NAT1 knockout cell lines (CRISPR 2–19 and CRISPR 5–50). This study provides further evidence that NAT1 functions as more than just a drug metabolizing enzyme given the observation that differences in NAT1 activity have significant impacts on global gene expression. Additionally, our data suggests the knockout of NAT1 results in transcription of its isozyme NAT2.

Effects of Genetic Polymorphisms of Cathepsin A on Metabolism of Tenofovir Alafenamide

Cathepsin A (CatA) is important as a drug-metabolizing enzyme responsible for the activation of prodrugs, such as the anti-human immunodeficiency virus drug Tenofovir Alafenamide (TAF). The present study was undertaken to clarify the presence of polymorphisms of the CatA gene in healthy Japanese subjects and the influence of gene polymorphism on the expression level of CatA protein and the drug-metabolizing activity. Single-strand conformation polymorphism method was used to analyze genetic polymorphisms in healthy Japanese subjects. Nine genetic polymorphisms were identified in the CatA gene. The polymorphism (85_87CTG>-) in exon 2 was a mutation causing a deletion of leucine, resulting in the change of the leucine 9-repeat (Leu9) to 8-repeat (Leu8) in the signal peptide region of CatA protein. The effect of Leu8 on the expression level of CatA protein was evaluated in Flp-In-293 cells with a stably expressed CatA, resulting in the expression of CatA protein being significantly elevated in variant 2 with Leu8 compared with Leu9. Higher concentrations of tenofovir alanine (TFV-Ala), a metabolite of TAF, were observed in the Leu8-expressing cells than in the Leu9-expressing cells using LC/MS/MS. Our findings suggest that the drug metabolic activity of CatA is altered by the genetic polymorphism.

Oxidative Stress Induced By The Metallic Oxide The Copper Oxide (Cuo-Nps) On Terrestrial Snail Helix Aspersa

Our study focused on the evaluation of the toxicity of copper oxide nanoparticles (CuO-NPs) on a bioindicator; the land snail Helix aspersa. Their effects were studied by a targeted approach in the laboratory, by evaluating the oxidative stress biomarkers in hepatopancreas and kidney (GSH, GST, GPx, CAT, and LPO). The snails were exposed to increasing concentrations (50, 100, 150, and 200 mg/kg) of CuO-NPs mixed in wheat flour during a sub-chronic treatment period of 45 days. Our results show that: CuO-NPs can induce oxidative stress, by producing reactive oxygen species (ROS), which was confirmed by the decrease in glutathione (GSH) level and reduction of its metabolizing enzyme glutathione-s-transferase (GST) in both organs, as they trigger the detoxification system resulting in increased activity of the glutathione peroxidase (GPx) and catalase defense enzyme and lipide peroxidation indices within the hepatopancreas.

CYP19A1 mediated sex hormone metabolism promotes severe SARS-CoV-2 disease outcome in males.

Male sex belongs to one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that could affect sex dependent disease outcome are yet unknown. Here, we identified the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (alias aromatase) as a male abundant host factor that contributes to worsened disease outcome in SARS-CoV-2 infected male hamsters. Pulmonary CYP19A1 transcription is further elevated upon viral infection in males correlating with reduced testosterone and increased estradiol levels. Dysregulated circulating sex hormone levels in male golden hamsters are associated with reduced lung function compared to females. Treatment of SARS-CoV-2 infected hamsters with letrozole, a clinically approved CYP19A1 inhibitor, supported recovery of dysregulated plasma sex hormone levels and was associated with improved lung function and health in male but not female animals compared to placebo controls. Whole human exome sequencing data analysis using a Machine Learning approach revealed a CYP19A1 activity increasing mutation being associated with the development of severe COVID-19 for men. In human autopsy-derived lungs CYP19A1 was expressed to higher levels in men who died of COVID-19, at a time point when most viral RNA was cleared. Our findings highlight the role of the lung as a yet unrecognized but critical organ regulating metabolic responses upon respiratory virus infection. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may pose a new therapeutic strategy to reduce poor long-term COVID-19 outcome.

Mitochondrial control of microglial phagocytosis in Alzheimer’s disease

Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic aggregates of beta amyloid (Aβ) in Alzheimer’s disease (AD). Recent evidence indicates that metabolic programming may breakdown in microglia in AD, thereby disrupting this important protective function. The mechanisms coordinating mitochondrial metabolism to fuel phagocytosis in microglia remain poorly understood, however. Here we demonstrate that mitochondrial displacement of the glucose metabolizing enzyme, hexokinase-II (HK) regulates microglial metabolism and phagocytosis, and that deletion of the translocator protein (TSPO) inhibits this. TSPO is a PET-visible inflammatory biomarker and therapeutic target in AD, previously shown to regulate microglial metabolism via an unknown mechanism. Using RNAseq and proteomic analyses, we found TSPO function in the brain to be linked with the regulation of mitochondrial bioenergetics, lipid metabolism and phagocytosis. In cultured microglia, TSPO deletion was associated with elevated mitochondrial recruitment of HK, which was associated with a switch to non-oxidative glucose metabolism, reduced mitochondrial energy production, lipid storage and impaired phagocytosis. Consistent with in vitro findings, TSPO expression was also associated with phagocytic microglia in both AD brain and AD mice. Conversely, TSPO deletion in AD mice reduced phagocytic microglia and exacerbated amyloid accumulation. Based on these findings we propose that microglial TSPO functions as an immunometabolic brake via regulation of mitochondrial HK recruitment, preventing hyperglycolysis and promoting phagocytosis in AD. Further, we demonstrate that targeting mitochondrial HK may offer a novel immunotherapeutic approach to promote microglial phagocytosis in AD.

Anti-inflammatory Effects in LPS-treated RAW 264.7 Cells and the Influences on Drug Metabolizing Enzyme Activities by the Traditional Herbal Formulas, Yongdamsagan-Tang and Paljung-san

Objectives: Yongdamsagan-tang (YST) and Paljung-san (PJS) in traditional medicine and finasteride in modern medicine are used to treat benign prostatic hyperplasia (BPH). In recent, the use of combination herbal remedies with conventional drugs has been increasing. Therefore, we investigated the anti-inflammatory effects of these drugs to treat BPH and the influence of herbal formulas on finasteride metabolism.Methods: The inhibitory effects of the herbal formulas and finasteride on the production of inflammatory mediators and cytokines were determined in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Additionally, the influence of herbal formulas on activities of human drug metabolizing enzymes (DMEs) was assessed using human microsomal enzymes.Results: We observed that YST, PJS and finasteride inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2) and interleukin-6 (IL-6) in RAW 264.7 cells. The half maximal inhibitory concentration (IC50) of YST on PGE2 production was calculated to be below 25 μg/mL. YST inhibited the activity of uridine diphosphateglucuronosyltransterase (UGT) 1A4 with an IC50 value of 49.35 μg/mL. The activities of cytochrome P450 (CYP) 1A2, CYP2B6, CYP2C19, CYP3A4, and UGT1A1 were inhibited by PJS (IC50 < 100 μg/mL, each). Although PJS and YST inhibited the activities of CYP3A4 and UGT1A4, respectively, these formulas may not influence the metabolism of finasteride because the IC50 values of herbal formulas on DMEs are too high to affect metabolism.Conclusions: Our results suggest that the combination of finasteride and YST or PJS might not influence their drug metabolism and that the drugs may have synergistic effects against BPH.

A Novel System for Evaluating the Inhibition Effect of Drugs on Cytochrome P450 Enzymes in vitro Based on Human-Induced Hepatocytes (hiHeps)

Cytochrome P450 (CYP) is the most important phase I drug-metabolizing enzyme, and the effect of drugs on CYP enzymes can lead to decreased pharmacological efficacy or enhanced toxicity of drugs, but there are many deficiencies in the evaluation models of CYP enzymes in vitro. Human-induced hepatocytes (hiHeps) derived from human fibroblasts by transdifferentiation have mature hepatocyte characteristics. The aim was to establish a novel evaluation system for the effect of drugs on CYP3A4, 1A2, 2B6, 2C9, and 2C19 in vitro based on hiHeps. Curcumin can inhibit many CYP enzymes in vitro, and so the inhibition of curcumin on CYP enzymes was compared by human liver microsomes, human hepatocytes, and hiHeps using UPLC-MS and the cocktail method. The results showed that the IC50 values of CYP enzymes in the hiHeps group were similar to those in the hepatocytes group, which proved the effectiveness and stability of the novel evaluation system in vitro. Subsequently, the evaluation system was applied to study the inhibitory activity of notoginseng total saponins (NS), safflower total flavonoids (SF), and the herb pair of NS–SF on five CYP enzymes. The mechanism of improving efficacy after NS and SF combined based on CYP enzymes was elucidated in vitro. The established evaluation system will become a powerful tool for the research of the effect of drugs on the activity of CYP enzymes in vitro, which has broad application prospects in drug research.

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic’s Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.