Exogenous Compounds Research Articles

CORM-3 alleviates the intestinal injury in a rodent model of hemorrhage shock and resuscitation: roles of GFAP-positive glia.

Hemorrhagic shock and resuscitation (HSR) can induce severe intestinal damages, thereby leading to sepsis and long-term complications including dysbacteriosis and pulmonary injury. The NOD-like receptor protein 3 (NLRP3) inflammasome facilitates inflammation-associated cell recruitment in the gastrointestinal tract, and participates in many inflammatory bowel diseases. Previous studies have shown that exogenous carbon monoxide (CO) exerts neuroprotective effects against pyroptosis after HSR. We aimed to investigate whether carbon monoxide-releasing molecules-3 (CORM-3), an exogenous CO compound, could attenuate HSR-induced intestinal injury and the potential underlying mechanism.Rats were subjected to a HSR model by bleeding and re-infusion. Following resuscitation, 4mg/kg of CORM-3 was administered intravenously into femoral vein. At 24h and 7 d after HSR modeling, the pathological changes in intestinal tissues were evaluated by H&E staining. The intestinal pyroptosis, glial fibrillary acidic protein (GFAP)-positive glial pyroptosis, DAO (diamine oxidase) content, intestine tight junction proteins including zonula occludens-1 (ZO-1) and claudin-1 were further detected by immunofluorescence, western blot and chemical assays at 7 d after HSR. CORM-3 administration led to significantly mitigated HSR-induced intestinal injury, aggravation of intestinal pyroptosis indicated by cleaved caspase-1, IL-1β and IL-18, upregulation of GFAP-positive glial pyroptosis, decreased intensity of ZO-1 and claudin-1 in the jejunum, and increased of DAO in the serum. Nigericin, an agonist of NLRP3, significantly reversed the protective effects of CORM-3. CORM-3 alleviates the intestinal barrier dysfunction in a rodent model of HSR, and the potential mechanism may be associated with inhibition of NLRP3-associated pyroptosis. CORM-3 administration could be a promising therapeutic strategy for intestinal injury after hemorrhagic shock.

Microbial transformation of artemisinin and its derivatives

Microbial transformation is an efficient enzymatic approach for the structural modification of exogenous compounds to obtain derivatives. Compared with traditional chemical synthesis, the microbial transformation has in fact the undoubtable advantages of strong region-and stereo-selectivity, and a low environmental and economic impact on the production process, which can achieve the reactions challenging to chemical synthesis. Because microbes are equipped with a broad-spectrum of enzymes and therefore can metabolize various substrates, they are not only a significant route for obtaining novel active derivatives, but also an effective tool for mimicking mammal metabolism in vitro. Artemisinin, a sesquiterpene with a peroxy-bridged structure serving as the main active functional group, is a famous antimalarial agent discovered from Artemisia annua L. Some sesquiterpenoids, such as dihydroartemisinin, artemether, and arteether, have been developed on the basis of artemisinin, which have been successfully marketed and become the first-line antimalarial drugs recommended by WHO. As revealed by pharmacological studies, artemisinin and its derivatives have exhibited extensive biological activities, including antimalarial, antitumor, antiviral, anti-inflammatory, and immunomodulatory. As an efficient approach for structural modification, microbial transformation of artemisinin and its derivatives is an increasingly popular strategy that attracts considerable attention recently, and numerous novel derivatives have been discovered. Herein, this paper reviewed the microbial transformation of artemisinin and its artemisinin, including microbial strains, culture conditions, product isolation and yield, and biological activities, and summarized the advances in microbial transformation in obtaining active derivatives of artemisinin and the simulation of in vivo metabolism of drugs.

Predicting Regioselectivity of Cytosolic Sulfotransferase Metabolism for Drugs.

Cytosolic sulfotransferases (SULTs) are a family of enzymes responsible for the sulfation of small endogenous and exogenous compounds. SULTs contribute to the conjugation phase of metabolism and share substrates with the uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes. UGTs are considered to be the most important enzymes in the conjugation phase, and SULTs are an auxiliary enzyme system to them. Understanding how the regioselectivity of SULTs differs from that of UGTs is essential from the perspective of developing novel drug candidates. We present a general ligand-based SULT model trained and tested using high-quality experimental regioselectivity data. The current study suggests that, unlike other metabolic enzymes in the modification and conjugation phases, the SULT regioselectivity is not strongly influenced by the activation energy of the rate-limiting step of the catalysis. Instead, the prominent role is played by the substrate binding site of SULT. Thus, the model is trained only on steric and orientation descriptors, which mimic the binding pocket of SULT. The resulting classification model, which predicts whether a site is metabolized, achieved a Cohen's kappa of 0.71.

Reactions of Diatom Ulnaria Ulna on Neurotransmitters and Their Antagonists

Exogenous compounds known as neurotransmitters acetylcholine, dopamine, histamine and serotonin and their antagonists d-tubocurarine, muscarine, yohimbine, tavegyl and inmecarb were tested on unicellular diatom algae Ulnaria ulna, where they demonstrated signaling and regulatory functions. The most primitive single-cellular organisms like diatoms exhibit a marked sensitivity to neurotransmitters and their antagonists, indicating a possible reception mechanism, similar to those observed in mammals.

The impacts of exposure to bisphenol A in the adult female prostate Meriones unguiculatus

The female prostate is associated with the urogenital system and presents homology in morphological terms with the male prostate. Due to its responsiveness to endogenous hormones, this gland is under a constant risk of developing prostatic pathologies and neoplasia when exposed to certain exogenous compounds. Bisphenol A (BPA) is an endocrine disruptor found in different plastic and resin products. Studies have emphasized the effects of perinatal exposure to this compound on different hormone-responsive organs. However, there have been few studies highlighting the influence on female prostate morphology of perinatal exposure to BPA. The objective of this study was to describe the histopathological alterations caused by perinatal exposure to BPA (50 µg/kg) and 17-β estradiol (E2) (35 µg/kg) in the prostate of adult female gerbils. The results showed that E2 and BPA induced proliferative lesions in the female prostate and acted along similar pathways by modulating steroid receptors in the epithelium. BPA was also found to be a pro-inflammatory and pro-angiogenic agent. The impacts of both agents were marked in the prostatic stroma. An increase in the thickness of the smooth muscle layer and a decrease in AR expression were observed, but no alterations in the expression of ERα and ERβ, leading to estrogenic sensitivity of the prostate. However, a peculiar response of the female prostate was to diminish the collagen frequency under BPA exposure correlated to smooth muscle layer. These data therefore indicate the development of features related to estrogenic and non-estrogenic tissue repercussions by BPA perinatally exposure in gerbil female prostate.

The fate of three typical persistent organic pollutants in bioretention columns as revealed by stable carbon isotopes

There is a lack of simple and effective methods to quantify the fate processes of persistent organic pollutants (POPs) in bioretention systems. In this study, the fate and elimination processes of three typical 13C-labeled POPs in regularly added bioretention columns were quantified using stable carbon isotope analysis techniques. The results showed that the modified media bioretention column removed more than 90% of Pyrene, PCB169 and p,p'-DDT. Media adsorption was the dominant removal mechanism for the reduction of the three exogenous organic compounds (59.1–71.8% of the input) although plant uptake (5.9–18.0%) was also important. Mineralization was effective in degrading pyrene (13.1%) but had a very limited effect on p,p'-DDT and PCB169 removal (<2.0%), the reason for which may be related to the aerobic conditions of the filter column. Volatilization was relatively weak and negligible (<1.5%). The presence of heavy metals inhibited the removal of POPs to some extent: media adsorption, mineralization and plant uptake were reduced by 4.3–6.4%, 1.8–8.3% and 1.5–3.6% respectively. This study suggests that bioretention systems are an effective measure for the sustainable removal of POPs from stormwater and that heavy metals can inhibit the overall performance of the system. Stable carbon isotope analysis techniques can help to investigate the migration and transformation of POPs in bioretention systems.

Biodegradation and metabolic pathway of quinalphos by Cunninghamella elegans ATCC36112.

Quinalphos is a long-term, wide-spectrum organophosphate insecticide with residual problems in the natural environment. Cunninghamella elegans (C. elegans) is a member of Mucoromycotina. Since the degradation products of its exogenous compounds are similar to those of mammals, it is often used to simulate the metabolism pathways of mammals. In this study, the detailed metabolic pathways of quinalphos were investigated with C. elegans. Quinalphos was degraded by 92% in 7days, while ten metabolites were produced. The metabolites were analyzed and identified by GC-MS. To determine the responsible enzymes in quinalphos metabolism, piperonyl butoxide (PB) and methimazole included in the culture flasks, and the kinetic responses of quinalphos and its metabolites by C. elegans were measured. Results indirectly demonstrated that cytochrome P450 monooxygenases were involved in the metabolism of quinalphos, but that methimazole inhibited the metabolism less efficiently. Comprehensive metabolic pathways can be deduced from the detailed analysis of metabolite profiles in control and inhibitor assays.

Blood–brain barrier microfluidic chips and their applications

As a natural dynamic barrier separating blood from brain parenchyma, the blood–brain barrier (BBB) is mainly composed of brain microvascular endothelial cells (BMECs), pericytes, astrocytes, and a variety of neurons. The BBB regulates the highly selective transport of various substances between the brain and blood and maintains the stability of the central nervous system (CNS). Owing to this tight control, the BBB represents a formidable challenge for the delivery of drugs and other exogenous compounds into the CNS, which has bottlenecked the development of many drugs for CNS diseases. Therefore, efficient and precise in vitro models of the BBB are needed to assess the efficacy and toxicity of drugs targeting the CNS to inform drug design and to improve the success rate of agents that enter clinical evaluation. In vitro BBB models have rapidly advanced from the early two-dimensional (2D) static models to the current three-dimensional (3D) dynamic microfluidic chips. Although the commonly used, static, in vitro BBB models are simple to construct and TEER values are convenient to detect, the static models do not provide an ideal (i.e., accurate) BBB environment, since they lack the correct physiological size/scale and hemodynamic shear stress, both of which play substantial roles in promoting and maintaining EC differentiation into a specific BBB phenotype. Compared with traditional static models, 3D microfluidic models thus enable cells to react in a manner more closely resembling in vivo behavior by simulating a microenvironment with more natural signal transduction. As a result, the dynamic 3D BBB model can more accurately recapitulate the structure and function of the human BBB. Here we summarize the recent progress in in vitro microfluidic BBB chips and their research applications as well as discuss the prospects and challenges for where the technology is headed.

Evidence for crosstalk between the aryl hydrocarbon receptor and the translocator protein in mouse lung epithelial cells

Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites. Many of these compounds are also linked to the translocator protein (TSPO), an outer mitochondrial membrane protein. Given a portion of the cellular pool of the AHR has also been localized to mitochondria and the overlap in putative ligands, we tested the hypothesis that crosstalk exists between the two proteins. CRISPR/Cas9 was used to create knockouts for AHR and TSPO in a mouse lung epithelial cell line (MLE-12). WT, AHR−/−, and TSPO−/− cells were then exposed to AHR ligand (TCDD), TSPO ligand (PK11195), or both and RNA-seq was performed. More mitochondrial-related genes were altered by loss of both AHR and TSPO than would have been expected just by chance. Some of the genes altered included those that encode for components of the electron transport system and the mitochondrial calcium uniporter. Both proteins altered the activity of the other as AHR loss caused the increase of TSPO at both the mRNA and protein level and loss of TSPO significantly increased the expression of classic AHR battery genes after TCDD treatment. This research provides evidence that AHR and TSPO participate in similar pathways that contribute to mitochondrial homeostasis.

GSTM1, GSTP1, p53 as some probable predictors of prognosis in primary and metastatic epithelial ovarian cancer

Objectives: Ovarian carcinomas are responsible for the death of more women than all other gynecologic malignancies in the Western world. Ovarian carcinomas are detected in an advanced stage of the disease in approximately 80% of the patients. Glutathione S-transferases (GSTs) are an important family involved in the detoxification of several xenobiotics. Thus, this mechanism protects tissues from the harmful effects of oxidative stress and chemical-induced damages. The expression of them may contribute to the characteristics of ovarian carcinoma as they can metabolise both exogenous and endogenous compounds, which are implicated in the development of ovarian cancer. Therefore, our aim was to determine the expressions of GST Mu 1 (GSTM1), GST Pi 1 (GSTP1), and also p53, which is a tumor suppressor gene, in benign and malign ovarian tumors and metastasis tissues. Methods: A total of the 99 patients with ovarian tumor enrolled in the study. Thirty-one of the tissues was benign tumor, 17 was malign tumor and 51 was metastasis. The immunohistochemical GSTM1, GSTP1, and p53 staining characteristics of these tissues were investigated. Results: The highest GSTM1, GSTP1, and p53 expression was noted in the malignant group followed by the metastasis group. GSTP1 expression was significantly higher in malignant tissues than benign ones (p = 0.015). No statistically significant difference was observed in the level of GSTM1 expression between groups (p = 0.524). p53 expression was significantly higher in the metastasis and malignant tissues than the benign ones (p &amp;lt; 0.001). Conclusions: The higher expressions of GSTP1 and p53 in malignant and metastasis tissues than benign ones indicate that these expressions could be important biomarkers in ovarian cancer development and progression. Further studies with more cases are required to confirm the results of our present study.

Ion suppression, reduced long-term robustness and leakage current of the spray voltage during the ionization of trichloroacetic acid; a case study with a methylmalonic acid assay

Sample clean-up with the protein precipitation solvent trichloroacetic acid (TCA), combined with a stable isotope labeled internal standard, is widely used for the analysis of endogenous and exogenous compounds in serum and plasma with liquid chromatography-tandem mass spectrometry (LC–MS/MS). During the application of an assay for methylmalonic acid (MMA), used for routine analysis in patient care, negative long-term side effects of TCA on assay performance were observed. Step-by-step extensive troubleshooting disclosed the limitations of using TCA in MS. After running over 2000 samples with the MMA assay over a course of one year, a black coating formed between the probe and the heater that was traced to the use of TCA. The MMA assay used a C18 column with an isocratic eluent of 95% water (0.1% formic acid) as starting condition, on which TCA was more retained than MMA. Next, concentrations of 2.2% TCA in the prepared serum or plasma sample caused a drop in spray voltage during ionization into the MS. This was caused by the strong acid properties of TCA, resulting in current loss of the spray voltage between the heated electrospray ionization (HESI) needle and the union holder, which had also a grounding function. Replacing the original metal HESI needle with a custom made fussed silica HESI needle or detaching the union from the union holder, eliminated the effect of the drop in spray voltage. In conclusion, TCA can seriously affect the long-term robustness by affecting the source of the MS. We recommend the use of a very low sample injection volume, and/or shifting the mobile phase to waste when TCA is eluting, when using TCA in LC-MS/MS analysis.

Potential and limitation of retrospective HRMS based data analysis: “Have meat-producing animals been exposed to illegal growth promotors such as SARMs?”

Targeted strategies using gas or liquid chromatography coupled to triple quadrupole mass analyzers are mostly used in food safety control laboratories. However, when a new chemical hazard emerges, the target list needs to be updated and thus the should be reanalyzed. Under this scenario, the constant emergence of compounds that can illegally be used as growth promotors in cattle, such as selective androgen receptor modulators (SARM), and the phasing out of older SARMs, would require routine control laboratories (RCL) to constantly re-develop and re-validate their once developed targeted methods. Nevertheless, high-resolution mass spectrometry (HRMS) is not limited to analyzing samples using only a predefined targeted list and therefore the implementation of untargeted HRMS methods in RCLs to detect SARMs would enhance the laboratories throughput. In this study, two HRMS profile databases of cattle urine samples gathered over the past years and collected by two independent laboratories, were retrospectively analyzed to find out if a new SARM has been used. This study assesses different retrospective screening approaches based on the Schymanski identification confidence levels. A screening purely based on identification confidence levels 4 and 5 does not permit reliable detection of any exogenous trace compound that does not belong to a certain food or environmental matrix sample due to the high number of false detects. Therefore, it is shown that the availability of a physical reference substance to increase the identification confidence up to level 1 is essential to discriminate a suspected detection between a false positive detection or a confirmed finding.

19 Effects of recombinant equine intra-articular IL-1 on synovial fluid PGE2 and movement asymmetry: A pilot study

Synovitis is a common feature of osteoarthritis (OA). Exogeneous intra-articular inflammatory compounds such as lipopolysaccharide or interleukin 1β (human or equine) produce a convenient, self-limiting, and reversible synovitis in horses, which can be useful to evaluate putative OA interventions. Although biochemical responses to this minimally invasive synovitis model have been described, its effects on functional adaptations in movement remains unclear. The aim of this pilot study was to correlate synovial fluid biochemical responses to intra-articular injection of recombinant equine IL-1b (IL-1) with objective measures of movement asymmetry. Horses (n = 3) randomly received 0, 50 or 75ng reIL-1 in 500μL of sterile saline into the left or right intercarpal joint in a 3-way crossover design, with 50 and 75ng treatments applied to the contralateral carpus and 24 h between treatments. Synovial fluid was collected by aseptic arthrocentesis at 0 (before IL-1 injection), 6, 12 and 24 h after IL-1 injection and analyzed for prostaglandin E2 (PGE2) by ELISA. To quantify movement asymmetry, MinDiff was calculated using withersvertical displacement data (2000 Hz) acquired from an inertial measurement unit (IMU) (Delsys Trigno) during in-hand trot trials at each time point/condition. PGE2 and MinDiff data were analyzed using 2-way repeated measures ANOVA with respect to IL-1 challenge dose and time. Correlations between variables were determined using the Spearman correlation. PGE2 significantly increased at 6 h (314.4 ± 136.4 pg/mL) compared with 0 h (128.0 ± 43.2 pg/mL) following challenge with 75ng IL-1 (P = 0.04). An increase in MinDiff 6 h following 75ng challenge was not significant (P = 0.13). MinDiff and PGE2 peaked simultaneously at 6 h after injection of 75ng IL-1, but the correlation was not significant (P = 0.27). Data from this small pilot study indicate that low intra-articular dose of reIL-1b induces measurable increase in local synovial fluid PGE2, which is a key pro-inflammatory biomarker. Effects on adaptations in gait asymmetry warrant further investigation.

The Specific Binding and Promotion Effect of Azoles on Human Aldo-Keto Reductase 7A2

Human AKR 7A2 broadly participates in the metabolism of a number of exogenous and endogenous compounds. Azoles are a class of clinically widely used antifungal drugs, which are usually metabolized by CYP 3A4, CYP2C19, and CYP1A1, etc. in vivo. The azole-protein interactions that human AKR7A2 participates in remain unreported. In this study, we investigated the effect of the representative azoles (miconazole, econazole, ketoconazole, fluconazole, itraconazole, voriconazole, and posaconazole) on the catalysis of human AKR7A2. The steady-state kinetics study showed that the catalytic efficiency of AKR7A2 enhanced in a dose-dependent manner in the presence of posaconazole, miconazole, fluconazole, and itraconazole, while it had no change in the presence of econazole, ketoconazole, and voriconazole. Biacore assays demonstrated that all seven azoles were able to specifically bind to AKR7A2, among which itraconazole, posaconazole, and voriconazole showed the strongest binding. Blind docking predicted that all azoles were apt to preferentially bind at the entrance of the substrate cavity of AKR7A2. Flexible docking showed that posaconazole, located at the region, can efficiently lower the binding energy of the substrate 2-CBA in the cavity compared to the case of no posaconazole. This study demonstrates that human AKR7A2 can interact with some azole drugs, and it also reveals that the enzyme activity can be regulated by some small molecules. These findings will enable a better understanding of azole-protein interactions.

1I. Vitamin D receptor-like receptors in GtoPdb v.2023.1

Vitamin D (VDR), Pregnane X (PXR) and Constitutive Androstane (CAR) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [50, 1]) are members of the NR1I family of nuclear receptors, which form heterodimers with members of the retinoid X receptor family. PXR and CAR are activated by a range of exogenous compounds, with no established endogenous physiological agonists, although high concentrations of bile acids and bile pigments activate PXR and CAR [50].

Novel perfluoroalkyl substances (PFAS) discovered in whole blood using automated non-targeted analysis of dried blood spots

A small subset of per- and polyfluoroalkyl substances (PFAS) are routinely screened in human blood. These compounds generally explain <50 % of the total PFAS in human blood. The percentage of known PFAS in human blood has been decreasing as replacement PFAS and more complex PFAS chemistries are introduced to the market. Most of these novel PFAS have not been previously identified. Non-targeted methods are required to characterize this “dark matter” PFAS. Our objective was to apply non-targeted PFAS analysis to human blood to gain an understanding about the sources, concentrations, and toxicity of these compounds. A high-resolution tandem mass spectrometry (HRMS) and software workflow for PFAS characterization in dried blood spots is reported. Dried blood spots are a less invasive collection technique compared to venous blood draws, allowing collection from vulnerable populations. Biorepositories of archived dried blood spots are available internationally from newborns and present opportunities to study prenatal exposure to PFAS. In this study, dried blood spot cards were analyzed using iterative MS/MS by liquid chromatography HRMS. Data processing was conducted using FluoroMatch Suite including a visualizer tool that presents homologous series, retention time vs m/z plots, MS/MS spectra, feature tables, annotations, and fragments for fragment screening. The researcher performing data-processing and annotation was blinded to the fact that standards were spiked in, and was able to annotate 95 % of standards spiked on dried blood spot samples, signifying a low false negative rate using FluoroMatch Suite. A total of 28 PFAS (20 standards and 4 exogenous compounds) were detected across five homologous series with Schymanski Level 2 confidence. Of these 4, 3 were perfluoroalkyl ether carboxylic acids (PFECA), a chemical class of PFAS which is increasingly being detected in environmental and biological matrices but is not currently screened in most targeted analysese. A further 86 potential PFAS were detected using fragment screening. PFAS are extremely persistent and widespread yet remain largely unregulated. Our findings will contribute to an improved an understanding of exposures. Application of these methods in environmental epidemiology studies have the potential to inform policy with regards to PFAS monitoring, regulation, and individual-level mitigation strategies.

Cytochrome P450 superfamily in spotted sea bass: Genome-wide identification and expression profiles under trichlorfon and environmental stresses

Cytochrome P450s (CYPs), as one of the most diverse enzyme superfamilies in nature, play critical functions in antioxidant reactions against endogenous and exogenous compounds. In this study, we performed genome-wide characterization of CYP superfamily members and analyzed their expression patterns under several abiotic stresses in spotted sea bass, which is known as an economically important fish species in the Chinese aquaculture industry. A total of 55 CYP genes were identified and divided into 17 families within 10 clans. The analysis of phylogeny, gene structure, and syntenic relationships provided evidence for the evolution of CYP genes and confirmed their annotation and orthology. The expression of CYP genes was examined in the liver during trichlorfon stress using quantitative real-time PCR. The results showed that 20 tested CYP genes displayed significant mRNA expression changes, indicating that they may play crucial roles in the metabolism of trichlorfon and can be potential biomarkers for trichlorfon pollution. Moreover, by screening transcriptomic databases, 10, 3 and 19 CYP genes exhibited differential expression patterns in response to hypoxia, alkalinity and heat stress, respectively. Taken together, this study provided insights into the regulation of CYP genes by toxicological and environmental stresses, laid basis for extensive functional studies of the CYP superfamily in spotted sea bass and other teleost species.

Application of the metal ions as potential population biomarkers for wastewater-based epidemiology: estimating tobacco consumption in Southern China.

Wastewater-based epidemiology (WBE) is an objective approach for the estimation of population-level exposure to a wide range of substances, in which the use of a population biomarker (PB) could significantly reduce back-calculation errors. Although some endogenous or exogenous compounds such as cotinine and other hormones have been developed as PBs, more PBs still need to be identified and evaluated. This study aimed to propose a novel method to estimate population parameters from the mass load of metal ion biomarkers in wastewater, and estimate the consumption of tobacco in 24 cities in Southern China using the developed method. Daily wastewater samples were collected from 234 wastewater treatment plants (WWTPs) in 24 cities in Southern China. Atomic absorption spectroscopy (AAS) was applied to determine the concentrations of common health-related metal ions in wastewater, including sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), and zinc (Zn), and compared them with the daily mass load of cotinine corresponding to catchment populations. The concentrations of cotinine in wastewater samples were measured using liquid chromatography-tandem mass spectrometry. There were clear and strong correlations between the target metal ion equivalent population and census data. The correlation coefficients (R) were RK = 0.78, RNa = 0.66, RCa = 0.81, RMg = 0.77, and RFe = 0.69, at p < 0.01 and R2 > 0.6. Subsequently, the combination of WBE and metal ion PBs was used to estimate tobacco consumption. Daily consumption of nicotine was estimated to be approximately 1.76 ± 1.19mg/d/capita, equivalent to an average of 13.0 ± 8.75 cigarettes/d being consumed by smokers. The data on tobacco consumption in this study were consistent with those in traditional surveys in Southern China. The metal ion potassium is an appropriate PB for reflecting the real-time population and could be used to evaluate the tobacco consumption in WBE study.

Abstract 3319: Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment

Abstract Introduction: Numerous studies have sought potential breath biomarkers for lung cancer and many have promising results but, to date, no validated biomarkers with clear connections to cancer metabolism have been discovered. We aim to show the feasibility of an Exogenous Volatile Organic Compounds (EVOC®) Probe targeting tumor-associated extracellular β-glucuronidase, a glycosidase enzyme that usually resides intracellularly within lysosomes in healthy tissues. Material and Methods: To further assess the suitability of β-glucuronidase as a target for a non-invasive breath test, the enzyme activity was evaluated by enzymatic assay in lung cancer PDX specimens and healthy tissues. In addition, β-glucuronidase was assessed by immunostaining a large panel of primary lung cancers, lymph node metastasis and normal lung tissue, including from smokers and those with chronic obstructive pulmonary disease (COPD). We developed a hydrophilic non-cell permeable substrate probe, D5-ethyl-βD-glucuronide (D5-EtGlu), that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule. This provides a readout of tumour-associated enzyme activity using breath analysis. Administering D5-EtGlu to mice resulted in tumour-specific release of D5-Ethanol enabling discrimination between healthy and tumour-bearing animals. D5-EtGlu has been administered intravenously to healthy and lung cancer patients followed by breath analysis. A phase 1a clinical trial administered D5-EtGlu to 21 healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. The phase 1b trial is a proof of mechanism study in humans, in which D5-ethanol breath levels in lung cancer patients will be compared to cancer-free individuals receiving the same D5-EtGlu dose. Results and Discussions: Using ex-vivo specimens, higher β-glucuronidase activity was detected in washouts of PDX specimens compared with healthy tissue samples from mice. Higher β-glucuronidase intracellular and extracellular expression was observed in lung cancers compared with lung normal tissue, with low to moderate expression intracellularly in bronchial tissue and in emphysema and COPD. These results reinforce the presence of β-glucuronidase in the tumour microenvironment and its suitability as a target for a breath-probe. A phase 1a resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular β-glucuronidase. In the phase 1b study, using the highest probe dose, we have detected a D5-ethanol signal in the majority of the cancer patients. Conclusion: β-glucuronidase can be targeted by the non-cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) with potential to be used as a non-invasive breath test for lung cancer diagnosis including a role in screening. Citation Format: Christiaan Labuschagne, Rob Smith, Neelam Kumar, Max Allsworth, Billy Boyle, Sam Janes, Philip Crosbie, Robert Rintoul. Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3319.

Organic anion-transporting polypeptide 2B1 knockout and humanized mice; insights into the handling of bilirubin and drugs

Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.