Metabolic Competence Research Articles

The Core Gut Microbiome of Black Soldier Fly (Hermetia illucens) Larvae Raised on Low-Bioburden Diets.

An organism’s gut microbiome handles most of the metabolic processes associated with food intake and digestion but can also strongly affect health and behavior. A stable microbial core community in the gut provides general metabolic competences for substrate degradation and is robust against extrinsic disturbances like changing diets or pathogens. Black Soldier Fly larvae (BSFL; Hermetia illucens) are well known for their ability to efficiently degrade a wide spectrum of organic materials. The ingested substrates build up the high fat and protein content in their bodies that make the larvae interesting for the animal feedstuff industry. In this study, we subjected BSFL to three distinct types of diets carrying a low bioburden and assessed the diets’ impact on larval development and on the composition of the bacterial and archaeal gut community. No significant impact on the gut microbiome across treatments pointed us to the presence of a predominant core community backed by a diverse spectrum of low-abundance taxa. Actinomyces spp., Dysgonomonas spp., and Enterococcus spp. as main members of this community provide various functional and metabolic skills that could be crucial for the thriving of BSFL in various environments. This indicates that the type of diet could play a lesser role in guts of BSFL than previously assumed and that instead a stable autochthonous collection of bacteria provides the tools for degrading of a broad range of substrates. Characterizing the interplay between the core gut microbiome and BSFL helps to understand the involved degradation processes and could contribute to further improving large-scale BSFL rearing.

Aerobic degradation of dichlorinated dibenzo-p-dioxin and dichlorinated dibenzofuran by bacteria strains obtained from tropical contaminated soil.

Bacterial diversity and aerobic catabolic competence of dioxin-degrading bacterial strains isolated from apolluted soil in the tropics were explored. Isolation of bacteria occurred after 12months of consecutive enrichment, with dioxin congeners serving as the only sources of carbon and energy. Seventeen strains that were isolated were subsequently screened for dioxin metabolic competence. Among these isolates, five had unique amplified ribosomal DNA restriction analysis (ARDRA) patterns out of which two exhibiting good metabolic competence were selected for further investigation. The two strains were identified as Bacillus sp. SS2 and Serratia sp. SSA1, based on their 16S rRNA gene sequences. Bacterial growth co-occurred with dioxin disappearance and near stoichiometric release of chloride for one ring of the chlorinated congeners. The overall percentage removal of dibenzofuran (DF) by strain SS2 was 93.87%; while corresponding values for 2,8-dichlorodibenzofuran (2,8-diCDF) and 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD) were 86.22% and 82.30% respectively. In the case of strain SSA1, percentage removal for DF, 2,8-diCDF and 2,7-diCDD were respectively 98.9%, 80.97% and 70.80%. The presence of two dioxin dioxygenase catabolic genes (dxnA1 and dbfA1) was investigated. Only the dbfA1 gene could be amplified in SS2 strain. Results further revealed that strain SS2 presented higher expression levels for the alpha-subunit of DF dioxygenase (dbfA1) gene during growth with dioxins. The expression level for dbfA1 gene was higher when growing on DF than on the other chlorinated analogs. This study gives an insight into dioxin degradation, with the catabolic potential of strains SS2 and SSA1 (an enteric bacterium) within the sub-Sahara Africa. It further shows that dioxin catabolic potential might be more prevalent in different groups of microorganisms than previously believed. Few reports have demonstrated the degradation of chlorinated congeners of dioxins, particularly from sub-Saharan African contaminated systems.

A Randomized, Placebo‐Controlled, Double‐Blind Clinical Investigation on a Patent‐Pending KD120 Metabolically Activated Enzyme Formulation, N‐SORB, in Healthy Young Volunteers

Impaired digestive competence and consequential sluggish immune and metabolic sequela are epidemic‐sized health problems in modern cultures. Since digestion is the first step of metabolism, implementing novel strategies to restore digestive and metabolic competence are important health priorities. N‐SORB is a patent‐pending KD120 multienzyme complex composed of metabolically activated natural digestive‐type enzymes encapsulated in a high‐potency phosphatidylcholine non‐GMO soy lecithin SK713 SLP absorption technology (Prodosome®). A randomized, double‐blind placebo‐controlled study was conducted in 46 male and female healthy volunteers (age: 25.8 ± 12.1 years) to assess the safety and efficacy of N‐SORB. Subjects received either N‐SORB (1 mL, twice daily) or the same amount of placebo for 90 consecutive days. Body weight, blood pressure, physical health, and complete blood chemistries including blood glucose, liver enzymes, and lipid profiles; and immune health, physical health, sleep quality and quality of life parameters were assessed pre‐ and post‐intervention. Serum cytokine levels were determined by using a Bio‐Plex Pro Human Cytokine 8‐plex assay and ELISA. Whole body composition analysis was performed by dual‐energy X‐Ray absorptiometry (DEXA) to determine body fat mass, lean mass and android and gynoid fat. Quality of life (QOL) and sleep quality were assessed using WHOQOL‐BREF and Pittsburgh Sleep Quality Index (PSQI) questionnaire. Adverse events were strictly monitored. 40 subjects completed the study. Hematocrit, hemoglobin, mean corpuscular volume, platelets and lymphocytes provide evidence indicating consistent trends of improvement in the N‐SORB group. QOL parameters showed a small but significant improvement in the N‐SORB group. A significant increase was observed in AST levels in the placebo group at the end of 90 days of treatment, indicating increased liver distress. However, no increase was observed in the N‐SORB group. No significant changes in BUN, serum creatinine, ALP, ALT, and lipid profile were observed between the placebo and treatment groups before and after the 90‐day intervention. No adverse effects were reported. This 90‐day intervention study with N‐SORB clearly exhibited trends of improvement in RBC and immune parameters and found improvements in QOL and sleep quality scivnin already healthy volunteers. The N‐SORB treatment group experienced significant improvement in gastrointestinal functions, immune and metabolic parameters and other systemic improvements. No adverse events were reported.Support or Funding InformationThe Project was partially sponsored by VNI Inc, Lederach, PA

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT)

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a “Use of 3D Tissues in Genotoxicity Testing” working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

Effect of 2-acetylaminofluorene and its genotoxic metabolites on DNA adduct formation and DNA damage in 3D reconstructed human skin tissue models.

In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.

Evaluation of circulating leukocyte transcriptome and its relationship with immune function and blood markers in dairy cows during the transition period

Dairy cows during the transition period are faced with important physiological changes which include a dysfunctional immune system and an increased inflammatory state. New data are necessary to understand the key factors involved in the immune system regulation. Six dairy cows were sampled during transition period to investigate the leukocyte transcriptome changes and its relationship with blood biomarkers. Blood samples were collected at - 20 ± 2, - 3 ± 1, 3, and 7 days from parturition (DFP). Leukocyte transcriptome was analyzed by deep sequencing technology (Hiseq1000 Illumina, USA). Plasma was analyzed for metabolic biomarkers. Differentially expressed genes (DEG) were used to run an enrichment analysis through the Dynamic Impact Approach (DIA). Considering - 20 DFP as references time, the main KEGG impacted pathways were activated before calving (- 3 DFP) and were connected to lipid metabolism, lipid transport in plasma, and phagosome. The greatest differences were found after parturition with 281 DEG (179 upregulated and 102 downregulated). The activated pathways were mainly related to immunity and endocrine aspects, while metabolic pathways related to lipid and amino acid metabolism were inhibited. Plasma BHBA had a substantial inhibitory impact on KEGG pathways related to DNA replication and cell cycle, while plasma IL-1β had an inhibitory impact on fatty acid elongation in mitochondria and an activated impact in several pathways related to cellular energy metabolism. Overall, this study confirmed that many changes in lipid metabolism and immune competence of the circulating leukocytes occurred in dairy cow around calving. Interestingly, BHBA and IL-1β connected with the transcriptome.

The functional repertoire contained within the native microbiota of the model nematode Caenorhabditis elegans

The microbiota is generally assumed to have a substantial influence on the biology of multicellular organisms. The exact functional contributions of the microbes are often unclear and cannot be inferred easily from 16S rRNA genotyping, which is commonly used for taxonomic characterization of bacterial associates. In order to bridge this knowledge gap, we here analyzed the metabolic competences of the native microbiota of the model nematode Caenorhabditis elegans. We integrated whole-genome sequences of 77 bacterial microbiota members with metabolic modeling and experimental characterization of bacterial physiology. We found that, as a community, the microbiota can synthesize all essential nutrients for C. elegans. Both metabolic models and experimental analyses revealed that nutrient context can influence how bacteria interact within the microbiota. We identified key bacterial traits that are likely to influence the microbe’s ability to colonize C. elegans (i.e., the ability of bacteria for pyruvate fermentation to acetoin) and affect nematode fitness (i.e., bacterial competence for hydroxyproline degradation). Considering that the microbiota is usually neglected in C. elegans research, the resource presented here will help our understanding of this nematode’s biology in a more natural context. Our integrative approach moreover provides a novel, general framework to characterize microbiota-mediated functions.

Performance of Three-Dimensional Rainbow Trout (Oncorhynchus mykiss) Hepatocyte Spheroids for Evaluating Biotransformation of Pyrene.

The aquatic bioconcentration of a chemical is typically determined using conventional fish tests. To foster the approach of alternatives to animal testing, a combination of computational models and in vitro substrate depletion bioassays (e.g., primary hepatocytes) can be used. One recently developed in vitro assay is the three-dimensional (3D) hepatic spheroid model from rainbow trout (Oncorhynchus mykiss). The aim of the present study was to evaluate the metabolic competence of the 3D spheroids from rainbow trout when exposed to pyrene, using 2 different sampling procedures (SP1 and SP2). The results were compared with previously published intrinsic clearance (CL) results from S9 fractions and primary hepatocyte assays. Extraction of pyrene using SP1 suggested that the spheroids had depleted 33% of the pyrene within 4 h of exposure, reducing to 91% after 30 h. However, when applying SP2 a substantial amount (36%) of the pyrene was bound to the exposure vial within 2 h, decreasing after 6 h of exposure. Formation of hydroxypyrene-glucuronide (OH-PYR-Glu) was obtained throughout the study, displaying the metabolic competence of the 3D spheroids. The 2 sampling procedures yielded different CLin vitro , where pyrene depletion using SP2 was very similar to published studies using primary hepatocytes. The 3D spheroids demonstrated reproducibile, log-linear biotransformation of pyrene and displayed formation of OH-PYR-Glu, indicating their metabolic competence for 30 h or more. Environ Toxicol Chem 2019;38:1738-1747. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Development of the Caecal Microbiota in Three Broiler Breeds.

The development of the caecal microbiota plays a role in the metabolism and immune competence of chickens. A detailed understanding of normal succession in the caecal microbiota can inform the use of probiotics and other interventions to optimize the caecal microbiota. The development of the microbiota in caecal mucus and lumen samples from three breeds of broiler chicken (Cobb 500, n = 36; Hubbard JA87, n = 38; and Ross 308, n = 36) was observed between 0 and 42 days post hatch. Chicks were housed in the same room of a climate-controlled, biosecure chicken housing unit. Between 0 and 14 days post hatch, chicks were kept in brooder pens ensuring a mixture of breeds in each brooder. From 22 days post hatch, chicks were removed from the brooders and kept in the same room. DNA was extracted from a pooled sample of caecal mucus and luminal contents from five birds of each breed at 0, 3, 7, 14, 21, 28, and 42 days post hatch. High-throughput Illumina sequencing was performed for the V4 hypervariable region of the 16S rRNA gene. The early caecal microbiota was characterized by poor diversity and dominance by one or two bacterial species. Early colonizers of the caecum included Bifidobacteriaceae, Lachnospiraceae, Bacteroidaceae and Burkholderiaceae with some amplicon sequence variants (ASVs) assigned to Ruminococcaceae. Later colonizers of the caecal microbiota were most apparent from 14 d.p.h and included Ruminococcaceae, Clostridiales vadin BB60 group, Christensenellaceae and Bacillaceae. The caecal microbiota continued to change until 42 d.p.h when the microbiota was characterized by a high abundance of Bacteroidaceae, Lachnospiraceae and Ruminococcaceae. The lumen microbiota was significantly different to the mucus with some ASVs assigned to Lachnospiraceae, Ruminococcaceae, Christensenellaceae and Bacillaceae showing increased abundance in the mucus. ASVs assigned to Bacteroidaceae, Lactobacillaceae and Burkholderiaceae showed a preference for the lumen. Analysis of five caecal mucus samples from each breed at 42 days post hatch showed differences in microbiota composition between Ross and Cobb as well as between Ross and Hubbard. Since performance data was not collected no functional inferences as to the significance of this finding can be made.

Validation of in vitro methods for human cytochrome P450 enzyme induction: Outcome of a multi-laboratory study

CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells.The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals.The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed.

Genome Analysis of Entomopathogenic Bacillus sp. ABP14 Isolated from a Lignocellulosic Compost.

We report the complete genome sequence of Bacillus sp. strain ABP14 isolated from lignocellulosic compost and selected by its ability in hydrolyzing carboxymethyl cellulose. This strain does not produce a Cry-like protein but showed an insecticidal activity against larvae of Anticarsia gemmatalis (Lepidoptera). Genome-based taxonomic analysis revealed that the ABP14 chromosome is genetically close to Bacillus thuringiensis serovar finitimus YBT020. ABP14 also carries one plasmid which showed no similarity with those from YBT020. Genome analysis of ABP14 identified unique genes related to cell surface structures, cell wall, metabolic competence, and virulence factors that may contribute for its survival and environmental adaptation, as well as its entomopathogenic activity.

Microfluidic Multitissue Platform for Advanced Embryotoxicity Testing In Vitro.

The integration of metabolic competence in developmental toxicity assays in vitro is of fundamental importance to better predict adverse drug effects. Here, a microfluidic hanging‐drop platform is presented that seamlessly integrates liver metabolism into the embryonic stem cell test (EST). Primary human liver microtissues (hLiMTs) and embryoid bodies (EBs) are combined in the same fluidic network, so that hLiMT‐generated metabolites are directly transported to the EBs. Gravity‐driven flow through the network enables continuous intertissue communication, constant medium turnover, and, most importantly, immediate exchange of metabolites. As a proof of concept, the prodrug cyclophosphamide is investigated and a fourfold lower ID50 concentration (50% inhibition of EB differentiation) is found after biotransformation, which demonstrates the potentially adverse effects of metabolites on embryotoxicity. The metaEST platform provides a promising tool to increase the predictive power of the current EST assay by more comprehensively including and better reflecting physiological processes in in vitro tests.

Metabolism of cyclic phenones in rainbow trout in vitro assays

1. Cyclic phenones are chemicals of interest to the USEPA due to their potential for endocrine disruption to aquatic and terrestrial species.2. Prior to this report, there was very limited information addressing metabolism of cyclic phenones by fish species and the potential for estrogen receptor (ER) binding and vitellogenin (Vtg) gene activation by their metabolites.3. The main objectives of the current research were to characterize rainbow trout (rt) liver slice-mediated in vitro metabolism of model parent cyclic phenones exhibiting disparity between ER binding and ER-mediated Vtg gene induction, and to assess the metabolic competency of fish liver in vitro tests to help determine the chemical form (parent and/or metabolite) associated with the observed biological response.4. GC-MS, HPLC and LC-MS/MS technologies were applied to investigate the in vitro biotransformation of cyclobutyl phenyl ketone (CBP), benzophenone (DPK), cyclohexyl phenyl ketone (CPK) mostly in the absence of standards for metabolite characterization.5. It was concluded that estrogenic effects of the studied cyclic phenones are mediated by the parent chemical structure for DPK, but by active metabolites for CPK. A definitive interpretation was not possible for CBP and CBPOH (alcohol), although a contribution of both structures to gene induction is suspected.

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their “normal” phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct‐acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration‐dependent increases in mutant frequency (MF) ranging from 2.6‐ to 14.4‐fold over the control. None of the four nonmutagens, including two misleading, or “false,” positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3‐nitrobenzanthrone [3‐NBA], benzo[a]pyrene [BaP], and aflatoxin B1 [AFB1]) to least (i.e., N‐ethyl‐N‐nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348–360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization.

To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their “normal” karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte‐like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte‐specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 ± 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to β‐naphthoflavone led to approximately 900‐ and 9‐fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that MutaMouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 60:331–347, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Characterisation of a functional rat hepatocyte spheroid model

Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo-like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.

Equine and Bovine Oocyte Maturation in a Novel Medium Without CO2 Gas Phase

Routinely, immature equine oocytes are placed in complex or simple embryo holding media (HM) at room temperature overnight, followed by a CO2 phase incubation before intracytoplasmic sperm injection (ICSI) and culture. The present study's objectives were to (1) evaluate effects of a novel shipping/maturation medium without CO2 phase (EqMM) on oocyte developmental and metabolic competence using bovine cumulus-oocyte complexes for initial validation and (2) evaluate developmental competence of immature equine oocytes matured in EqMM in a clinical program. In experiment 1, bovine oocytes were aspirated from follicles of abattoir-derived ovaries, matured for 22–24 hours in M199 at 6% CO2 in air (control-M), or in EqMM in a portable incubator at 38.5°C in atmospheric air, fertilized in vitro, and cultured for 7.5 days. Cleavage and blastocyst rates were evaluated and a subset of blastocysts were stained for lipid content or for mitochondrial activity. In experiment 2, immature equine oocytes recovered by ovum pick up were placed either in EqMM for 30 hours at 38.5°C in atmospheric air, or in HM for 18 hours at 23°C and then in control-M for 30 hours followed by ICSI and cultured for 8 days. Resulting blastocysts were transferred into recipients. In experiment 1, cleavage, blastocyst rates, and lipid content were similar for control-M and EqMM groups. However, mitochondrial activity was higher in embryos obtained in EqMM. In experiment 2, for EqMM and control-M, maturation, cleavage, blastocyst, and pregnancy rates were similar. In conclusion, EqMM supports developmental competence of bovine and equine immature oocytes without CO2 gas phase.

The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells.

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/−), TP53(−/−), TP53(R248W/+) or TP53(R248W/−)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/−) and TP53(−/−) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/−) and TP53(−/−) cells compared to TP53(+/+) cells, and p53’s impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/−) cells but not in TP53(R248W/+) cells. Our results show that p53’s influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.

Xenobiotic Metabolism in Alginate-Encapsulated Primary Human Hepatocytes Over Long Timeframes

Introduction: Application of in vitro-to-in vivo extrapolation to chemical safety assessment has gained significant attention in the past few years. Accurate prediction of hepatic metabolic clearance from in vitro data is critical in this regard as it determines compound pharmacokinetics and exposure at sites of action. To estimate hepatic metabolic clearance, intrinsic clearance (CLint) is typically measured using in vitro systems such as primary hepatocytes or subcellular fractions derived from liver tissues. However, currently available in vitro systems used in these studies often lack long-term metabolic competence, making it challenging to estimate CLint for poorly metabolized and slowly cleared chemicals. Materials and Methods: To address this challenge, we developed a three-dimensional (3D) primary human hepatocyte cell culture system using alginate hydrogels (“alginate bead culture”) with extended viability and metabolic competence. Results: Alginate bead culture generates comparable estimates of metabolism to other in vitro and in vivo systems. In addition, expression of several xenobiotic metabolizing enzymes over long timeframes in beads is comparable to freshly isolated hepatocytes in suspension culture. Discussion: We compare CLint estimated from traditional suspension versus alginate bead culture for two chemicals (S-warfarin and coumarin) and show that alginate bead culture holds promise to increase confidence in estimates of CLint for slowly cleared chemicals. Conclusions: In addition to providing an improved in vitro tool to estimate metabolic clearance for a broad range of chemicals, this simple 3D hepatocyte culture can be adapted to a continuous flow-based bioreactor system allowing repeated exposure experiments over long periods in vitro, tackling an important challenge in in vitro-based chemical safety assessment.

Using bisphenol A and its analogs to address the feasibility and usefulness of the CALUX-PPARγ assay to identify chemicals with obesogenic potential

Environmental chemical exposures have been implicated in the obesity epidemic as potential mis-regulators of a variety of metabolic pathways. As agonism of the peroxisome proliferator-activated nuclear hormone receptor γ (PPARγ) is one of the suspected mechanisms involved, a PPARγ screening assay may have relevance for the biodetection of such effects of environmental chemicals. To test this hypothesis, we established the PPARγ2-CALUX® assay in-house and tested it against a number of known and suspected PPARγ modulators. Furthermore, we added a rat liver S9 metabolizing system to the protocol to introduce metabolic competence to the assay. Our results confirmed the responsiveness of the cell line to the known PPARγ agonists and antagonists: rosiglitazone, tributyltin, 15-deoxy-Δ12,14-prostaglandin J2, GW9662 and diclofenac. These data are in agreement with previous studies in various models. Seven bisphenol analogs tested induced little to no agonist activity, but all demonstrated antagonistic properties. These findings were contrary to both our assumptions and literature reports. Addition of the S9-metabolizing system to each of these tests did not alter any of the measured activities. Taken together, it seems probable that there are additional obesogenic effects of these chemicals which would not be detected by this assay.