Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Transcriptomic profile of human iPSC-derived podocyte-like cells exposed to a panel of xenobiotics.

Podocytes play a critical role in the formation and maintenance of the glomerular filtration barrier and injury to these cells can lead to a breakdown of the glomerular barrier causing permanent damage leading to progressive chronic kidney disease. Matured podocytes have little proliferative potential, which makes them critical cells from a health perspective, but also challenging cells to maintain in vitro. Differentiating podocyte-like cells from induced pluripotent stem cells (iPSC) provides a novel and continuous source of cells. Here, we investigated the effect of a 24-h exposure to eight compounds, including the known glomerular toxins doxorubicin and pamidronate, on transcriptomic alterations in iPSC derived podocytes. Doxorubicin (50 nM), pamidronate (50 µM), sodium arsenite (10 µM), and cyclosporine A (15 µM) had a strong impact on the transcriptome, gentamicin (450 µg/ml), lead chloride (15 µM) and valproic acid (500 µM) had a mild impact and busulfan (50 µM) exhibited no impact. Gene alterations and pathways analysis provided mechanistic insight for example, doxorubicin exposure affected the p53 pathway and dedifferentiation, pamidronate activated several pathways including HIF1alpha and sodium arsenite up-regulated oxidative stress and metal responses. The results demonstrate the applicability of iPSC derived podocytes for toxicological and mechanistic investigations.

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Endogenous production of 2-phenylethanol by Cunninghamella echinulata inhibits biofilm growth of the fungus.

The filamentous fungus Cunninghamella echinulata is a model of mammalian xenobiotic metabolism. Under certain conditions it grows as a biofilm, which is a natural form of immobilisation and enables the fungus to catalyse repeated biotransformations. Putative signalling molecules produced by other Cunninghamella spp., such as 3-hydroxytyrosol and tyrosol, do not affect the biofilm growth of C. echinulata, suggesting that it employs a different molecule to regulate biofilm growth. In this paper we report that 2-phenylethanol is produced in higher concentrations in planktonic cultures of C. echinulata than when the fungus is grown as a biofilm. We demonstrate that exogenously added 2-phenylethanol inhibits biofilm growth of C. echinulata but has no effect on planktonic growth. Furthermore, we show that addition of 2-phenylethanol to established C. echinulata biofilm causes detachment. Therefore, we conclude that this molecule is produced by the fungus to regulate biofilm growth.

Aryl (ß,ß',ß″-Trifluoro)-tert-butyl: A Candidate Motif for the Discovery of Bioactives.

The (ß,ß',ß″-trifluoro)-tert-butyl (TFTB) group has received very little attention in the literature. This work presents a direct synthesis of this group and explores its properties. The TFTB group arises when the methyl groups of a tert-butyl moiety are exchanged for fluoromethyl groups. Sequential fluoromethylations result in a decrease of Log P (increasing hydrophilicity), ultimately by 1.7 Log P units in the TFTB group relative to that of tert-butyl benzene itself. A focus is placed on synthetic transformations, conformational analysis, and metabolism of the TFTB group in the context of presenting a favorable profile as a motif for the discovery of bioactives.

Recent advances in fungal xenobiotic metabolism: enzymes and applications.

Fungi have been extensively studied for their capacity to biotransform a wide range of natural and xenobiotic compounds. This versatility is a reflection of the broad substrate specificity of fungal enzymes such as laccases, peroxidases and cytochromes P450, which are involved in these reactions. This review gives an account of recent advances in the understanding of fungal metabolism of drugs and pollutants such as dyes, agrochemicals and per- and poly-fluorinated alkyl substances (PFAS), and describes the key enzymes involved in xenobiotic biotransformation. The potential of fungi and their enzymes in the bioremediation of polluted environments and in the biocatalytic production of important compounds is also discussed.

Enhanced removal of perfluorooctanoic acid with sequential photocatalysis and fungal treatment.

In this paper, we report the degradation of perfluorooctanoic acid (PFOA), which is a persistent contaminant in the environment that can severely impact human health, by exposing it to a photocatalyst, bismuth oxyiodide (BiOI), containing both Bi4O5I2 and Bi5O7I phases and a fungal biocatalyst (Cunninghamella elegans). Individually, the photocatalyst (after 3 h) and biocatalyst (after 48 h) degraded 35-40% of 100 ppm PFOA with 20-30% defluorination. There was a marked improvement in the degree of degradation (90%) and defluorination (60%) when PFOA was first photocatalytically treated, then exposed to the fungus. GC- and LC-MS analysis identified the products formed by the different treatments. Photocatalytic degradation of PFOA yielded short-chain perfluorocarboxylic acids, whereas fungal degradation yielded mainly 5:3 fluorotelomer carboxylic acid, which is a known inhibitor of cytochrome P450-catalysed degradation of PFAS in C. elegans. The combined treatment likely resulted in greater degradation because photocatalysis reduced the PFOA concentration without generating the inhibitory 5:3 fluorotelomer carboxylic acid, enabling the fungus to remove most of the remaining substrate. In addition, new fluorometabolites were identified that shed light on the initial catabolic steps involved in PFOA biodegradation.

Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Fluorotelomer alcohols are efficiently biotransformed by Cunninghamella elegans.

Cunninghamella elegans is a well-studied fungus that biotransforms a range of xenobiotics owing to impressive cytochrome P450 (CYP) activity. In this paper, we report the biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) by the fungus, yielding a range of fluorinated products that were detectable by fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Upon incubation with the pre-grown cultures, the substrate (100 mg/L) was completely consumed within 48 h, which is faster biotransformation than other fungi that have hitherto been studied. The main metabolite formed was the 5:3 fluorotelomer carboxylic acid (5:3 FTCA), which accumulated in the culture supernatant. When the cytochrome P450 inhibitor 1-aminobenzotriazole was included in the culture flasks, there was no biotransformation of 6:2 FTOH, indicating that these enzymes are key to the catalysis. Furthermore, when exogenous 5:3 FTCA was added to the fungus, the standard biotransformation of the drug flurbiprofen was inhibited, strongly suggesting that the main fluorotelomer alcohol biotransformation product inhibits CYP activity and accounts for its accumulation.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation.

Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is MatrigelR, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited characterizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (≥ 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.

Cytochrome P450 5208A3 is a promiscuous xenobiotic biotransforming enzyme in Cunninghamella elegans.

Cunninghamella elegans is a long-established microbial model of mammalian drug and xenobiotic metabolism enabled by the actions of cytochrome P450 enzymes that are poorly characterised. In this paper we describe the identification of a new cytochrome P450 (CYP) monooxygenase in the fungus that catalyses the biotransformation of a range of structurally distinct xenobiotic substrates. The fungal enzyme was heterologously expressed in the yeast Pichia pastoris X-33 alone and in combination with previously identified C. elegans CYP reductases (CPRs A, B and C). Enzyme activity was assessed against a panel of drugs (flurbiprofen, diclofenac and ibuprofen), pesticides (transfluthrin, ß-cyfluthrin and λ-cyhalothrin) and a perfluoroalkyl substance (6:2 fluorotelomer alcohol) that were incubated with whole yeast cells expressing CYP5208A3. The biotransformation products were determined by gas chromatography-mass spectrometry (GC-MS) revealing the same metabolites that had been previously observed in the fungus. Co-expression of the CPRs improved metabolite production and the degree of improvement depended on the substrate and the CYP/CPR combination. Optimal pyrethroid biotransformation was achieved with CYP/CPR_C, whereas the best combination for non-steroidal anti-inflammatory drug hydroxylation was CYP/CPR_A; fluorotelomer alcohol oxidation was only observed with CYP/CPR_B. The change in substrate specificity observed with CYP5208A3 in combination with the different CPRs might help explain how C. elegans can biotransform such a broad spectrum of xenobiotics.

Improving the specificity of E. coli acetate/propionate exclusion biosensors via iterative engineering.

Real-time monitoring of key performance indicator analytes such as acetate and propionate within anaerobic digestors (AD) is required for optimum biogas production. In this paper the further refinement of acetate and propionate whole cell (E. coli) exclusion biosensors is reported following an iterative process in which key metabolites that might interfere with O2-uptake measurements are identified and genes required for their catabolism are knocked out (exclusion). Analysis of biological leachate from an AD reactor treating lignocellulosic material revealed the presence of formate, which was subsequently shown to elicit a response in previously developed E. coli biosensor strains. P1 phage transduction was employed to delete two genes encoding formate dehydrogenase, fdoH and fdnH, to eliminate formate catabolism. Deletion of these genes from the propionate biosensor strain W:ldgyepak abolished interference from formate and enabled accurate determination of propionate concentrations in biological leachate. However, the acetate biosensing strain E1/pGDR11-acs, despite not having any response to formate, responded to propionate. It was likely that this was a result of the promiscuity of the wild type acetyl CoA synthetase, which was replaced with Acs2 from Saccharomyces cerevisiae, resolving the problem and enabling acetate determination with the biosensor. Acetate and propionate concentrations in authentic leachate influent were estimated to be 26.5 mM and 65.5 mM, respectively, using the biosensor, and 26.6 and 70 mM, respectively, by HPLC, demonstrating the accuracy and specificity of the refined biosensor.

An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase.

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase. The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.

Bacterial degradation of the anti-depressant drug fluoxetine produces trifluoroacetic acid and fluoride ion.

Fluoxetine (FLX) is a blockbuster drug with annual sales in the billions of dollars. Its widespread use has resulted in its detection in water courses, where it impacts aquatic life. Investigations on the biodegradation of FLX by microorganisms are important, since augmentation of secondary wastewater treatment by an effective degrader may be one method of improving the drug's removal. In this paper, we demonstrate that common environmental bacteria can use FLX as a sole carbon and energy source. Investigations into the metabolites formed using fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR) and gas chromatography-mass spectrometry indicated that the drug was initially hydrolysed to yield 4-(trifluoromethyl)phenol (TFMP) and 3-(methylamino)-1-phenylpropan-1-ol. Since the fluorometabolite accumulated, the bacteria presumably used the latter compound for carbon and energy. Further growth studies revealed that TFMP could also be used as a sole carbon and energy source and was most likely catabolised via meta-cleavage, since semialdehyde products were detected in culture supernatants. The final products of the degradation pathway were trifluoroacetate and fluoride ion; the former is a dead-end product and was not further catabolised. Fluoride ion most likely arises owing to spontaneous defluorination of the meta-cleavage products that were shown to be photolabile.Key points• Bacteria can use FLX and TFMP as sole carbon and energy sources for their growth.• Biodegradation produces fluorometabolites that were detected by 19F NMR and GC-MS.• Trifluoroacetic acid and fluoride ion were identified as end products.

Cunninghamella spp. produce mammalian-equivalent metabolites from fluorinated pyrethroid pesticides.

Cunninghamella spp. are fungi that are routinely used to model the metabolism of drugs. In this paper we demonstrate that they can be employed to generate mammalian-equivalent metabolites of the pyrethroid pesticides transfluthrin and ß-cyfluthrin, both of which are fluorinated. The pesticides were incubated with grown cultures of Cunninghamella elegans, C. blakesleeana and C. echinulata and the biotransformation monitored using fluorine-19 nuclear magnetic resonance spectroscopy. Transfluthrin was initially absorbed in the biomass, but after 72 h a new fluorometabolite appeared in the supernatant; although all three species yielded this compound, it was most prominent in C. blakesleeana. In contrast ß-cyfluthrin mostly remained in the fungal biomasss and only minor biotransformation was observed. Gas chromatography-mass spectrometry (GC-MS) analysis of culture supernatant extracts revealed the identity of the fluorinated metabolite of transfluthrin to be tetrafluorobenzyl alcohol, which arose from the cytochrome P450-catalysed cleavage of the ester bond in the pesticide. The other product of this hydrolysis, dichlorovinyl-2,2-dimethylcyclopropane carboxylic acid, was also detected by GC-MS and was a product of ß-cyfluthrin metabolism too. Upon incubation with rat liver microsomes the same products were detected, demonstrating that the fungi can be used as models of mammalian metabolism of fluorinated pesticides.