Induction of drug metabolizing enzymes, such as the cytochromes P450 (CYP) is known to cause drug-drug interactions due to increased elimination of co-administered drugs. This increased elimination may lead to significant reduction or complete loss of efficacy of the co-administered drug. Due to the significance of such drug interactions, many pharmaceutical companies employ screening and characterization models which predict CYP enzyme induction to avoid or attenuate the potential for drug interactions with new drug candidates. The most common mechanism of CYP induction is transcriptional gene activation. Activation is mediated by nuclear receptors, such as AhR, CAR, and PXR that function as transcription factors. Early high throughput screening models utilize these nuclear hormone receptors in ligand binding or cell-based transactivation/reporter assays. In addition, immortalized hepatocyte cell lines can be used to assess enzyme induction of specific drug metabolizing enzymes. Cultured primary human hepatocytes, the best established in vitro model for predicting enzyme induction and most accepted by regulatory agencies, is the predominant assay used to evaluate induction of a wide variety of drug metabolizing enzymes. These in vitro models are able to appropriately predict enzyme induction in patients when compared to clinical drug-drug interactions. Finally, transgenic animal models and the cynomolgus monkey have also been shown to recapitulate human enzyme induction and may be appropriate in vivo animal models for predicting human drug interactions.

Rhazya stricta Decne. (Apocynaceae) is a common medicinal plant in the Arabian Peninsula, Pakistan and India. Rhazya stricta has been used traditionally to treat several diseases including tumors; however, the underlying mechanism is still not fully elucidated.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse) of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.

Treatment with carbamazepine (CBZ), a potent enzyme inducer, is known to affect the lipid profile, steroid, and vitamin D metabolism. Consequently, it has been postulated that patients on CBZ should be switched to noninducing antiepileptic drugs (AEDs). However, little is known about the seizure outcome following a CBZ switch in seizure-free patients. We aimed to address this issue using a controlled observational study design.

Prostate cancer is the most commonly diagnosed malignancy in men and is thought to arise as a result of endogenous oxidative stress in the face of compromised carcinogen defenses. We tested whether carcinogen defense (phase 2) enzymes could be induced in the prostate tissues of rats after oral feeding of candidate phase 2 enzyme inducing compounds.

Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

The single-stranded DNA cytidine deaminases APOBEC3B, APOBEC3H haplotype I, and APOBEC3A can contribute to cancer through deamination of cytosine to form promutagenic uracil in genomic DNA. The enzymes must access single-stranded DNA during the dynamic processes of DNA replication or transcription, but the enzymatic mechanisms enabling this activity are not known. To study this, we developed a method to purify full length APOBEC3B and characterized it in comparison to APOBEC3A and APOBEC3H on substrates relevant to cancer mutagenesis. We found that the ability of an APOBEC3 to cycle between DNA substrates determined whether it was able to efficiently deaminate single-stranded DNA produced by replication and single-stranded DNA bound by replication protein A (RPA). APOBEC3 deaminase activity during transcription had a size limitation that inhibited APOBEC3B tetramers, but not APOBEC3A monomers or APOBEC3H dimers. Altogether, the data support a model in which the availability of single-stranded DNA is necessary, but alone not sufficient for APOBEC3-induced mutagenesis in cells because there is also a dependence on the inherent biochemical properties of the enzymes. The biochemical properties identified in this study can be used to measure the mutagenic potential of other APOBEC enzymes in the genome.

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [14 C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis-Menten constant (Km ) and ataluren protein binding but had a minimal effect on maximum velocity (Vmax ) of glucuronidation. Due to the decreased unbound Michaelis-Menten constant (Km,u ), the ataluren unbound intrinsic clearance (CLint,u ) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7-fold more active than human liver microsomes, in terms of CLint,u /mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes.

1. At premature birth, man and animals are exposed to relatively high oxygen levels, compared with intra-uterine conditions, at a time when their antioxidant enzyme (AOE) system is still immature. Using the chick embryo as a study model, we investigated changes in the AOE system in response to hyperoxia applied at different time points during the incubation period. Relations between hyperoxia and AOE activity were studied in selected organs (brain, heart, liver, intestine and lungs) of developing chick embryos (during the second half of the incubation period). 2. Incubated White Leghorn eggs were divided into four groups: control (n = 100) and three test groups exposed for 48 h to 60 % O2 on day 10 (test group 1, n = 80), day 14 (test group 2, n = 60) and day 18 (test group 3, n = 30). Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) enzyme activities were measured in homogenates of the brain, heart, liver, intestine and lungs. 3. Exposure to hyperoxia at different time points during incubation resulted in a 2- to 10-fold increase in SOD activity in all organs except the brain. Catalase and GPx enzyme activities were only induced in test group 1, 48 h after initiation of hyperoxia. 4. In the developing chick embryo, hyperoxia can produce a temporary induction of AOE activity, which is dependent on the AOE, organ, incubation time and time point of exposure.

Enzyme replacement therapy with laronidase is an established treatment for Mucopolysaccharidosis type I (MPS I), but its efficacy may be limited by the development of anti-drug antibodies, which inhibit cellular uptake of the enzyme. In a related disorder, infantile Pompe disease, immune tolerance induction with low-dose, short-course methotrexate appears to reduce antibody formation. We investigated a similar regimen using oral methotrexate in three MPS I patients. All patients developed anti-laronidase immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, and they had clinically relevant levels of cellular uptake inhibition. We then explored several immune tolerance induction strategies in MPS I mice: (1) methotrexate, (2) combination of non-depleting anti-CD4 and anti-CD8 monoclonal antibodies, (3) methotrexate with anti-CD4 and anti-CD8 monoclonals, (4) anti-CD4 monoclonal, and (5) anti-CD8 monoclonal. Treated mice received 10 weekly laronidase injections, and laronidase was delivered with adjuvant on day 49 to further challenge the immune system. Most regimens were only partially effective at reducing antibody responses, but two courses of non-depleting anti-CD4 monoclonal antibody (mAb) ablated immune responses to laronidase in seven of eight MPS I mice (87.5%), even after adjuvant stimulation. Immune tolerance induction with methotrexate does not appear to be effective in MPS I patients, but use of non-depleting anti-CD4 monoclonal is a promising strategy.

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.

Anemia is a very common blood disorder that affects the lives of billions of people worldwide. Anemia is caused by the loss of blood, increased destruction of red blood cells (RBCs), or reduced production of RBCs. Erythropoiesis is the complex process of RBC differentiation and maturation, in which protein degradation plays a crucial role. Protein ubiquitination regulates programmed protein degradation, which can be reversed by deubiquitinating enzymes (DUBs); however, the role of DUBs in erythropoiesis has not been well studied. We examined the expression of DUBs during erythropoiesis using an ex vivo human CD34+ hematopoietic progenitor cell culture system. Here we show that ubiquitin-specific protease 50 (USP50) levels are increased during erythropoiesis. USP50 mRNA levels are significantly increased on day 3 and protein levels are elevated on day 9 of erythroid differentiation. Coimmunoprecipitation and proteomics analyses reveal that Ku70, a DNA-binding protein, is associated with USP50. Overexpression of USP50 has no effect on Ku70 mRNA levels, while it reduces Ku70 protein levels by promoting Ku70 degradation, suggesting that USP50 may indirectly regulate Ku70 protein stability. USP50 protein is also not stable. USP50 protein degradation is independent of the proteasomal and the lysosomal degradation systems. This study suggests that DUBs like USP50 may regulate protein stability during erythropoiesis; however, more investigation is warranted.

Zea mays L. is one of the most produced crops, and there are still parts of the world where maize is the basic staple food. To improve agriculture, mankind always looks for new, better methods of growing crops, especially in the current changing climatic conditions. Cold atmospheric pressure plasma (CAPP) has already showed its potential to enhance the culturing of crops, but it still needs more research for safe implementation into agriculture. In this work, it was shown that short CAPP treatment of maize grains had a positive effect on the vitality of grains and young seedlings, which may be connected to stimulation of antioxidant and lytic enzyme activities by short CAPP treatment. However, the prolonged treatment had a negative impact on the germination, growth, and production indexes. CAPP treatment caused the increased expression of genes for heat shock proteins HSP101 and HSP70 in the first two days after sowing. Using comet assay it was observed that shorter treatment times (30-120 s) did not cause DNA damage. Surface diagnostics of plasma-treated grains showed that plasma increases the hydrophilicity of the surface but does not damage the chemical bonds on the surface.

Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous chemicals, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction/inhibition can bring about significant alterations in the level of in vivo exposure to CYP substrates and metabolites, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. On the basis of available human in vivo pharmaceutical data, a draft Organisation for Economic Co-operation and Development Test Guideline (TG) for an in vitro CYP HepaRG test method that is capable of detecting the induction of four human CYPs (CYP1A1/1A2, 2B6, and 3A4), has been developed and validated for a set of pharmaceutical proficiency chemicals. However to support TG adoption, further validation data was requested to demonstrate the ability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals, together with an indication on regulatory uses of the test method. As part of "GOLIATH", a European Union Horizon-2020 funded research project on metabolic disrupting chemical testing approaches, work is underway to generate supplemental validated data for an additional set of chemicals with sufficient diversity to allow for the approval of the guideline. Here we report on the process of proficiency chemical selection based on a targeted literature review, the selection criteria and considerations required for acceptance of proficiency chemical selection for OECD TG development (i.e. structural diversity, range of activity, relevant chemical sectors, global restrictions etc). The following 13 proposed proficiency chemicals were reviewed and selected as a suitable set for use in the additional validation experiments: tebuconazole, benfuracarb, atrazine, cypermethrin, chlorpyrifos, perfluorooctanoic acid, bisphenol A, N,N-diethyl-m-toluamide, benzo-[a]-pyrene, fludioxonil, malathion, triclosan, and caffeine. Illustrations of applications of the test method in relation to endocrine disruption and non-genotoxic carcinogenicity are provided.

During a survey from 26 August through 13 September 1991, specimens of the flatfish, Limanda limanda (dab), and the asteroid echinoderm Asterias rubens (seastar), were collected at sampling locations along transects radiating into the North Sea from the coastal zone of The Netherlands. In homogenates of liver tissue from male dab and the digestive gland (pyloric caeca) of female seastar, DNA damage (strand breaks) and induction of the cytochrome P450-dependent monooxygenase system (MO) were determined. Areas could be described with significantly increased percentages of strand breaks (lower integrity) both in dab and seastar. However, enhanced DNA strand breaks did not correspond with contamination gradients, expressed as concentrations of polychlorinated biphenyls (PCBs) or polyaromatic hydrocarbons. MO enzyme induction in the hepatic 13,000g fraction of male dab, measured as 7-ethoxyresorufin-O-deethylase activity, was significantly enhanced in response to low ambient temperatures. Some evidence was found for the facilitation of benzo[a]pyrene hydroxylase activity expressing the enzyme induction in the microsomal fraction of pyloric caeca of seastars, at increasing PCB concentrations. DNA integrity and enzyme induction elucidate the physiologic status and might be indicative for ambient impairment within restricted areas, and not necessarily related to the presence of anthropogenic or xenobiotic substances.

Removal of recalcitrant lignin from wastewater remains a critical bottleneck in multiple aspects relating to microbial carbon cycling ranging from incomplete treatment of biosolids during wastewater treatment to limited conversion of biomass feedstock to biofuels. Based on previous studies showing that the white rot fungus Phanerochaete chrysosporium and Fenton chemistry synergistically degrade lignin, we sought to determine optimum levels of Fenton addition and the mechanisms underlying this synergy. We tested the extent of degradation of lignin under different ratios of Fenton reagents and found that relatively low levels of H2O2 and Fe(II) enhanced fungal lignin degradation, achieving 80.4 ± 1.61 % lignin degradation at 1.5 mM H2O2 and 0.3 mM Fe(II). Using a combination of whole-transcriptome sequencing and iron speciation assays, we determined that at these concentrations, Fenton chemistry induced the upregulation of 80 differentially expressed genes in P. ch including several oxidative enzymes. This study underlines the importance of non-canonical, auxiliary lignin-degrading pathways in the synergy between white rot fungi and Fenton chemistry in lignin degradation. We also found that, relative to the abiotic control, P. ch. increases the availability of Fe(II) for the production of hydroxyl radicals in the Fenton reaction by recycling Fe(III) (p < 0.001), decreasing the Fe(II) inputs necessary for lignin degradation via the Fenton reaction.

Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.