Search

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

Clozapine is the most effective antipsychotic for patients with treatment-resistant schizophrenia. However, response is highly variable and possible genetic underpinnings of this variability remain unknown. Here, we performed polygenic risk score (PRS) analyses to estimate the amount of variance in symptom severity among clozapine-treated patients explained by PRSs (R2) and examined the association between symptom severity and genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activity. Genome-wide association (GWA) analyses were performed to explore loci associated with symptom severity. A multicenter cohort of 804 patients (after quality control N = 684) with schizophrenia spectrum disorder treated with clozapine were cross-sectionally assessed using the Positive and Negative Syndrome Scale and/or the Clinical Global Impression-Severity (CGI-S) scale. GWA and PRS regression analyses were conducted. Genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activities were calculated. Schizophrenia-PRS was most significantly and positively associated with low symptom severity (p = 1.03 × 10−3; R2 = 1.85). Cross-disorder-PRS was also positively associated with lower CGI-S score (p = 0.01; R2 = 0.81). Compared to the lowest tertile, patients in the highest schizophrenia-PRS tertile had 1.94 times (p = 6.84×10−4) increased probability of low symptom severity. Higher genotype-predicted CYP2C19 enzyme activity was independently associated with lower symptom severity (p = 8.44×10−3). While no locus surpassed the genome-wide significance threshold, rs1923778 within NFIB showed a suggestive association (p = 3.78×10−7) with symptom severity. We show that high schizophrenia-PRS and genotype-predicted CYP2C19 enzyme activity are independently associated with lower symptom severity among individuals treated with clozapine. Our findings open avenues for future pharmacogenomic projects investigating the potential of PRS and genotype-predicted CYP-activity in schizophrenia.

The currently used pharmacogenetic genotyping assays offer limited haplotype information, which can potentially cause specific functional effects to be missed. This study tested if Targeted Locus Amplification (TLA), when using non-patient-specific primers combined with Illumina or Nanopore sequencing, can offer an advantage in terms of accurate phasing. The TLA method selectively amplifies and sequences entire genes based on crosslinking DNA in close physical proximity. This way, DNA fragments that were initially further apart in the genome are ligated into one molecule, making it possible to sequence distant variants within one short read. In this study, four pharmacogenes, CYP2D6, CYP2C19, CYP1A2 and BRCA1, were sequenced after enrichment using different primer pairs. Only 24% or 38% of the nucleotides mapped on target when using Illumina or Nanopore sequencing, respectively. With an average depth of more than 1000X for the regions of interest, none of the genes were entirely covered with either sequencing method. For three of the four genes, less than half of the variants were phased correctly compared to the reference. The Nanopore dataset with the optimized primer pair for CYP2D6 resulted in the correct haplotype, showing that this method can be used for reliable genotyping and phasing of pharmacogenes but does require patient-specific primer design and optimization to be effective.

Herbicides are key weed-control tools, but their repeated use across large areas has favored the evolution of herbicide resistance. Although target-site has been the most prevalent and studied type of resistance, non-target-site resistance (NTSR) is increasing. However, the genetic factors involved in NTSR are widely unknown. In this study, four gene groups encoding putative NTSR enzymes, namely, cytochrome-P450, glutathione-S-transferase (GST), uridine 5′-diphospho-glucuronosyltransferase (UDPGT), and nitronate monooxygenase (NMO) were analyzed. The monocot and dicot gene sequences were downloaded from publicly available databases. Phylogenetic trees revealed that most of the CYP450 resistance-related sequences belong to CYP81 (5), and in GST, most of the resistance sequences belonged to GSTU18 (9) and GSTF6 (8) groups. In addition, the study of upstream promoter sequences of these NTSR genes revealed stress-related cis-regulatory motifs, as well as eight transcription factor binding sites (TFBS) were identified. The discovered TFBS were commonly present in both monocots and dicots, and the identified motifs are known to play key roles in countering abiotic stress. Further, we predicted the 3D structure for the resistant CYP450 and GST protein and identified the substrate recognition site through the homology approach. Our description of putative NTSR enzymes may be used to develop innovative weed control techniques to delay the evolution of NTSR.

Insecticide resistant pests become increasingly difficult to control in current day agriculture. Because of environmental and health concerns, the insecticide portfolio to combat agricultural pests is gradually decreasing. It is therefore crucial to make rational decisions on insecticide use to assure effective resistance management. However, resistance monitoring programs that inform on pest susceptibility and resistance are not yet common practice in agriculture. Molecular markers of resistance that are turned into convenient diagnostic tools are urgently needed and will only increase in importance. This review investigates which factors determine the strength, diagnostic value, and success of a diagnostic marker, and in which cases recent technical advances might provide new opportunities for decision making in an operational meaningful way.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

Stress-responsive microRNAs (miRNAs) contribute to the regulation of cellular homeostasis or pathological processes, including carcinogenesis, by reprogramming target gene expression following human exposure to environmental or dietary xenobiotics. Herein, we predicted the targets of carcinogenic mycotoxin-responsive miRNAs and analyzed their association with disease and functionality. miRNA target-derived prediction indicated potent associations of oncogenic mycotoxin exposure with metabolism- or hormone-related diseases, including sex hormone-linked cancers. Mechanistically, the signaling network evaluation suggested androgen receptor (AR)-linked signaling as a common pivotal cluster associated with metabolism- or hormone-related tumorigenesis in response to aflatoxin B1 and ochratoxin A co-exposure. Particularly, high levels of AR and AR-linked genes for the retinol and xenobiotic metabolic enzymes were positively associated with attenuated disease biomarkers and good prognosis in patients with liver or kidney cancers. Moreover, AR-linked signaling was protective against OTA-induced genetic insults in human hepatocytes whereas it was positively involved in AFB1-induced genotoxic actions. Collectively, miRNA target network-based predictions provide novel clinical insights into the progression or intervention against malignant adverse outcomes of human exposure to environmental oncogenic insults.

Mycotoxins, fungal secondary metabolites, are ubiquitously present in food commodities. Acute exposure to high levels or chronic exposure to low levels has an impact on the human body. The phase I metabolism in the human liver, performed by cytochrome P450 (CYP450) enzymes, is accountable for more than 80% of the overall metabolism of exogenous and endogenous compounds. Mycotoxins are (partially) metabolized by CYP450 enzymes. In this study, in vitro research was performed on CYP450 probes and aflatoxin B1 (AFB1), a carcinogenic mycotoxin, to obtain pharmacokinetic data on AFB1, required for further experimental work. The CYP450 probes of choice were a CYP3A4 substrate, midazolam (MDZ) and a CYP1A2 substrate, phenacetin (PH) since these are the main metabolizing phase I enzymes of AFB1. Linearity experiments were performed on the three substrates indicating that linear conditions were achieved at a microsomal protein concentration and incubation time of 0.25 mg/ml and 5 min, 0.50 mg/ml and 20 min and 0.25 mg/ml and 5 min for MDZ, PH and AFB1, respectively. The Km was determined in human liver microsomes and was estimated at 2.15 μM for MDZ, 40.0 μM for PH and 40.9 μM for AFB1. The associated Vmax values were 956 pmol/(mg.min) (MDZ), 856 pmol/(mg.min) (PH) and 11,536 pmol/(mg.min) (AFB1). Recombinant CYP systems were used to determine CYP450-specific Michaelis–Menten values for AFB1, leading to a CYP3A4 Km of 49.6 μM and an intersystem extrapolation factor (ISEF) corrected Vmax of 43.6 pmol/min/pmol P450 and a CYP1A2 Km of 58.2 μM and an ISEF corrected Vmax of 283 pmol/min/pmol P450. An activity adjustment factor (AAF) was calculated to account for differences between microsome batches and was used as a correction factor in the determination of the human in vivo hepatic clearance for MDZ, PH and AFB1. The hepatic blood clearance corrected for the AAF CLH,B,MDZ,AAF, CLH,B,PH,AAF CLH,B,AFB1,AAF(CYP3A4) and CLH,B,AFB1,AAF(CYP1A2) were determined in HLM at 44.1 L/h, 21.7 L/h, 40.0 L/h and 38.5 L/h. Finally, inhibition assays in HLM showed that 45% of the AFB1 metabolism was performed by CYP3A4/3A5 enzymes and 49% by CYP1A2 enzymes.

BACKGROUND: Maternal transplacental administration of sildenafil is being considered for a variety of fetal conditions. Clinical translation also requires evaluation of fetal safety in a higher species, such as the fetal lamb. Experiments with the pregnant ewe are curtailed by minimal transplacental transfer as well as limited access to the fetus. The EXTra-uterine Environment for Neonatal Development (EXTEND) model renders the isolated fetal lamb readily accessible and allows for direct fetal administration of sildenafil. METHODS: Five fetal lambs were placed on extracorporeal support in the EXTEND device and received continuous intravenous (IV) sildenafil (0.3-0.5-0.7 mg/kg/24hr) for a duration of one to seven days. Plasma sildenafil concentrations were sampled at regular intervals to establish the pharmacokinetic profile using population pharmacokinetic modeling. Serial Doppler ultrasound examination, continuous non-invasive hemodynamic monitoring and blood gas analysis were done to evaluate the pharmacodynamic effects and fetal response. FINDINGS: The target concentration range (47-500 ng/mL) was attained with all doses. Sildenafil induced an immediate and temporary reduction of pulmonary vascular resistance, mean arterial pressure and circuit flow, without change in fetal lactate levels and acid-base status. The duration of the systemic effects increased with the dose. INTERPRETATION: Immediate temporary pulmonary vascular and systemic hemodynamic changes induced by sildenafil were biochemically well tolerated by fetal lambs on extracorporeal support, with the 0.5 mg/kg/24 h dose balancing rapid attainment of target concentrations with short-lived systemic effects. RESEARCH IN CONTEXT: None. SEARCH STRATEGY BEFORE UNDERTAKING THE STUDY: A literature review was conducted searching online databases (Medline, Embase and Cochrane), using search terms: fetal OR prenatal OR antenatal AND sildenafil, without time-limit and excluding human studies. Where relevant, investigators were contacted in order to avoid duplication of work. EVIDENCE BEFORE THIS STUDY: Prenatal therapy with sildenafil, a phosphodiesterase-5 inhibitor with vasodilatory and anti-remodeling effects on vascular smooth muscle cells, has been considered for a variety of fetal conditions. One multicenter clinical trial investigating the benefit of sildenafil in severe intrauterine growth restriction (the STRIDER-trial) was halted early due to excess mortality in the sildenafil-exposed arm at one treatment site. Such findings demonstrate the importance of extensive preclinical safety assessment in relevant animal models. Transplacentally administered sildenafil leads to decreased pulmonary arterial muscularization, preventing or reducing the occurrence of pulmonary hypertension in rat and rabbit fetuses with diaphragmatic hernia (DH). Validation of these results in a higher and relevant animal model, e.g. fetal lambs, is the next step to advance clinical translation. We recently demonstrated that, in contrast to humans, transplacental transfer of sildenafil in sheep is minimal, precluding the in vivo study of fetal effects at target concentrations using the conventional pregnant ewe model. ADDED VALUE OF THIS STUDY: We therefore used the extracorporeal support model for fetal lambs, referred to as the EXTra-uterine Environment for Neonatal Development (EXTEND) system, bypassing placental and maternal metabolism, to investigate at what dose the target concentrations are reached, and what the fetal hemodynamic impact and response are. Fetal hemodynamic and metabolic tolerance to sildenafil are a crucial missing element on the road to clinical translation. This is therefore the first study investigating the pharmacokinetics, hemodynamic and biochemical effects of clinical-range concentrations of sildenafil in fetal lambs, free from placental and maternal interference. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: We demonstrated self-limiting pulmonary vasodilation, a decrease of both systemic arterial pressures and circuit flows, induced by clinical range concentrations of sildenafil, without the development of fetal acidosis. This paves the way for further investigation of prenatal sildenafil in fetal lambs on extracorporeal support. A dose of 0.5 mg/kg/24 h offered the best trade-off between rapid achievement of target concentrations and shortest duration of systemic effects. This is also the first study using the EXTEND as a model for pharmacotherapy during pregnancy. ispartof: BIOMEDICINE & PHARMACOTHERAPY vol:143 ispartof: location:France status: published

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Extensive migration has led to the necessity of knowledge regarding the treatment of migrants with different ethnical backgrounds. This is especially relevant for pharmacological treatment, because of the significant variation between migrant groups in their capacity to metabolize drugs. For psychiatric medications, CYP2D6 and CYP2C19 enzymes are clinically relevant. The aim of this meta-analysis was to analyze studies reporting clinically useful information regarding CYP2D6 and CYP2C19 genotype frequencies, across populations and ethnic groups worldwide. To that end, we conducted a comprehensive meta-analysis using Embase, PubMed, Web of Science, and PsycINFO (>336,000 subjects, 318 reports). A non-normal metabolizer (non-NM) probability estimate was introduced as the equivalent of the sum-prevalence of predicted poor, intermediate, and ultrarapid metabolizer CYP2D6 and CYP2C19 phenotypes. The probability of having a CYP2D6 non-NM predicted phenotype was highest in Algeria (61%) and lowest in Gambia (2.7%) while the probability for CYP2C19 was highest in India (80%) and lowest in countries in the Americas, particularly Mexico (32%). The mean total probability estimates of having a non-NM predicted phenotype worldwide were 36.4% and 61.9% for CYP2D6 and CYP2C19, respectively. We provide detailed tables and world maps summarizing clinically relevant data regarding the prevalence of CYP2D6 and CYP2C19 predicted phenotypes and demonstrating large inter-ethnic differences. Based on the documented probability estimates, pre-emptive pharmacogenetic testing is encouraged for every patient who will undergo therapy with a drug(s) that is metabolized by CYP2D6 and/or CYP2C19 pathways and should be considered in case of treatment resistance or serious side effects.

Extensive migration has led to the necessity of knowledge regarding the treatment of migrants with different ethnical backgrounds. This is especially relevant for pharmacological treatment, because of the significant variation between migrant groups in their capacity to metabolize drugs. For psychiatric medications, CYP2D6 and CYP2C19 enzymes are clinically relevant. The aim of this meta-analysis was to analyze studies reporting clinically useful information regarding CYP2D6 and CYP2C19 genotype frequencies, across populations and ethnic groups worldwide. To that end, we conducted a comprehensive meta-analysis using Embase, PubMed, Web of Science, and PsycINFO (>336,000 subjects, 318 reports). A non-normal metabolizer (non-NM) probability estimate was introduced as the equivalent of the sum-prevalence of predicted poor, intermediate, and ultrarapid metabolizer CYP2D6 and CYP2C19 phenotypes. The probability of having a CYP2D6 non-NM predicted phenotype was highest in Algeria (61%) and lowest in Gambia (2.7%) while the probability for CYP2C19 was highest in India (80%) and lowest in countries in the Americas, particularly Mexico (32%). The mean total probability estimates of having a non-NM predicted phenotype worldwide were 36.4% and 61.9% for CYP2D6 and CYP2C19, respectively. We provide detailed tables and world maps summarizing clinically relevant data regarding the prevalence of CYP2D6 and CYP2C19 predicted phenotypes and demonstrating large inter-ethnic differences. Based on the documented probability estimates, pre-emptive pharmacogenetic testing is encouraged for every patient who will undergo therapy with a drug(s) that is metabolized by CYP2D6 and/or CYP2C19 pathways and should be considered in case of treatment resistance or serious side effects.

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.

The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but a simple consistent positive or negative impact on a basic cellular parameter was not identified. In this study, we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wild type. The resultant datasets demonstrate that brassinosteroid signaling affects neither cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react non-cell-autonomously and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion., This study combines digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize roots of brassinosteroid-blind mutants. The data demonstrate that brassinosteroid signaling is essential for precise cell division plane orientation and the extent and timing of anisotropic cell expansion. These cell-aligning brassinosteroid effects can propagate non-cell-autonomously, manifesting in arabinogalactan expression as sentinels of brassinosteroid-dependent anisotropic cell expansion.

Brassinosteroid hormones are indispensable for root growth and they control both cell division and cell elongation through the establishment of an increasing signaling gradient along the longitudinal root axis. Because of their limited mobility, the importance of brassinosteroid distribution for achieving the signaling maximum is largely overlooked. Expression pattern analysis of all known brassinosteroid biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery and completion of biosynthesis relies on cell-to-cell movement of the hormone precursors. We demonstrate that brassinosteroid biosynthesis is largely restricted to the root elongation zone where it overlaps with brassinosteroid signaling maxima. Moreover, optimal root growth requires hormone concentrations, low in the meristem and high in the root elongation zone attributable to an increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in a local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as “highways” for transport, and companion cells (CCs), which serve as “gates” to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. Here we develop a new culture system for CC differentiation in Arabidopsis named VISUAL-CC, which almost mimics the process of the SE–CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess many more SEs and fewer CCs, whereas gsk3 gain-of-function mutants partially increase the CC number. Taken together, GSK3 activity appears to function as a cell-fate switch in the phloem, thereby balancing the SE/CC ratio., Tamaki et al. develop VISUAL-CC to study SE–CC (sieve elements–companion cells) complex formation. They show that the balance in the SE/CC ratio is dependent on GSK3 activity using different genetic backgrounds. Their work provides insights on the role of GSK3 as a cell-fate switch in the phloem.

In vitro, esomeprazole is a time-dependent inhibitor of CYP2C19. Additionally, racemic omeprazole induces CYP1A2 and omeprazole and its metabolites inhibit CYP3A4 in vitro. In this 5-phase study, 10 healthy volunteers ingested 20 mg pantoprazole, 0.5 mg midazolam, and 50 mg caffeine as respective index substrates for CYP2C19, 3A4, and 1A2 before and 1, 25, 49 (pantoprazole only), and 73 hours after an 8-day pretreatment with 80 mg esomeprazole twice daily. The area under the plasma concentration-time curve (AUC) of R-pantoprazole increased 4.92-fold (90% confidence interval (CI) 3.55-6.82), 2.31-fold (90% CI 1.85-2.88), and 1.33-fold (90% CI 1.06-1.68) at the 1-hour, 25-hour, and 73-hour phases, respectively, consistent with a substantial and persistent inhibition of CYP2C19. The AUC of midazolam increased up to 1.44-fold (90% CI 1.22-1.72) and the paraxanthine/caffeine metabolic ratio up to 1.19-fold (90% CI 1.04-1.36), when the index substrates were taken 1 hour after esomeprazole. Based on the recovery of R-pantoprazole oral clearance, the turnover half- life of CYP2C19 was estimated to average 53 hours. Pharmacokinetic simulation based on the observed concentrations of esomeprazole and its metabolites as well as their published CYP2C19 inhibitory constants was well in line with the observed changes in R-pantoprazole pharmacokinetics during the course of the study. Extrapolations assuming linear pharmacokinetics of esomeprazole suggested weak to moderate inhibition at 20 and 40 mg twice daily dosing. In conclusion, high-dose esomeprazole can cause strong inhibition of CYP2C19, but only weakly inhibits CYP3A4 and leads to minor induction of CYP1A2. The enzymatic activity of CYP2C19 recovers gradually in similar to 3-4 days after discontinuation of esomeprazole treatment.

With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology., The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3′-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

Background: Phenobarbital and midazolam are commonly used drugs in (near-)term neonates treated with therapeutic hypothermia for hypoxic-ischaemic encephalopathy, for sedation, and/or as anti-epileptic drug. Phenobarbital is an inducer of cytochrome P450 (CYP) 3A, while midazolam is a CYP3A substrate. Therefore, co-treatment with phenobarbital might impact midazolam clearance. Objectives: To assess pharmacokinetics and clinical anti-epileptic effectiveness of phenobarbital and midazolam in asphyxiated neonates and to develop dosing guidelines. Methods: Data were collected in the prospective multicentre PharmaCool study. In the present study, neonates treated with therapeutic hypothermia and receiving midazolam and/or phenobarbital were included. Plasma concentrations of phenobarbital and midazolam including its metabolites were determined in blood samples drawn on days 2–5 after birth. Pharmacokinetic analyses were performed using non-linear mixed effects modelling; clinical effectiveness was defined as no use of additional anti-epileptic drugs. Results: Data were available from 113 (phenobarbital) and 118 (midazolam) neonates; 68 were treated with both medications. Only clearance of 1-hydroxy midazolam was influenced by hypothermia. Phenobarbital co-administration increased midazolam clearance by a factor 2.3 (95% CI 1.9–2.9, p < 0.05). Anticonvulsant effectiveness was 65.5% for phenobarbital and 37.1% for add-on midazolam. Conclusions: Therapeutic hypothermia does not influence clearance of phenobarbital or midazolam in (near-)term neonates with hypoxic-ischaemic encephalopathy. A phenobarbital dose of 30 mg/kg is advised to reach therapeutic concentrations. Phenobarbital co-administration significantly increased midazolam clearance. Should phenobarbital be substituted by non-CYP3A inducers as first-line anticonvulsant, a 50% lower midazolam maintenance dose might be appropriate to avoid excessive exposure during the first days after birth.

Chemical warfare including insecticidal secondary metabolites is a well-known strategy for environmental microbes to monopolize a food source. Insects in turn have evolved behavioural and physiological defences to eradicate or neutralize the harmful microorganisms. We studied the defensive repertoire of insects in this interference competition by combining behavioural and developmental assays with whole-transcriptome time-series analysis. Confrontation with the toxic filamentous fungus Aspergillus nidulans severely reduced the survival of Drosophila melanogaster larvae. Nonetheless, the larvae did not behaviourally avoid the fungus, but aggregated at it. Confrontation with fungi strongly affected larval gene expression, including many genes involved in detoxification (e.g., CYP, GST and UGT genes) and the formation of the insect cuticle (e.g., Tweedle genes). The most strongly upregulated genes were several members of the insect-specific gene family Osiris, and CHK-kinase-like domains were over-represented. Immune responses were not activated, reflecting the competitive rather than pathogenic nature of the antagonistic interaction. While internal microbes are widely acknowledged as important, our study emphasizes the underappreciated role of environmental microbes as fierce competitors.

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses., Maize is the highest yielding cereal crop grown worldwide. Here Sun et al. show that maize growth can be further enhanced by prolonging the duration of leaf elongation by targeted ectopic expression of the PLASTOCHRON1 gene and show that this leads to increased yield in field trials.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids., Cytochrome P450 family enzymes have an essential role in the creation of triterpenoid diversity in plants. Here, the authors describe triterpenoid synthesis as mediated by CYP716 enzymes in medicinal plant species, and perform phylogenetic analysis to describe CYP716 molecular evolution in plants.

Aromatic hydroxylation reactions, which are mainly catalyzed by cytochrome P450 (CYP) enzymes in vivo, are some of the most important reactions of Phase I metabolism, because insertion of a hydroxyl group into a lipophilic drug compound increases its hydrophilicity and prepares it for subsequent Phase II metabolic conjugation reactions as a prerequisite to excretion. Aromatic hydroxylation metabolites of pharmaceuticals may be obtained through various synthetic and enzymatic methods Electrochemical oxidation is an alternative with advantages in terms of mild reaction conditions and less hazardous chemicals. In the present study, we report that aromatic hydroxylation metabolites of lidocaine can be readily obtained electrochemically under aqueous acidic conditions at platinum electrodes. Our results show that the dominant N-deallcylation reaction can be suppressed by decreasing the solution pH below 0.5 resulting in selective 3-hydroxylidocaine, which is an in vivo metabolite of lidocaine. Experiments in O-18 labelled water indicated that water is the primary source of oxygen, while dissolved molecular oxygen contributes to a minor extent to the hydroxylation reaction. (C) 2016 Elsevier Ltd. All rights reserved.

The juvenile conventional pig has been suggested as a preclinical animal model to evaluate pharmacokinetic (PK), pharmacodynamic (PD), and safety parameters in children. However, a lot of developmental changes in pig physiology still need to be unraveled. While the in vitro ontogeny of pig biotransformation enzymes is getting more attention in literature, the in vivo developmental changes have not yet been investigated. Therefore, the aim of the current study was to evaluate the biotransformation of ibuprofen (IBU) in conventional pigs aged 1 week, 4 weeks, 8 weeks, and 6-7 months after a single intravenous and oral administration of 5 mg/kg body weight (BVV) of IBU, using a PK approach in a crossover design for each age group. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine 2-hydroxyibuprofen (2OH-IBU), carboxyibuprofen (COOH-IBU), and ibuprofen glucuronide (IBU-GICA) in pig plasma. All three metabolites could be quantified in plasma and the following PK parameters were determined: C-max, T-max, AUC(0 -> 6h), area under plasma concentration-time curve (AUC) ratio between parent drug and metabolite, and the absolute oral bioavailability of the parent drug IBU. The plasma concentrations of the metabolites were always lower than those of IBU. The bioavailability was high, indicating limited pre-systemic biotransformation. The AUC ratio of 2OH-IBU and COOH-IBU/IBU showed a significant increase at 4 weeks of age compared to the 1-week-old and 6- to 7-month-old pigs. Interestingly, the IBUGIcA/IBU AUC ratio did not change with age. The present study demonstrated that the main metabolites of IBU in human are also present in growing pigs. The oxidative phase I metabolism of IBU in growing conventional pigs did change with age. In contrast, age did not seem to affect the glucuronidation capacity of IBU in conventional pigs, although more studies with other substrate drugs are needed to confirm this.

Many drug-drug interactions (DDIs) are based on alterations of the plasma concentrations of a victim drug due to another drug causing inhibition and/or induction of the metabolism or transporter-mediated disposition of the victim drug. In the worst case, such interactions cause more than tenfold increases or decreases in victim drug exposure, with potentially life-threatening consequences. There has been tremendous progress in the predictability and modeling of DDIs. Accordingly, the combination of modeling approaches and clinical studies is the current mainstay in evaluation of the pharmacokinetic DDI risks of drugs. In this paper, we focus on the methodology of clinical studies on DDIs involving drug metabolism or transport. We specifically present considerations related to general DDI study designs, recommended enzyme and transporter index substrates and inhibitors, pharmacogenetic perspectives, index drug cocktails, endogenous substrates, limited sampling strategies, physiologically-based pharmacokinetic modeling, complex DDIs, methodological pitfalls, and interpretation of DDI information.

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.

Excessive levels of Cu2+ are phytotoxic and exposure of Chinese cabbage to elevated Cu2+ concentrations led to reduction of the plant biomass. To get more insight into the role of glucosinolates upon copper stress, the impact of elevated Cu2+ levels on glucosinolates biosynthesis were studied in Chinese cabbage. The content of total glucosinolates was only elevated in the roots, mostly due to indolic and aromatic glucosinolates. The results showed a higher contribution of indolic glucosinolates, notably glucobrassicin, a 2- and 4-fold increase in Chinese cabbage exposed to 5 and 10 µM Cu2+, respectively. Furthermore, the increase in the indolic glucosinolates was accompanied by enhanced transcript levels of CYP79B2 and CYP83B1, two genes involved in biosynthesis of indolic glucosinolates, and that of the MYB51, a transcription factor involved in regulation of indolic glucosinolate biosynthesis pathway, at elevated Cu2+ concentrations. In addition, total sulfur and nitrogen remained unaffected in the root, but total glucosinolate was significantly enhanced upon exposure to elevated Cu2+. This result may show that relatively more sulfur and nitrogen was channeled into glucosinolates in the root. In conclusion, accumulation of indolic glucosinolates in the root can be considered as a strategy for Chinese cabbage to combat elevated Cu2+ concentrations.

Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D3 metabolites are of pharmaceutical importance and are applied for the treatment of vitamin D3 deficiency and other disorders. However, the chemical synthesis of vitamin D3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified and shown to oxidize vitamin D3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24h and allowed to identify the main product by NMR analysis as 25-hydroxylated vitamin D3 . Product yield amounted to 54.9 mg L(-1) day(-1) , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for vitamin D3 binding as the related vitamin D3 hydroxylases CYP109E1 from Bacillus megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of vitamin D3 hydroxylation. This article is protected by copyright. All rights reserved.

Aim: This study was aimed to asses the prevalence of CYP2D6 and CYP2C19 polymorphisms in psychiatric patients and in volunteers from Dutch caribbean origin. Methods: In total, 435 individuals were genotyped for CYP2D6 and CYP2C19. Of these, 269 were psychiatric patients on psychotropic medication, living in Curacao and 166 were volunteers from the Dutch Caribbean population. Results: No differences in prevalence of alleles were found. Conclusion: Although prevalence of alleles appeared to be very different from African and Caucasian populations, the distribution into predicted phenotypes shows an equal distribution as in Caucasians.

Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed(®), and Cellartis(®), and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis(®) medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis(®) medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis(®) medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure. CC BY 4.0Erratum in: Archives of Toxicology, volume 91, issue 7, page 2711 (2017). doi:10.1007/s00204-016-1895-4Received: 7 July 2016 / Accepted: 28 September 2016 / Published online: 7 October 2016The authors thank Prof. Dr. Robert Porte and all the surgeons of the University Medical Center Groningen for providing the human liver tissue. This study was financially supported by the EU-funded project NanoBio4Trans (Grant No. 304842).

1. Humans and animals are commonly exposed to indole-3-carbinol (I3C) and resveratrol (RES) via food or beverages. Moreover, these compounds have been demonstrated to potentially cause food–drug interactions. However, information about their combined effects is limited. Therefore, we investigated the effects of I3C and RES, both as single compounds and in combination, on cytochrome P450 1A and 3A activity and gene expression. 2. Using porcine microsomes, we demonstrated that RES caused non-competitive inhibition of CYP1A activity and un-competitive inhibition of CYP3A activity. Compared to the effect of single compounds, co-treatment with I3C and RES increased a degree of inhibition of CYP1A activity. 3. In porcine primary hepatocytes, treatment with I3C and RES resulted in induction of CYP1A1, CYP1A2 and CYP3A29 mRNA expression. 4. In conclusion, we demonstrated that both RES and I3C could cause food–drug interactions and that the combined effect could be more potent in doing so.

Background: Schizophrenia is a psychiatric disorder that affects approximately 0.4%–1% of the population worldwide. Diagnosis of schizophrenia is based primarily on Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria. Clozapine is an antipsychotic drug that is mainly used in the treatment of schizophrenia patients who are refractory or intolerant to at least 2 other antipsychotics. Due to the high variability in pharmacokinetics of clozapine, therapeutic drug monitoring (TDM) is highly recommended for clozapine therapy. Objective: To develop and clinically validate a novel sampling method using dried blood spot (DBS) to support TDM of clozapine and norclozapine. Methods: From June 2014 to September 2014, 15 schizophrenia patients (18–55 years) treated with clozapine were included. Plasma, DBS samples made from venous samples (VDBS), and finger prick DBS (DBS) samples were obtained before administration and 2, 4, 6, and 8 hours after clozapine intake. The study was repeated in 6 Russian patients for external validation. Passing-Bablok regression and Bland-Altman analysis were used to compare the DBS, VDBS, and plasma results for clozapine and norclozapine. Results: The DBS validation results showed good linearity over the concentration time curve measured for clozapine and norclozapine. The accuracy and between- and within-day precision variation values were within accepted ranges. Different blood spot volumes and hematocrit values had no significant influence on the results. The DBS samples were stable at 20°C and 37°C for 2 weeks and at −20°C for 2 years. The mean clozapine and norclozapine DBS/plasma ratios were, respectively, 0.80 (95% CI, 0.76 to 0.85) and 1.063 (95% CI, 1.027 to 1.099) in Dutch patients. The mean clozapine DBS/DPS ratio in Russian patients was 0.70 (95% CI, 0.64 to 0.76). Conclusion: DBS analysis is a reliable tool for blood sampling and performing TDM of clozapine and norclozapine in daily practice and substantially extends the opportunities for TDM of clozapine.

Biotransformation is one of the processes which influence the bioaccumulation of chemicals. The enzymatic action of metabolism involves two processes, i.e. the binding of the substrate to the enzyme followed by a catalytic reaction, which are described by the Michaelis–Menten constant (Km) and the maximum rate (Vmax). Here, we developed Quantitative Structure–Activity Relationships (QSARs) for Log(1/Km) and LogVmax for substrates of four enzyme classes. We focused on oxidations catalysed by alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), flavin-containing monooxygenase (FMO) and cytochrome P450 (CYP) in mammals. The chemicals investigated were xenobiotics, including alcohols, aldehydes, pesticides and drugs. We applied general linear models for this purpose, employing descriptors related to partitioning, geometric characteristics, and electronic properties of the substrates, which can be interpreted mechanistically. The explained variance of the QSARs varied between 20% and 70%, and it was larger for Log(1/Km) than for LogVmax. The increase of 1/Km with compound logP and size suggests that weak interactions are important, e.g. by substrate binding via desolvation processes. The importance of electronic factors for 1/Km was described in relation to the catalytic mechanism of the enzymes. Vmax was particularly influenced by electronic properties, such as dipole moment and energy of the lowest unoccupied molecular orbital. This can be explained by the nature of the catalysis, characterised by the cleavage and formation of covalent or ionic bonds (strong interactions). The present study may be helpful to understand the underlying principles of the chemical specific activity of four important oxidising enzymes.

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso -hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino- N -phenylbenzenesulfonamide, and N -(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso -hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para -position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drugdrug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drugdrug interaction possibilities, the inducing properties of artemisinin metabolites should be considered. German Federal Ministry of Education and Research (BMBF) HepatosSys network [0313081B, 0313080F, 0313080I]; Deutsche Forschungsgemeinschaft (Germany) [KE 1629/1-1]; Robert Bosch Foundation, Stuttgart, Germany info:eu-repo/semantics/publishedVersion

Natural Selection at Work Catching the evolution of a novel function and determining its selective parameters in nature remains an extremely difficult task. Prasad et al. (p. 1081 ) have undertaken this quest documenting the molecular basis of a natural allelic polymorphism and its effects on herbivory and survival in the Arabidopsis relative, Boechera stricta , living in the Rocky Mountains.

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can be metabolized to 1′-hydroxyestragole by cytochrome P450 enzymes. Basil extract addition to incubations of rat and human liver S9 homogenates with 1′-hydroxyestragole, the sulfotransferase cofactor PAPS, and 2′-deoxyguanosine resulted in a dose-dependent inhibition of N2-(trans-isoestragol-3′-yl)-2′-deoxyguanosine formation. Because the inhibition resembled the inhibition by the sulfotransferase inhibitor pentachlorophenol and since the inhibition was not observed in incubations with the direct electrophile 1′-acetoxyestragole it is concluded that the inhibition occurs at the level of the sulfotransferase mediated bioactivation step. Additional experiments in HepG2 cells revealed the same protective effect of basil extract in intact cells, demonstrating that the inhibitors are able to enter the cells. The results of this study suggest that bioactivation and subsequent adverse effects of 1′-hydroxyestragole might be lower in a matrix of other basil ingredients than what would be expected on the basis of experiments using 1′-hydroxyestragole as a single compound. © 2008 Elsevier Ltd. All rights reserved. Chemicals / CAS: 1' hydroxyestragole, 51410-44-7; cytochrome P450, 9035-51-2; deoxyguanosine, 961-07-9; DNA, 9007-49-2; estragole, 140-67-0; sulfotransferase, 9023-09-0; 1'-hydroxyestragole, 51410-44-7; Anisoles; Deoxyguanosine, 961-07-9; DNA Adducts; Enzyme Inhibitors; Plant Extracts; Sulfotransferases, EC 2.8.2.-; Tetrazolium Salts; Thiazoles; thiazolyl blue, 298-93-1