Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.

Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation.

Frequent herbicide use selects for herbicide resistance in weeds. Cytochrome P450s are important detoxification enzymes responsible for herbicide resistance in plants. We identified and characterized a candidate P450 gene (BsCYP81Q32) from the problematic weed Beckmannia syzigachne to test whether it conferred metabolic resistance to the acetolactate synthase-inhibiting herbicides mesosulfuron-methyl, bispyribac-sodium, and pyriminobac-methyl. Transgenic rice overexpressing BsCYP81Q32 was resistant to the three herbicides. Equally, rice overexpressing the rice ortholog gene OsCYP81Q32 was more resistant to mesosulfuron-methyl. Conversely, an OsCYP81Q32 gene knockout generated using CRISPR/Cas9 enhanced mesosulfuron-methyl sensitivity in rice. Overexpression of the BsCYP81Q32 gene resulted in enhanced mesosulfuron-methyl metabolism in transgenic rice seedlings via O-demethylation. The major metabolite, demethylated mesosulfuron-methyl, was chemically synthesized and displayed reduced herbicidal effect in plants. Moreover, a transcription factor (BsTGAL6) was identified and shown to bind a key region in the BsCYP81Q32 promoter for gene activation. Inhibition of BsTGAL6 expression by salicylic acid treatment in B. syzigachne plants reduced BsCYP81Q32 expression and consequently changed the whole plant response to mesosulfuron-methyl. Sequence polymorphisms in an important region of the BsTGAL6 promoter may explain the higher expression of BsTGAL6 in resistant versus susceptible B. syzigachne plants. Collectively, the present study reveals the evolution of an herbicide-metabolizing and resistance-endowing P450 and its transcription regulation in an economically important weedy plant species.

An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase.

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H2 O2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2 O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H2 O2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2 O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.

Distribution and insecticide resistance profile of the major malaria vector Anopheles funestus group across the African continent.

There has been significant progress in malaria control in the last 2 decades, with a decline in mortality and morbidity. However, these gains are jeopardised by insecticide resistance, which negatively impacts the core interventions, such as insecticide-treated nets (ITN) and indoor residual spraying (IRS). While most malaria control and research efforts are still focused on Anopheles gambiae complex mosquitoes, Anopheles funestus remains an important vector in many countries and, in some cases, contributes to most of the local transmission. As countries move towards malaria elimination, it is important to ensure that all dominant vector species, including An. funestus, an important vector in some countries, are targeted. The objective of this review is to compile and discuss information related to A. funestus populations' resistance to insecticides and the mechanisms involved across Africa, emphasising the sibling species and their resistance profiles in relation to malaria elimination goals. Data on insecticide resistance in An. funestus malaria vectors in Africa were extracted from published studies. Online bibliographic databases, including Google Scholar and PubMed, were used to search for relevant studies. Articles published between 2000 and May 2023 reporting resistance of An. funestus to insecticides and associated mechanisms were included. Those reporting only bionomics were excluded. Spatial variation in species distribution and resistance to insecticides was recorded from 174 articles that met the selection criteria. It was found that An. funestus was increasingly resistant to the four classes of insecticides recommended by the World Health Organisation for malaria vector control; however, this varied by country. Insecticide resistance appears to reduce the effectiveness of vector control methods, particularly IRS and ITN. Biochemical resistance due to detoxification enzymes (P450s and glutathione-S-transferases [GSTs]) in An. funestus was widely recorded. However, An. funestus in Africa remains susceptible to other insecticide classes, such as organophosphates and neonicotinoids. This review highlights the increasing insecticide resistance of An. funestus mosquitoes, which are important malaria vectors in Africa, posing a significant challenge to malaria control efforts. While An. funestus has shown resistance to the recommended insecticide classes, notably pyrethroids and, in some cases, organochlorides and carbamates, it remains susceptible to other classes of insecticides such as organophosphates and neonicotinoids, providing potential alternative options for vector control strategies. The study underscores the need for targeted interventions that consider the population structure and geographical distribution of An. funestus, including its sibling species and their insecticide resistance profiles, to effectively achieve malaria elimination goals.

Des progrès importants ont été réalisés dans le contrôle du paludisme au cours des deux dernières décennies, qui se traduisent par une baisse de la mortalité et de la morbidité. Cependant, ces gains sont compromis par la résistance aux insecticides, ce qui a un impact négatif sur les interventions de base, telles que les moustiquaires imprégnées d'insecticides et la pulvérisation intradomicilliare (PID). Alors que la plupart des efforts de contrôle et de recherche sur le paludisme sont toujours axés sur les moustiques du complexes Anopheles gambiae, Anopheles funestus reste un vecteur important dans de nombreux pays et, dans certains cas, contribue à la majeure partie de la transmission locale. Au moment où certains pays se dirigent vers l'élimination du paludisme, il serait important de prendre en considération toutes les espèces vectrices dominantes, y compris An. funestus. L'objectif de cette revue est de compiler et de discuter des informations liées à la résistance des populations d'An. funestus aux insecticides et les mécanismes impliqués à travers l'Afrique, en mettant l'accent sur les sous espèces et leurs profils de résistance en relation avec les objectifs d'élimination du paludisme. Les données sur la résistance aux insecticides chez An. funestus vecteurs du paludisme en Afrique ont été extraites d'études publiées dans des bases de données bibliographiques comme Google Scholar et PubMed. Les articles publiés entre 2000 et mai 2023, rapportant la résistance de An. funestus aux insecticides et les mécanismes associés ont été inclus. Ceux portant uniquement sur la bionomie ont été exclus. Au total 174 articles portant sur la variation spatiale de la résistance des espèces du groupe An. funestus aux insecticides répondaient aux critères de sélection. De ces analyses, il ressort qu'An. funestus était de plus en plus résistant aux quatre classes d'insecticides recommandées par l'Organisation Mondiale de la Santé (OMS) pour le contrôle des vecteurs du paludisme ce qui semble réduire l'efficacité des méthodes de contrôle des vecteurs, en particulier les moustiquaires imprégnées d'insecticide et la pulvérisation intradomiciliaire. avec des variations en fonction des pays. Les mécanismes de résistance aux insecticides de type biochimique liée aux enzymes de détoxification (P450S et GST) ont été largement rapportés chez An. funestus. De nombreux gènes P450 associés à la résistance métabolique ont été mis en évidence chez An. funestus collecté sur le terrain. Cependant, An. funestus en Afrique reste sensible à d'autres classes d'insecticides, telles que les organophosphorés et les néonicotinoïdes. La résistance aux insecticides. Cette revue met en évidence la résistance croissante aux insecticides chez les moustiques du groupe Funestus, un vecteur important du paludisme en Afrique, posant ainsi un défi important aux efforts de contrôle du paludisme. Tandis que An. funestus a montré une résistance aux classes d'insecticide recommandées, notamment les pyréthroïdes et, dans certains cas, les organochlorés et les carbamates, il reste sensible à d'autres classes d'insecticides tels que les organophosphorés et les néonicotinoïdes, offrant des options alternatives potentielles de contrôle des vecteurs. L'étude souligne la nécessité d'interventions ciblées qui considèrent la structure de la population et la distribution géographique d'An. funestus, y compris ses sous espèces et leurs profils de résistance aux insecticides, pour atteindre efficacement les objectifs d'élimination du paludisme.

Regulation of T16H subcellular localization for promoting its catalytic efficiency in yeast cells.

To investigate the effect of subcellular localization on the transformation efficiency of heterologous expressed functional P450s in yeast. Microbial biotransformation offers a promising substitute for the direct extraction of natural products, but its viability in industrial applications depends on achieving high transformation efficiencies. To investigate the influence of subcellular microenvironments on the activity of heterologously expressed P450s, Catharanthus roseus tabersonine 16-hydroxylase (T16H) was chosen, and its subcellular localization was regulated by fusing organelle-localization signals. Interestingly, this manipulation had no effect on the gene expression levels of T16H, but resulted in varying conversion rates from tabersonine to 16-hydroxy tabersonine. Notably, the highest transformation efficiency was observed in yeast cells expressing peroxisome-localized T16H. Given the alkaline pH optimum for P450s, the alkaline peroxisomal lumen could be a suitable compartment for P450s reactions to achieve high transformation efficiency using yeast cells. Different organelle-localization of T16H in yeast cells resulted in varying conversion rates, suggesting that compartmentalizing the expression of target enzymes could be a viable approach to increase transformation efficiency in yeast.

Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

Identification of inducible CYP3 and CYP4 genes associated with abamectin tolerance in the fat body and Malpighian tubules of Spodoptera litura.

Abamectin, as a broad-spectrum bioinsecticide, has been widely used for the control of Lepidoptera insects, resulting in different levels of resistance to abamectin in Spodoptera litura. Cytochrome P450 monooxygenases (P450s) are known for their important roles in insecticide detoxification. In this study, the expression of SlCYP6B40, SlCYP4L12 and SlCYP9A32 in the fat body, and SlCYP4S9, SlCYP6AB12, SlCYP6AB58, SlCYP9A75a and SlCYP9A75b in Malpighian tubules was found to be significantly upregulated after abamectin exposure. SlCYP6AE44 and SlCYP6AN4 were simultaneously upregulated in these two tissues after abamectin exposure. Ectopically overexpressed SlCYP6AE44, SlCYP9A32 and SlCYP4S9 in transgenic Drosophila conferred tolerance to abamectin. In addition, homology modeling and molecular docking results suggested that SlCYP6AE44, SlCYP9A32 and SlCYP4S9 may be capable of binding with abamectin. These results demonstrate that upregulation of CYP3 and CYP4 genes may contribute to abamectin detoxification in S. litura and provide information for evidence-based insecticide resistance management strategies.

An Asp376Glu substitution and P450s-involved metabolism endow resistance to ALS inhibitors in an Ammannia auriculata Willd. Population.

Ammannia auriculata Willd. is a noxious broadleaf weed, commonly infesting rice ecosystems across southern China. A putative resistant A. auriculata population (AHSC-5) was sampled from a rice field of Anhui Province, where bensulfuron-methyl (BM) was unable to control its occurrence. This study aimed to determine the sensitivities of the AHSC-5 population to common-use herbicides, and to investigate the underlying resistance mechanisms. The bioassays showed that the AHSC-5 population was 138.1-fold resistant to BM, compared with the susceptible population (JSGL-1). Pretreatment of malathion reduced the resistance index to 19.5. ALS sequencing revealed an Asp376Glu substitution in the AHSC-5 population, and in vitro ALS activity assays found that 50% activity inhibition (I50) of BM in AHSC-5 was 75.4 times higher than that of JSGL-1. Moreover, the AHSC-5 population displayed cross-resistance to pyrazosulfuron-ethyl (10.6-fold), bispyribac­sodium (3.6-fold), and imazethapyr (2.2-fold), and was in the process of evolving multiple resistance to synthetic auxin herbicides fluroxypyr (2.3-fold) and florpyrauxifen-benzyl (3.1-fold). This study proved the BM resistance in A. auriculata caused by the Asp376Glu mutation and P450-regulated metabolism. This multi-resistant population can still be controlled by penoxsulam, MCPA, bentazone, and carfentrazone-ethyl, which aids in developing targeted and effective weed management strategies.

Engineering Electron Transfer Pathway of Cytochrome P450s.

Cytochrome P450s (P450s), a superfamily of heme-containing enzymes, existed in animals, plants, and microorganisms. P450s can catalyze various regional and stereoselective oxidation reactions, which are widely used in natural product biosynthesis, drug metabolism, and biotechnology. In a typical catalytic cycle, P450s use redox proteins or domains to mediate electron transfer from NAD(P)H to heme iron. Therefore, the main factors determining the catalytic efficiency of P450s include not only the P450s themselves but also their redox-partners and electron transfer pathways. In this review, the electron transfer pathway engineering strategies of the P450s catalytic system are reviewed from four aspects: cofactor regeneration, selection of redox-partners, P450s and redox-partner engineering, and electrochemically or photochemically driven electron transfer.

Discovery and biosynthesis of bacterial drimane-type sesquiterpenoids from Streptomyces clavuligerus.

Drimane-type sesquiterpenoids (DMTs) are characterized by a distinctive 6/6 bicyclic skeleton comprising the A and B rings. While DMTs are commonly found in fungi and plants, their presence in bacteria has not been reported. Moreover, the biosynthetic pathways for DMTs have been primarily elucidated in fungi, with identified P450s only acting on the B ring. In this study, we isolated and characterized three bacterial DMTs, namely 3ß-hydroxydrimenol (2), 2α-hydroxydrimenol (3), and 3-ketodrimenol (4), from Streptomyces clavuligerus. Through genome mining and heterologous expression, we identified a cav biosynthetic gene cluster responsible for the biosynthesis of DMTs 2-4, along with a P450, CavA, responsible for introducing the C-2 and C-3 hydroxy groups. Furthermore, the substrate scope of CavA revealed its ability to hydroxylate drimenol analogs. This discovery not only broadens the known chemical diversity of DMTs from bacteria, but also provides new insights into DMT biosynthesis in bacteria.

Mutual Modulation of the Activities of Human CYP2D6 and Four UGTs during the Metabolism of Propranolol.

Cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) are two enzyme families that play an important role in drug metabolism, catalyzing either the functionalization or glucuronidation of xenobiotics. However, their mutual interactions are poorly understood. In this study, the functional interactions of human CYP2D6 with four human UGTs (UGT1A7, UGT1A8, UGT1A9, and UGT2A1) were investigated using our previously established co-expression model system in the fission yeast Schizosaccharomyces pombe. The substrate employed was propranolol because it is well metabolized by CYP2D6. Moreover, the CYP2D6 metabolite 4-hydroxypropranolol is a known substrate for the four UGTs included in this study. Co-expression of either UGT1A7, UGT1A8, or UGT1A9 was found to increase the activity of CYP2D6 by a factor of 3.3, 2.1 or 2.8, respectively, for the conversion of propranolol to 4-hydroxypropranolol. In contrast, UGT2A1 co-expression did not change CYP2D6 activity. On the other hand, the activities of all four UGTs were completely suppressed by co-expression of CYP2D6. This data corroborates our previous report that CYP2D6 is involved in functional CYP-UGT interactions and suggest that such interactions can contribute to both adverse drug reactions and changes in drug efficacy.

CYPArm2 is a CYP450 Monooxygenase with Protoilludene 13-Hydroxylase Activity Involved in the Biosynthesis of Armillyl Orsellinate-Type Sesquiterpenoids.

The melleolides are a family of structurally and functionally diverse sesquiterpenoids with potential applications as fungicides, antimicrobials, and cancer therapeutics. The initial and terminal steps of the biosynthesis pathway in Armillaria spp. have been characterized, but the intermediate steps are unclear. Biosynthetic gene clusters in A. mellea and A. gallica were shown to encode a terpene cyclase, a polyketide synthase, and four CYP450 monooxygenases. We have characterized CYPArm3, which is responsible for the hydroxylation of Δ-6-protoilludene, but the functions of the other CYP450s remain to be determined. Here we describe CYPArm2, which accepts Δ-6-protoilludene and 8α-hydroxy-6-protoilludene as substrates. To investigate the products in more detail, we generated recombinant Saccharomyces cerevisiae strains overexpressing CYPArm2 in combination with the previously characterized protoilludene synthase and 8α-hydroxylase. Using this total biosynthesis approach, sufficient quantities of product were obtained for NMR spectroscopy. This allowed the identification of 8α,13-dihydroxy-protoilludene, confirming that CYPArm2 is a protoilludene 13-hydroxylase.

Spinosad-associated modulation of select cytochrome P450s and glutathione S-transferases in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect pest that threatens potato crops. Multiple options exist to limit the impact of this pest even though insecticides remain a primary option for its control. Insecticide resistance has been reported in Colorado potato beetles and a better understanding of the molecular players underlying such process is of utmost importance to optimize the tools used to mitigate the impact of this insect. Resistance against the insecticide spinosad has been reported in this insect and this work thus aims at exploring the expression of targets previously associated with insecticide response in Colorado potato beetles exposed to this compound. Amplification and quantification of transcripts coding for cytochrome P450s and glutathione S-transferases were conducted via qRT-PCR in insects treated with varying doses of spinosad and for different time duration. This approach notably revealed differential expression of CYP6a23 and CYP12a5 in insects exposed to low doses of spinosad for 4 h as well as modulation of CYP6a13, CYP6d4, GST, GST1, and GST1-Like in insects treated with high doses of spinosad for the same duration. RNAi-based targeting of CYP4g15 and CYP6a23 was associated with marked reduction of transcript expression 7 days following dsRNA injection and reduction of the former had a marked impact on insect viability. In general, results presented here provide novel information regarding the expression of transcripts relevant to spinosad response in Colorado potato beetles and reveal a novel target to consider in the development of RNAi-based strategies aimed at this potato pest.

Ectopic expression of MmCYP1A1, a mouse cytochrome P450 gene, positively regulates stress tolerance in apple calli and Arabidopsis.

KEY MESSAGE: Ectopic expression of MmCYP1A1 gene from Mus musculus in apple calli and Arabidopsis increased the levels of melatonin and 6-hydroxymelatonin, and improved their stress resistance. Melatonin occurs widely in organisms, playing a key regulatory role. CYP1A1 is a cytochrome P450 monooxygenase, involved in the melatonin metabolism, and is responsible for the synthesis of 6-hydroxymelatonin from melatonin. Melatonin and 6-hydroxymelatonin have strong antioxidant activities in animals. Here, we cloned MmCYP1A1 from Mus musculus and found that ectopic expression of MmCYP1A1 improved the levels of melatonin and 6-hydroxymelatonin in transgenic apple calli and Arabidopsis. Subsequently, we observed that MmCYP1A1 increased the tolerance of transgenic apple calli and Arabidopsis to osmotic stress simulated by polyethylene glycol 6000 (PEG 6000), as well as resistance of transgenic Arabidopsis to drought stress. Further, the number of lateral roots of MmCYP1A1 transgenic Arabidopsis were enhanced significantly after PEG 6000 treatment. The expression of MmCYP1A1 remarkably reduced malondialdehyde (MDA) content, electrolyte leakage, accumulation of H2O2 and O2- during stress treatment. Moreover, MmCYP1A1 enhanced stress tolerance in apple calli and Arabidopsis by increasing the expression levels of resistance genes. MmCYP1A1 also promoted stomatal closure in transgenic Arabidopsis to reduce leaf water loss during drought. Our results indicate that MmCYP1A1 plays a key role in plant stress tolerance, which may provide a reference for future plant stress tolerance studies.

Cytochrome P450 CYP709C56 metabolizing mesosulfuron-methyl confers herbicide resistance in Alopecurus aequalis.

Multiple herbicide resistance in diverse weed species endowed by enhanced herbicide detoxification or degradation is rapidly growing into a great threat to herbicide sustainability and global food safety. Although metabolic resistance is frequently documented in the economically damaging arable weed species shortawn foxtail (Alopecurus aequalis Sobol.), relevant molecular knowledge has been lacking. Previously, we identified a field population of A. aequalis (R) that had evolved metabolic resistance to the commonly used acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl. RNA sequencing was used to discover potential herbicide metabolism-related genes, and four cytochrome P450s (CYP709C56, CYP71R18, CYP94C117, and CYP94E14) were identified with higher expressions in the R vs. susceptible (S) plants. Here the full-length P450 complementary DNA transcripts were each cloned with identical sequences between the S and R plants. Transgenic Arabidopsis overexpressing CYP709C56 became resistant to the sulfonylurea herbicide mesosulfuron-methyl and the triazolo-pyrimidine herbicide pyroxsulam. This resistance profile generally but does not completely in accordance with what is evident in the R A. aequalis. Transgenic lines exhibited enhanced capacity for detoxifying mesosulfuron-methyl into O-demethylated metabolite, which is in line with the detection of O-demethylated herbicide metabolite in vitro in transformed yeast. Structural modeling predicted that mesosulfuron-methyl binds to CYP709C56 involving amino acid residues Thr-328, Thr-500, Asn-129, Gln-392, Phe-238, and Phe-242 for achieving O-demethylation. Constitutive expression of CYP709C56 was highly correlated with the metabolic mesosulfuron-methyl resistance in A. aequalis. These results indicate that CYP709C56 degrades mesosulfuron-methyl and its up-regulated expression in A. aequalis confers resistance to mesosulfuron-methyl.

Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used ß-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

Decreased plasma exposure of clopidogrel active metabolite in rats after long-term treatment with clopidogrel.

Clopidogrel (Clop) is oxidized by cytochrome P450s (CYPs) to an active thiol metabolite, Clop-AM, to inhibit platelet activation and aggregation. As an irreversible inhibitor of CYP2B6 and CYP2C19, clopidogrel may inhibit its own metabolism after long-term administration. The study compared the pharmacokinetic profiles of clopidogrel and its metabolites in rats receiving a single or a 2 week administration of Clop. The mRNA and protein levels of hepatic clopidogrel-metabolizing enzymes and their enzymatic activities were analyzed to explore their contribution to any altered plasma exposure of Clop and its metabolites. The results showed that long-term treatment with clopidogrel significantly decreased the AUC(0-t) and Cmax values of Clop-AM in rats, accompanied with markedly impaired catalytic activities of Clop-metabolizing CYPs including CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4. It suggests that consecutive administration of Clop to rats decreases hepatic CYPs activities, which may, in turn, inhibit clopidogrel metabolism and then reduce Clop-AM plasma exposure. Therefore, long-term treatment with clopidogrel has the potential to reduce its anti-platelet activity and to increase the risk of drug-drug interaction.

Enhanced metabolic ability enabled wild panicgrass (Panicum miliaceum L. var. ruderale kit.) resistance to ALS inhibitor herbicide.

Wild panicgrass (Panicum miliaceum L. var. ruderale kit.) is an annual grass weed that primarily occurs in maize fields. Nicosulfuron is a widely used selective herbicide that effectively controls gramineous weeds in maize fields. However, owing to its long-term and extensive application, the control of P. miliaceum has been substantially reduced. The objective of this study was to determine the resistance pattern to ALS inhibitors in P. miliaceum and investigate the underlying resistance mechanisms. These are important for guiding the prevention and eradication of resistant weeds. Whole plant bioassays showed P. miliaceum had evolved high levels of resistance to nicosulfuron and multiple resistance to atrazine and mesotrione. The ALS gene sequence results indicated the absence of mutations in the resistant population. Additionally, there was no significant difference found in the inhibition rate of the ALS enzyme activity (I50) between the resistant and sensitive populations. Following the application of malathion the resistant P. miliaceum population became more sensitive to nicosulfuron. At 96 h after application of nicosulfuron, glutathione-S-transferase activity in the resistant population was significantly higher than that in the susceptible population. The study reveals that the main cause of resistance to ALS inhibitor herbicide in P. miliaceum is likely increased metabolism of herbicides. These findings may assist in devising effective strategies for preventing and eliminating resistant P. miliaceum.

Function of Cytochrome P450s and Gut Microbiome in Biopesticide Adaptation of Grapholita molesta on Different Host Diets.

Insects that feed on various host plants possess diverse xenobiotic adaptations; however, the underlying mechanisms are poorly understood. In the present study, we used Grapholita molesta, which shifts feeding sites from peach shoots to apple fruits, as a model to explore the effects of shifts in host plant diet on the profiles of cytochrome P450s and the gut bacteria microbiome, as well as their effects on biopesticide adaptation. We found that the sensitivity of the fruit-feeding G. molesta to emamectin benzoate biopesticide was significantly lower than that of the shoot-feeding larvae. We also found that the P450 enzyme activity and the expression of nine cytochrome P450s were enhanced in G. molesta fed on Fuji apples compared to those fed on peach shoots. The survival rates of G. molesta exposed to emamectin benzoate significantly decreased as each of three of four emamectin benzoate-inducted cytochrome P450 genes were silenced. Furthermore, we discovered the gut bacteria dynamics of G. molesta changed with the host shift and the structure of the gut bacteria microbiome was determined by the final diet ingested; additionally, the dysbiosis of the gut microbiota induced by antibiotics could significantly increase the sensitivity to emamectin benzoate. Taken together, our results suggest that the expression of P450s and the composition of the gut bacteria microbiome promote adaptation to emamectin benzoate in G. molesta, providing new insights into the molecular mechanisms underlying xenobiotic adaptation in this notorious pest.