Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Improved UAV Opium Poppy Detection Using an Updated YOLOv3 Model.

Rapid detection of illicit opium poppy plants using UAV (unmanned aerial vehicle) imagery has become an important means to prevent and combat crimes related to drug cultivation. However, current methods rely on time-consuming visual image interpretation. Here, the You Only Look Once version 3 (YOLOv3) network structure was used to assess the influence that different backbone networks have on the average precision and detection speed of an UAV-derived dataset of poppy imagery, with MobileNetv2 (MN) selected as the most suitable backbone network. A Spatial Pyramid Pooling (SPP) unit was introduced and Generalized Intersection over Union (GIoU) was used to calculate the coordinate loss. The resulting SPP-GIoU-YOLOv3-MN model improved the average precision by 1.62% (from 94.75% to 96.37%) without decreasing speed and achieved an average precision of 96.37%, with a detection speed of 29 FPS using an RTX 2080Ti platform. The sliding window method was used for detection in complete UAV images, which took approximately 2.2 sec/image, approximately 10× faster than visual interpretation. The proposed technique significantly improved the efficiency of poppy detection in UAV images while also maintaining a high detection accuracy. The proposed method is thus suitable for the rapid detection of illicit opium poppy cultivation in residential areas and farmland where UAVs with ordinary visible light cameras can be operated at low altitudes (relative height < 200 m).

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.

Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis.

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence.

Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.

Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

BACKGROUND: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. RESULTS: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. CONCLUSIONS: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Confirmation of recent heroin abuse: Accepting the challenge.

Confirmation or exclusion of recent heroin consumption is still one of the major challenges for forensic and clinical toxicologists. A great variety of biomarkers is available for heroin abuse confirmation, including various opium alkaloids (eg, morphine, codeine), street heroin impurities (eg, 6-acetylcodeine [6-AC], noscapine, papaverine) as well as associated metabolites (eg, 6-monoacetylmorphine [6-MAM], morphine glucuronides). However, the presence of most of these biomarkers cannot solely be attributed to a previous heroin administration but can, among other things, also be due to consumption of poppy seed products ('poppy seed defense'), opium preparations or specific medications, respectively. A reliable allocation is of great importance in different contexts, for instance in the case of DUID (driving under the influence of drugs) investigations, in driving licence re-granting processes, in workplace drug testing (WDT), as well as in post-mortem identification of illicit opiate use. Additionally, differentiation between illicit street heroin abuse and pharmaceutical heroin administration is also important, especially within the frame of heroin-assisted treatments. Therefore, analysis of multiple biomarkers is recommended when illicit opiate consumption is assumed to obtain the most reliable results possible. Beyond that, interpretation of positive opiate test results requires a profound insight into the great variety of biomarkers available and their validity regarding the alleged consumption. This paper aims to provide an overview of the wide variety of heroin abuse biomarkers described in the literature and to review them regarding their utility and reliability in daily routine analysis.

T-helper 1 and 2 serum cytokine assay in chronic opioid addicts.

There are a few studies with conflicting results on the effects of opioids on the functioning of immune system. This study was performed to investigate the in vitro production of interferon-gamma and interleukin-10 after antigenic stimulation of cells using whole blood from opioid addicts. Blood samples were taken from 20 chronically opioid-addicted persons, who voluntarily enrolled for detoxification (10 opium and 10 heroin addicts). Blood samples were also taken from 10 healthy individuals with no history of drug abuse as the control. Cell culture was performed in a whole blood culture assay. Diluted blood samples were stimulated with phytohemagglutinin or with lipopolysaccharide and the supernatants were collected to measure cytokine production. The results demonstrated a significant decrease in interferon-gamma production and an increase in interleukin-10 secretion in heroin addicts, relative to the control group (35.9+/-26.3 versus 110.2+/-60.3 pg/mL, p<0.01 and 71.8+/-28.4 versus 17.1+/-13.5 pg/mL, p<0.01, respectively), however the changes in these values in opium addicts were not significant compared to healthy individuals. The results could suggest that opioid addiction leads to a shift in the Th1/Th2 cytokine balance of peripheral CD4+ cells towards the Th2 response, and opioid addicts demonstrate reduced mitogenic responsiveness of lymphocytes relative to healthy individuals.

Honeybees foraging response in genetically diversified opium poppy.

Studies were carried out on honeybees foraging on plant flowers. Results showed significantly higher foraging response of honeybees (Apis mellifera) in genetically divergent narcotic plant opium poppy (Papaver somniferum). Of the 18 mutants and two locally adapted cultivars of diverse genotypes screened, eight revealed significantly greater foraging response manifesting honeybee's preference towards specific plant morphotypes. The number of flower bloom did not correspond to number of foraging bees in both mutant and cultivar plant types of opium poppy. The genotype specific foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honeys for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops.

Anesthesiologist suicide with atracurium.

Atracurium is a nondepolarizing skeletal muscle relaxant used to facilitate endotracheal intubation and to induce skeletal muscle relaxation during surgery or mechanical ventilation. The drug undergoes a spontaneous non-enzymatic biotransformation, yielding laudanosine and an acrylate moiety. This report documents the case of a 45-year-old anesthesiologist who was found dead at the hospital where he worked. The victim was known to be depressed and undergoing treatment with venlafaxine. An empty syringe was found near the body. Toxicological analysis revealed the presence of laudanosine in the syringe, 0.6 mg/L of laudanosine in heart blood, 0.3 mg/L in urine, and 0.02 mg/L in vitreous humor. Meanwhile, concentrations of venlafaxine and O-desmethyl-venlafaxine, its active metabolite, were 0.7 and 1.1 mg/L in heart blood, 1.7 and 5.2 mg/L in urine, 0.5 and 0.7 mg/L in vitreous humor, and 400 and 20 mg in gastric content, respectively. All drugs and metabolites involved in the case were detected using gas chromatography with nitrogen-phosphorus detection (GC-NPD) and confirmed using GC-mass spectrometry in full scan mode after solid-phase extraction using Bond-Elut Certify columns. Additional high-performance liquid chromatography coupled to diode-array detection screening also obtained the same results. Quantitation of laudanosine and venlafaxine together with its metabolite was carried out using GC-NPD. No other drugs, including ethanol, were detected. Recoveries for laudanosine and venlafaxine were 89% and 86%, respectively, at 0.5 mg/L; intraday and interday precisions were 2% and 6%, and 3% and 7%, respectively; and limits of detection and quantitation were 6 and 20 ng/mL and 18 and 59 ng/mL, respectively. The linearity of the blood calibration curves was excellent for both drugs with r(2) values of > 0.999 (range 0.1-2.0 mg/L). Based on the autopsy findings, case history, and toxicology results, the forensic pathologists ruled that the cause of death was an overdose of atracurium, and the manner of death was suicide.

Concentration profiling of minerals in iliac crest bone tissue of opium addicted humans using inductively coupled plasma and discriminant analysis techniques.

Opium addiction is one of the main health problems in developing countries and induces serious defects on the human body. In this work, the concentrations of 32 minerals including alkaline, heavy and toxic metals have been determined in the iliac crest bone tissue of 22 opium addicted individuals using inductively coupled plasma-optical emission spectroscopy (ICP-OES). The bone tissues of 30 humans with no physiological and metabolomic diseases were used as the control group. For subsequent analyses, the linear and quadratic discriminant analysis techniques have been used for classification of the data into "addicted" and "non-addicted" groups. Moreover, the counter-propagation artificial neural network (CPANN) has been used for clustering of the data. The results revealed that the CPANN is a robust model and thoroughly classifies the data. The area under the curve for the receiver operating characteristic curve for this model was more than 0.91. Investigation of the results revealed that the opium consumption causes a deficiency in the level of Calcium, Phosphate, Potassium and Sodium in iliac crest bone tissue. Moreover, this type of addiction induces an increment in the level of toxic and heavy metals such as Co, Cr, Mo and Ni in iliac crest tissue. The correlation analysis revealed that there were no significant dependencies between the age of the samples and the mineral content of their iliac crest, in this study. The results of this work suggest that the opium addicted individuals need thorough and restricted dietary and medical care programs after recovery phases, in order to have healthy bones.

Gene actions for yield and its attributes and their implications in the inheritance pattern over three generations in opium poppy (Papaver somniferum L.).

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.

Mammalian opiate alkaloid synthesis: lessons derived from plant biochemistry.

The presence of opiate receptors in mammalian tissues has stimulated the search for endogenous ligands to these receptors and has led to the discovery and characterization of endogenous opioid peptides. However, recent studies have provided evidence for the presence of opiate alkaloids in mammalian tissues and for their endogenous synthesis. The study of their origin and synthetic pathway has been significantly influenced by the early classical biochemical studies performed in plants. This review is a historical account of the use and abuse of opiates, the elucidation of morphine's synthetic pathway in the poppy plant, and the subsequent characterization of its presence in mammalian tissues. Clearly, our understanding of its synthetic pathway and regulation is a reflection of observations originally made in plant biochemistry.

Hmong folk remedies: limited acetylation of opium by aspirin and acetaminophen.

The traditional folk medicine of the Hmong and other Southeast Asian refugees has accompanied them during their immigration in this country over the past two decades. In two recent cases involving Hmong defendants, unknown solids, resembling charcoal in consistency and purported to be "backache remedies," were analyzed and found to be complex mixtures of aspirin, acetaminophen, caffeine and partly acetylated opium. In particular, significant amounts of acetylacetaminophen, 3-O-acetylmorphine, 6-O-acetylcodeine, 6-O-acetylmorphine, and heroin were identified by gas chromatography/mass spectrometry. Heating approximately equal weights of solid opium, aspirin, and acetaminophen at 130 degrees C for several hours produced a mixture of compounds showing a similar acetylation pattern.

Application of membrane and ozonation technologies to remove color from agro-industry effluents.

The results of membrane and ozonation experiments carried out on various agro-industry effluents including fermentation (baker's yeast), corrugated board, opium alkaloid and textile dying industries are presented. The experiments were performed using lab-scale membrane and ozonation reactors. Color removals were in the range of 80 to 99% for the membrane treatment studies. Ozonation experiments have shown that color removals in the range of 83 to 98% are possible for the investigated wastewaters. Final color levels were lower than 100 Pt-Co unit, which is quite acceptable aesthetically. The relative unit treatment costs of ozonation were about two times higher than membrane systems especially for very strong colored effluents including fermentation and opium alkaloid industries. The study has demonstrated that both membrane and ozonation technologies are viable options for color removal.