Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes.

There is an active and growing interest in cannabis female inflorescence (Cannabis sativa) for medical purposes. Therefore, a definition of its quality attributes can help mitigate public health risks associated with contaminated, substandard, or adulterated products and support sound and reproducible basic and clinical research. As cannabis is a heterogeneous matrix that can contain a complex secondary metabolome with an uneven distribution of constituents, ensuring its quality requires appropriate sampling procedures and a suite of tests, analytical procedures, and acceptance criteria to define the identity, content of constituents (e.g., cannabinoids), and limits on contaminants. As an independent science-based public health organization, United States Pharmacopeia (USP) has formed a Cannabis Expert Panel, which has evaluated specifications necessary to define key cannabis quality attributes. The consensus within the expert panel was that these specifications should differentiate between cannabis chemotypes. Based on the secondary metabolite profiles, the expert panel has suggested adoption of three broad categories of cannabis. These three main chemotypes have been identified as useful for labeling based on the following cannabinoid constituents: (1) tetrahydrocannabinol (THC)-dominant chemotype; (2) intermediate chemotype with both THC and cannabidiol (CBD); and (3) CBD-dominant chemotype. Cannabis plants in each of these chemotypes may be further subcategorized based on the content of other cannabinoids and/or mono- and sesquiterpene profiles. Morphological and chromatographic tests are presented for the identification and quantitative determination of critical constituents. Limits for contaminants including pesticide residues, microbial levels, mycotoxins, and elemental contaminants are presented based on toxicological considerations and aligned with the existing USP procedures for general tests and assays. The principles outlined in this review should be able to be used as the basis of public quality specifications for cannabis inflorescence, which are needed for public health protection and to facilitate scientific research on cannabis safety and therapeutic potential.

Cross talk between Leishmania donovani CpG DNA and Toll-like receptor 9: an immunoinformatics approach.

The precise and potential contribution of Toll-like receptors (TLRs) signaling pathways in fighting parasitic infections of Leishmania spp., an intracellular protozoan parasite, has gained significant attention during the last decades. Although it is well established that TLR9 recognizes CpG motifs in microbial genomes, the specificity of the CpG DNA pattern of Leishmania parasite interacting with endosomal TLR9 is still unknown. Hence in our study to identify the CpG DNA pattern of Leishmania donovani acting as ligand for TLR9, consecutive homology searches were performed using known CpG ODN 2216 as initial template until a consistent CpG pattern in L. donovani was found. A reliable model of TLR9 ectodomains (ECDs) as well as CpG DNA patterns was predicted to develop the 3D structural complexes of TLR9 ECD-CpG DNA utilizing molecular modeling and docking approaches. The results revealed the preferential specificity of L. donovani CpG DNA to TLR9 compared to control ODN and other CpG patterns. The interface between TLR9 and L. donovani CpG DNA was also found to be geometrically complementary with the LRR11 region of TLR9, acting as the critical region for ligand recognition. The L. donovani CpG pattern identified can be employed to derive a platform for development of an innate immunomodulatory agent for deadly disease.

Ovulation but not milt production is inhibited in fathead minnows (Pimephales promelas) exposed to a reproductively inhibitory pulp mill effluent.

BACKGROUND: A 5-day fathead minnow (FHM) spawning assay is used by industry to monitor pulp mill effluent quality, with some mill effluents capable of completely inhibiting spawning. The purpose of this report is to characterize the effect of an inhibitory effluent on egg and milt production in FHM. METHODS: Eight tanks were treated with an inhibitory effluent while eight were kept with clean water. Each tank contained two males and four females as per the 5-day FHM spawning assay used by industry. Females were stripped of ovulated eggs and males of milt in four effluent-exposed and four control tanks. Eggs oviposited in every tank were also counted and checked for fertilization and data analyzed with 2-way ANOVA. RESULTS: We show that female, but not male, fathead minnow reproductive function is impaired in the 5-day fathead minnow spawning assay used by industry to evaluate pulp mill effluent quality in Canada. Milt production was not changed in the control or exposed males mid-way and at the end of the five day exposure (p > 0.05; n = 8). Total egg production (stripped + oviposited) was impaired (p < 0.05) in fathead minnows exposed to effluent (288 eggs/tank, n = 4 tanks) compared to those in control tanks (753 eggs/tank, n = 4 tanks). CONCLUSIONS: Our results indicate that males are able to detect female signals and prepare appropriately for spawning while in females inhibition of ovulation is occurring somewhere along the hypothalamus-pituitary-gonad reproductive axis. These results suggest female-specific neuroendocrine disruption and provide mechanistic insight into an assay used by industry to assess pulp mill effluent quality.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

Evaluating the potential of effluents and wood feedstocks from pulp and paper mills in Brazil, Canada, and New Zealand to affect fish reproduction: chemical profiling and in vitro assessments.

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

Evidence That Metal Particles in Cannabis Vape Liquids Limit Measurement Reproducibility.

Cannabis vaping involves the vaporization of a cannabis vaping liquid or solid via a vaping accessory such as a vape pen constructed of various metals or other parts. An increasing number of reports advocate for expansion of the testing and regulation of metal contaminants in cannabis vape liquids beyond the metals typically tested such as arsenic, cadmium, mercury, and lead to reflect the possibility of consumers' exposure to other metal contaminants. Metal contaminants may originate not only from the cannabis itself but also from the vape devices in which the cannabis vape liquid is packaged. However, metal analyses of cannabis vape liquids sampled from cannabis vaping devices are challenged by poor precision and reproducibility. Herein, we present data on the metal content of 12 metals in 20 legal and 21 illegal cannabis vape liquids. The lead mass fraction in several illegal samples reached up to 50 µg g-1. High levels of nickel (max 677 µg g-1) and zinc (max 426 µg g-1) were found in illegal samples, whereas the highest copper content (485 µg g-1) was measured in legal samples. Significant differences in metal mass fractions were observed in the legal cannabis vape liquid taken from two identical devices, even though the liquid was from the same lot of the same cannabis product. Metal particles in the vape liquids were observed by scanning electron microscopy, and laser ablation inductively coupled plasma mass spectrometry confirmed the presence of copper-, zinc-, lead-, and manganese-bearing particles, metals that are in common alloys that may be used to make vape devices. Colocalized particles containing aluminum, silica, and sodium were also detected. These results suggest that metal particles could be a contributing factor to poor measurement precision and for the first time, to the best of our knowledge, provide evidence of metal particles in cannabis vape liquids contained in unused cannabis vape pens.

Neuroendocrine disruption: more than hormones are upset.

Only a small proportion of the published research on endocrine-disrupting chemicals (EDC) directly examined effects on neuroendocrine processes. There is an expanding body of evidence that anthropogenic chemicals exert effects on neuroendocrine systems and that these changes might impact peripheral organ systems and physiological processes. Neuroendocrine disruption extends the concept of endocrine disruption to include the full breadth of integrative physiology (i.e., more than hormones are upset). Pollutants may also disrupt numerous other neurochemical pathways to affect an animal's capacity to reproduce, develop and grow, or deal with stress and other challenges. Several examples are presented in this review, from both vertebrates and invertebrates, illustrating that diverse environmental pollutants including pharmaceuticals, organochlorine pesticides, and industrial contaminants have the potential to disrupt neuroendocrine control mechanisms. While most investigations on EDC are carried out with vertebrate models, an attempt is also made to highlight the importance of research on invertebrate neuroendocrine disruption. The neurophysiology of many invertebrates is well described and many of their neurotransmitters are similar or identical to those in vertebrates; therefore, lessons learned from one group of organisms may help us understand potential adverse effects in others. This review argues for the adoption of systems biology and integrative physiology to address the effects of EDC. Effects of pulp and paper mill effluents on fish reproduction are a good example of where relatively narrow hypothesis testing strategies (e.g., whether or not pollutants are sex steroid mimics) have only partially solved a major problem in environmental biology. It is clear that a global, integrative physiological approach, including improved understanding of neuroendocrine control mechanisms, is warranted to fully understand the impacts of pulp and paper mill effluents. Neuroendocrine disruptors are defined as pollutants in the environment that are capable of acting as agonists/antagonists or modulators of the synthesis and/or metabolism of neuropeptides, neurotransmitters, or neurohormones, which subsequently alter diverse physiological, behavioral, or hormonal processes to affect an animal's capacity to reproduce, develop and grow, or deal with stress and other challenges. By adopting a definition of neuroendocrine disruption that encompasses both direct physiological targets and their indirect downstream effects, from the level of the individual to the ecosystem, a more comprehensive picture of the consequences of environmentally relevant EDC exposure may emerge.

Canadian boreal pulp and paper feedstocks contain neuroactive substances that interact in vitro with GABA and dopaminergic systems in the brain.

Pulp and paper wood feedstocks have been previously implicated as a source of chemicals with the ability to interact with or disrupt key neuroendocrine endpoints important in the control of reproduction. We tested nine Canadian conifers commonly used in pulp and paper production as well as 16 phytochemicals that have been observed in various pulp and paper mill effluent streams for their ability to interact in vitro with the enzymes monoamine oxidase (MAO), glutamic acid decarboxylase (GAD), and GABA-transaminase (GABA-T), and bind to the benzodiazepine-binding site of the GABA(A) receptor (GABA(A)-BZD). These neuroendocrine endpoints are also important targets for treatment of neurological disorders such as anxiety, epilepsy, or depression. MAO and GAD were inhibited by various conifer extracts of different polarities, including major feedstocks such as balsam fir, black spruce, and white spruce. MAO was selectively stimulated or inhibited by many of the tested phytochemicals, with inhibition observed by a group of phenylpropenes (e.g. isoeugenol and vanillin). Selective GAD inhibition was also observed, with all of the resin acids tested being inhibitory. GABA(A)-BZD ligand displacement was also observed. We compiled a table identifying which of these phytochemicals have been described in each of the species tested here. Given the diversity of conifer species and plant chemicals with these specific neuroactivities, it is reasonable to propose that MAO and GAD inhibition reported in effluents is phytochemical in origin. We propose disruption of these neuroendocrine endpoints as a possible mechanism of reproductive inhibition, and also identify an avenue for potential research and sourcing of conifer-derived neuroactive natural products.

Extracts from hardwood trees used in commercial paper mills contain biologically active neurochemical disruptors.

Following on our discovery that pulp and paper mill effluents can interact with, and disrupt, various neurotransmitter receptors and enzymes important to fish reproduction, we tested wood and bark extracts of 14 Eastern North American hardwood trees used in pulp and paper production. Radioligand binding to neurotransmitter receptors, including the dopamine-2 receptor (D2), the gamma aminobutyric acid receptor A (GABA(A)), N-methyl-D-aspartic acid (NMDA) receptor, and muscarinic cholinergic receptor (mACh-R), were significantly changed following in vitro incubations with many but not all extracts. Activities of neurotransmitter-related enzymes monoamine oxidase (MAO), GABA-transaminase (GABA-T), acetylcholinesterase (AChE) and glutamic acid decarboxylase (GAD) were also significantly altered. Butternut wood extracts and the isolated compound juglone significantly inhibited the enzymatic activities of MAO and GAD which we suggest may be part of a mechanism that may negatively affect fish reproduction. Besides giving credence to the hypothesis that neuroactive compounds in pulp and paper effluent may originate in the trees used by mills, the results reported here also indicate important neuropharmacological activities in hardwoods which may help identify new sources of biologically active natural products.

Pulp and paper mill effluents contain neuroactive substances that potentially disrupt neuroendocrine control of fish reproduction.

Here we show for the first time that components of pulp and paper mill effluents contain neuroactive substances that may impair fish reproduction. Grab samples of primary and secondary effluent were obtained from a representative pulp and paper mill in Eastern Canada. Effluents were fractionated using classic polarity and polyphenolic extraction methods into solvents of selected polarities (water, ethanol, ethyl acetate, and hexane). By means of in vitro, competitive assays on goldfish (Carassius auratus) brain tissues, the extracts were screened for their ability to interact with enzymes and receptors involved in gamma-aminobutyric acid (GABA), dopamine, glutamate, and acetylcholine-dependent neurotransmission. These neurotransmission pathways have essential regulatory roles in fish reproduction. Radioligand binding to the following neurotransmitter receptors were significantly impacted following in vitro incubations with extracts (percentage change from controls indicated in brackets): dopamine-2 (D2; 21-48% increase), GABA(A) receptor binding (65-67% decrease and 189% increase), N-methyl-o-aspartic acid (NMDA; 26-75% decrease), and muscarinic cholinergic (mACh; 42% increase). Activities of the following neurotransmitter-related enzymeswere significantly impacted: monoamine oxidase (MAO; 14-48% decrease), GABA-transaminase activity (33% decrease and 21-69% increase), and acetylcholinesterase (AChE; 21-50% decrease). No changes in glutamic acid decarboxylase (GAD) activity were detected. These findings provide a novel and plausible mechanism by which pulp and paper mills effluents impair fish reproduction by interacting with neurotransmitter systems. Furtherwork is required to identify the active compounds and explore whether these changes occur in vivo.