Comparison of State-Level Regulations for Cannabis Contaminants and Implications for Public Health.

BACKGROUND: The presence of contaminants in cannabis presents a potential health hazard to recreational users and susceptible patients with medical conditions. Because of the federally illegal status of cannabis, there are no unified regulatory guidelines mitigating the public health risk of cannabis contaminants. OBJECTIVE: To inform further research and provide solutions to the public health risk of cannabis contaminants at a national level, we examined the current landscape of state-level contaminant regulations, and cannabis contaminants of concern, as well as patient populations susceptible to contaminants. METHODS: We examined the regulatory documents for medical and recreational cannabis in all legalized U.S. jurisdictions and compiled a complete list of regulated contaminants, namely, pesticides, inorganics, solvents, microbes, and mycotoxins. We data mined the compliance testing records of 5,654 cured flower and 3,760 extract samples that accounted for ∼6% of California's legal cannabis production in 2020-2021. We also reviewed the publicly available medical cannabis use reports to tabulate the susceptible patient populations. RESULTS: As of 18 May 2022, 36 states and the District of Columbia listed a total of 679 cannabis contaminants as regulated in medical or recreational cannabis, including 551 pesticides, 74 solvents, 12 inorganics, 21 microbes, 5 mycotoxins, and 16 other contaminants. Different jurisdictions showed significant variations in regulated contaminants and action levels ranging up to four orders of magnitude. A failure rate of 2.3% was identified for flowers and 9.2% for extracts in the California samples. Insecticides and fungicides were the most prevalent categories of detected contaminants, with boscalid and chlorpyrifos being the most common. The contaminant concentrations fell below the regulatory action levels in many legalized jurisdictions, indicating a higher risk of contaminant exposure. Cannabis use reports indicated usage in several patient populations susceptible to contamination toxicity, including cancer (44,318) and seizure (21,195) patients. DISCUSSION: Although individual jurisdictions can implement their policies and regulations for legalized cannabis, this study demonstrates the urgent need to mitigate the public health risk of cannabis contamination by introducing national-level guidelines based on conventional risk assessment methodologies and knowledge of patients' susceptibility in medical use. https://doi.org/10.1289/EHP11206.

Cannabis Chemovar Nomenclature Misrepresents Chemical and Genetic Diversity; Survey of Variations in Chemical Profiles and Genetic Markers in Nevada Medical Cannabis Samples.

Introduction: Medical cannabis patients receive clinical benefits from the secondary metabolites of the plant, which contain a variety of cannabinoids and terpenoids in combinations that can be used to classify the chemovars. State-regulated medical cannabis programs rely on breeder-reported "strain" names both within diversion control systems and to describe the medical cannabis products that are sold to patients in medical cannabis dispensaries. In state-regulated medical cannabis programs, there is no conventional nomenclature system that correlates the breeder-reported names with their profiles of active ingredients, and these "strain" names are invalid as they refer to chemical differences properly referred to as to chemovars. Materials and Methods: To determine the actual levels of chemical diversity represented in 2662 samples of Cannabis flower collected between January 2016 and June of 2017 in Nevada, chemical profile data were measured from these samples by a state-qualified third-party testing laboratory. Principal component analysis (PCA) was used to define clusters in data sets representing both cannabinoids and terpenoids, cannabinoids only, or terpenoids only. Results: The PCA of the terpenoid only data set revealed three well-defined clusters. All three terpenoids only data clusters had high tetrahydrocannabinolic acid synthase, but the terpene profiles listed in reverse-order of abundance best defined these chemovars. The three chemovars in Nevada were labeled with 396 breeder-reported sample names, which overestimate the diversity and do not inform patients regarding chemical properties. Representative DNA samples were taken from each chemovar to determine whether the genetic diversity was greater than the chemical diversity. The limited genotyping experiment was based on DNA sequence polymorphisms. The genetic analysis revealed twelve distinct genetic clades, which still does not account for the entirety of the 396 reported sample names. The finite genotypes did not correlate with the chemotypes determined for the samples. This suggests that either the DNA-markers used were too narrowly restricted for factual separation or that environmental factors contributed more significantly to the chemical profiles of cannabis than genetics. Conclusion: The three chemovars and twelve genotypes reflect low medical diversity on the market in Nevada during its "medical use only" phase. Furthermore, the 396 breeder-reported sample names within this set imply a false sense of diversity of products in Nevada dispensaries.

A single nucleotide polymorphism assay sheds light on the extent and distribution of genetic diversity, population structure and functional basis of key traits in cultivated north American cannabis.

BACKGROUND: The taxonomic classification of Cannabis genus has been delineated through three main types: sativa (tall and less branched plant with long and narrow leaves), indica (short and highly branched plant with broader leaves) and ruderalis (heirloom type with short stature, less branching and small thick leaves). While still under discussion, particularly whether the genus is polytypic or monotypic, this broad classification reflects putative geographical origins of each group and putative chemotype and pharmacologic effect. METHODS: Here we describe a thorough investigation of cannabis accessions using a set of 23 highly informative and polymorphic SNP (Single Nucleotide Polymorphism) markers associated with important traits such as cannabinoid and terpenoid expression as well as fibre and resin production. The assay offers insight into cannabis population structure, phylogenetic relationship, population genetics and correlation to secondary metabolite concentrations. We demonstrate the utility of the assay for rapid, repeatable and cost-efficient genotyping of commercial and industrial cannabis accessions for use in product traceability, breeding programs, regulatory compliance and consumer education. RESULTS: We identified 5 clusters in the sample set, including industrial hemp (K5) and resin hemp, which likely underwent a bottleneck to stabilize cannabidiolic acid (CBDA) accumulation (K2, Type II & III). Tetrahydrocannabinolic acid (THCA) resin (Type I) makes up the other three clusters with terpinolene (K4 - colloquial "sativa" or "Narrow Leaflet Drug" (NLD), myrcene/pinene (K1) and myrcene/limonene/linalool (K3 - colloquial "indica", "Broad Leaflet Drug" (BLD), which also putatively harbour an active version of the cannabichrometic acid Synthase gene (CBCAS). CONCLUSION: The final chemical compositions of cannabis products have key traits related to their genetic identities. Our analyses in the context of the NCBI Cannabis sativa Annotation Release 100 allows for hypothesis testing with regards to secondary metabolite production. Genetic markers related to secondary metabolite production will be important in many sectors of the cannabis marketplace. For example, markers related to THC production will be important for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

Detection of misfolded prion protein in blood with conformationally sensitive peptides.

BACKGROUND: The long-standing goal of a preclinical diagnostic test for transmissible spongiform encephalopathy (TSE) has recently become urgent because of the discovery that humans with variant Creutzfeldt-Jakob disease can transmit disease via blood transfusions. STUDY DESIGN AND METHODS: The misfolded protein diagnostic (MPD) assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to beta-sheet conversion in the presence of the misfolded prion protein (PrP(TSE)). The ability of the assay to detect PrP(TSE) in brain, serum, and plasma was tested. The basic protocol involved a several-hour incubation of 200-microL sample volumes with the peptide reagent in 96-well plates, after which fluorescence was monitored by a fluorescence plate reader with an excitation wavelength of 350 nm and emission scanning wavelength range of 365 to 600 nm. RESULTS: Target specificity for PrP(TSE) was documented by correlation of assay signal with Western blot signals in brain tissue from TSE-infected, normal, and knockout mice and negative assay signals by use of reagents with different peptide sequences. When applied to plasma or serum, the assay discriminated between samples from a variety of experimental and natural TSE infections compared to uninfected controls, with a sensitivity threshold of approximately 1 infectious dose per mL in pooled plasma from TSE-infected mice. CONCLUSIONS: The MPD assay is a sensitive and specific test for the detection of PrP(TSE) that may be useful in both preclinical and clinical diagnosis of TSE diseases of animals and humans.

Trehalose lyophilized platelets for wound healing.

Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets' ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.

Wound-healing properties of trehalose-stabilized freeze-dried outdated platelets.

BACKGROUND: The wound-healing applications of platelet (PLT)-derived cytokines, proteins, and membranes is accepted but continues to be investigated. In this study, it is demonstrated that stabilized freeze-dried PLTs prepared from outdated PLTs (FDPOs) accelerate wound healing and form tube structures as well as stabilized indated freeze-dried PLTs (FDPIs) and room-temperature fresh PLTs (RT-PLTs). STUDY DESIGN AND METHODS: Experiments were designed to compare in vitro and in vivo wound-healing properties of FDPI, FDPO, and RT-PLT preparations. The concentration of PLT-derived growth factor (PDGF)-betabeta and transforming growth factor (TGF)-beta1 was determined, and the abilities of FDPIs, FDPOs and RT-PLTs to induce endothelial cell proliferation and promote endothelial cell tube formation (cells formed solid spouts connecting neighboring cells to form tube structures) were observed. Wound-healing characteristics were measured by surgically inducing 1-cm(2), full-thickness wounds on db/db mice (n = 10 per group). The wounds were treated with single or multiple doses of FDPIs and FDPOs. Wound closure rate was determined, and histology samples were evaluated for cellular makeup. RESULTS: FDPOs retained the same levels of PDGF-betabeta and TGF-beta1 and were able to promote endothelial cell proliferation and tube formation in vitro as well as FDPIs or RT-PLTs. Multiple applications of FDPO accelerated wound closure and enhanced reepithelialization when compared to untreated wounds in db/db mice. CONCLUSION: FDPOs enhanced wound healing in db/db mice as well as FDPIs and RT-PLTs. Wound closure was obtained 6 days earlier than untreated wounds and histologic examination revealed reduced granulation and increased cellular angiogenesis.

Conformational biosensor for diagnosis of prion diseases.

A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-beta-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.

Production of Se-methylselenocysteine in transgenic plants expressing selenocysteine methyltransferase.

BACKGROUND: It has become increasingly evident that dietary Se plays a significant role in reducing the incidence of lung, colorectal and prostate cancer in humans. Different forms of Se vary in their chemopreventative efficacy, with Se-methylselenocysteine being one of the most potent. Interestingly, the Se accumulating plant Astragalus bisulcatus (Two-grooved poison vetch) contains up to 0.6% of its shoot dry weight as Se-methylselenocysteine. The ability of this Se accumulator to biosynthesize Se-methylselenocysteine provides a critical metabolic shunt that prevents selenocysteine and selenomethionine from entering the protein biosynthetic machinery. Such a metabolic shunt has been proposed to be vital for Se tolerance in A. bisulcatus. Utilization of this mechanism in other plants may provide a possible avenue for the genetic engineering of Se tolerance in plants ideally suited for the phytoremediation of Se contaminated land. Here, we describe the overexpression of a selenocysteine methyltransferase from A. bisulcatus to engineer Se-methylselenocysteine metabolism in the Se non-accumulator Arabidopsis thaliana (Thale cress). RESULTS: By over producing the A. bisulcatus enzyme selenocysteine methyltransferase in A. thaliana, we have introduced a novel biosynthetic ability that allows the non-accumulator to accumulate Se-methylselenocysteine and gamma-glutamylmethylselenocysteine in shoots. The biosynthesis of Se-methylselenocysteine in A. thaliana also confers significantly increased selenite tolerance and foliar Se accumulation. CONCLUSION: These results demonstrate the feasibility of developing transgenic plant-based production of Se-methylselenocysteine, as well as bioengineering selenite resistance in plants. Selenite resistance is the first step in engineering plants that are resistant to selenate, the predominant form of Se in the environment.

The role of hydrophobic interactions in amyloidogenesis: example of prion-related polypeptides.

Conversion of the non-infectious, cellular form of the prion protein (PrP(C)) to the infectious form (PrP(Sc)) is thought to be driven by an alpha-helical to beta-sheet conformational transition. To reveal the sequence determinants which encourage the transition to beta-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and steady state fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant beta-strand formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichroism. At moderate concentrations of trifluoroethanol and/or acetonitrile, the conformational ensemble shifts towards beta-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid beta-sheet structures. Encouragement of beta-folds requires the destabilization of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms.