Opportunities, Challenges, and Scientific Progress in Hemp Crops.

The resurgence of cannabis (Cannabis sativa L.) has been propelled by changes in the legal framework governing its cultivation and use, increased demand for hemp-derived products, and studies recognizing the industrial and health benefits of hemp. This has led to the creation of novel high-cannabidiol, low-Δ9-tetrahydrocannabinol varieties, enabling hemp crop expansion worldwide. This review elucidates the recent implications for hemp cultivation in Europe, with a focus on the legislative impacts on the cultivation practices, prospective breeding efforts, and dynamic scientific landscape surrounding this crop. We also review the current cultivars' cannabinoid composition of the European hemp market and its major differences with that of the United States.

Fractionation and Extraction Optimization of Potentially Valuable Compounds and Their Profiling in Six Varieties of Two Nicotiana Species.

There is an increasingly urgent call to shift industrial processes from fossil fuel feedstock to sustainable bio-based resources. This change becomes of high importance considering new budget requirements for a carbon-neutral economy. Such a transformation can be driven by traditionally used plants that are able to produce large amounts of valuable biologically relevant secondary metabolites. Tobacco plants can play a leading role in providing value-added products in remote areas of the world. In this study, we propose a non-exhaustive list of compounds with potential economic interest that can be sourced from the tobacco plant. In order to optimize extraction methodologies, we first analyzed their physico-chemical properties using rapid solubility tests and high-resolution microfractionation techniques. Next, to identify an optimal extraction for a selected list of compounds, we compared 13 different extraction method-solvent combinations. We proceeded with profiling some of these compounds in a total of six varieties from Nicotiana tabacum and Nicotiana rustica species, identifying the optimal variety for each. The estimated expected yields for each of these compounds demonstrate that tobacco plants can be a superior source of valuable compounds with diverse applications beyond nicotine. Among the most interesting results, we found high variability of anatabine content between species and varieties, ranging from 287 to 1699 µg/g. In addition, we found that CGA (1305 µg/g) and rutin (7910 µg/g) content are orders of magnitude lower in the Burley variety as compared to all others.

Systems toxicology study reveals reduced impact of heated tobacco product aerosol extract relative to cigarette smoke on premature aging and exacerbation effects in aged aortic cells in vitro.

Aging and smoking are major risk factors for cardiovascular diseases (CVD). Our in vitro study compared, in the context of aging, the effects of the aerosol of Tobacco Heating System 2.2 (THS; an electrically heated tobacco product) and 3R4F reference cigarette smoke (CS) on processes that contribute to vascular pathomechanisms leading to CVD. Young and old human aortic smooth muscle cells (HAoSMC) were exposed to various concentrations of aqueous extracts (AE) from 3R4F CS [0.014-0.22 puffs/mL] or THS aerosol [0.11-1.76 puffs/mL] for 24 h. Key markers were measured by high-content imaging, transcriptomics profiling and multianalyte profiling. In our study, in vitro aging increased senescence, DNA damage, and inflammation and decreased proliferation in the HAoSMCs. At higher concentrations of 3R4F AE, young HAoSMCs behaved similarly to aged cells, while old HAoSMCs showed additional DNA damage and apoptosis effects. At 3R4F AE concentrations with the maximum effect, the THS AE showed no significant effect in young or old HAoSMCs. It required an approximately ten-fold higher concentration of THS AE to induce effects similar to those observed with 3R4F. These effects were independent of nicotine, which did not show a significant effect on HAoSMCs at any tested concentration. Our results show that 3R4F AE accelerates aging in young HAoSMCs and exacerbates the aging effect in old HAoSMCs in vitro, consistent with CS-related contributions to the risk of CVD. Relative to 3R4F AE, the THS AE showed a significantly reduced impact on HAoSMCs, suggesting its lower risk for vascular SMC-associated pathomechanisms leading to CVD.

A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model.

Cigarette smoking is one major modifiable risk factor in the development and progression of chronic obstructive pulmonary disease and cardiovascular disease. To characterize and compare cigarette smoke (CS)-induced disease endpoints after exposure in either whole-body (WB) or nose-only (NO) exposure systems, we exposed apolipoprotein E-deficient mice to filtered air (Sham) or to the same total particulate matter (TPM) concentration of mainstream smoke from 3R4F reference cigarettes in NO or WB exposure chambers (EC) for 2 months. At matching TPM concentrations, we observed similar concentrations of carbon monoxide, acetaldehyde, and acrolein, but higher concentrations of nicotine and formaldehyde in NOEC than in WBEC. In both exposure systems, CS exposure led to the expected adaptive changes in nasal epithelia, altered lung function, lung inflammation, and pronounced changes in the nasal epithelial transcriptome and lung proteome. Exposure in the NOEC caused generally more severe histopathological changes in the nasal epithelia and a higher stress response as indicated by body weight decrease and lower blood lymphocyte counts compared with WB exposed mice. Erythropoiesis, and increases in total plasma triglyceride levels and atherosclerotic plaque area were observed only in CS-exposed mice in the WBEC group but not in the NOEC group. Although the composition of CS in the breathing zone is not completely comparable in the two exposure systems, the CS-induced respiratory disease endpoints were largely confirmed in both systems, with a higher magnitude of severity after NO exposure. CS-accelerated atherosclerosis and other pro-atherosclerotic factors were only significant in WBEC.

A 6-month systems toxicology inhalation study in ApoE-/- mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE-/-) mice. The mice were exposed to aerosols from three different E-vapor formulations: 1) carrier (propylene glycol and vegetable glycerol), 2) base (carrier and nicotine), or 3) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases.NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

Toxicological assessment of Tobacco Heating System 2.2: Findings from an independent peer review.

Offering safer alternatives to cigarettes, such as e-cigarettes and heated tobacco products, to smokers who are not willing to quit could reduce the harm caused by smoking. Extensive and rigorous scientific studies are conducted to assess the relative risk of such potentially modified risk tobacco products compared with that of smoking cigarettes. In addition to the peer review of publications reporting individual studies, we aimed to gauge the plausibility of the evidence to the scientific community and appreciate likely necessary additions prior to regulatory submission. Therefore, we sponsored a two-tier peer review organized by an independent third party who identified, recruited, and managed 7 panels of 5-12 experts whose identity remains unknown to us. The reviewers had access to all publications and raw data from preclinical and clinical studies via a web portal. The reviewers were asked questions regarding study design, methods, quality of data, and interpretation of results to judge the validity of the conclusions regarding the relative effects of the Tobacco Heating System 2.2 compared with cigarettes. Once their conclusions were submitted, the experts had the opportunity to participate in an anonymized online debate with their fellow panel members. We present here the results obtained from this innovative peer review effort which revealed supportive or very supportive of the study methods and results, and support the robustness of the studies and validity of the conclusions.

Physicochemical studies of direct interactions between lung surfactant and components of electronic cigarettes liquid mixtures.

Direct physicochemical interactions between the major components of electronic cigarette liquids (e-liquids): glycerol (VG) and propylene glycol (PG), and lung surfactant (LS) were studied by determining the dynamic surface tension under a simulated breathing cycle using drop shape method. The studies were performed for a wide range of concentrations based on estimated doses of e-liquid aerosols (up to 2500 × the expected nominal concentrations) and for various VG/PG ratios. The results are discussed as relationships among mean surface tension, surface tension amplitude, and surface rheological properties (dilatational elasticity and viscosity) versus concentration and composition of e-liquid. The results showed that high local concentrations (>200 × higher than the estimated average dose after a single puffing session) may induce measurable changes in biophysical activity of LS; however, only ultra-high e-liquid concentrations inactivated the surfactant. Physiochemical characterization of e-liquids provide additional insights for the safety assessment of electronic nicotine delivery systems (ENDS).

The Apoe(-/-) mouse model: a suitable model to study cardiovascular and respiratory diseases in the context of cigarette smoke exposure and harm reduction.

Atherosclerosis-prone apolipoprotein E-deficient (Apoe(-/-)) mice display poor lipoprotein clearance with subsequent accumulation of cholesterol ester-enriched particles in the blood, which promote the development of atherosclerotic plaques. Therefore, the Apoe(-/-) mouse model is well established for the study of human atherosclerosis. The systemic proinflammatory status of Apoe(-/-) mice also makes them good candidates for studying chronic obstructive pulmonary disease, characterized by pulmonary inflammation, airway obstruction, and emphysema, and which shares several risk factors with cardiovascular diseases, including smoking. Herein, we review the results from published studies using Apoe(-/-) mice, with a particular focus on work conducted in the context of cigarette smoke inhalation studies. The findings from these studies highlight the suitability of this animal model for researching the effects of cigarette smoking on atherosclerosis and emphysema.

Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures.

Cigarette smoke (CS) has been reported to increase predisposition to oral cancer and is also recognized as a risk factor for many conditions including periodontal diseases, gingivitis, and other benign mucosal disorders. Smoking cessation remains the most effective approach for minimizing the risk of smoking-related diseases. However, reduction of harmful constituents by heating rather than combusting tobacco, without modifying the amount of nicotine, is a promising new paradigm in harm reduction. In this study, we compared effects of exposure to aerosol derived from a candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, with those of CS generated from the 3R4F reference cigarette. Human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) were exposed for 28 min to 3R4F CS or THS2.2 aerosol, both diluted with air to comparable nicotine concentrations (0.32 or 0.51 mg nicotine/L aerosol/CS for 3R4F and 0.31 or 0.46 mg/L for THS2.2). We also tested one higher concentration (1.09 mg/L) of THS2.2. A systems toxicology approach was employed combining cellular assays (i.e., cytotoxicity and cytochrome P450 activity assays), comprehensive molecular investigations of the buccal epithelial transcriptome (mRNA and miRNA) by means of computational network biology, measurements of secreted proinflammatory markers, and histopathological analysis. We observed that the impact of 3R4F CS was greater than THS2.2 aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. Analysis of the transcriptomic changes in the exposed oral cultures revealed significant perturbations in various network models such as apoptosis, necroptosis, senescence, xenobiotic metabolism, oxidative stress, and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) signaling. The stress responses following THS2.2 aerosol exposure were markedly decreased, and the exposed cultures recovered more completely compared with those exposed to 3R4F CS.

Evaluation of the Tobacco Heating System 2.2. Part 7: Systems toxicological assessment of a mentholated version revealed reduced cellular and molecular exposure effects compared with mentholated and non-mentholated cigarette smoke.

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M.

Transcriptional profiling and targeted proteomics reveals common molecular changes associated with cigarette smoke-induced lung emphysema development in five susceptible mouse strains.

BACKGROUND: Mouse models are useful for studying cigarette smoke (CS)-induced chronic pulmonary pathologies such as lung emphysema. To enhance translation of large-scale omics data from mechanistic studies into pathophysiological changes, we have developed computational tools based on reverse causal reasoning (RCR). OBJECTIVE: In the present study we applied a systems biology approach leveraging RCR to identify molecular mechanistic explanations of pathophysiological changes associated with CS-induced lung emphysema in susceptible mice. METHODS: The lung transcriptomes of five mouse models (C57BL/6, ApoE (-/-) , A/J, CD1, and Nrf2 (-/-) ) were analyzed following 5-7 months of CS exposure. RESULTS: We predicted 39 molecular changes mostly related to inflammatory processes including known key emphysema drivers such as NF-κB and TLR4 signaling, and increased levels of TNF-α, CSF2, and several interleukins. More importantly, RCR predicted potential molecular mechanisms that are less well-established, including increased transcriptional activity of PU.1, STAT1, C/EBP, FOXM1, YY1, and N-COR, and reduced protein abundance of ITGB6 and CFTR. We corroborated several predictions using targeted proteomic approaches, demonstrating increased abundance of CSF2, C/EBPα, C/EBPß, PU.1, BRCA1, and STAT1. CONCLUSION: These systems biology-derived candidate mechanisms common to susceptible mouse models may enhance understanding of CS-induced molecular processes underlying emphysema development in mice and their relevancy for human chronic obstructive pulmonary disease.

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague-Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.

Toxicity of nebulized nicotine (Nic) and nicotine/pyruvic acid mixtures (Nic/Pyr) was characterized in a 28-day Organization for Economic Co-operation and Development 412 inhalation study with additional transcriptomic and lipidomic analyses. Sprague-Dawley rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, saline, nicotine (50 µg/l), sodium pyruvate (NaPyr, 33.9 µg/l) or equimolar Nic/Pyr mixtures (18, 25 and 50 µg nicotine/l). Saline and NaPyr caused no health effects, but rats exposed to nicotine-containing aerosols had decreased body weight gains and concentration-dependent increases in liver weight. Blood neutrophil counts were increased and lymphocyte counts decreased in rats exposed to nicotine; activities of alkaline phosphatase and alanine aminotransferase were increased, and levels of cholesterol and glucose decreased. The only histopathologic finding in non-respiratory tract organs was increased liver vacuolation and glycogen content. Respiratory tract findings upon nicotine exposure (but also some phosphate-buffered saline aerosol effects) were observed only in the larynx and were limited to adaptive changes. Gene expression changes in the lung and liver were very weak. Nic and Nic/Pyr caused few significant changes (including Cyp1a1 gene upregulation). Changes were predominantly related to energy metabolism and fatty acid metabolism but did not indicate an obvious toxicity-related response. Nicotine exposure lowered plasma lipids, including cholesteryl ester (CE) and free cholesterol and, in the liver, phospholipids and sphingolipids. Nic, NaPyr and Nic/Pyr decreased hepatic triacylglycerol and CE. In the lung, Nic and Nic/Pyr increased CE levels. These data suggest that only minor biologic effects related to inhalation of Nic or Nic/Pyr aerosols were observed in this 28-day study.

A vascular biology network model focused on inflammatory processes to investigate atherogenesis and plaque instability.

BACKGROUND: Numerous inflammation-related pathways have been shown to play important roles in atherogenesis. Rapid and efficient assessment of the relative influence of each of those pathways is a challenge in the era of "omics" data generation. The aim of the present work was to develop a network model of inflammation-related molecular pathways underlying vascular disease to assess the degree of translatability of preclinical molecular data to the human clinical setting. METHODS: We constructed and evaluated the Vascular Inflammatory Processes Network (V-IPN), a model representing a collection of vascular processes modulated by inflammatory stimuli that lead to the development of atherosclerosis. RESULTS: Utilizing the V-IPN as a platform for biological discovery, we have identified key vascular processes and mechanisms captured by gene expression profiling data from four independent datasets from human endothelial cells (ECs) and human and murine intact vessels. Primary ECs in culture from multiple donors revealed a richer mapping of mechanisms identified by the V-IPN compared to an immortalized EC line. Furthermore, an evaluation of gene expression datasets from aortas of old ApoE-/- mice (78 weeks) and human coronary arteries with advanced atherosclerotic lesions identified significant commonalities in the two species, as well as several mechanisms specific to human arteries that are consistent with the development of unstable atherosclerotic plaques. CONCLUSIONS: We have generated a new biological network model of atherogenic processes that demonstrates the power of network analysis to advance integrative, systems biology-based knowledge of cross-species translatability, plaque development and potential mechanisms leading to plaque instability.

In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

Recent advances in the development of portable technologies and commercial products to detect Δ9-tetrahydrocannabinol in biofluids: a systematic review.

BACKGROUND: The primary components driving the current commercial fascination with cannabis products are phytocannabinoids, a diverse group of over 100 lipophilic secondary metabolites derived from the cannabis plant. Although numerous phytocannabinoids exhibit pharmacological effects, the foremost attention has been directed towards Δ9-tetrahydrocannabinol (THC) and cannabidiol, the two most abundant phytocannabinoids, for their potential human applications. Despite their structural similarity, THC and cannabidiol diverge in terms of their psychotropic effects, with THC inducing notable psychological alterations. There is a clear need for accurate and rapid THC measurement methods that offer dependable, readily accessible, and cost-effective analytical information. This review presents a comprehensive view of the present state of alternative technologies that could potentially facilitate the creation of portable devices suitable for on-site usage or as personal monitors, enabling non-intrusive THC measurements. METHOD: A literature survey from 2017 to 2023 on the development of portable technologies and commercial products to detect THC in biofluids was performed using electronic databases such as PubMed, Scopus, and Google Scholar. A systematic review of available literature was conducted using Preferred Reporting Items for Systematic. Reviews and Meta-analysis (PRISMA) guidelines. RESULTS: Eighty-nine studies met the selection criteria. Fifty-seven peer-reviewed studies were related to the detection of THC by conventional separation techniques used in analytical laboratories that are still considered the gold standard. Studies using optical (n = 12) and electrochemical (n = 13) portable sensors and biosensors were also identified as well as commercially available devices (n = 7). DISCUSSION: The landscape of THC detection technology is predominantly shaped by immunoassay tests, owing to their established reliability. However, these methods have distinct drawbacks, particularly for quantitative analysis. Electrochemical sensing technology holds great potential to overcome the challenges of quantification and present a multitude of advantages, encompassing the possibility of miniaturization and diverse modifications to amplify sensitivity and selectivity. Nevertheless, these sensors have considerable limitations, including non-specific interactions and the potential interference of compounds and substances existing in biofluids. CONCLUSION: The foremost challenge in THC detection involves creating electrochemical sensors that are both stable and long-lasting while exhibiting exceptional selectivity, minimal non-specific interactions, and decreased susceptibility to matrix interferences. These aspects need to be resolved before these sensors can be successfully introduced to the market.

Δ9-Tetrahydrocannabinol (THC): A Critical Overview of Recent Clinical Trials and Suggested Guidelines for Future Research.

In this overview, we seek to appraise recent experimental and observational studies investigating THC and its potential role as adjunctive therapy in various medical illnesses. Recent clinical trials are suggestive of the diverse pharmacologic potentials for THC but suffer from small sample sizes, short study duration, failure to address tolerance, little dose variation, ill-defined outcome measures, and failure to identify and/or evaluate confounds, all of which may constitute significant threats to the validity of most trials. However, the existing work underscores the potential therapeutic value of THC and, at the same time, calls attention to the critical need for better-designed protocols to fully explore and demonstrate safety and efficacy. In the most general sense, the present brief review illuminates some intriguing findings about THC, along with the basic threats to the validity of the research that supports those findings. The intent is to highlight existing generic weaknesses in the existing randomized controlled trial literature and, most importantly, provide guidance for improved clinical research.

Quantitative assessment of biological impact using transcriptomic data and mechanistic network models.

Exposure to biologically active substances such as therapeutic drugs or environmental toxicants can impact biological systems at various levels, affecting individual molecules, signaling pathways, and overall cellular processes. The ability to derive mechanistic insights from the resulting system responses requires the integration of experimental measures with a priori knowledge about the system and the interacting molecules therein. We developed a novel systems biology-based methodology that leverages mechanistic network models and transcriptomic data to quantitatively assess the biological impact of exposures to active substances. Hierarchically organized network models were first constructed to provide a coherent framework for investigating the impact of exposures at the molecular, pathway and process levels. We then validated our methodology using novel and previously published experiments. For both in vitro systems with simple exposure and in vivo systems with complex exposures, our methodology was able to recapitulate known biological responses matching expected or measured phenotypes. In addition, the quantitative results were in agreement with experimental endpoint data for many of the mechanistic effects that were assessed, providing further objective confirmation of the approach. We conclude that our methodology evaluates the biological impact of exposures in an objective, systematic, and quantifiable manner, enabling the computation of a systems-wide and pan-mechanistic biological impact measure for a given active substance or mixture. Our results suggest that various fields of human disease research, from drug development to consumer product testing and environmental impact analysis, could benefit from using this methodology.

Cigarette smoke-induced morphological transformation of Bhas 42 cells in vitro.

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha-ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60µg TPM/ml, with a peak at 20µg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20µg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose-response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor∗ml/mg TPM; mean ± SD) ; and the goodness of fit (r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-genotoxic carcinogenic activity of tobacco smoke-related test substances.

Deconvolution of Systemic Pharmacokinetics Predicts Inhaled Aerosol Dosimetry of Nicotine.

Absorption of inhaled compounds can occur from multiple sites based on upper and lower respiratory tract deposition, and clearance mechanisms leading to differential local and systemic pharmacokinetics. Deriving inhaled aerosol dosimetry and local tissue concentrations for nose-only exposure in rodents and inhaled products in humans is challenging. In this study we use inhaled nicotine as an example to identify regional respiratory tract deposition, absorption fractions, and their contribution toward systemic pharmacokinetics in rodents and humans. A physiologically based pharmacokinetic (PBPK) model was constructed to describe the disposition of nicotine and its major metabolite, cotinine. The model description for the lungs was simplified to include an upper respiratory tract region with active mucociliary clearance and a lower respiratory tract region. The PBPK model parameters such as rate of oral absorption, metabolism and clearance were fitted to the published nicotine and cotinine plasma concentrations post systemic administration and oral dosing. The fractional deposition of inhaled aerosol in the upper and lower respiratory tract regions was estimated by fitting the plasma concentrations. The model predicted upper respiratory tract deposition was 63.9% for nose-only exposure to nicotine containing nebulized aqueous aerosol in rats and 60.2% for orally inhaled electronic vapor product in humans. A marked absorption of nicotine from the upper respiratory tract and the gastrointestinal tract for inhaled aqueous aerosol contributed to the differential systemic pharmacokinetics in rats and humans. The PBPK model derived dosimetry shows that the current aerosol dosimetry models with their posteriori application using independent aerosol physicochemical characterization to predict aerosol deposition are insufficient and will need to consider complex interplay of inhaled aerosol evolutionary process. While the study highlights the needs for future research, it provides a preliminary framework for interpreting pharmacokinetics of inhaled aerosols to facilitate the analysis of in vivo exposure-responses for pharmacological and toxicological assessments.