The profile of the changing genes was comparable between tobacco and marijuana exposed cells.Many of the genes that were differentially expressed in TSC exposed cells are among those that have been typically observed to be induced by cigarette smoke [e.g., Nqo1,Esd,Hmox1,Cyp1a1 and Cyp1b1 ]. Moreover, the concentration response patterns support the assertion of initial metabolic responses,followed by responses to toxic insult  and secondary metabolism.Similar concentration response trends were noted in our previous toxicogenomics analysis of three different TSCs.Although very few studies have been conducted with marijuana smoke, Roth et al.  demonstrated the induction of cytochrome P450 genes following exposure of Hepa-1 cells to marijuana tar extracts. Furthermore, the authors showed that tar from marijuana cigarettes tends to be more effective than tar from tobacco at inducing Cyp1a1 gene expression. Since the cannabinoids present in marijuana are capable of acting through the aryl hydrocarbon receptor to induce cytochrome P450 enzymes,and Cyp1a1 is known to bioactivate procarcinogens such as PAHs,questions have been raised about the role of cannabinoids in augmenting the carcinogenic risk posed by marijuana smoke. The question becomes increasingly complex as the cannabinoids THC, CBD and CBN have also been shown to competitively inhibit Cyp1a1, potentially decreasing the production of carcinogens and curtailing negative consequences.In the present study, however, substantial differences in the expression profiles of cytochrome P450 genes between the two smoke types were not observed. The expression of Cyp1a1 following exposure to MSC was comparable to that following TSC exposure, and the microarray results were supported by RT-PCR.One of the differences in the xenobiotic metabolism responses for the two condensate types is that Hsp90 and Rras2 were only upregulated following MSC exposure. Despite these findings, Hsp90 has been previously observed to be induced following cigarette smoke exposure,dry rack cannabis and mutations in genes from the Ras family are known to be associated with cigarette-induced cancers .

The IPA Canonical Pathway most significantly affected by exposure to TSC was the NRF2-Mediated Oxidative Stress Response Pathway. In this pathway, the transcription factor Nrf2 is phosphorylated following exposure to reactive oxygen, and translocates to the nucleus where it binds to antioxidant response elements.It then activates the expression of detoxification and antioxidant genes that protect the cell against oxidative damage. Of the 192 genes in this pathway, 6–18 genes were perturbed by TSC at the various time points in a concentration dependent manner. The largest expression changes and number of genes were associated with the 6 h time point. Nrf2-regulated antioxidant genes have been shown to play an important role in protection against the toxic effects of tobacco smoke. Iizuka et al. showed that neutrophilic lung inflammation was significantly enhanced in Nrf2-knockout mice following cigarette smoke exposure.In addition, emphysema was observed 8 and 16 weeks following cigarette smoke exposure in the knockout mice, whereas no pathological abnormalities were observed in wild-type mice. Similarly, Gebel et al. confirmed the protective nature of Nrf2 against the development of emphysema in cigarette smoke exposed wild type mice versus Nrf2 knockout mice, and further investigated the relationships between Nrf2 and inflammation and cell cycle arrest.Comandini et al. conducted a meta-analysis of eight genomic studies on the mechanisms of smoke-induced lung damage in healthy smokers, COPD smokers and non-smokers.They found the Nrf2-mediated oxidative stress response Pathway to be the most significantly altered pathway in healthy smokers compared to non-smokers. In contrast, the Nrf2 pathway was not significantly differentially expressed in COPD smokers, indicating that Nrf2-regulated genes play a key role in protecting against the toxic effects of TSC. The authors suggest that the response of Nrf2- regulated genes may potentially be used as a biomarker for COPD susceptibility. In the present study, we found that the NRF2-Mediated Oxidative Stress Response Pathway is also an important component of the toxicological response to MSC.

IPA analyses identified it as one of the top five pathways for both time points and all concentrations of MSC, except for the lowest concentration at the 6 + 4 h time point.A comparison of the Nrf2 pathway at the 6 h time point for the highest exposure concentrations of TSC and MSC shows many similarities.The Nrf2 gene itself was up-regulated along with several basic leucine zipper family transcription factors such as Jun, Atf4, and Maff. In addition, several antioxidant and stress response proteins such as Nqo1, Prdx1, Hmox1, Sod, Txnrd1, Herpud1, Dnajb1/9 were up-regulated. Other studies have also noted that these genes are up-regulated following cigarette smoke exposure.However, a notable difference between the two condensates studied here is that Gclc and Gclm, the rate limiting enzymes in glutathione synthesis, were significantly upregulated by TSC,but were not statistically significantly affected in MSC exposed cells.Furthermore Gsta genes were up-regulated in TSC and Gstm genes were down-regulated in MSC exposed cells. These findings were further confirmed by the significant up-regulation of the Glutathione Metabolism Pathway in tobacco exposed cells at all times and concentrations and the significant down-regulation of this pathway in marijuana exposed cells, particularly at the high concentration at the 6 + 4 h time point. These results suggest that exposure to MSC elicits more severe oxidative stress than exposure to TSC. The relative difference between the two condensates to mount an antioxidant defense may account for the greater cytotoxicity of MSC observed here and in our earlier genotoxicity study, where it appeared that the acute toxicity of MSC prevented the manifestation of micronucleus induction.The assertion regarding the relative severity of oxidative stress induced by MSC and TSC is supported by published results from other studies. In a previous study, Sarafian et al. examined reactive oxygen species production and reduced glutathione levels as indicators of oxidative damage following exposure to marijuana smoke.They showed that exposure of human endothelial cells to marijuana smoke resulted in an 80% increase in ROS over control levels, and these levels were as much as three times higher than those resulting from tobacco smoke. Moreover, intracellular glutathione levels following marijuana exposure were lower than for tobacco, and were reduced by 81% relative to controls. The authors argued that the products produced by the pyrolysis of the cannabinoids were likely responsible for the oxidative damage.

The same authors also conducted preliminary studies with cultured lung alveolar macrophages from non-smokers and marijuana smokers, and found that marijuana smokers had lower levels of GSH than non-smokers, suggesting a decrease in GSH dependent oxidative defenses in habitual marijuana smokers.In a previous study, Sarafian et al. investigated the effects of marijuana smoke and tobacco smoke on apoptosis and necrosis in A549 lung tumor cells.They found that both tobacco and marijuana whole smoke inhibited Fasmediated apoptosis but promoted necrotic cell death. In addition, particulate phase smoke  from marijuana was a more potent inhibitor of Fas-induced caspase-3 activity than tobacco. In a later study, the authors also noted the decreased expression of Bax and caspase-8 in human small airway epithelial cells exposed to THC, which they suggest could have accounted for the previously observed suppression in Fas-mediated apoptosis.Although apoptotic pathways were not significantly perturbed following TSC exposure in our present study, Sarafian et al. and other investigators of tobacco smoke effects have found this to be a commonly disrupted pathway.It is suspected that the gene expression fold change cutoff of 2 used in the present study likely prevented a number of apoptotic genes from being included in the analyses.It is importantto note thatthe marijuana used for this study was obtained from a contracted supplier that provides marijuana for therapeutic use in Canada. It is grown under strictly controlled and documented conditions. Although this study has only examined smoke condensate from a single lot of marijuana, the quality control measures would be expected to minimise differences between marijuana harvests. The TSC used in this study was generated from cigarettes containing Virginia flue-cured tobacco, the type of tobacco typically contained in Canadian cigarettes. This is distinct from the mixed tobacco blends  typically found in American cigarettes.

Our earlier toxicogenomic examination of TSC from three Canadian cigarette brands containing either Virginia flue-cured or mixed tobacco blends failed to show any appreciable brand-driven differences in gene expression profiles elicited by in vitro exposures.Therefore, we contend that the similarities and difference between MSC and TSC noted in this study can be cautiously extended to other types of tobacco. Nevertheless, it should also be noted that some toxicogenomic studies have shown that cigarette brand  can have a significant effect on gene expression signatures elicited by in vitro CSC exposures,and moreover, many aspects of cigarette design  and smoking method  have been shown to influence the composition and toxicological activity of TSC .The pivotal role of protein in human health and the environment is becoming clear in several ways. Lack of protein is a key metric of malnutrition, while a dietary transition from primarily animal towards plant protein products is required to avoid biodiversity loss and climate change . However, diet is a cultural attitude that is difficult to change despite the potential health and environmental benefits . In this way, meat-eating consumers would prefer products that strongly resemble real meat , which triggers the search for more sustainable proteins that can be used to produce meat-resembling structures. Although successful plant-based meat substitutes, such as burger patties, are already available in the supermarkets of many countries, the development of whole-muscle cuts using plant proteins still remains a major challenge. Hemp is an unconventional crop with a broader spectrum of adaptation to colder and warmer conditions than other plants typical from moderate climates . Its increasing legalization and the diverse range of products that a hemp plant can produce has drawn the attention of a variety of industries . In addition, hemp grows prolifically with no pesticides and is good for soil phytoremediation, roll bench owing to its long roots growing and penetrating deep into the soil . Hemp seeds are particularly abundant in nutritionally relevant oil and proteins , with 25–30% oil, 20–30% protein, 30–40% fibre, and 5.0–5.8% of minerals on a dry basis  depending on genotype and growing factors . Hemp oil is extracted from the seeds by screw pressing, resulting in a residue after extraction ambiguously called meal or cake, with a protein fraction that mainly consists of globular globulins and albumins characterized by an amino acid profile that complements perfectly that from other plant proteins, such that from pea proteins . The amount of protein in the hemp seed cake/meal can be increased to over 60%  by removing 1) the carbohydrate-rich hull , and 2) the oil fraction through further pelletizing or using organic solvents .

Higher protein enrichment  can be attained by tandem alkaline extraction plus isoelectric precipitation , which still remains the most utilized aqueous extraction method to extract protein from oilseeds . It usually involves an alkaline solubilisation of the proteins, the removal of insoluble material by centrifugation and the isoelectric precipitation of the protein, followed by its separation by centrifugation . Nevertheless, the omission of aqueous extraction steps and, particularly, the omission of protein precipitation and final drying steps, can have a notorious reduction of the environmental footprint during protein fractionation, e.g., by decreasing the consumption of energy, water and chemical reagents, and the generation of chemical waste . With the amount of recovered protein as the functional unit , processes that have less refinement still have a lower negative impact than the conventional wet extraction method. In addition, AE-IP can result in differential extraction of storage proteins of different types , disrupt the native oligomeric state of proteins, and lead to thermodynamically favourable organized assemblies  that affect the functional properties of the resulting protein concentrates. Moreover, compared to lab-scale protein concentrates, industrial processes are performed under compromised, and often more complex, processing conditions to ensure economic viability. For example, processing at higher total solids  and temperatures  could explain the particularly poor solubility of commercial concentrates compared to their lab-scale counterparts . There is still, however, very little information about the technological functionality of commercial hemp protein concentrates. In addition, the effect of hemp protein industrial-scale enrichment technologies on the anisotropic structuring behaviour of hemp protein concentrates has not been reported.