Exposure to tobacco smoke can also trigger an inflammatory response and induce oxidative stress through increased levels of reactive oxygen species.Persistent induction of these processes following repeated exposure contributes to loss of normal growth control mechanisms,which is a key step in cancer development.Our study supports many of these findings,with exposure to TSC inducing the expression of genes involved in xenobiotic metabolism,oxidative stress,and DNA damage response as evidenced by changes in the expression of genes involved in cell cycle arrest,protein unfolding,transcription regulation,and inflammation.These same pathways were also significantly affected following MSC exposure,indicating that,as expected,MSC impacts many of the same molecular processes and functions as TSC.Although the effects of the condensates were largely similar,dose–response analysis indicates that the MSC is substantially more potent than TSC,with BMDs that in many instances are an order of magnitude lower than those for TSC.In addition,the results also highlighted some differences in steroid biosynthesis,apoptosis and inflammation,which were more significantly affected following MSC exposure,and cell cycle,which was more affected following TSC exposure.IPA canonical pathways related to the metabolism of xenobiotics were significantly affected in both TSC and MSC exposed cells at both time points.These pathways included Xenobiotic Metabolism Signaling,Metabolism of Xenobiotics by CYP450,and AHR Signaling.For both TSC and MSC,the number of genes that were significantly affected increased with increasing concentration and the greatest number of genes changing occurred at the 6 + 4 h time point.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,cannabis grow tray 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 bio-activate 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 micro-array 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,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 atall 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 micro-nucleus 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.M phase pathways,including the Mitotic Roles of Polo-like Kinase and G2/M DNA Damage Checkpoint Regulation pathways,were significantly perturbed in TSC exposed cells.At the highest concentration,TSC affects Ccnb1,Cdk1,Plk1,Plk2,Plk3,Prc1,Gadd45,Cdc20 and Mdm2 expression at the 6 h time point and Ccnb1,Cdk1,Plk1,Prc1,Gadd45,Ccnb2,Ppp2r2b and Top2a at the 6 + 4 h time point.Some of these genes are p53 responsive genes which could indicate a DNA damage response regulated by p53.The genes in these pathways are involved in checkpoint regulation and,by providing time for DNA repair,they prevent cells with DNA damage from entering mitosis.
Similar genes have also been found to be down-regulated in a study by Nordskog et al..Following exposure of primary cultures of human aorticendothelial cells to cigarette smoke condensate,they noted the down-regulation of cell cycle genes including Top2a,Ccnb1,Ccna,and Cdkn3.In contrast to TSC exposed cells,the above M phase pathways were not significantly perturbed in the marijuana exposed cells.Rather,the Cell Cycle Regulation by BTG Family Proteins Pathway was significantly disrupted,particularly for cells exposed to the highest MSC concentrations.The BTG proteins act as growth arrest genes and prevent G1 to S phase transition by inhibiting Ccnd1 and maintaining a quiescent state.In the present study,Btg1,Btg2 and Hoxb9 were up-regulated at both time points,whereas Ccrn4l was also up-regulated for the 6 h time point,vertical grow systems for sale and Ccnd1 and Ppp2r2b were down-regulated for the 6 + 4 h time point.Interestingly,this BTG regulated cell cycle pathway was also significant for cells exposed to the highest concentration of TSC at the 6 h time point,with Btg1,Btg2 and Ccrn4l being up-regulated.In our earlier toxicogenomic analyses of three cigarette smoke condensates Btg2 was also found to be among the most up-regulated genes.Fig.7 shows a comparison of the significantly altered cell cycle genes following exposure to the two smoke condensates.Although many of the same genes are affected and cell cycle appears to be a commonly disrupted function,there appears to be subtle differences in how this disruption occurs.Furthermore,cluster analyses of cell cycle genes confirms the importance of the smoke condensate type since cell cycle genes cluster primarily by smoke type,and subsequently by concentration.The Biosynthesis of Steroids Pathway was among the most significantly affected IPA Canonical Pathways for MSC exposed cells.This held true both when all of the significantly altered MSC genes were taken into account,and when only the genes unique to MSC were considered.The Biosynthesis of Steroids Pathway is a lipid metabolism pathway that controls the synthesis of cholesterol,which is an essential component of cell membranes and a precursor in the production of bile acids,steroid hormones,and vitamin D.This pathway was significantly down-regulated for all concentrations of the MSC at both time points,and the number of genes that were significantly affected increased with increasing concentration.The greatest number of genes was affected at the 6 + 4 h time point,and these included Dhcr7,Fdft1,Fdps,Hmgcr,Idi1,Mvd,Mvk,Nqo1,Pmvk,Sc5dl,and Sqle.The majority of these genes are involved in the mevalonate and squalene synthesis portions of the pathway.Although no studies have been conducted to specifically investigate the effect of marijuana smoke on lipid metabolism and steroid biosynthesis,early investigations using rodent cells have shown that cannabinoids can affect lipid metabolism,and the effects include an increase in lipolysis in adipose tissue,the inhibition of corticosteroidogenesis,and the reduced testosterone and progesterone production.The cannabinoid CBD has also been shown to affect cholesterol metabolism in human fibroblasts and aortic medial cells through the inhibition of cholesteryl ester formation.In the present study,HMG-CoA reductase,which is the rate-limiting enzyme for cholesterol synthesis,was notably down-regulated for the medium and high concentrations of MSC at both time points.
Previous in vitro investigations with THC have shown that this cannabinoid reduces Hmgcr by 29%,whereas CBD had no effect on Hmgcr levels.When comparing TSC and MSC exposed cells,the Biosynthesis of Steroids Pathway was also significant for TSC,particularly for the 6 + 4 h time point.However,only one to three genes were perturbed,depending on the concentration.These genes included Fdps,Ggps1,Nqo1,and Hmgcr.The LXR/RXR pathway,which is involved in the regulation of lipid metabolism and cholesterol to bile acid catabolism,was also significantly down-regulated at the 6 + 4 h time point in both MSC and TSC exposed cells.Of note in this pathway is Ldlr,which is the greatest down-regulated gene in MSC exposed cells.This gene was down-regulated 10 fold following the highest MSC exposure concentration but only 1.6 fold following the highest TSC exposure.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.