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 ( Fig. 8). 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 (Wing and Paton, 1978), the inhibition of corticosteroidogenesis (Warner et al., 1977), and

the reduced testosterone and progesterone production (Burstein et al., 1979 and Burstein et al., Roscovitine clinical trial 1978). 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 (Cornicelli et al., 1981). In the present study, HMG-CoA reductase (Hmgcr), 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% ( Rimmerman et al., 2011), whereas CBD had no effect on Hmgcr levels ( Cornicelli et al., 1981 and Rimmerman et al., 2011). When comparing TSC and MSC exposed cells, the Biosynthesis of Steroids Pathway was also significant for TSC, particularly for the 6 + 4 h Olopatadine time point. However, only one to three Sorafenib chemical structure 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. Exposure to MSC but not TSC appears to have affected apoptosis pathways. Genes in the TWEAK Signaling, TNFR1 and TNFR2 Signaling Pathways (Birc3, Nfkbia, Tnfaip3, Pak3, Fos, Jun, Tnfrsf12a) were significantly up-regulated following exposure to MSC particularly at the 6 h time point. The up-regulation of these particular genes suggests that MSC inhibits apoptosis and may promote a TNF receptor mediated survival pathway. 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 (Sarafian et al., 2001). They found that both tobacco and marijuana whole smoke inhibited Fas-mediated apoptosis but promoted necrotic cell death.