Metals and organic compounds in the biosynthesis of cannabinoids: a chemometric approach to the analysis of <i>Cannabis sativa</i> samples

<div><p>Illicit production and trade of <i>Cannabis sativa</i> affect many societies. This drug is the most popular and easy to produce. Important information for the authorities is the production locality and the indicators of a particular production. This work is an attempt to recognise correlations between the metal content in the different parts of<i> C. sativa</i> L., in soils where plants were cultivated and the cannabinoids content, as a potential indicator. The organic fraction of the leaves of <i>Cannabis</i> plants was investigated by GC-FID analysis. In addition, the determination of Cu, Fe, Cr, Mn, Zn, Ca and Mg was realised by spectroscopic techniques (FAAS and GFAAS). In this study, numerous correlations between metal content in plants and soil, already confirmed in previous publications, were analysed applying chemometric unsupervised methods, that is, principal component analysis, factor analysis and cluster analysis, in order to highlight their role in the biosynthesis of cannabinoids.</p></div

The relationship of the nuclear GSH level and the rhythm of cell growth.

<p>The consequences of the GSH depletion by DEM and BSO on the cell growth are shown on the panel B and C, respectively. Control cells are presented at the panel A. The mean CMFDA fluorescence in nuclear area (defined by Hoechst staining, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) was obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">materials and methods</a>, and the profile of the cell growth was characterized by cell number. The results are presented as mean values of at least 4 experiments.</p

Effect of nuclear GSH depletion by DEM and BSO on cell cycle.

<p>Cells were plated and treated as described previously. The cell cycle was studied by flow cytometry, using the level of the fluorescence of the DNA dye propidium iodide (final concentration, 5 µg/mL) at 630 nm fluorescence emission as a measure of the DNA content per cell, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">materials and methods</a>. The cells were detached, fixed in ethanol and stained along the proliferation curve of the 3T3 fibroblasts. The histograms corresponding to each experimental group at 6 h, 24 h, 48 h, and 5 days of culture (of the same representative experiment) were overlaid and presented at the panel A. The effect of the GSH depletion on the cell proliferation, defined by mean percentages of cells in phases S+M/G2±SD, is presented at the panel B, and the effect of the depletion on the cell death, i.e. the percentage of apoptotic and necrotic cells is shown on the panel C. The results presented are mean±SD of 5–17 different experiments.</p

The expression of the cell proliferation marker, Id2, after the depletion of the GSH.

<p>The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The protein extracts were obtained at 6 h, 24 h, and 72 h of culture and western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">materials and methods</a>. At upper panel, western blot analysis of Id2 and β-tubulin in 3T3 fibroblasts at 6, 24, and 72 h of culture is shown. Lower panel shows the relative Id2 to β-tubulin band intensity [Mean±SD (n = 4)] derived from densitometry.</p

The GSH distribution after its depletion with DEM and BSO.

<p>The cells were plated as usual and after attaching, 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. At 24 h after plating the cells were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a> and observed by confocal microscopy in the chamber provided with 5% CO₂ and at 37°C. Images were taken by light microscopy (transmission) and by confocal microscopy, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>, to capture blue fluorescence of nuclei (Hoechst-nuclei), green fluorescence that marks GSH (CMFDA-GSH) and red fluorescence of dead cells (PI-dead cells) (results not shown). Z series of at least 8 planes were obtained and maximum projection images were created and analysed. The representative experiment (of five) is presented.</p

The effect of DEM and BSO treatment on the nuclear and cytoplasmic pool of GSH.

<p>The maximum projection images (as presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) were analysed by area, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>”. CMFDA fluorescence in nuclear area (defined by Hoechst staining, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g004" target="_blank">fig. 4A</a> and the CMFDA fluorescence of the cytoplasm area (defined by transmission images, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g003" target="_blank">fig. 3</a>) is presented at <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#pone-0006413-g004" target="_blank">fig. 4B</a>. The results are mean values of at least 4 different experiments (50–100 cells per experiment). The level of CMFDA fluorescence in the nuclear, cytoplasmic and mitochondrial area after treatment with BSO and DEM at 24 h of culture is presented at the panel C. The analysis of nuclear and cytoplasmic area was performed as described. Mitochondrial area was considered to be marked by perinuclear green fluorescence, as demonstrated previously (4). The results are presented as mean of 3–5 different experiments.</p

Effect of glutathione depletion on Glutathione S-Transferase (GST) activity.

<p>The 3T3 fibroblasts were treated with 100 µM diethylmaleate (DEM) or with 10 µM buthionine sulfoximine (BSO) immediately after attaching. The activity of the GST was determined at 24 h and 6 days of culture, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and methods</a>.</p

Effect of glutathione depletion on Glutathione reductase (GR) activity.

<p>The 3T3 fibroblasts were treated with 100 µM diethylmaleate (DEM) or with 10 µM buthionine sulfoximine (BSO) immediately after attaching. The activity of the GR was determined at 24 h and 6 days of culture, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and methods</a>.</p

GSH depletion in 3T3 fibroblasts. The total cellular GSH concentration was assessed.

<p>The cells were plated as described previously (4) and after attaching 100 µM DEM, or 10 mM BSO, or 100 µM DEM+1 mM GSHe were added. The total cellular concentration of GSH was determined spectrophotometrically as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">Materials and Methods</a>”. The results are presented as mean±SD of 5–12 different experiments.</p

The pattern of overall S-glutathionylated and oxidized nuclear proteins induced by the two GSH depleting agents at 6 h, 24 h and 6 days in culture.

<p>A.- Glutathionylated proteins: equal amount of nuclear extracts were loaded and separated by a 12% SDS gel under non-reducing conditions. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. B.- Oxidized proteins: equal amounts of nuclear extracts were derivatized and separated by electrophoresis in an acrilamide gel. Western blotting and subsequent immunodetection of carbonylated proteins were performed according to the protocol recommended by the manufacturer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006413#s2" target="_blank">material and methods</a>). Right panels shows the densitometry results for the level of glutathyolation and oxidation of nuclear proteins, respectively [Mean±SD (n = 3)].</p