On-site test for cannabinoids in oral fluid.
(17/25)
BACKGROUND: Oral fluid (OF) testing offers noninvasive sample collection for on-site drug testing; however, to date, test performance for Delta(9)-tetrahydrocannabinol (THC) detection has had unacceptable diagnostic sensitivity. On-site tests must accurately identify cannabis exposure because this drug accounts for the highest prevalence in workplace drug testing and driving under the influence of drugs (DUID) programs. METHODS: Ten cannabis smokers (9 males, 1 female) provided written informed consent to participate in this institutional review board-approved study and smoked 1 6.8%-THC cigarette ad libitum. OF was collected with the Draeger DrugTest((R)) 5000 test cassette and Quantisal device 0.5 h before and up to 22 h after smoking. Test cassettes were analyzed within 15 min (n = 66), and Quantisal GC-MS THC results obtained within 24 h. Final THC detection times and test performances were assessed at different cannabinoid cutoffs. RESULTS: Diagnostic sensitivity, diagnostic specificity, and efficiency at DrugTest 5000's 5 mug/L screening cutoff and various THC confirmation cutoffs were 86.2-90.7, 75.0-77.8, and 84.8-87.9%, respectively. Last detection times were >22 h, longer than previously suggested. Confirmation of 11-nor-9-carboxy-THC, absent in THC smoke, minimized the potential for passive OF contamination and still provided 22-h windows of detection, appropriate for workplace drug testing, whereas confirmation of cannabidiol, and/or cannabinol yielded shorter 6-h windows of detection, appropriate for DUID OF testing. CONCLUSIONS: The DrugTest 5000 on-site device provided high diagnostic sensitivity for detection of cannabinoid exposure, and the selection of OF confirmation analytes and cutoffs provided appropriate windows of detection to meet the goals of different drug testing programs. (+info)
Cannabinoids induce incomplete maturation of cultured human leukemia cells.
(22/25)
Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 microM delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody or the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 microM THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. Pronounced among these changes was an increase in the synthesis of at least 10 proteins that are found abundantly in monocytes. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype; the THC-treated cells failed to exhibit other monocyte markers such as attachment to the surface of tissue culture dishes or morphological maturation beyond the promonocyte stage. However, treatment of these "incompletely" matured cells with either phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. Two other cannabinoids, cannabidiol and cannabinol, which were more cytotoxic than THC at comparable doses, also caused an increase in the expression of maturation markers, but at doses higher than those required for THC. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced "incomplete" cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells. (+info)
Cannabinoids impair the formation of cholesteryl ester in cultured human cells.
(23/25)
The ability of cultured human fibroblasts to form cholesteryl esters from 14C-oleate is impaired by delta'-tetrahydrocannabinol, cannabidiol, and cannabinol, a group of natural products isolated from Cannabis sativa. This inhibition is compound and dose-related; 30 microM cannabidiol reduced esterification to less than 20% of the control values. The esterification of endogenous and exogenous cholesterol was affected, since inhibition was seen with either low density lipoproteins (200 micrograms/ml) or 25-hydroxycholesterol (5 micrograms/ml) as esterification stimuli. Cells treated with these compounds at doses of from 1 to 30 microM showed no impairment of protein synthesis, triglyceride or phospholipid formation, or ability to metabolize 125I-low density lipoproteins. An inhibition of cholesterol esterification was seen in human aortic medial cells. With increasing doses of these compounds, low density lipoproteins (25 micrograms/ml) became progressively less effective in suppressing HMG-CoA reductase in cultured human fibroblasts; with 30 microM cannabidiol the enzyme suppression was only 24% of that found in cells incubated with low density lipoproteins in the absence of drugs. Based on these data, we conclude that the cannabinoids "compartmentalize" cholesterol and, thus, make is unavailable for regulating cellular cholesterol metabolism. This may occur as a result of enhanced sterol efflux. (+info)
Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.
(24/25)
Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription. (+info)