Reactive metabolites profiling

Most of the tests that were developed for detection of cannabinoids in plants have shown that antibodies are specific for the cannabinoid structure. Because of this specifity these tests can be extensively applied for the detection of cannabinoids and metabolites in human body fluids such as plasma, urine, and oral fluids. Many different kits based on these methods were developed and they are commercially available, for example Oratect, Branan or Uplink, and OraSure. We must consider, however, that no humans have the same metabolite profile in their blood and that cross-reactivity may always occur [122,123]. Nevertheless, these tests offer a simple way of excluding most of the suspicious samples, but the results still have to be confirmed with a second method such as GC-MS [124,125]. 

A number of chemicals with demonstrable suppression of immune function produce this action via indirect effects. By and large, the approach that has been most frequently used to support an indirect mechanism of action is to show immune suppression after in vivo exposure but no immune suppression after in vitro exposure to relevant concentrations. One of the most often cited mechanisms for an indirect action is centered around the limited metabolic capabilities of immunocompetent cells and tissues. A number of chemicals have caused immune suppression when administered to animals but were essentially devoid of any potency when added directly to suspensions of lymphocytes and macrophages. Many of these chemicals are capable of being metabolized to reactive metabolites, including dime-thylnitrosamine, aflatoxin Bi, and carbon tetrachloride. Interestingly, a similar profile of activity (i.e., suppression after in vivo exposure but no activity after in vitro exposure) has been demonstrated with the potent immunosuppressive drug cyclophosphamide. With the exception of the PAHs, few chemicals have been demonstrated to be metabolized when added directly to immunocompetent cells in culture. A primary role for a reactive intermediate in the immune suppression by dimethylnitrosamine, aflatoxin Bi, carbon tetrachloride, and cyclophosphamide has been confirmed in studies in which these xenobiotics were incubated with suspensions of immunocompetent cells in the presence of metabolic activation systems (MASs). Examples of MASs include primary hepatocytes, liver microsomes, and liver homogenates. In most cases, confirmation of a primary role for a reactive metabolite has been provided by in vivo studies in which the metabolic capability was either enhanced or suppressed by the administration of an enzyme inducer or a metabolic inhibitor, respectively. 

FIGURE 10.10 Analysis of [ H]GSH trapped reactive metabolites by HPLC with TopCount (Zhu et al., 2005b). A nonlabeled drug (50 mM) was incubated with a mixture of GSH (1 mM) and trace [ H]GSH (1-2 p,Ci/mL) in human liver microsomes. After precipitating proteins, the samples were analyzed by HPLC (1 mL/min) with TopCount (4wlls/min, 10-min counting time) (a) Radioactivity profile of the incubation. Ml and M2 were GSH-trapped reactive metabolites and (b) Radioactivity profile of a control incubation sample (without NADPH). 

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