Detection of Cutting Agents in Drug-Positive Seized Exhibits within the United States.

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute-and-shoot procedure. Initial analysis was performed using gas chromatography-mass spectrometry (GC-MS) followed by analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.

Arsenic decreases RXRα-dependent transcription of CYP3A and suppresses immune regulators in hepatocytes.

Arsenite is critical pharmacologically as a treatment for advanced stage blood cancer. However, environmental exposure to arsenic results in multiple diseases. Previous studies have shown that arsenic decreases expression of CYP3A, a critical drug metabolizing enzyme in human and rat liver. In addition, acute and chronic arsenic exposure in liver stimulates an inflammatory response. Our work has shown that arsenite decreases nuclear levels of RXRα the nuclear receptor that, as a heterodimer partner with PXR, transactivates the CYP3A gene. These results suggest that arsenite decreases transcription of CYP3A by decreasing RXRα. The present report shows that exposure to 5 µM arsenite decreased the activity of a rat CYP3A promoter luciferase reporter in HepG2 cells. The activity of a RARE-luciferase reporter, that is transactivated by the retinoic acid receptor (RAR)/RXRα, was also decreased. Previous studies have shown that arsenic in the concentration range of 2-5 µM affects CYP3A mRNA. When rifampicin-treated primary human hepatocyte cultures were exposed to arsenite concentrations as low as 50 nM, CYP3A mRNA was decreased. Treatment of primary human hepatocytes with the proteasome inhibitor MG132 increased RXRα suggesting the involvement of the proteasome pathway in regulation of RXRα. Finally, arsenic induces a pro-inflammatory response in liver. Surprisingly, we show that in hepatocytes arsenite decreases expression of two inflammatory mediators, TNF and VEGF, an effect that is not predicted from suppression of RXRα activity.

Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs.

A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.

Development of a real-time method to detect DNA degradation in forensic samples.

Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real-time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.

Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: comparison with arsenite.

Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.