Prevalence and drug use correlates of inadvertent fentanyl exposure among individuals misusing drugs in seven U.S. states.

Fentanyl has emerged as the leading cause of fatal drug overdoses in the U.S. Individuals misusing drugs may not always be aware of exposure to fentanyl.To determine the prevalence of fentanyl use and extent of awareness of fentanyl exposure among a national sample of treatment-seeking individuals with opioid use disorder (n = 1098).Participants provided oral fluid and urine specimens, which were tested for drugs by liquid chromatography/tandem mass spectrometry. Participants also provided self-reports of fentanyl use.49.5% tested positive for fentanyl in oral fluid, urine, or both. Of those testing positive for fentanyl, 29.8% were unaware that they had been exposed to fentanyl. Participants testing positive for opioids methadone, and specifically 6-monoacetylmorphine (6-MAM), a unique metabolite of heroin, were significantly more likely to be unaware of fentanyl exposure than participants testing negative for these substances, with a similar trend for oxycodone and tramadol.These findings may be due to fentanyl's effect being difficult to distinguish from that of other opioids, whereas when other types of drugs are adulterated with fentanyl, the differences in effects are likely to be readily discernable. These results support the importance of expanded drug-checking services.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Inactivation of wheat-germ aspartate transcarbamoylase by the triazinyl dye, procion red HE3B.

Aspartate transcarbamoylase from wheat germ is irreversibly inactivated by the triazinyl dye Procion Red HE3B. Since triazinyl dyes may mimic nucleotides, and UMP is a known allosteric modifier of this enzyme, the reaction was studied to elucidate whether the dye is an 'affinity label' for the enzyme. The reaction is apparently first order in the first 5-10 min, but is more complex in the longer term and does not go to completion. Kinetic analysis of the initial phase suggests that there are two parallel reactions, one saturable (dye binds reversibly before reaction) and one non-saturable (biomolecular). The apparent rate constant kapp (i.e. the sum of the rate constants for the parallel reactions) varies only slightly over the pH range 7-10. In the presence of a number of active centre ligands, as well as the allosteric ligand UMP, there is a clear increase in kapp. This finding is contrary to the reduction in rate of inactivation (protection) normally provided by ligands against active-site directed reagents, suggesting that in the saturable reaction, there is a conformational change upon dye-binding that increases the exposure of the essential residue(s) with which the dye reacts. These results show that, although it probably inactivates by reaction with specific amino-acid residues, the dye is not bound at the substrate-binding or allosteric sites, i.e. it is not an affinity-labeling reagent in the usual sense.