Interactions Between Ephedra sinica and Prunus armeniaca: From Stereoselectivity to Deamination as a Metabolic Detoxification Mechanism of Amygdalin

Mahuang–Xingren (MX, Ephedra sinica Stapf-Prunus armeniaca L.) is a classic herb pair used in traditional Chinese medicine. This combined preparation reduces the toxicity of Xingren through the stereoselective metabolism of its main active ingredient amygdalin. However, whether stereoselectivity is important in the pharmacokinetic properties of amygdalin either in the traditional decoction or in the dispensing granules is unclear. Amygdalin is hydrolyzed to its metabolite, prunasin, which produces hydrogen cyanide by degradation of the cyano group. A comprehensive study of the metabolic pathway of amygdalin is essential to better understand the detoxification process. In this article, the potential detoxification pathway of MX is further discussed with regard to herb interactions. In this study, the pharmacokinetic parameters and metabolism of amygdalin and prunasin were investigated by comparing the traditional decoction and the dispensing granule preparations. In addition, several potential metabolites were characterized in an incubation system with rat liver microsomes or gut microbial enzymes. The combination of Xingren with Mahuang reduces exposure to D-amygdalin in vivo and contributes to its detoxification, a process that can be further facilitated in the traditional decoction. From the in vitro co-incubation model, 15 metabolites were identified and classified into cyanogenesis and non-cyanogenesis metabolic pathways, and of these, 10 metabolites were described for the first time. The level of detoxified metabolites in the MX traditional decoction was higher than that in the dispensing granules. The metabolism of amygdalin by the gut microbial enzymes occurred more rapidly than that by the rat liver microsomes. These results indicated that combined boiling both herbs during the preparation of the traditional decoction may induce several chemical changes that will influence drug metabolism in vivo. The gut microbiota may play a critical role in amygdalin metabolism. In conclusion, detoxification of MX may result 1) during the preparation of the decoction, in the boiling phase, and 2) from the metabolic pathways activated in vivo. Stereoselective pharmacokinetics and deamination metabolism have been proposed as the detoxification pathway underlying the compatibility of MX. Metabolic detoxification of amygdalin was quite different between the two combinations, which indicates that the MX decoctions should not be completely replaced by their dispensing granules.

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu1+ to the less toxic Cu2+. Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb3-type cytochrome oxidase (cbb3-Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb3-Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF.

Molecular docking approach to elucidate metabolic detoxification pathway of polycyclic aromatic hydrocarbons

This study assessed the molecular interactions of (±)-anti-and-syn- dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide (DBPDE), 7,12- dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (DMBADE), N2- hydroxylated-PhIP(N2-OH-PhIP), (±)-anti-and-syn-benzo[a]pyrene-7,8-diol- 9,10-epoxide (BPDE) with various Glutathione S-transferase (GST) and N- acetyltransferase (NAT) isozymes. Our in-silico data revealed that GSTP1 (- 8.83 kcal/mol), showing more plausible binding as compared to GSTM1 (-8.74 kcal/mol) and GSTA1 (ΔG: -8.03 kcal/mol) against (-)-anti-DBPDE and (+)- syn-DBPDE. We also investigated the involvement of GST and NAT isozymes in the conjugation of DMBADE andN2-OH-PhIP as a control despite their preferred routes sulfonation and glucuronidation for detoxification. The findings exhibited feeble binding of different classes of GSTs with metabolites of DMBA and PhIP, as highlighted by their free energy of binding. The enzymatic activity of GSTM1 against the most potent diol-epoxide of benzo[a]pyrene (BP), (+)-anti-BPDE, and (+)-syn-BPDE followed by GSTP1 and GSTA1 has well documented. In addition, these findings provide new perspectives for most probable mechanistic details of the detoxification pathway of PAHs and xenobiotics useful in combination therapy for future ligand-based drug discovery and development.

Artemisinin – Based Drugs Target the Plasmodium falciparum heme-detoxification pathway

Artemisinin – based antimalarial drugs are believed to exert lethal effects on malarial parasites by alkylating a variety of intracellular molecular targets. Recent work with live parasites has shown that one of the alkylated targets is free heme within the parasite digestive vacuole, which is liberated upon hemoglobin catabolism by the intraerythrocytic parasite, and that reduced levels of heme alkylation occur in artemisinin resistant parasites. One implication of heme alkylation is that these drugs may inhibit parasite detoxification of free heme via inhibition of heme to hemozoin crystallization; however, previous reports that have investigated this hypothesis present conflicting data. By controlling reducing conditions and hence the availability of ferrous vs ferric forms of free heme, we modify a previously reported hemozoin inhibition assay to quantify the ability of ART – based drugs to target the heme detoxification pathway under reduced vs oxidizing conditions. Contrary to some previous reports, we find that artemisinins are potent inhibitors of hemozoin crystallization, with effective half maximal concentrations approximately an order of magnitude lower than those for most quinoline – based antimalarial drugs. We also examine hemozoin and in vitro parasite growth inhibition for drug pairs found in the most commonly used ART – based combination therapies (ACTs). All ACTs examined inhibit hemozoin crystallization in an additive fashion, and all but one inhibit parasite growth in additive fashion.

N-phenyl-1-naphthylamine (PNA) Accumulates in Snapping Turtle (Chelydra serpentina) Liver Activating the Detoxification Pathway

Substituted phenylamine antioxidants (SPAs) are used in Canadian industrial processes. SPAs, specifically N-phenyl-1-naphthylamine (PNA), have received very little attention despite their current use in Canada and their expected aquatic and environmental releases. There is a research gap regarding the effects of PNA in wildlife; therefore, Chelydra serpentina (common snapping turtle) was studied due to its importance as an environmental indicator species. A chronic experiment was performed using PNA spiked food (0 to 3446 ng/g) to determine its toxicity to juvenile C. serpentina. A significant increase in cyp1a mRNA level was observed in the liver of turtles exposed to 3446 ng/g PNA, suggesting that phase I detoxification is activated in the exposed animals. Additionally, a significant decrease in cyp2b transcript level was observed at the two lowest PNA doses, likely indicating another metabolic alteration for PNA. This study helped determine the molecular effects associated with a PNA exposure in reptiles.