Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Synthesis of typical sulfonamide antibiotics with [14C]- and [13C]-labelling on phenyl ring for environmental studies

Background As a kind of widely used antibiotics, sulfonamide antibiotics (SAs) has become ubiquitous environmental contaminants that caused public concerns. The behavior of SAs in complex environmental system need to be elucidated, which is hampered by unavailability or high cost of isotope-labelled SAs. Results Using commercially available uniformly [l4C]- and [l3C]-labelled aniline as starting material, we synthesized [phenyl-ring-14C]- and [phenyl-ring-l3C]-labelled sulfamethoxazole (SMX), sulfamonomethoxine (SMM), and sulfadiazine (SDZ) using four-step (via condensation of labelled N-acetylsulfanilyl chloride and aminoheterocycles) or five-step (via condensation of labelled N-acetylsulfonamide and chloroheterocycles) reactions in good yields (5.0−22.5% and 28.1−54.1% for [14C]- and [13C]-labelled SAs, respectively) and high purities (> 98.0%). Conclusion The synthesis of [l4C]-labelled SAs could be completed on milligram-level, being feasible for preparation of labelled SAs with high specific radioactivity. This study provides efficient and maneuverable methods to obtain a variety of [14C]- or [13C]-labelled SAs for studies on their environmental behavior, such as fate, transformation, and bioaccumulation.

Comparison of chronic low-dose effects of alpha- and beta-emitting radionuclides on marine bacteria

Effects of Americium-241 (241Am), alpha-emitting radionuclide of high specific radioactivity, and tritium (3H), beta-emitting radionuclide, on luminous bacteria Photobacterium phosphoreum were compared. Bioluminescence intensity served as a marker of bacterial physiological activity. Three successive stages in the bioluminescence response to 241Am and 3H were found under conditions of lowdose irradiation: (1) absence of effects, (2) activation, and (3) inhibition. They were interpreted in terms of bacterial response to stressfactor as stress recognition, adaptive response/syndrome, and suppression of physiological function (i.e. radiation toxicity). Times of bioluminescence activation (TBA) and inhibition (TBI) were suggested as parameters to characterize hormesis and toxic stages in a course of chronic low-dose irradiation of the microorganisms. Values of TBA and TBI of 241Am were shorter than those of 3H, revealing higher impact of alpha-irradiation (as compared to beta-irradiation) under comparable radiation doses. Increases of peroxide concentration and NADH oxidation rates in 241Am aquatic solutions were demonstrated; these were not found in tritiated water. The results reveal a biological role of reactive oxygen species generated in water solutions as secondary products of the radioactive decay. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha- and beta-emitting radionuclides in aquatic solutions.