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A Goldfish Model for Evaluation of the Neurotaxicity of \(\omega\)-Conotoxin GVI A and Screening of Monoclonal Antibodies. ADEYEMO, 0. M .. SHAPIRA, S., TOMBACCINI, D., POLLARD, H. 8 .• FEUERSTEIN, G .. AND SIREN, A-L. ( 1991 ). Toxicol. App/. Pharmaco/. 108, 489-496. The neurotoxicity of \(\omega\)-conotoxin (\(\omega\)-CgTx), a potent neuronal voltage-sensitive calcium channel blocker, was measured using a new bioassay. \(\omega\)-CgTx was administered intraperitoneally (ip) to goldfish weighing approximately 1.6 g, and dose-related changes were observed over a 2-hr period. \(\omega\)CgTx induced time- and dose-dependent abnormal swimming behavior (ASB) and mortality. The antitoxin activity of the antiborlies was investigated in vivo by either ( l) preincubation of the antibody with w-CgTx at 4°C overnight, or (2) pretreatment with antibody, 30 min before \(\omega\)CgTx injection in a 10:1 antibody/\(\omega\)-CgTx molar ratio. The LD50 dose of \(\omega\)-CgTx in goldfish was 5 nmol/kg ip, and preincubation of monoclonal antibody (50 nmol/kg ip) with \(\omega\)-CgTx (5 nmol/kg ip) significantly (p < 0.05) reduced mortality. ASB, and toxicity time. The antitoxin activity of the monoclonal antiborlies evidenced in the goldfish bioassay was further tested in the conscious rat. In the rat, the increases in mean arterial pressure and heart rate induced by \(\omega\)-CgTx (0.03 nmol/rat icv) were significantly (p < 0.02 and p < 0.0 l, respectively) attenuated by preincubation of the toxin with the antibody (0.3 nmol/rat). We conclude that the goldfish bioassay provides a simple. accurate, and inexpensive in vivo model for the study of the toxicity of \(\omega\)CgTx
The hyperfine coupling constants (isotropic hfcc and four Cartesian components of the ani~ tropic tensor) are calculated for all three atoms of C\(_2\)H in its three lowest-lying electronic states at various molecu)ar geometries by means of the ab initio configuration interaction ( MRO.CI) method. The off-diagonal electronic matrix elements involving the two species ofthe A' symmetry are also computed. A diabatic transforrnation is perforrned Jeading to simple geometrical depen· dences of the hyperline coupling constants.
The vibronically averaged values for tbe hyperfine coupling constants in the X\(^2 \sum\)-A\(^2 \Pi\) system of the ethynyl radical are computed by means of tbe ab initio metbod calculations. The results point at tbe importance of taking into account the coupling of a1l tbree electronic states in question ( I\(^2\)A', 2\(^2\)A', and 1\(^2\)A") for a reliable explanation of the available experimental findings. The mean values of the hfcc's for K = 0 and 1 levels in \(^{13}\)C\(_2\)H and \(^{13}\)C\(_2\)D in the energy range up to 6000 cm\(^{-1}\) are predicted.
Tbe alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, 1)rr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, l)T-'I)T, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present durlog the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artiticial sweetener aspartame); only Met under these conditions bad a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Metproduces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the tirst-order reaction rate for nitrite. A decrease in nitrite concentration from the millimolar concentrations ofthe in-vitro assay to the micromolar concentrations in the stomach reduces the reaction rate by a factor of 1000 for the side-chain nitrosation, whereas a million-fold reduction will be observed for nitrosation of the amino group.