Biochemical and toxicological evaluation of agent-cofactor reactivity as a mechanism of action for osteolathyrism

In vitro reactivity for each of four osteolathyrogens with a model compound for the lysyl oxidase (LO) cofactor was evaluated and coupled with mixture toxicity testing to evaluate agent-cofactor reactivity as a potential mechanism of action for osteolathyrism. Reactivity of the model cofactor (mLTQ: 4-butylamino-5-methyl-o-quinone), with each of two ureides, semicarbazide (SC) and thiosemicarbazide (TSC), and each of two aminonitriles, aminoacetonitrile (AAN) and beta-aminopropionitrile (betaAPN), was assessed using UV-vis spectrophotometry; both in the absence and presence of Cu(II)-bipyridine (bipy) complex. Two sets of mixture toxicity experiments were conducted using a frog embryo assay that assessed the incidence of osteolathyrism in the notochord of tadpoles after 96-h exposure. The resulting concentration-response curves for each set were evaluated (chi(2) goodness-of-fit test) against theoretical curves for two combined effects models: dose-addition and independence, to determine the combined effect of each osteolathyrogen combination. The agents SC, TSC and AAN each showed rapid, irreversible reactivity with mLTQ, both in the absence and presence of Cu(II)-bipy complex, as indicated by bleaching of the mLTQ peak (504 nm) and formation of an adduct at 350 nm. betaAPN showed no apparent reactivity in the absence of prolonged incubation with mLTQ, whether Cu(II)-bipy complex was present or not. After prolonged incubation (24-144 h) a new peak formed at 350 nm, suggesting that betaAPN reacts weakly with the cofactor, but in a manner different from the other agents examined. The toxicity tests indicated a dose-additive combined effect for the SC:TSC, AAN:SC and AAN:SC:TSC mixtures (0.1 < P < 0.9; with slope and EC50 additivity quotient values between 0.93-1.14), suggesting these agents induce osteolathyrism via a common mechanism. The betaAPN:SC and betaAPN: SC:TSC mixtures. showed a greater-than dose-additive combined effect (P < 0.001; slope and EC50 additivity quotient values from 0.74 to 0.89), suggesting a different or additional mechanism of osteolathyrism for βAPN. Taken together the results indicate that SC, TSC and AAN induce osteolathyrism primarily, if not completely, by binding to the LO cofactor, thereby inhibiting proper connective tissue fiber cross-linking. While βAPN also has the potential to react with the cofactor, the nature of the reactivity observed suggests that βAPN-cofactor binding is, at best, a secondary mechanism for induction of osteolathyrism in vivo. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.