Thalidomide teratogenesis: evidence for a toxic arene oxide metabolite.

Abstract

It was postulated that thalidomide causes birth defects by being metabolized to a toxic electrophilic intermediate. This hypothesis was tested by using an in vitro assay in which drug toxicity to human lymphocytes was assessed in the presence of a hepatic microsomal drug metabolizing system. Maternal hepatic microsomes from pregnant rabbits mediated the production of a metabolite that was toxic to lymphocytes. Toxicity was enhanced by inhibitors of epoxide hydrolase (EC 3.3.2.3) and abolished by adding the purified enzyme to the incubation medium. The metabolite thus appears to be an arene oxide, consistent with the previously reported isolation of phenolic metabolites of thalidomide from the urine of treated animals. Two teratogenic analogs of thalidomide (phthalimidophthalimide and phthalimidinoglutarimide) were also toxic in the system; two nonteratogenic analogs (phthalimide and hexahydrothalidomide) were not toxic, even in the presence of epoxide hydrolase inhibitors. The toxic metabolite of thalidomide was not produced by rat liver microsomes (the rat is not sensitive to thalidomide teratogenesis) but was produced by hepatic preparations from maternal rabbits, and rabbit, monkey, and human (all sensitive species) fetuses. A toxic arene oxide therefore may be involved in the teratogenicity of thalidomide.Thalidomide was identified as a human teratogen 20 years ago (1-3). Compared to other teratogens, thalidomide's selective toxicity in the embryo, particularly for the developing limbs, and its relative lack oftoxicity in the adult is striking (4). Despite intensive investigation, however, the mechanism of the fetal toxicity of the drug remains unknown.An interesting early observation was that rats were resistant to the teratogenic effects of thalidomide but rabbits and monkeys were sensitive (5-7). Differences in species susceptibility could result from differences in biotransformation of the compound. It was noted that rabbit liver homogenates enhanced the rate of disappearance of the drug from incubation mixtures whereas rat liver homogenates did not (8). Similarly, in vivo, more thalidomide metabolites were bound to liver macromolecules in the rabbit than in the rat, suggesting that a metabolite might interact covalently with macromolecules important in morphogenesis. Furthermore, after thalidomide treatment, 4-and 5-hydroxylated metabolites of thalidomide were recovered from the urine of rabbits but not from rats (9).The presence ofphenolic derivatives ofthalidomide suggests that the drug might undergo oxidative metabolism via an arene oxide intermediate. Arene oxides have been implicated as mutagens, cytotoxins, and teratogens (10-12). Thus, birth defects caused by the anticonvulsant phenytoin may result from an arene oxide metabolite which covalently binds to critical structures in the developing fetus (12). Therefore, we have attempted to look for a possible toxic arene oxide metabolite ofthalidomide. We have used an in vitro assay system in which human lymphocytes are the target of metabol...

Epoxidation of olefins by cytochrome P450: Evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant

“…Some examples in which the formation of an epoxide has been implicated are the conversion of arachidonic acid to a vasoactive metabolite (26), of benzo [a]pyrene to the ultimate carcinogenic diol-epoxide (27,28), and of urethane and vinyl carbamate to a carcinogenic product (29,30), as well as drug metabolism leading to the hepatotoxicity of furosemide (31) and the teratogenicity of phenytoin (32) and thalidomide (33). For this study, however, model olefin substrates were selected for the ease with which the products could be quantitated, the availability of cis and trans isomers in one case, and the choice of compounds that undergo hydroxylation as well as epoxidation.…”

Development of an In Vitro System Detecting Pro-embryotoxin

“…However, many mammalian cell lines have poor metabolic activation potencies, so the cell lines generally display limited sensitivities to toxic compounds. External metabolic activating capacity has been introduced to detect the cytotoxicity and mutagenicity of chemicals in test systems with mammalian cells (4,5). In these systems, cells were directly contacted with microsomal reaction mixtures containing an NADPH generating system and test compound.…”

Pathogenesis of Thalidomide Teratogenesis in the Marmoset (Callithrix jacchus): Evidence Suggesting a Possible Trophic Influence of Cholinergic Nerves in Limb Morphogenesis

“…This emphasises the inadequacy of the rabbit as a model of thalidomide teratogenesis. GORDON et a1 (19) have stated that metabolism to an arene oxide may be the reason for the selective toxicity to rabbits and monkeys. It may also be that a relative difference in such metabolism Thalidomide will produce limb malformations only if ingested before the 51st day (18).…”