Teratogenicity of chlorambucil in rat embryos in vitro

Abstract

Chlorambucil, a bifunctional alkylating agent and antitumor drug, is a potent teratogen in rodents and possibly teratogenic in humans. Unlike cyclophosphamide, a related bifunctional alkylating agent, chlorambucil is a direct-acting cytotoxic agent. However, several studies indicate that the cytotoxic potential of chlorambucil is enhanced by metabolism of the parent compound. Using day-10 rat embryos cultured in vitro, we have demonstrated that chlorambucil is a direct acting teratogen but that its teratogenicity can be enhanced by the addition of rat liver S-9 preparations to culture medium containing the drug. Our data also suggest that this enhancement is not related to cytochrome P-450 monooxygenases. This conclusion is based upon three lines of evidence. First, enhancement occurs under culture conditions which lack cofactors necessary for cytochrome P-450 monooxygenases. Second, metyrapone, a known inhibitor of cytochrome P-450, did not abolish the enhancement. Third, induction of cytochrome P-450 by two specific inducers of monoxygenase activity had no effect on the observed enhancement. We postulate that the observed enhancement of chlorambucil teratogenicity is related to the beta-oxidation of the butyric acid side chain of chlorambucil generating the more cytotoxic (teratogenic) metabolite, phenylacetic acid mustard. Finally, our data indicate that the teratogenic effects elicited by chlorambucil are indistinguishable from those elicited by cyclophosphamide.

Culture of whole rodent embryos in teratogen screening

“…Most workers have found at least reasonable correspondence between the two sets of curves, but there are clear exceptions. We have found good correspondence for cyclophosphamide [14,16} and chlorambucil [16,26]. By contrast, we have been unable to detect clear evidence of teratogenicity with thalidomide.…”

“…We and a number of other workers have found that extraembryonic (maternal) metabolism, but not fetal metabolism is instrumental in the teratogenicity of a number of compounds. These compounds include cyclophosphamide [ 14,22,23], cytochalasin D [24], N-2-fluorenylacetamide [25], chlorambucil [26], rifampicin [27], and procarbazine (Fantel, unpublished data). These findings are consistent with the inability of a number of workers to detect significant cytochrome P-450-dependent activity in embryos [28-321. Other studies have shown that as for many mutagens and carcinogens, inducing agents and metabolic inhibitors are effective modifiers of teratogenicity [ 1 5,221.…”

The Role of Mammalian Embryo Culture in Developmental Biology and Teratology

“…The second involves isolating rat liver supernatants (S-9) or microsomes and adding them, together with the cofactor NADPH, to embryo cultures. Results show that these activating systems do not impede or alter normal development in culture and are capable of initiating both activation and inactivation of teratogens (Fantel et at., 1979(Fantel et at., , 1981Kitchin et at., 1981a,b;Mirkes and Greenaway, 1982;Mirkes et at., 1983). Finally there is the potential of monitoring the teratogenicity of sera from animals treated with a potential teratogen, which presumably would contain metabolites, the parent compound, or both (Sadler, 1980b;Klein and Pierro, 1983).…”

“…Vitamin A (Morriss and Steele, , 1977Davis and Sadler, 1981), hydroxyurea (Warner et at., 1983), cadmium , alcohol (Brown et at., 1979), valproic acid and diphenylhydantoin (Bruckner et a!., 1983), glucose (Cockroft and Coppola, 1977;Sadler, 1980a), increased O 2 (Morriss and New, 1979), chlorambucil (Mirkes and Greenaway, 1982), cyclophosphamide (Fantel et at." 1979;Mirkes et at., 1983), and hyperthermia New, 1975, 1978) have caused abnormalities in growth and development.…”

Reactions of {4‐[Bis(2‐chloroethyl)amino]phenyl}acetic Acid (Phenylacetic Acid Mustard) with 2′‐Deoxyribonucleosides

“…It was shown with murine sarcoma cells that PAM (2) has a stronger acute toxicity and antitumor activity than CLB (1) [20]. Similarly, 2 was more cytotoxic and also more teratogenic than 1 in a rat-embryo model [21].…”