Jean C. Greenaway

Teratogenic bioactivation of cyclophosphamide in vitro

Abstract Day 10 rat embryos grown as cultured explants in vitro developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.

Teratogenicity of cyclophosphamide metabolites: Phosphoramide mustard, acrolein, and 4-ketocyclophosphamide in rat embryos cultured in vitro

Abstract Phosphoramide mustard, acrolein, and 4-ketocyclosphosphamide, known stable metabolic products of bioactivated cyclophosphamide, were tested for their teratogenicity against rat embryos grown in vitro from Day 10 to Day 11 of gestation. Results indicate that phosphoramide mustard is the teratogenic metabolite of cyclophosphamide, since the effects of phosphoramide mustard exactly parallel those of bioactivated cyclophosphamide. These effects are reductions in total embryo protein content, crown-rump length and number of somites, characteristic malformations, and pattern of cell necrosis. Acrolein at a dose equimolar to a dose of bioactivated cyclophosphamide which produced malformations in 100% of treated embryos had no effect on any of the parameters measured. An equimolar dose of 4-ketocyclophosphamide had no effect on total embryo protein content or crown-rump length and number of somites, but consistently produced some malformed embryos. The kinds of malformations observed, however, are not seen in embryos treated with phospharamide mustard or bioactivated cyclophosphamide.

Role of acrolein in cyclophosphamide teratogenicity in rat embryos in vitro

To elucidate the role of acrolein in cyclophosphamide (CP) teratogenesis, we used the dechloro derivative of cyclophosphamide (D-CP). After activation, D-CP spontaneously breaks down to yield acrolein and dechlorophosphoramide mustard (D-PM), the nonalkylating derivative of phosphoramide mustard. At concentrations ranging from 6.25 to 50 micrograms/ml (33 to 262 microM), D-CP produced concentration-dependent cell death, growth retardation, and malformations in rat embryos cultured in vitro from Day 10 to 11 of gestation. D-PM, however, even at a concentration of 238 micrograms/ml (950 microM), had no effect on embryonic growth and development when added directly to standard culture medium containing Day 10 rat embryos. When embryos were exposed to acrolein (0.025 to 0.1 microgram/ml) directly in serum-free medium, this metabolite produced concentration-dependent decreases in growth parameters and abnormal flexion in some embryos. In no case, however, did acrolein-treated embryos resemble D-CP-treated embryos in terms of morphological malformations. Although we were able to show that D-CP was teratogenic in vitro, D-CP doses up to 50 mg/kg administered on Day 11 in vivo had no effect (with the exception of decreased growth at the highest dose) on growth or development at Day 20 of gestation. Our results suggest that acrolein plays a role in CP teratogenesis, but that the form in which it arrives at the teratogenically sensitive sites within the embryo is an important consideration in terms of the relative roles of acrolein and phosphoramide mustard in CP teratogenicity.

Effects of 3-methylcholanthrene and phenobarbital on the capacity of embryos to bioactivate teratogens during organogenesis☆

Pregnant Sprague-Dawley rats were divided into four groups and given ip injections of 3-methylcholanthrene (MC) in corn oil, corn oil only, phenobarbital (PB) in Hanks balanced salt solution (HBSS), or HBSS only. Maternal animals were killed on Day 10 of gestation, and embryos from each group were explanted in medium containing cyclophosphamide (CP), 2-acetylaminofluorene (AAF), or dimethylsulfoxide vehicle. After a 24-hr culture period, embryos from dams treated with HBSS, corn oil, or PB/HBSS exhibited no increase in abnormalities (as compared with controls) when either CP or AAF were added to the media. However, embryos transplacentally preexposed to MC and subsequently treated during culturing with AAF (but not CP) exhibited striking increases in malformation incidence. Commonly observed malformations included abnormally open neural tubes, abnormal flexure rotation, and prosencephalic defects. Homogenates of Day 10 embryos transplacentally preexposed to MC exhibited readily measurable oxidative biotransformation of AAF as assessed with HPLC. Biotransformation of AAF by embryos from the other three groups was virtually undetectable. Incorporation of exogenously supplemented bioactivating systems from livers of mature animals indicated that postmitochondrial supernatant fractions (S-9) from male, MC-pretreated rats effectively catalyzed the conversion of AAF (but not CP) to embryotoxic metabolites. Conversely, hepatic S-9 from adult, male, PB-pretreated rats was highly effective in converting CP (but not AAF) to embryotoxic metabolites. The results indicated the inducerspecific occurrence of embryonic bioconversion of AAF to embryotoxic metabolites via MC-inducible, P-450-dependent, embryonic enzyme systems.

Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system: Relationship of biotransformation to teratogenicity in vitro

2-Acetylaminofluorene (AAF) produced abnormal, open neural tubes in cultured whole rat embryos only in the presence of an added, NADPH-dependent monooxygenase system. Reactive intermediary metabolites, including N-hydroxy-AAF, N-hydroxy-2-aminofluorene, 2-nitrosofluorene and N-acetoxy-AAF, each elicited embryonic malformations under culture conditions, but a statistically significant increase in the incidence of abnormal neurulation was not observed. Using [14C]AAF and high pressure liquid chromatography (HPLC) separation techniques, the biotransformation of AAF was studied under conditions in which embryos and the monooxygenase system were coincubated. The major metabolites produced cochromatographed with 5-hydroxy-AAF, 7-hydroxy-AAF, 9-hydroxy-AAF and 3-hydroxy-AAF. Other metabolic products also were detected. The embryonic effects of these major AAF metabolites were tested singly and in combination in the embryo culture system. Addition of 7-hydroxy-AAF to the embryo culture system resulted in open neural tubes in the absence of an added monooxygenase system. Other individual ring-hydroxylated metabolites produced retarded growth, but neurulation appeared normal. Ring-hydroxylated metabolites, added to the embryo culture system in combination in the same proportions as were formed during biotransformation in culture, also produced a marked increase in incidence of neural tube defects in the absence of an exogenous (added) biotransforming system. In combination with 3-, 5- and 9-hydroxy-AAF, 7-hydroxy-AAF exposure (86 microM) resulted in a 47% incidence of abnormal, open neural tubes. When tested individually, higher concentrations of 7-hydroxy-AAF (104 microM) produced a lower percentage of malformed embryos (13%). The results suggested that 7-hydroxy-AAF was principally responsible for the neural tube defects caused by AAF following monooxygenase-dependent bioactivation, but that other metabolites also appeared to contribute to the observed effect.

Teratogenicity in vitro of two deacetylated metabolites of N-hydroxy-2-acetylaminofluorene

In previous studies [E. Faustman-Watts, J. C. Greenaway, M. J. Namkung, A. G. Fantel, and M. R. Juchau (1983) Teratology 27, 19-28] an embryo culture system was utilized to investigate the role of biotransformation in the embryotoxicity of 2-acetylaminofluorene. For this investigation, the capacity of two deacetylated metabolites of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to produce malformations in cultured whole rat embryos is reported. The relative capacities of N-hydroxy-2-aminofluorene (N-OH-AF) and 2-nitrosofluorene (NF) to elicit embryotoxic effects, including embryolethality, malformations, growth retardation, and alterations in macromolecular content, were assessed and compared with effects produced by N-OH-AAF and bioactivated 2-acetylaminofluorene (AAF). Qualitatively similar patterns of malformations were produced by NF and N-OH-AF. At initial concentrations greater than 60 microM, both deacetylated compounds caused abnormalities in axial rotation (flexure), decreased viability, and decreases in embryonic DNA and protein content. Both chemicals were active in the absence of a bioactivating system. AAF produced a different spectrum of defects, and was active only in the presence of a complete monooxygenase system. The malformations produced by bioactivated AAF included abnormally open neural tubes; flexure abnormalities were rarely observed. The primary defect elicited by N-OH-AAF was prosencephalic hypoplasia. This chemical was active without an added bioactivating system. Temporal studies demonstrated that exposure of embryos to NF (128 microM) for as little as 2 hr was sufficient to elicit embryotoxic effects. None of the individual metabolites appeared to be solely responsible for the interruptions of neural tube closure produced by bioactivated AAF.

The embryotoxicity of adriamycin in rat embryos in vitro.

Adriamycin (ADR) is a widely used and highly valued antineoplastic agent but chronic treatment is limited by cardiotoxicity. To investigate its embryotoxic potential, cultured rat embryos were exposed to ADR at concentrations ranging from 0.4 to 1.2 microM. Within this range, ADR elicited decreases in growth parameters (somite numbers, embryonic length, and protein and DNA content), malformations involving the prosencephalic region, and embryolethality at the higher concentrations. Embryotoxicity was significantly increased (p less than 0.05) when cultures included cofactors for cytochrome P-450-dependent biotransformation and a hepatic microsomal preparation (S-9) prepared from animals pretreated with 3-methylcholanthrene or a mixture of polychlorinated biphenyls (Aroclor 1254). When S-9 from control animals or from rats pretreated with phenobarbital was used, significant increases in ADR-elicited embryotoxicity were not observed. Substitution of NADH for NADPH as a cofactor reduced the incidence of malformations from 100 to 60% at ADR concentrations of 0.5 microM. Increasing O2 concentrations partially counteracted the embryotoxic effects of ADR. Several other agents [including various antioxidants, compounds bearing free sulfhydryl groups, coenzyme Q10, and superoxide dismutase (with or without catalase)] that prevent or reduce the cardiotoxicity of ADR without impairing its antineoplastic properties, failed to modify the embryotoxicity significantly. This suggested that the embryopathic and antineoplastic properties of ADR may share a common mechanism which is distinct from that responsible for cardiotoxicity.

Generation of reactive dysmorphogenic intermediates by rat embryos in culture: Effects of cytochrome P-450 inducers☆

We have investigated the capacity of cultured whole rat embryos to convert 2-acetylaminofluorene (AAF) to reactive metabolites capable of eliciting dysmorphogenic effects in the same embryos. Cultured embryos (Sprague-Dawley) were exposed to AAF for periods of 2 or 24 hr, after which metabolites were isolated from the culture medium and identified with HPLC. Embryotoxic effects were evaluated in the same embryos. Day 10 embryos preexposed in utero to pregnenolone-16 alpha-carbonitrile (PCN) exhibited marked increases in capacity to convert AAF to a variety of hydroxylated metabolites. 3-Methylcholanthrene (3MC) was also a very effective inducer in utero but Aroclor 1254 (PCB), and isosafrole (ISF) evoked only minimal induction while phenobarbital (PB) was not demonstrably effective. Exogenously added hepatic postmitochondrial supernatant (S9) fractions from adult male rats pretreated with PCB, 3MC, or ISF exhibited induced monooxygenase activities as well as increased capacity to convert AAF to dysmorphogenic intermediates in the culture system. PB and PCN displayed much lesser effects. PCN was a very effective inducer of hepatic monooxygenases of pregnant rats but, when this tissue was utilized as an enzyme source, no significant increase in malformations was observed. Embryos with relatively high monooxygenase activities also displayed a high incidence of embryonic abnormalities when cocultured with AAF. Malformation incidence was strongly correlated with hydroxy metabolite generation, suggesting that induction in utero of P-450-dependent, embryonic monooxygenases resulted in the production of embryotoxic metabolites by the embryos own enzymes. The data also indicated that endogenous bioactivation (within the conceptus) was considerably more effective than bioactivation effected by an exogenous (hepatic) enzyme source.

Embryotoxic effects of salicylates: Role of biotransformation☆

The three major metabolites of salicylate, o- hydroxyhippurate ( salicylglycine , salicyluric acid), 2,5-dihydroxybenzoate (gentisic acid), and 2,3-dihydroxybenzoate, were examined for their capacities to elicit dysmorphogenesis, embryolethality , and growth retarding effects in an embryo culture system. The effects were compared with those produced by the parent salicylate. At the highest concentrations tested (1.9 mM), none of the three metabolites produced significant increases in the number of malformed embryos or in embryolethality . At the same concentration, all three agents reduced crown-rump lengths and somite numbers slightly but significantly (p less than 0.01), and the dihydroxy metabolites also reduced the embryonic protein content (p less than 0.01). In contrast, the parent salicylate produced large increases in embryolethality ( embryolethality in controls was 6% or less) and malformed embryos at equivalent or lower concentrations. Preincubation of the parent salicylate with various biotransforming systems did not affect embryotoxicity significantly. The most rapid biotransformation of salicylate in vitro was achieved with mitochondrial preparations of monkey kidney as the enzyme source but quantities metabolized were not sufficient to prevent malformations in the culture system. Increased serum protein concentration in the culture medium, however, markedly reduced the capacity of added salicylate to cause malformations. An examination of the kinetics of the dysmorphogenic effects of parent salicylate indicated that 5 hr of exposure elicited nonsignificant increases in numbers of malformations. A significant malformation rate was produced by 9 hr of exposure. In contrast, effects on embryonic growth parameters and embryolethality were greatest after a 24-hr exposure period. The results strongly suggest that the parent salicylate, rather than generated metabolites, was primarily or solely responsible for the malformations observed and that the duration of exposure of embryos to unmetabolized salicylate may be the critical factor for determining teratogenic outcome.

Comparison of cytochalasins (A, B, D, and E) in chick explant teratogenicity and tissue culture systems

Summary The four cytochalasins studied were found to have quantitative biologic activity at low concentrations, but elicited marked variation in ordering of response when compared in two in vitro systems. All four cytochalasins were teratogenic in vitro. Cytochalasin D was teratogenic at the lowest concentration, while cytochalasin E was the strongest inhibitor of cytokinesis. Since these compounds have been recovered from food and are stable to heat, their biologic activity in man should be studied.