In vitro embryotoxicity of N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB): evidence for N-dehydroxylated metabolite as a proximate dysmorphogen.

The rat conceptus biotransforms N-methyl-N-(7-propoxynaphthalene-2-ethyl)hydroxylamine (QAB) in vitro to 7-propoxynaphthalen-2-ylacetic acid (QAA) and six more (M1 to M6) metabolites. Thus far, M4 has been identified as N-demethyl-QAB and M6 as N-desoxy-QAB. We investigated which of these two metabolites might be involved in QAB-embryotoxicity in vitro. Conceptuses were cultured from day 9.5 to 11.5 post-coitum, and were exposed to N-demethyl-QAB or N-desoxy-QAB either in the culture medium or by microinjection directly into the amniotic cavity. When added to the culture medium, N-demethyl-QAB (No Observed Adverse Effect Level, NOAEL, for growth 122 microM and for differentiation 41 microM) was less active than QAB itself (NOAEL for growth and differentiation 12 microM). N-desoxy-QAB caused severe growth retardation and an impairment of differentiation at a concentration of 11 microM (NOAEL 3.6 microM). As regards causing anomalies, the NOAEL of N-demethyl-QAB (41 microM) was 10-fold higher than that of QAB (NOAEL 3.9 microM) and that of N-desoxy-QAB (NOAEL 3.6 microM). At an intraamniotic concentration of 0.7 mM, N-demethyl-QAB caused no effects on growth and differentiation and no increase of anomalies was observed, whereas QAB and N-desoxy-QAB each elicited an increase in dysmorphogenic embryos at equimolar concentrations without affecting growth and differentiation. It is, therefore, concluded that N-desoxy-QAB, but not N-demethyl-QAB, could be a proximate dysmorphogen responsible for the embryotoxicity/teratogenicity of QAB in vitro.

Cytochrome P-450-dependent biotransformation of QA 208-199 in cultured rat conceptuses.

It has been shown, using the method of rat post-implantation embryo culture, that the rat conceptus metabolizes the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphthalenethan-amine (QA 208-199, QAB) in vitro. The capacity to metabolize QAB and accumulate its main metabolites depends on the developmental stage and length of exposure. In the present study, pregnant Han Wistar dams were given ip injections of either Aroclor 1254 (AC), 3-methylcholanthrene (3-MC) or phenobarbital (PB) before the dissection and culture of 9.5- and 10.5-day-old conceptuses in order to identify possible inducible conceptal cytochrome P-450 species. Conceptuses preinduced in utero were exposed to QAB in vitro for 48 hr, after which metabolites in the culture medium and conceptual tissues were determined with HPLC. Embryotoxic effects were evaluated in the same conceptuses. Preinduction in utero with AC, 3-MC or PB revealed that pretreatment with PB led, after 48 hr of culture, to a statistically significantly increased impairment of embryonic differentiation in 9.5-day-old conceptuses in vitro in comparison with vehicle-induced QAB-exposed conceptuses. Preinduction with PB led to higher levels of QAA, the main metabolite in vivo, in the culture medium. In the conceptal tissues, metabolite levels were significantly lower after preinduction with AC. In vehicle-induced 10.5-day-old embryos QAB caused less embryotoxicity in comparison with 9.5-day-old ones. In all preinduced conceptuses cultured from 10.5 to 12.5 days, yolk-sac vascularization was less affected and fewer embryonic anomalies were found in comparison with conceptuses cultured from 9.5 to 11.5 days. AC and PB pretreatment caused a decreased morphological score without changes in overall growth. Analysis of QAB and its metabolites in culture media and conceptal tissues at 12.5 days showed different levels of metabolites depending on the inducer used. To find further evidence for the involvement of cytochrome P-450 enzymes in the conceptal metabolism of QAB, conceptuses preinduced in utero (3-MC or PB) were exposed to QAB in vitro and gassed during the second half of the culture period with a mixture containing 35% carbon monoxide (CO), an inhibitor of cytochrome P-450 enzymes. CO treatment led to an inhibition of conceptal metabolism of QAB in comparison with that in conceptuses cultured under normal gassing conditions (without CO). These results strongly suggest the involvement of cytochrome P-450-dependent monooxygenases in the conceptal metabolism of QAB in vitro. 10.5-day-old conceptuses, after preexposure in utero to PB, were less susceptible to QAB embryotoxicity in vitro than were 9.5-day-old ones. In addition, the response to preinduction appeared to be age dependent.

Strain differences in the control incidences of morphological abnormalities in the rat whole embryo culture.

Since the animal supplier to this laboratory stopped carrying the rat strain used for the previous 7 yr, a number of other strains from this supplier were tested to determine their suitability for use in the whole embryo culture system. The in vitro development from prenatal days 9.5 to 11.5 of rat conceptuses of the strain Kfm:WSA (A) was compared with that of strains Ibm:WSA (B), HanIbm:WIST (C) and Ibm:RORO (D) cultured under identical methodological conditions. The parameters for conceptal growth (yolk-sac diameter, embryonic crown-rump and head lengths) after 48 hr of culture of strains A, B and D were similar. These measurements were smaller in the conceptuses of strain C than in those of strain A, but embryonic differentiation (morphological score and somite numbers) was not delayed in comparison with strain A. The overall frequencies of conceptuses with dysmorphogenic events in strains A and B were 3.1 and 4.4%, respectively. In contrast, in strains C and D 30.0 and 22.6% of the conceptuses assessed had morphologically abnormal features. Both values were statistically significantly higher than the incidence in strain A. The types and frequencies of anomalies observed were different in the different strains tested. The results from conceptuses of strain B (derived from strain A) indicate that this strain is suitable for the culture model established in this laboratory, whereas strains C and D are not useful without methodological changes or adaptations. This study indicates that strain differences should be kept in mind when setting up the whole embryo culture system.

An immunohistochemical investigation into the presence and localization of cytochrome P-450 isozymes in organogenesis-stage rat conceptal tissue.

To investigate the presence and localization of a variety of xenobiotic biotransforming isozymes of the cytochrome P-450 superfamily in the organogenesis-stage rat conceptal tissue, pregnant female rats were dosed with one of two inducing agents, 3-methylcholanthrene [3MC, 40 mg/kg body weight, ip, day 7 post coitum (pc)] and phenobarbital (PB, 40 mg/kg, ip, days 5, 6, 7 and 8 pc), or with their vehicles (3MC, olive oil; PB, 0.9% NaCl) as controls. The conceptuses were allowed to grow either in vivo, or in vitro, using the whole embryo culture system, from days 9.5 to 11.5 pc. The embryos and isolated visceral yolk sacs were submitted to immunohistochemical investigation using light microscopy. The livers of the dams served as positive controls. Polyclonal and monoclonal antibodies raised against a variety of cytochrome P-450 isozymes were used in the alkaline phosphatase-anti-alkaline phosphatase enzyme immune complex method. All pre-induced dam livers showed positive staining with all polyclonal and monoclonal antibodies tested. The presence of P450IA1 was detected in the visceral yolk sac of both ex vivo and cultured conceptuses, preinduced in utero with 3MC, with the appropriate polyclonal antibodies but not with the monoclonal antibodies. P450IIB1/2 was detected in the visceral yolk sac of both ex vivo and cultured conceptuses, pre-induced in utero by phenobarbital, with the appropriate polyclonal antibodies, but not with the monoclonal antibodies. No staining was seen in any embryo proper, with any vehicle-treated conceptal tissue, or with antibodies raised against P-450s IIE1, IIIA or IVA. Our results support the hypothesis that the organogenesis-stage rat conceptus contains, in the visceral yolk sac, a 3MC-inducible P-450 isozyme similar, but not identical, to adult IA1. They also provide evidence that a PB-inducible isozyme similar, but not identical, to adult IIB1/2, is present in the visceral yolk sac at this stage of conceptus development.

A model combining the whole embryo culture with human liver S-9 fraction for human teratogenic prediction.

The whole embryo culture system has proved particularly useful in evaluating the role of biotransformation in dysmorphogenic processes. Using the traditional method, information on the teratogenic potential of chemicals can be obtained, but the results are difficult to extrapolate to humans. In this study, a human liver S-9 fraction was used as an enzyme source for the bioactivation of cyclophosphamide (CP) in vitro, to assess whether this model mimics more nearly the human situation. CP is a well known rodent teratogen but probably is not teratogenic in humans. The effects were compared of Aroclor-1254-induced rat liver S-9, non-induced rat liver S-9 and human liver S-9 fractions on CP teratogenicity in vitro. CP (30 mug/ml) with non-induced rat S-9 (50 mul) did not cause a significant increase in adverse effects (46.7%), whereas 100% dysmorphogenic effects were shown with induced rat S-9 (30 mul, 50 mul). CP (30-150 mug/ml) did not produce dysmorphogenesis (below 35.5%) in the presence of human S-9 (50 mul).

Teratogenicity of arotinoids (retinoids) in the rat whole embryo culture.

Structural modifications of the arotinoid molecule RO 13-7410 led to a difference in the teratogenic potencies of more than five orders of magnitude in mice in vivo and in micromass cultures of rat embryonic limb bud cells (Kistler et al. 1990). Five of these retinoids were selected and tested in rat whole embryo culture to determine the suitability of this in vitro test system for the identification of potentially non-teratogenic derivatives among this class of chemicals. The highest concentrations of the compounds with no effects (NOAEL) on general conceptus growth, on differentiation and on the frequency of dysmorphogenic embryos in vitro were compared with the lowest effective teratogenic doses in vivo (LOAEL) or with the concentrations leading to 50% inhibition of limb bud cell differentiation (IC50) in vitro. NOAEL's for the parameters of conceptus development ranged from 10(-5) micrograms/ml (0.03 nM) to 10 micrograms/ml (28.7 microM) for the compounds tested. These correlated very well with LOAEL and IC50 (R greater than 0.95). The types of dysmorphogenesis in vitro were those typical for retinoids, and for the most part resembled the malformations found in vivo. We conclude that the whole embryo culture system is a useful tool for the preliminary testing of retinoids.

Comparison of the metabolic activity of yolk sac tissue in the whole embryo and isolated yolk sac culture.

In rat visceral yolk sac tissue cultured from 10.5 to 12.5 days of prenatal age, metabolism of the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphtalinethanamine (QA 208-199, QAB) and the accumulation of its metabolites have been shown previously. In this study, the metabolic activities of visceral yolk sac tissues cultured either alone or together with the embryo were compared. The metabolite patterns in medium and visceral yolk sac tissue generated by intact conceptuses or by isolated visceral yolk sac tissues were similar. After 24 as well as 48 h of culture, the major in vivo metabolite, 7-propoxy-naphthalene-2-ylacetic acid (QAA) and other, as yet unidentified metabolites, accumulated in embryo proper and visceral yolk sac tissues. QAB was not found in the embryo proper, and was only found in yolk sac tissues using the higher concentration. In particular the metabolites M5 and M6 exhibited a massive accumulation in the visceral yolk sac, whereas QAA, M3, and M4 accumulated to a much lesser degree. In isolated yolk sacs cultured for 48 h, tissue levels of QAA and M4 were similar to those in yolk sacs of cultured whole conceptuses, whereas M5 and M6 exhibited twofold higher levels in isolated yolk sacs. These findings were in agreement with the distinct increase of myeloid figures containing partly fragmented inclusion bodies in yolk sac tissues. These results suggest that the visceral yolk sac may be the major site of QAB metabolism in cultured rat conceptuses in vitro.

Effects of QA 208-199 and its metabolite 209-668 on embryonic development in vitro after microinjection into the exocoelomic space or into the amniotic cavity of cultured rat conceptuses.

The objective of this study was to determine the direct embryotoxic effects in vitro of N-hydroxy-N-methyl-7-propoxy-2-naphthalene-ethanamine (QA 208-199, QAB) and of one of its metabolites, 7-propoxy-naphthalene-2-ylacetic acid (209-668, QAA), after circumventing the bioconverting conceptual membranes. The compounds were, therefore, microinjected either into the exocoelomic space or into the amniotic cavity of rat conceptuses of 10 d at prenatal age at doses of up to 84.9 ng (QAA) and 180 ng (QAB) per conceptus respectively. The conceptuses were subsequently cultured for 28 h after which their development was assessed. QAB produced marginal effects on embryonic differentiation only after microinjection of the compound into the amniotic cavity. Dysmorphogenic effects, however, occurred in a dose-dependent fashion after either exocoelomic or intraamniotic microinjections of the compound. The frequencies and types of anomalies were similar after either exposure route and consisted predominantly of anomalies associated with axial rotation. QAA also impaired embryonic differentiation at only the high dose level of 84.9 ng per embryo and after intraamniotic injections only. Dysmorphogenic effects were observed in all experimental groups, although the differences were not statistically significant when compared with the concomitant controls. An increased proportion of anomalies observed were in the cephalic region as compared to the defects produced by QAB. These data suggest that QAA most probably is not the QAB metabolite responsible for the embryotoxic action of QAB in vitro. Furthermore, the results tend to confirm the suggested involvement of the visceral yolk sac membrane in mediating QAB embryotoxicity.

Xenobiotic metabolism in the isolated conceptus.

The development of culture systems using either pre- or post-implantation embryos has made it possible to study the metabolizing capacity of the isolated conceptus in vitro. In the rodent pre-implantation embryo and post-implantation conceptus (embryo and its membranes), constitutive levels and inducibility of different enzyme systems involved in drug metabolism have been shown in vitro to lead to the formation of embryotoxic metabolites of different xenobiotics. This indicated the presence of enzyme systems during early organogenesis. For example, using the rat post-implantation embryo culture, we could show that incubation with the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphthalenethanamine (QAB) led to high levels of the main in vivo metabolite 7-propoxy-naphthalene-2-ylacetic acid (QAA) and two as yet unidentified products, M5 and M6, in the conceptus. QAB was not found in tissues and QAA itself did not enter the embryonic compartments. In addition, accumulation in tissue was dependent on the time and duration of exposure. It started at 10.5 days of development. A similar metabolite pattern was obtained after yolk-sac tissue had been cultured alone, which suggests metabolizing capacity of mainly the yolk-sac tissue. The enzyme reactions involved might have included oxidative N-demethylation and oxidative deamination, probably also including the formation of reactive intermediate metabolites. In conclusion, our data demonstrate that not only maternal metabolism may play an important role in the toxic action of xenobiotics, but also the metabolizing capacity of the conceptus itself may be crucial, since the formation of (intermediate, highly reactive) metabolites takes place at the target site.