Craig Harris

Regulation of intracellular glutathione in rat embryos and visceral yolk sacs and its effect on 2-nitrosofluorene-induced malformations in the whole embryo culture system

The dysmorphogenic effects of 2-nitrosofluorene (NF) in vitro were modulated in Day 10 rat embryos by agents which regulate intracellular glutathione (GSH) levels. The incidence of abnormal axial rotation caused by NF alone increased in a dose-dependent manner at NF concentrations in excess of 25 microM. No effects were observed at 15 microM NF and doses of 100 microM resulted in a 100% incidence of mortality. L-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, produced malformations (50%) in embryos exposed to 15 microM NF but produced no additional effects on embryos at higher NF concentrations. BSO treatment alone resulted in a greater than 50% decrease in GSH content in visceral yolk sacs and had a lesser but likewise significant effect (15% decrease) on the GSH content of embryos. Protein content was inversely affected as embryonic levels were increased by 20% and yolk sac levels were unchanged. When BSO was added in combination with NF at the onset of the culture period, embryonic GSH decreased in a dose-dependent manner, suggesting a relatively low rate of embryonic GSH turnover that could be increased by addition of an exogenous substrate capable of forming adducts with and removing GSH from the cells. 2-Oxothiazolidine-4-carboxylate (OTC), a compound which is enzymatically modified to provide an additional source of intracellular cysteine and increase GSH synthesis, produced no significant changes in embryonic or yolk sac GSH when added alone to the culture medium. When OTC (5 mM) was added in combination with NF, however, NF-elicited malformations were eliminated. This was also the case at 100 microM NF in which OTC not only prevented malformations but completely protected embryos against the loss in viability. The GSH and protein levels were indistinguishable from controls when OTC and NF were added simultaneously except for the 41 microM NF dose at which a highly significant increase in both embryonic and yolk sac protein was observed. This study clearly demonstrates the potential importance of GSH in the modulation of chemical dysmorphogenesis and provides an important new tool for the study of mechanisms of developmental toxicity.

Modulation of the embryotoxicity and cytotoxicity elicited by 7-hydroxy-2-acetylaminofluorene and acetaminophen via deacetylation

Acetaminophen (APAP) and 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) each produced a similar incidence of, as well as a qualitatively similar, abnormal closure of the anterior neuropore at similar concentrations when added to the medium of cultured rat embryos. At concentrations producing a 50-65% incidence of abnormal neurulation, the affected embryos displayed relatively complete embryonic development as assessed from measurements of protein, axial rotation, and embryonic length. The neural tube defect produced by these agents consisted of elevated neural folds remaining separated by approximately 45 degrees as well as the presence of a mitotically active neural epithelium. In contrast, the nonacetylated structures, p-aminophenol (PAP) and 7-hydroxyaminofluorene (7-OH-AF), were embryotoxic at concentrations 10-fold lower than the corresponding acetylated compounds; each produced a greater incidence of abnormal axial rotation and a greater decrease in embryonic protein than APAP or 7-OH-AAF. In addition, the embryos exposed to PAP or 7-OH-AF were morphologically and histologically dissimilar to those exposed to the acetylated compounds. The neural folds of the latter remained elevated and in apposition, but lacked complete fusion of the folds of neural epithelium and were accompanied by marked cytotoxicity. Addition of active deacetylase sources (guinea pig liver microsomes or commercially obtained, purified carboxylic-ester hydrolase) to the culture medium of conceptuses exposed to 7-OH-AAF or APAP resulted in an increased embryotoxicity which was indistinguishable from that produced by the nonacetylated compounds alone. The increases in toxicity were effectively blocked by the addition of paraoxon, indicating that catalysis of the deacetylation of APAP and 7-OH-AAF was the causative factor. No evidence could be found for deacetylation of 7-OH-AAF or APAP mediated by the Day 10 conceptus itself. When examined for cytotoxicity in F9 embryonal carcinoma cells, APAP and 7-OH-AAF each produced observable cell death only if reduced glutathione (GSH) had previously been depleted and if a deacetylase source were present; this cytotoxicity was also blocked by addition of paraoxon. The nonacetylated metabolites were directly cytotoxic, although GSH depletion greatly increased the incidence of cell death. Therefore, deacetylation of APAP and 7-OH-AAF produced an increase in generalized embryotoxicity and cytotoxicity relative to abnormal neurulation, suggesting that APAP and 7-OH-AAF are capable of eliciting neural tube defects via a mechanism(s) that is distinguishable from the generalized embryotoxicity or cytotoxicity produced by their nonacetylated counterparts.

Abnormal neurulation induced by 7-hydroxy-2-acetylaminofluorene and acetaminophen: Evidence for catechol metabolites as proximate dysmorphogens

Direct additions to culture media of either acetaminophen (APAP) or 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) resulted in abnormal closure of the anterior neuropores of cultured rat embryos in the absence of an exogenous bioactivation system. Concentrations required to produce a 50% incidence of the defect were approximately 500 and 250 microM for APAP and 7-OH-AAF, respectively. Losses of viability were not evident at these concentrations but 7-OH-AAF elicited a somewhat greater effect on growth parameters and generalized embryotoxicity. Transplacental induction with 3-methylcholanthrene (MC) of P450IA1 in subsequently cultured rat embryos did not detectably alter the capacity of APAP or 7-OH-AAF to effect embryotoxicity or neuropore closure. However, additions to the culture medium of exogenous hepatic bioactivating systems (S9) from MC-induced vs phenobarbital (PB)-induced adult rats produced profoundly different effects on neuropore closure. Coincubation with S9 from MC-induced rats reduced the incidence of 7-OH-AAF-elicited abnormal neuropores from 45 to 19%, whereas coincubation with S9 from PB-induced rats increased the incidence to 77%. Coincubation with MC-induced S9 produced no statistically significant effect on APAP-elicited neuropore abnormalities but, with PB-induced S9, resulted in a significant increase from 60 to 86%. Additions of 3-OH-APAP (0.1-0.2 mM) but not N-acetyl-p-benzoquinoneimine (NAPQI, 0.1-0.5 mM) to the culture medium elicited the typical neuropore abnormality. Experiments with APAP and 7-OH-AAF as substrates demonstrated that embryonic enzymes catalyzed their conversion to the corresponding catechols. Considered together, the results provided evidence that embryonic conversion of APAP or 7-OH-AAF to the corresponding catechol metabolites may be instrumental in effecting the abnormal anterior neuropore closure observed after exposure of embryos to the respective parent compounds.

Embryotoxicity elicited by inhibition of γ-glutamyltransferase by Acivicin and transferase antibodies in cultured rat embryos☆

Acivicin (also known as AT-125) and IgG isolated from goat anti-gamma-glutamyltransferase antiserum were used to inhibit the activity of gamma-glutamyltransferase (GGT, EC 2.3.2.2) in rat conceptuses cultured from Days 10 to 11 of gestation. Inhibition of GGT by either Acivicin or anti-GGT IgG produced embryotoxicity and malformations, although each compound produced a unique spectrum of effects. Acivicin, at an initial concentration in the culture medium of 5 microM, produced a marked decrease in yolk sac vasculature and was associated with embryonic malformations such as neural tube necrosis, microophthalmia, and cephalic edema after 24 hr exposure. These malformations were accompanied by significant decreases in both embryonic and yolk sac protein, yolk sac GGT activity, as well as embryonic glutathione (GSH) levels. In contrast, anti-GGT IgG produced no apparent effects on yolk sac vasculature or protein after exposure of conceptuses to an initial concentration of 50 micrograms IgG/ml culture medium, even though equal inhibition of yolk sac GGT (30%) was achieved by each inhibitor. Exposure to IgG (50 micrograms/ml) for 24 hr was associated with decreased embryonic protein; decreased levels of GSH in the embryo were observed after both 3 and 24 hr. The dichotomy of effects on the yolk sac by the two compounds indicates that Acivicin produced these effects by mechanisms other than by GGT inhibition alone. These results demonstrate that inhibition of GGT in rat embryos undergoing organogenesis can elicit embryotoxic effects and produce alterations in GSH levels. The capacity of the anti-GGT antibody to inhibit the GGT activity in the yolk sac (while having no apparent effect on yolk sac morphology), and yet influence the embryo by decreasing protein and GSH levels, underscores the important role of the yolk sac during the highly sensitive stages of organogenesis.

Differential glutathione depletion by L-buthionine-S,R-sulfoximine in rat embryo versus visceral yolk sac in vivo and in vitro

L-Buthionine-S,R-sulfoximine (L-BSO), a selective inhibitor of glutathione synthesis, exhibited the capacity to deplete embryonic and visceral yolk sac glutathione (GSH) after administration in vivo and also after addition of L-BSO to a whole embryo culture system. Administration of L-BSO to pregnant dams at 2.5 or 18 hr prior to the explantation of day 10 conceptuses resulted in greater than 55% GSH depletion relative to control values in both the yolk sac and the embryo proper. The levels of GSH returned to normal in all embryos and in the corresponding visceral yolk sacs pretreated at 2.5 hr (but not at 18 hr) after culturing for 24 hr in L-BSO-free medium. GSH content was significantly lower in yolk sacs but not in embryos of conceptuses cultured for 24 hr in medium containing 1 mM L-BSO. This treatment, however, significantly increased the amount of protein and DNA in the embryo. The differential sensitivity of the yolk sac versus embryo and the demonstration of the ability to modulate tissue levels of GSH during organogenesis promise to provide important new tools for the study of embryonic protective mechanisms in chemical teratogenesis.

Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos

To examine the importance of reduced intracellular glutathione (GSH) in the modulation of dysmorphogenesis and to gain insight into the electrophilic character of the embryotoxic intermediates generated in the rat embryo from N-acetoxy-2-acetylaminofluorene (AAAF) and acetaminophen (APAP) in cultured embryos, the effects of GSH depletion on the embryotoxicity, dysmorphogenesis and covalent binding of these agents were examined. Both AAAF (90 microM) and APAP (500 microM) produced concentration-dependent, statistically significant (P less than or equal to 0.05) decreases in embryonic length as well as embryonic and visceral yolk sac protein content when rat embryos were exposed in vitro between days 10 and 11 of gestation. The predominant malformations observed upon exposure to AAAF and APAP were prosencephalic hypoplasia and abnormal neurulation respectively. Exposure of conceptuses to [3H]APAP followed by separation and fractionation of the cellular RNA, DNA and protein via density gradient centrifugation resulted in detectable binding in fractions that contained protein, but not DNA or RNA. This suggested that the rat conceptus is capable of bioactivating APAP to a soft electrophile that selectively arylates protein. In contrast, conceptuses exposed to [3H]AAAF exhibited detectable binding to RNA, DNA and protein, indicative of conversion to both hard and soft electrophiles. Depletion of GSH was accomplished by pretreating conceptuses with 500 microM L-buthionine-S,R-sulfoximine (BSO) from the start of the culture period (day 9.5) until the morning of day 10. When conceptuses were depleted previously of GSH by BSO, exposure to APAP resulted in significant potentiation (relative to APAP alone) of the observed embryotoxicity. These conceptuses displayed further decreases in both embryonic size and protein content of the embryo and yolk sac, as well as increased incidence of abnormally open anterior neuropores and increased binding (3-fold) of [3H]APAP to protein. In contrast, pretreatment with BSO did not potentiate the AAAF-elicited decreases in embryonic size or protein content, nor the severity of prosencephalic hypoplasia, although a slight increase in binding of [3H]AAAF to DNA was observed. Taken together, these data are consistent with the concept that abnormal neurulation elicited by APAP results from the generation of one or more soft electrophilic species, whereas elicitation of prosencephalic hypoplasia by AAAF appears to be a consequence of conversion to a relatively hard electrophile(s).

Cytochrome P450-dependent bioactivation of prodysmorphogens in cultured conceptuses

These investigations were undertaken to determine the extent to which tissues of cultured rat conceptuses contain cytochrome P450 isoforms in sufficient quantities to significantly influence the capacity of certain chemicals to elicit dysmorphogenic effects in vitro. Investigations with highly sensitive probe substrates/inhibitors and with immunologic methods enabled the detection of at least four separate P450 isoforms in tissues of the visceral yolk sac, ectoplacental cone, and embryo proper. One of the isoforms was identified as P450IA1 and was found to be inducible by polycyclic aromatic hydrocarbons in all three tissues. Other isoforms exhibited properties differing from characterized adult rat hepatic isoforms. Each of the isoforms was detectable in conceptuses on gestational days 10, 11, 12, and 14 and was present in the highest concentrations in the visceral yolk sac. Conceptal P450IA1 catalyzed the conversion of dysmorphogenically inactive 2-acetylaminofluorene to 7-hydroxy-2-acetylaminofluorene, a proximate dysmorphogen. Investigations with microinjections suggested that visceral yolk sac hydroxylation was largely responsible for the bioactivation reaction in vitro. The same isoform exhibited no capacity to influence the dysmorphogenic activity of cyclophosphamide. The results demonstrated that tissues of cultured rat conceptuses may contain P450 isoforms in sufficient amounts to markedly influence the dysmorphogenic activity of substrates of the corresponding isoforms.