Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death.

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four-week treatment of male rats with a low dose of cyclophosphamide causes a dramatic, dose-dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague-Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark-stained pyknotic nuclei. Very few or no inner cell mass-derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass-derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide-treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.     

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

Effect of feeding pattern on the sensitivity of hepatic carnitine palmitoyl-transferase to inhibition by malonyl-CoA in the rat

1. The feeding pattern influences the inhibitory effects of malonyl-CoA on carnitine palmitoyltransferase-I. 2. The sensitivity of liver carnitine palmitoyltransferase-I to malonyl-CoA is increased in rats meal-fed when compared to rats fed ad libitum. 3. Moreover, liver carnitine palmitoyltransferase-I of meal-fed rats remains more sensitive to inhibition by malonyl-CoA during a 24 hour fast than liver carnitine palmitoyltransferase-I of rats previously fed ad libitum.     

Application of an epithelial liver cell line, metabolically competent, for mutation studies of promutagens.

Recently numerous attempts have been made to reduce the use of vertebrate animals in laboratory experiments to evaluate general and acute toxicity, mutagenesis and teratogenesis of new drugs or chemicals. One common approach is to use established, proliferating cell lines that preserve differentiated functions such as the competence to metabolize xenobiotics. To this end a continuous Chinese hamster epithelial liver cell line (CHEL cells) was established, cultured as used for mutagenesis studies. Structurally different promutagens, such as 7,12-dimethylbenz[a]anthracene (7,12-DMBA), benzo[a]pyrene (B(a)P), aflatoxin B1 (AB1) and cyclophosphamide (CP), were used in order to check and validate the test system. anti-Chrysene-1,2-diol 3,4-epoxide (CDE) and mitomycin C (MMC) were taken as representatives of direct mutagens. The genetic change induced by the mutagens was quantified by measuring mutation frequencies at the HGPRT locus. Several parameters, such as mutant expression time for each chemical, cell density for selection of mutants and enzymatic characterization for HGPRT phenotype, were examined to establish the optimal assay conditions. All promutagens analyzed significantly affected either the cloning efficiency and/or the mutant frequency of CHEL cells after 24 h of exposure. In addition, various enzyme activities involved in the metabolism of the promutagens were determined in CHEL cells, under the experimental conditions of chemical exposure used in the mutagenesis assay. The enzyme activities were compared with those found in uninduced Chinese hamster liver.