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B F Hales

Publications and source records attributed to B F Hales.

At least 37 records · Page 2Linked to original sources
PubMed1995

Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa.

Previous studies from our laboratory have shown that chronic exposure of the father to low doses of cyclophosphamide results in a significant increase in early embryonic death with little effect on the male reproductive system in rats. Such effects on progeny outcome are hypothesized to be mediated by an action of the drug on the nucleus of spermatozoa. The purpose of the present studies was to investigate the effects of cyclophosphamide treatment for 1 or 6 wk on the pattern of decondensation of sperm nuclei and on the sulfhydryl content of sperm nuclear proteins. Adult male rats were treated with cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected, demembranated, and then incubated with dithiothreitol (DTT) and proteinase K. The in vitro decondensation pattern of the nuclei of spermatozoa was divided into two phases: nuclear swelling and nuclear elongation. Spermatozoa from animals treated for 1 wk with cyclophosphamide showed the same decondensation pattern as those treated with vehicle (saline) alone. However, spermatozoa from animals treated for 6 wk with cyclophosphamide showed normal initial nuclear swelling but had a markedly affected nuclear elongation pattern. The changes with time in the decondensation pattern of these spermatozoa were quantitated by morphometric analysis of the head region of the spermatozoa. The nuclear area, curvature, and length of spermatozoa obtained from chronically drug-treated males were all significantly smaller than for those obtained from controls, while cell diameter was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Antisense oligonucleotide down-regulation of E-cadherin in the yolk sac and cranial neural tube malformations.

The cadherins are a family of calcium-dependent cell adhesion molecules that are regulated both spatially and temporally during development. Epithelial cadherin (E-cadherin) is present in epithelial cells in both the embryo and yolk sac during organogenesis. The consequences of disrupting the expression of E-cadherin at this stage of development are poorly understood. We report here our studies on the effects of antisense oligonucleotides on E-cadherin in the rat whole embryo culture system. Four 18-base single strand phosphorothioate oligodeoxynucleotides (AS-oligos), complementary to various regions of the mouse E-cadherin cDNA sequence, were dissolved in saline and injected into the amniotic cavities of 5-7 somite rat embryos; a sense (S-oligo) to oligo-1, an 18-base random sequence oligo (C-oligo), and PBS were used as controls. Embryos were cultured for up to 45 h; embryo morphology and the relative concentrations of E-cadherin protein were examined. All six oligonucleotides (AS-oligos and control oligos) induced malformations when amounts ranging from 25 to 50 pmol of oligonucleotide were injected per embryo. The malformations induced by all the oligos included craniofacial hypoplasia, an enlarged pericardium, twisted spinal cord, swelling of the rhombencephalon, and underdeveloped forelimb. Injection of AS-oligo-1, a sequence starting at the tenth base downstream from the translation initiation codon (ATG), resulted in malformed embryos with a high incidence of cranial neural tube malformations. The effects of AS-oligo-1 on the relative abundance of E- and neural (N)-cadherin proteins were examined by Western blot analysis. In the AS-oligo-1-exposed malformed embryos, the relative abundance of E- and N-cadherin proteins was not altered up to 24 h after injection; E- and N-cadherin concentrations in the embryo were decreased at 45 h postinjection. In contrast, the relative abundance of the E-cadherin protein in the yolk sac was reduced at 1-2 h after injection of AS oligo-1 and returned to control levels by 4 h. S-oligo-1 did not induce any change in the relative abundance of E- or N-cadherins. Thus, there was a tissue-specific and temporary "knockdown" of E-cadherin expression in the yolk sac of embryos exposed to antisense (AS-oligo-1); the down-regulation of yolk sac E-cadherin appears to lead to the induction of neural tube defects in the embryo. The exposure of whole embryos in culture to antisense oligonucleotides provides a model system in which the roles of developmentally important molecules and their spatial and temporal contributions to embryogenesis can be elucidated.

Animals↗
PubMed1995

Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure.

Treatment of male rats with low dosages of cyclophosphamide causes a dramatic increase in early embryo death among their progeny without significantly affecting the general health of the male. It is hypothesized that cyclophosphamide exerts its effects by targeting specific components of spermatozoal nuclei. The purpose of the present studies was to investigate the effects of chronic cyclophosphamide treatment on spermatozoal DNA. Two approaches were pursued. The first was to determine total DNA damage by using the alkaline elution method. The second was to study spermatozoal DNA template function by using an in vitro DNA synthesis system. Adult male rats were treated with saline or cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected and subjected to alkaline elution using DNA-DNA dot hybridization to quantify the fractionated DNA. One week of treatment with cyclophosphamide caused DNA single strand breaks that could be detected only in the presence of proteinase K in the lysis solution; no DNA cross-links were observed in the animals that received 1-wk drug treatment. In contrast, 6 wk of treatment with cyclophosphamide induced a significant increase in both DNA single strand breaks and cross-links in spermatozoal nuclei; the cross-links were attributable primarily to DNA-DNA linkages. The availability of spermatozoal DNA for template function was not affected by 1 wk of treatment with cyclophosphamide but was markedly affected after 6 wk of treatment with this drug. It is proposed that during chromatin transition processes the male genome may be in an open dynamic state with many exposed sites that are vulnerable to alkylating agents. Since there is no DNA repair during spermiogenesis, damage to the genome by alkylation at this stage may be cumulative, resulting in the production of dysfunctional germ cells.

Animals↗
PubMed1994

Regulation of the Yp subunit of glutathione S-transferase P in rat embryos and yolk sacs during organogenesis.

Manipulation of the glutathione status of an embryo during organogenesis leads to abnormal development, as well as increasing the susceptibility of the embryo to insult by either xenobiotic or endogenous electrophiles. The glutathione S-transferases are a family of drug-metabolizing enzymes that catalyze the conjugation of reactive chemicals with glutathione, playing an important role in protecting cells against attack. The purpose of this study was to investigate the presence and regulation of one glutathione S-transferase, glutathione S-transferase P, a homodimer of the Yp subunit, in the conceptus during organogenesis. Northern blot analysis of the RNA isolated from rat embryos and their yolk sacs on days 10, 11 and 12 of gestation revealed a single Yp transcript. Steady-state concentrations of the Yp mRNA in embryos did not change with either gestational age or culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). In contrast, concentrations of this transcript in yolk sac increased 3-fold from day 10 to 12 of gestation and a further 3-fold with culture (day 12 in vivo compared with in vitro). Transcription of the rat Yp subunit gene in cell lines is induced by treatment with phorbol esters. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 or 100 nM) to embryos in culture had no effect on the steady-state concentrations of the Yp transcript. Thus, the glutathione S-transferase Yp message is subject to tissue- and development-specific regulation in the conceptus during organogenesis. Moreover, culture of the embryos resulted in a further up-regulation of the steady-state concentrations of the Yp transcript in yolk sac. Western blot analysis demonstrated that a single immunoreactive Yp subunit band of 26 kDa was found in both embryos and yolk sacs. Neither age nor culture appeared to affect the concentrations of immunoreactive Yp subunit in the yolk sac. Thus, glutathione S-transferase Yp mRNA is translated in the conceptus during organogenesis. The apparent differences between the relative amounts of the message and immunoreactive protein in yolk sac suggest that this subunit may be subject to post-transcriptional as well as transcriptional regulation in this tissue. Immunohistochemical analysis of embryos cultured for 45 hr (day 12 in vitro) revealed that the Yp reaction product was localized over the hepatic primordia, mesonephric ducts, otocyst, yolk sac and ectoplacental cone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Role of apoptosis in mediating phosphoramide mustard-induced rat embryo malformations in vitro.

Phosphoramide mustard, an active metabolite of the anticancer drug cyclophosphamide, causes malformations in rat embryos undergoing organogenesis in vitro. The purpose of the present study was to investigate the hypothesis that apoptosis plays an important role in mediating the teratogenicity of phosphoramide mustard. Apoptosis is a process of active or programmed cell death which is characterized by internucleosomal DNA fragmentation and de novo RNA and protein synthesis. Sulphated glycoprotein-2 (SGP-2) or clusterin is induced in some models of apoptosis and is one of the proteins likely to be involved in the maintenance of cell integrity. In the present study, day 10 rat embryos were cultured for 6, 12, 24, and 45 hr, with or without the addition of 10 microM phosphoramide mustard. After culture for 24 or 45 hr with exposure to 10 microM phosphoramide mustard, the embryos were both growth-retarded and malformed. Exposure to phosphoramide mustard for 6 or 12 hr did not significantly alter the relative amounts of either the mRNA or protein for SGP-2; this treatment also had no effect on DNA fragmentation in embryos or their yolk sacs. After 24 hr in culture, the relative amounts of SGP-2 protein, but not mRNA, were increased 2-fold in the yolk sacs of the phosphoramide mustard-exposed embryos, but not in the embryos themselves. At this time, DNA fragmentation was detected in phosphoramide mustard-exposed embryos, but not in their yolk sacs or in control embryos. After 45 hr in culture, SGP-2 protein and mRNA levels were increased 2-4-fold above the controls in the phosphoramide mustard-exposed embryos and their yolk sacs. Immunohistochemical analysis revealed that in control embryos cultured for 45 hr, the SGP-2 reaction product was localized in the heart, hindgut, and yolk sac. In contrast, in phosphoramide mustard-treated embryos cultured for 45 hr, SGP-2 immunostaining was found throughout the embryo, with a strong immunoreaction in the mesenchyme and ectoplacental cone. DNA fragmentation in the embryos exposed to phosphoramide mustard for 45 hr was more extensive than that found after 24 hr, but fragmentation was still not detected in the yolk sac. Thus exposure in vitro to a teratogenic concentration of phosphoramide mustard resulted in DNA fragmentation and an increased expression of SGP-2 in the embryo. These data suggest that apoptosis is involved in mediating the teratogenicity of phosphoramide mustard.

Abnormalities, Drug-Induced↗
PubMed1994

12-O-tetradecanoyl-phorbol-13-acetate-induced rat embryo malformations in vitro are associated with an increased relative abundance of embryonic E-cadherin mRNA.

Epithelial-cadherin (E-cadherin) is a member of a family of Ca(2+)-dependent cell adhesion molecules which are localized in zonulae adherens and play an important role during development. E-cadherin is abundant in rat embryos and their yolk sacs during organogenesis. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been reported to disrupt the morphology and functional development of the rat embryonic visceral yolk sac. The present study investigated the possibility that the effect of TPA on yolk sac development may be due to the altered expression of E-cadherin. Rat embryos, with their yolk sacs intact, were cultured on day 10 of gestation for 1 hr. At this time the vehicle, dimethyl sulfoxide (DMSO), or TPA (at different concentrations) was added to the culture medium; the cultures were continued for up to 24 hr. Embryos and yolk sacs were collected separately at the end of each culture period. The relative abundances of E-cadherin mRNA and protein were analyzed with Northern and Western blot analyses. Despite the TPA-induced abnormalities in yolk sac development, the relative abundance of E-cadherin mRNA or protein in the yolk sac was not altered by TPA exposure. However, in embryos exposed to dysmorphogenic concentrations of TPA, the relative abundance of E-cadherin mRNA was significantly increased after 24 hr in culture, compared to either controls or embryos exposed to non-dysmorphogenic concentrations of TPA. The magnitude of the increase in embryonic E-cadherin mRNA appeared to correlate with the severity of the embryo malformations.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMed1994

Cadmium-induced rat embryotoxicity in vitro is associated with an increased abundance of E-cadherin protein in the yolk sac.

Cadmium, a cytotoxic heavy metal, is embryotoxic. Cadmium interferes with the functions of other cations such as zinc and calcium. Cadherins, calcium-dependent cell adhesion molecules, are expressed and spatiotemporally regulated in embryos and their yolk sacs during organogenesis. Cadmium has been shown to disrupt calcium-dependent cell-cell interactions and to alter the distribution of epithelial or E-cadherin in cultured cells. The purpose of this study was to determine whether the embryotoxicity of cadmium is mediated through an effect on E-cadherin. Day 10 rat embryos were cultured for 1 hr without cadmium and then cultured for another 2, 6, or 20 hr in the presence or absence of 2.5 microM CdCl2. Embryos and yolk sacs were collected and analyzed separately. The growth of embryos and yolk sacs after exposure to cadmium for 2 or 6 hr was not different from that of controls. After 20 hr exposure to cadmium, embryos were growth retarded, with morphological scores 10-15% lower than those of controls; more dramatically, their yolk sacs were thickened and decreased in diameter to only half of that of control yolk sacs. Northern blot analysis revealed no significant differences in the relative amounts of E-cadherin mRNA between control and cadmium-treated embryos or their yolk sacs. There was also no alteration in the abundance of E-cadherin protein in cadmium-treated embryos at any of the times examined. There was a gradual decline in the relative amount of E-cadherin protein in control yolk sacs with time in culture; interestingly, cadmium treatment appeared to prevent this decline, resulting in significantly higher concentrations of E-cadherin protein in the cadmium-exposed yolk sacs after 6 hr (1.7-fold) or 20 hr (2.3-fold) of culture. The relative abundance of other structural proteins such as alpha- or beta-tubulin and actin was unchanged. Exposure of embryos for 20 hr to concentrations of cadmium varying from nonembryotoxic to embryotoxic resulted in concentration-dependent increases in E-cadherin protein in the yolk sac; E-cadherin was only increased in the yolk sac of growth-retarded embryos. Thus, an increase in yolk sac E-cadherin protein is associated with the induction of embryotoxicity by cadmium. To determine the ability of cadmium to interact directly with the cadherins, the binding of radioactive 109Cd to embryo and yolk sac proteins was assessed. 109Cd bound to an unidentified 87-kDa protein; binding to this protein was attenuated by 1 mM zinc, but not by 1 mM calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1994

Paternal cyclophosphamide exposure causes decreased cell proliferation in cleavage-stage embryos.

Exposure of the male germ cell to cyclophosphamide during spermatogenesis and sperm maturation can interfere with development of the embryo. When male rats were treated with a chronic low dose of cyclophosphamide for 4 wk there was a dramatic increase in early postimplantation loss in their progeny, characterized by implantation sites selectively lacking in embryonic tissues. The present study was designed to determine the earliest appearance of a paternal effect of cyclophosphamide treatment and to examine whether the embryonic lineage was selectively affected. Male Sprague-Dawley rats were orally dosed for 4-5 wk with saline or 6 mg/kg per day of cyclophosphamide; their progeny were obtained on Days 2, 2.5, 3, 4, and 4.5 of gestation. Paternal cyclophosphamide treatment had no effect on the mean number of embryos per pregnant female. However, as early as Day 3 of gestation, there was a significant decrease in cell number among the embryos sired by cyclophosphamide-treated males, increasing to a greater than 50% decrease in cell number by Day 4. The cell doubling time in embryos sired by treated males (16 h) was longer than that of controls (12 h). This decreased proliferation rate was confirmed by a dramatic decrease in the capacity of both Day 3 and Day 4 embryos sired by cyclophosphamide-treated males to incorporate [3H]thymidine over a 26-h culture period. Cytogenetic analysis in a limited number of blastomeres entering metaphase revealed no evidence of chromosomal abnormalities. Both the trophectoderm and the inner cell mass cells were proportionally decreased in Day 4.5 embryos sired by cyclophosphamide-treated males. Thus, paternal cyclophosphamide exposure affected both cell lineages in the conceptus as early as Day 3 of gestation.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Increased postimplantation loss and malformations among the F2 progeny of male rats chronically treated with cyclophosphamide.

Cyclophosphamide, administered to the male rat, produces increased pre- and postimplantation loss in the progeny as well as an increase in the numbers of malformed and growth retarded fetuses. The purpose of this study was to determine whether the adverse effects of chronic paternal cyclophosphamide exposure are transmissible to the next generation, the F2 progeny. Adult male rats were treated by gavage daily with saline or with cyclophosphamide (3.4 or 5.1 mg/kg) for 4 or 18 weeks and mated. The male and female offspring in each treatment group (F1 generation) were randomly mated. The resulting pregnant females were killed on day 20 of gestation to evaluate progeny outcome in the F2 generation. There was a significant increase in postimplantation loss among the offspring of the group whose fathers had been treated with cyclophosphamide at a dose of 5.1 mg/kg/day. Exposure to a dose of 5.1 mg/kg/day of cyclophosphamide also resulted in an F2 generation with a significantly decreased mean fetal weight per litter and a significant increase in the number of malformed fetuses. The malformations observed among the F2 progeny included open eyes, omphalocele, generalized edema, syndactyly, gigantism, and dwarfism. Thus, exposure of the father to cyclophosphamide does result in a specific and heritable alteration in the fertility of the surviving "apparently normal" F1 progeny. Interestingly, the adverse consequences of exposure of male rats to cyclophosphamide are similar in the F2 generation to those previously reported for the F1 progeny.

Animals↗
PubMed1992

Genetic differences in heat-induced tolerance to cadmium in cultured mouse embryos are not correlated with changes in a 68-kD heat shock protein.

Heat-induced cross-tolerance to cadmium was investigated in two inbred strains of mice, BALB/c and SWV, using a whole embryo culture system. Embryos were exposed to a pretreatment of 5 min at 43 degrees C and subsequently to an embryotoxic concentration of cadmium, 1.75 microM. The two types of embryos responded differently to the heat pretreatment, as cross-tolerance was induced in SWV but not in BALB/c mice. In SWV embryos, prior exposure to 43 degrees C for 5 min essentially eliminated the negative effects of cadmium on embryonic development and growth. However, in BALB/c embryos, no protection was observed. The variation in development of cross-tolerance in embryos from the two strains of mice was not correlated with differences in the induction of a 68-kD heat-shock protein (hsp68). There was a rapid increase in this protein in both strains after the initial heat exposure but not excess induction in the SWV strain that developed tolerance. The induction of hsp68 is therefore not sufficient to elicit cross-tolerance, and other mechanisms are likely to be important in the protective response of the embryo.

Animals↗
PubMed1992

Adverse effects of cyclophosphamide on progeny outcome can be mediated through post-testicular mechanisms in the rat.

Previous studies from our laboratory have suggested that, in addition to an effect on spermatozoa in the testis, cyclophosphamide may have an adverse effect on spermatozoa after they leave the testis, during epididymal transit. To elaborate on this post-testicular effect on germ cells and to determine at which site(s) in the epididymis germ cells are most sensitive to cyclophosphamide treatment, three experiments were undertaken. First, the time course of the effect of treatment of male rats with cyclophosphamide on the outcome of their progeny was determined. Male rats were treated daily by gavage with saline or one of two doses of cyclophosphamide (6.8 mg/kg or 10.0 mg/kg) for 1, 4, or 7 days. At the end of each treatment period, males were mated to assess the effect on pregnancy outcome. No effect was observed on pre-implantation loss at any time among any of the groups, but there was a time-dependent and dose-related increase in post-implantation loss. Post-implantation loss was significantly increased after 4 days of treatment and reached nearly 40% after 7 days of drug exposure (10.0 mg/kg). Second, the effect of treatment with single high doses of cyclophosphamide was studied. Male rats were treated with a single dose of cyclophosphamide (10, 30, or 70 mg/kg) and bred 1 day and 4 days post-treatment. No significant change in pre-implantation loss was observed at either time point; no change in post-implantation loss was found after 1 day post-treatment. However, a significant increase in post-implantation loss was observed in the two high-dose groups 4 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Heat-shock induced tolerance to the embryotoxic effects of hyperthermia and cadmium in mouse embryos in vitro.

Mammalian embryos growing in vitro are harmed by short elevations in the culture temperature. However, a relatively mild hyperthermic exposure can induce thermotolerance, a transient state of resistance to the effects of a subsequent heat exposure. The present study examines the induction of tolerance to heat and cross-tolerance to another teratogen, cadmium, in day 8 CD-1 mouse embryos in vitro. The ability of a mild heat pretreatment (5 min at 43 degrees C) to partially protect embryos against an embryotoxic heat exposure (20 min at 43 degrees C) was demonstrated. The frequency of death was reduced from 43% to 20%, abnormal branchial arches from 44% to 13.2%, and retarded turning from 22% to 5% in pretreated embryos. Other malformations, such as small forebrains and microphthalmia, were not affected, and the rate of exencephaly was significantly increased. The same heat pretreatment (5 min at 43 degrees C) was also found to reduce the damaging effects of a subsequent exposure to 1.75 microM cadmium. In the absence of pretreatment, cadmium caused 55% embryo deaths and 87% malformations, but prior heat exposure caused significant reductions in these frequencies to 29% and 55%. The total morphological score was higher in the pretreated group, as were the measurements of the yolk sac diameter, crown-rump length, and head length. Thus, embryos that have developed resistance to hyperthermia are also partially protected against the harmful effects of a second teratogen, cadmium. The response of the embryo to elevated temperatures may be involved in the development of tolerance to a variety of stresses.

Abnormalities, Drug-Induced↗
PubMed1991

The effect of in vivo glutathione depletion with buthionine sulfoximine on rat embryo development.

Glutathione is an abundant endogenous nucleophile whose depletion may have adverse effects on a number of cellular processes, including protein and DNA synthesis, amino acid transport, and detoxification of reactive electrophiles. Previous studies have indicated that a certain basal level of glutathione is essential for normal development in vitro in the whole rat embryo culture system. The objective of this study was to determine the effect of glutathione depletion with buthionine sulfoximine on the development of rat embryos in vivo. Timed pregnant Sprague-Dawley rats were treated by gavage on days 10 and 11 of gestation with saline (control) or with L-buthionine-(S,R)-sulfoximine (4 or 8 mmol/kg). Pregnancy outcome was assessed on day 12 or day 20 of gestation. The glutathione content of embryos and of maternal organs was measured on day 12. Glutathione concentrations were significantly decreased in the embryos and in maternal muscle and ovary but not in maternal liver and kidney. Glutathione depletion, when assessed on day 12 of gestation, was found to result in an increase in the numbers of dead and malformed embryos. Treatment with the higher dose of buthionine sulfoximine resulted in the death of 13.2% of the total implanted embryos; of the surviving embryos, 21.7% were malformed. There was also a significant decrease on day 12 after treatment with the higher dose of buthionine sulfoximine in the embryonic total morphological score, a measure of the developmental stage of the embryos (control, 57.7 +/- 1.0; 8 mmol/kg buthionine sulfoximine, 52.8 +/- 1.8).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.

Animals↗
PubMed1990

Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats.

Low-dose chronic treatment of the male rat with the antitumor drug cyclophosphamide causes a time- and dose-dependent increase in pre- and post-implantation loss in the untreated females to which he is mated. The objective of the present study was to determine whether such effects are reversed, and if so at what time after cessation of drug treatment. Adult male Sprague-Dawley rats were gavage fed daily, 6 times per week for 9 weeks, with saline (control) or with 1 of 3 doses of cyclophosphamide, 1.4, 3.4 or 5.1 mg/kg/day. After the 9 weeks of treatment and at 2-week intervals thereafter, each male was mated with 2 females in proestrus. The females were caesarian sectioned 20 days later and pregnancy outcome assessed. After 9 weeks of drug treatment, pre-implantation loss increased more than 3-fold from 6% in the control group to 21% in the 5.1 mg/kg/day cyclophosphamide treatment group. Post-implantation loss increased in a dose dependent fashion from 5% in the control group to 74% in the 5.1 mg/kg/day cyclosphosphamide treatment group. Pre-implantation loss rapidly decreased upon cessation of treatment with cyclophosphamide: within 2 weeks it had returned to within the control range. Within just 2 weeks after termination of drug treatment in the 5.1 mg/kg/day cyclophosphamide treatment group, post-implantation loss decreased by half to 44%; it had decreased to 11% by 4 weeks and then was maintained at 4-6% thereafter. In the 3.4 mg/kg/day cyclophosphamide treatment group, post-implantation loss returned to the control range by 4 weeks. Thus, the effects of paternally administered cyclophosphamide on progeny outcome are reversible. The timing of reversal suggests that the effects on pre-implantation loss are due to a drug effect on spermatozoa either in the epididymis or near the time of spermiation while those on post-implantation loss are due to an additional effect on spermatids in the seminiferous tubules.

Abnormalities, Drug-Induced↗
PubMed1989

Embryotoxicity of phenyl ketone analogs of cyclophosphamide.

Phenylketophosphamide and phenylketoisophosphamide are preactivated acyclic ketone analogs of cyclophosphamide and isophosphamide with antitumor activity. These compounds undergo an elimination reaction to yield phosphoramide or isophosphoramide mustard and phenyl vinyl ketone. In this study, the embryotoxicity of phenylketophosphamide, phenylketoisophosphamide, and phenyl vinyl ketone were determined. Embryotoxicity was assessed in vitro in whole rat embryos cultured on day 10.5 of gestation in the absence and presence of an activating system derived from maternal liver. Both phenylketophosphamide and phenylketoisophosphamide were embryotoxic in the absence of metabolic activation. Moreover, there was no enhancement of this embryotoxicity in the presence of an activating system. A 10-microM concentration of phenylketophosphamide produced 100% malformed embryos, while this concentration of phenylketoisophosphamide was not teratogenic. At 25 microM phenylketoisophosphamide, all the surviving exposed embryos were malformed. Phenylketophosphamide was embryolethal to more than 50% of the exposed embryos at a concentration of 50 microM. In contrast, a concentration of phenylketoisophosphamide of 100 microM was required to produce significant embryolethality. Phenyl vinyl ketone was not embryotoxic at any of the concentrations tested. The major malformation observed, a hypoplastic prosencephalon, and the growth retardation effects were not only similar for phenylketophosphamide and phenylketoisophosphamide, but also similar to those previously reported for "activated" cyclophosphamide. Unlike the results with cyclophosphamide, where both phosphoramide mustard and the aldehydic metabolite of cyclophosphamide, acrolein, are toxic, the embryotoxic effects of phenylketophosphamide and phenylketoisophosphamide are mediated only by the mustard metabolite.

Animals↗