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R G Skalko

Publications and source records attributed to R G Skalko.

At least 19 recordsLinked to original sources
PubMed1993

Occurrence of embryotoxicity in mouse embryos following in utero exposure to 2'-deoxycoformycin (pentostatin).

Previous investigations had shown that i.p. injection of 2'-deoxycoformycin (dCF; pentostatin; 5 mg/kg) on either E7 or E8 into pregnant mice results in a 61-81% resorption rate at E17. The incidence of visible gross malformations among the surviving conceptuses was exceptionally low (3%) at the time of necropsy on E17 and was unrelated to dCF dose (Knudsen et al., Teratology, 40:5-626, '89; Teratology, 45:91-103, '92). These findings demonstrated the embryotoxicity of dCF but provided no clues as to the site(s) of dCF action. To define the lesion site(s), we have now examined embryos at 72 h (E10), 96 h (E11), and 120 h (E12) following administration of a highly embryotoxic dose of 5 mg dCF/kg to dams on E7. Deoxycoformycin caused multiple abnormalities and growth retardation, and the temporal sequence between maximal abnormal embryo incidence and resorption frequency was established. The quantitative data show that the maximal occurrence of abnormal embryos on E10 (71%) was followed by a maximal resorption rate on E12 (78%). There was a strong correlation (r = -0.82; P < 0.05) between the rapid decline of percent abnormal embryos over E10-E12 and the simultaneous increase in resorption rate, with linear regression analysis showing nearly equal but opposite slopes (-31.2% vs. +35.8% per gestational day, respectively). This suggests that one or more of the abnormalities seen at E10 is associated with the death and resorption of the embryo at E12. The dCF treatment perturbed a wide spectrum of developmental events, including neural tube closure, craniofacial and limb development, turning of the embryo, and growth retardation. None of the individual abnormalities, however, can quantitatively account for the high percentage of dead and resorbed embryos. Therefore, the specific cause of dCF-induced embryolethality is not clear. There is evidence both for direct dCF toxicity at specific embryonic sites as well as for a generalized retardation in the rate of development.

Abnormalities, Drug-Induced↗
PubMed1993

Reproductive and developmental toxicity of the components of gasoline.

The reproductive, developmental, and postnatal toxicity of 14 select chemicals and mixtures that are components of gasoline has been reviewed. The majority of experimental analyses have been performed as either variations of the accepted segment 2 protocol or as traditional teratology studies. Specific deficiencies in the present database have been identified and are most obvious in the evaluation of reproductive and postnatal effects. It is recommended that future studies address the continuing need for assessment in multiple species and over a range of dosages with specific emphasis on the impact of route of administration on the results obtained.

Animals↗
PubMed1992

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

5'-Nucleotidase↗
PubMed1992

Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse.

The viability of early mouse embryos is acutely sensitive to (R)-deoxycoformycin (pentostatin), a tight-binding inhibitor of adenosine deaminase (ADA). Previous studies have shown that a single 5-mg/kg dose on day 7 (plug = day 0) of gestation fully inhibits uteroplacental ADA activity within 0.5 h; causes massive cell death in the neural plate and primary mesenchyme by 6 h, major craniofacial anomalies by day 10, and resorption by day 12 (Knudsen et al., '89; Airhart et al., '91). The present study has examined further the developmental toxicity and early effects of this inhibitor on ADA metabolism. (R)-Deoxycoformycin was administered to pregnant CD-1 (ICR) mice as a single intraperitoneal dose of 0.5-10 mg/kg total body weight on days 6-11 of gestation. The major adverse effect, early resorption, was dose dependent and specific to day 7-8 exposure. Treatment with 5 mg/kg on day 7 resulted in 85% resorptions, 15% malformations, and a 24% reduction in mean fetal weight, whereas the same dose of (S)-deoxycoformycin had no effect. Levels of adenosine and 2'-deoxyadenosine, which are the endogenous substrates of ADA, were monitored in the embryo/decidual unit (E/D) by reversed-phase high-performance liquid chromatography (RP-HPLC). In response to the inhibitor, both nucleosides increased transiently in the antimesometrial compartment (antimesometrial decidua + embryo). Peak levels (Cmax) of adenosine and 2'-deoxyadenosine were dose dependent over the range tested (0.05-10 mg/kg). Exposure to 5 mg/kg on day 7 raised adenosine levels within 0.5 h to 42-fold over the basal level of 0.06 nmol/mg protein. There was an even stronger effect on 2'-deoxyadenosine levels, which were elevated 674-fold over the detection limit of 0.0005 nmol/mg protein. Direct exposure to the inhibitor in serum-free E/D culture produced similar results: 50 microM (R)-deoxycoformycin within 1 h raised adenosine levels 26-fold and 2'-deoxyadenosine levels 410-fold. In vivo studies also showed a general correlation between embryolethality and the length of adenine nucleoside pool expansion, apparent for exposure on day 7, 8, or 9 but not on day 6, suggesting that the embryo becomes sensitive to adenosine or 2'-deoxyadenosine once the neural plate has formed.

Adenosine↗
PubMed1990

Axonal guidance of adenosine deaminase immunoreactive primary afferent fibers in developing mouse spinal cord.

This study examined the precision of central fiber growth in a subpopulation of dorsal root ganglion neurons in developing mouse spinal cord. Immunohistochemical techniques using a monospecific, polyclonal antiserum to mouse adenosine deaminase (ADA) were utilized to label a population of primary sensory afferents that have been found to exclusively innervate laminae I and II of the dorsal horn in adult mice. Initial growth of ADA-immunoreactive (ADA-IR) primary afferents occurred very early in development, embryonic day 10 (E10), a time coincident with the earliest settling time of dorsal root ganglion neurons. Adenosine deaminase immunoreactive primary afferents were observed throughout the cross-sectional area of the primordial dorsal funiculus (DF) as early as E10. Immunostained fibers remained quiescent in the DF during its growth and separation into the tract of Lissauer and dorsal column pathway. By E15, the two pathways had formed and ADA-IR fibers were observed exclusively in the tract of Lissauer. This segregation of fibers remained throughout development and reflected the adult pattern. Growth was reinitiated at E16 when the fibers advanced into the dorsal horn and proceeded directly to laminae I and II mimicking their adult distribution. Exuberant fiber growth was not detected throughout their development. These results strongly suggest that ADA-IR fibers exhibit precise fiber guidance to a preferred pathway, the tract of Lissauer, and accurate laminar innervation of the dorsal horn.

Adenosine Deaminase↗
PubMed1989

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin.

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.

Adenosine Deaminase↗
PubMed1989

Pharmacological concepts and developmental toxicology.

This communication provides evidence to support the concept that developmental toxicants (teratogens) produce their effect by either interfering with or enhancing the time-dependent signal-response mechanisms within the embryo. Essential to this hypothesis is the need to show that an observed effect is a function of the administered dose, that there is a positive correlation between the observed effect and pharmacokinetic parameters and that there is evidence for the existence of a specific receptor for the toxicant. While extensive effort is required for ultimate validation of this concept, it serves to emphasize the value of applying known pharmacological principles in defining a mechanistic framework for the biological activity of developmental toxicants.

Animals↗
PubMed1987

Consensus workshop on the evaluation of maternal and developmental toxicity work group I report: end points of maternal and developmental toxicity.

It was concluded that this conference, sponsored by the EPA, has provided an opportunity to state current ideas regarding the utility of data from studies in developmental toxicology and that the deliberations have highlighted the need to consider fundamental relationships between maternal and developmental effects in a more comprehensive manner in the future.

Animals↗
PubMed1986

Retinoic acid enhances and depresses in vitro development of cartilaginous bone anlagen in embryonic mouse limbs.

Forelimbs of Day 11 and Day 12 embryonic mice were excised and cultured for 3 d in the presence of either 0.25 microgram (8 X 10(-7) M), 0.5 microgram (1.7 X 10(-6) M), or 1.0 microgram (3.3 X 10(-6) M) of all-trans retinoic acid (RA) per milliliter of culture medium. Cultured limbs were fixed, stained, and mounted whole on glass slides and evaluated with computerized optical image analysis for RA-induced effects on the area and shape of the total limb and individual bone anlagen. Relative effects of RA on total bone, soft tissue, long bone, and paw regions were also examined. With Day 11 forelimbs total bone area was increased by 10.5% by the low dose of RA. The increase was mostly in long bones and at the expense of soft tissue. Total bone area was increased 9.3% with Day 12 forelimbs. This increase was primarily in the paw. The high dose of RA decreased Day 11 forelimb area, primarily affecting long bones. Day 12 forelimbs were not significantly affected by the high dose of RA. Effects of the intermediate dose were primarily limited to reduction in soft tissue area. Long bone:paw and soft tissue:bone ratios reflected these effects. The high dose produced a consistent rounding or shortening of Day 11 forelimb bones. On Day 12 0.5 microgram/ml RA produced an inconsistent pattern of rounding of bone anlagen. Treatment with the high dose on Day 12 produced angular rather than rounded contours in many cases, as indicated by shape factor values closer to zero than obtained with controls. These data show that direct exposure to RA can affect both the size and shape of bone anlagen of the developing limb; the low dose enhances and the high dose depresses development. The results support previous studies which suggest that RA may play a critical role in the control of cell activities such as cell migration, proliferation, and cytodifferentiation in the development of the cartilaginous bone anlagen.

Animals↗
PubMed1985

The teratogenic interaction of hydroxyurea and 5-bromodeoxyuridine examined with the aid of limb culture and image analysis.

Pregnant mice were treated on gestation Day 11 (E11) with a single dose of either hydroxyurea (HU, 250 mg/kg) or 5-bromo-2'deoxyuridine (BrdU, 500 mg/kg), or with a combination of the two agents. The dams were either allowed to go to term, when the fetuses were examined for cleft palate (CP) and digital anomalies, or killed 24 hr after the treatment. In the latter case, limbs from the embryos of control and treated dams were excised and cultured for 6 days in submerged culture. At the end of the culture period, the limb explants were stained for cartilage. The various cartilaginous components were subsequently analyzed using image analysis methods. CP was observed in 2.4 and 22.9% of the fetuses, respectively, after a single dose of HU or BrdU on E11. Simultaneous HU + BrdU treatment decreased the incidence of BrdU-induced CP to 3.6%, while treatment with BrdU 3 hr after HU resulted in 33.3% CP. In addition, syndactyly and ectrodactyly, not seen after the treatment with either HU or BrdU alone, were observed when the two agents were administered simultaneously, 1 hr apart or with a 3-hr delay. The teratogenic response was enhanced when limbs were cultured for 6 days. Digital anomalies were observed in the limb explants of BrdU-pretreated embryos; such abnormalities were not observed in vivo. Image analysis of cultured limb explants revealed that with HU-3 hr-BrdU pretreatment, the total limb area occupied by the long bones was increased at the cost of the paw area. However, no consistent changes in the shape or form of individual bones were observed. HU and BrdU given together produced a teratogenic response which was significantly different from that observed following the administration of HU or BrdU alone. This is considered to be due to an interference with the incorporation of BrdU in the DNA, since HU is known to block DNA synthesis. When HU was given before BrdU, the effects of BrdU were enhanced, probably due to the reported synchronizing action of HU on dividing cells. We also found that after a similar in utero exposure, the culture conditions themselves enhanced the frequency of teratogenic expression of BrdU.

Abnormalities, Drug-Induced↗
PubMed1984

The toxicology of chemical interactions during pregnancy in the mouse: caffeine and phenytoin.

The toxic interaction of caffeine and phenytoin during pregnancy was investigated in mice of the ICR strain on E10 of gestation. Caffeine, over a range of dosages, showed limited embryotoxic activity. Phenytoin was also weakly teratogenic and dosages needed to elicit embryotoxicity were accompanied by a significant increase in maternal lethality. Pretreatment with caffeine enhanced phenytoin-induced toxicity and teratogenicity and these observations confirm that caffeine has the ability to function as a co-teratogen. Pretreatment with phenytoin produced a significant increase in maternal lethality following caffeine administration but no co-teratogenic effect. It is suggested that these results are the consequence of a yet undefined interaction at critical receptor sites in the maternal-embryo unit.

Animals↗
PubMed1984

Mouse limb bud development in submerged culture: quantitative assessment of the effects of in vivo exposure to retinoic acid.

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose-dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid-induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose-dependent decrease in area of both fore- and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

Abnormalities, Drug-Induced↗
PubMed1984

Estimation of creatine kinase isoenzymes: the effects of caffeine and retinoic acid on CK levels in fetal mouse limbs.

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

Animals↗
PubMed1983

The interaction of chemicals during pregnancy: an update.

The role of chemical interactions in the genesis of induced embryotoxicity in humans and in experimental animals is a point of current concern and interest. This review summarizes the current knowledge in this field and demonstrates that, under appropriate conditions, the observed results of chemical interactions can be explained, in part, through an understanding of the underlying pharmacokinetic and pharmacodynamic parameters. These define the concentration of the "primary" teratogen at its receptor site in cells and tissues that are particularly sensitive to its embryotoxic action. The interacting "secondary" or "coteratogen" is viewed as an agent that is capable of altering these parameters in a way that is correlated with an observed alteration in the embryotoxic response. A series of recent experiments (diazouracil: 5-fluouracil; hydroxyurea: 5-bromodeoxyuridine; caffeine: 5-bromodeoxyuridine; caffeine: phenytoin) are reported which demonstrate both the validity and the limitations of such an approach and attempt to interpret observations, which are primarily empirical in nature, in pharmacologic terms.

Abnormalities, Drug-Induced↗
PubMed1980

A combination staining method for fibers and cell bodies of the urodele central nervous system.

A simple method for staining nerve cells and fibers of the salamander central nervous system is described. The procedure employs Carnoy's fixation followed by Protargol inpregnation and Nissl staining. This technique permits the simultaneous observation of intracellular neurofibrils, neuronal processes and basophilic components of the neuron. In addition, it eliminates the need to stain alternate sections with separate procedures to view the various components of the urodele central nervous system.

Animals↗