Sensitivity of early mouse embryos to [H]thymidine

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to [³H]thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of [³H]thymidine, concentrations as low as 0.1 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs was reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highest concentration tested (100 nCi/ml). When embryos were cultured from the 2-cell stage for 3 days in the presence of [³H]thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to [³H]thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages; the lowest concentration that interfered with development was 10 nCi/ml, and exposure at the morula stage was most detrimental to the subsequent development of embryos, particularly that of ICMs. The 24-h exposure of immunosurgically isolated ICMs to [³H]thymidine revealed that the high sensitivity of the ICM to [³H]thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate [³H]thymidine into the DNA.     

Distribution of 3H in rat conceptus cultured in vitro following brief administration of [3H]thymidine

Rat embryos with intact visceral yolk sacs, explanted at 12 1/2 days of gestation, were cultured in vitro for up to 60 min in medium consisting of fetal calf serum, Eagle's MEM, and [3H]thymidine (1.2 kBq ml-1), using the roller bottle method. The total amount of 3H incorporated into the conceptus during the 60-min incubation was 79.2 Bq, and approximately 33, 23, and 44% of this activity was distributed to the embryo, the yolk sac, and the fluid in the exocoelom and amniotic cavity, respectively. The rate of 3H accumulation in conceptuses decreased with time in culture. It appeared that the decrease in the viability of the conceptus was not responsible for this phenomenon. The concentration of 3H in the yolk sac, i.e., 3H activity per gram wet weight, was 2.1 times that in the medium at the end of culture. In contrast, the 3H concentration in the embryo was significantly lower than that in the medium. These findings suggest that the visceral yolk sac of rat conceptuses may act as a barrier to the transport of tritiated thymidine between the medium and embryo.     

Differentiation of cartilage cells studied by quantitative [3H]thymidine autoradiography in vitro

The apical segments of the mandibular condylar cartilage of newborn ICR mice, containing the intact zones of progenitor cells along with a few rows of chondroblasts were initially prelabelled in vitro with [3H]thymidine and were subsequently chased and cultured for as long as eight days. Such explants underwent a process of tissue regeneration, as after three days in culture they reconstituted the original structure of the organ, thus resembling the in vivo appearance of neonatal mandibular condylar cartilage. Cellular proliferation with subsequent differentiation in the regenerating tissue was followed by means of quantitative autoradiography. Immediately after labelling, the autoradiography-positive grains were confined exclusively to progenitor cells. The latter revealed a substantial ability to proliferate in vitro, a fact that was manifested by a progressive increase in the labelling index along the course of the culture. The latter process was followed by cellular differentiation thereby obtaining hypertrophic chondrocytes. The increase in the rate of labelling index and in the total number of [3H]thymidine-labelled cells was significantly correlated with the overall growth of the regenerating explants.     

Uptake and binding of tritium from [chloroethyl 3H] cyclophosphamide by rat embryos in vitro

Day 10 rat embryos were exposed in vitro to [chloroethyl 3H] cyclophosphamide (3H-CP) at 4 micrograms/ml over a 24-hour period and the uptake and binding of labeled drug were monitored autoradiographically and biochemically. Autoradiographic analysis revealed that embryos exposed to 3H-CP and a complete activating system exhibited radioactivity distributed throughout the embryo. Subsequent analysis indicated that the distribution of autoradiographic grains on a per cell basis ranged from 7.7 in surface ectoderm to 13.4 in the neuroepithelium. No correlation was found between the sensitivity of various embryonic tissues to the cytotoxic effects of CP and the number of grains per cell. Direct radiochemical analysis of the amount of tritium taken up and bound by embryos under bioactivating conditions (3H-CP + S-9 + cofactors) confirmed the autoradiographic analysis. Autoradiographic and radiochemical analyses demonstrate that embryos exposed under bioactivating conditions take up and bind approximately three times more tritium than embryos exposed under nonactivating conditions (3H-CP + S-9 without cofactors). Additional studies have demonstrated that uptake and binding of tritium from bioactivated 3H-CP only are linear over the first 10 hours of incubation with no detectable increases thereafter.     

Potential pitfalls of [3H]thymidine techniques to measure cell proliferation

Pitfalls and artifacts in the use of [3H]thymidine in the measurement of cell proliferation kinetics in vitro and in vivo are reviewed. These pitfalls are of particular significance for the study of inhibitors of cell proliferation including chalones.     

Carboxy-terminal PTH fragments stimulate [3H]thymidine incorporation in vascular endothelial cells

We have previously reported that the intact PTH molecule (1-84) stimulates proliferation of human umbilical vein endothelial cells (HUVECs). To define the bioactive portion of the PTH molecule we utilized amino, mid and carboxy-terminal PTH fragments. Carboxy- but not amino-terminal fragments were equivalent to the intact PTH molecule in stimulating [3H]thymidine incorporation in HUVEC. Carboxy- but not amino-terminal PTH fragments increased intracellular calcium. Blocking the rise in intracellular calcium with calcium chelators abolished PTHs proliferative effect on HUVEC. In contrast to PTH 1-84, the carboxy-terminal fragment effect on [3H]thymidine incorporation was blocked by KN-93 an inhibitor of CaM kinase II. Taken together, these data suggest that the carboxy-terminal PTH is (or contains) the bioactive fragment responsible for the changes in intracellular calcium and thymidine incorporation in HUVEC stimulated with the intact PTH molecule.     

Resistance of 1-deamino-[8-D-arginine]-vasopressin to in vitro degradation as compared with arginine vasopressin

In order to elucidate the mechanism(s) responsible for the prolonged antidiuretic activity of 1-deamino-[8-D-arginine]-vasopressin (dDAVP), antidiuretic activities of dDAVP and arginine vasopressin (AVP) were determined in the rat following either oral administration or incubation with AVP-degrading enzymes and reagents. Oral administration of dDAVP to conscious water-loaded rats resulted in significant antidiuresis while AVP resulted in slight and transient antidiuresis. In the ethanol anesthetized water-loaded rats, antidiuretic activities of 136pg of AVP and 50pg of dDAVP, which were found to be equipotent, were compared after incubation with digestive enzymes (pepsin, trypsin, alpha-chymotrypsin), late pregnancy plasma, or sodium thioglycollate. The antidiuretic activity of AVP was completely destroyed by 30-min incubation with trypsin, alpha-chymotrypsin, or late pregnancy plasma and almost all AVP was inactivated by 0.2 M sodium thioglycollate. On the other hand, the antidiuretic activity of dDAVP was not destroyed by trypsin or pregnancy plasma but was partly destroyed by alpha-chymotrypsin and sodium thioglycollate. Neither the antidiuretic activity of AVP nor that of dDAVP was affected by pepsin. Thus, the antidiuresis observed after oral administration of dDAVP might be brought about by the resistance to digestive enzymes. Furthermore, the resistance of dDAVP to digestive enzymes, late pregnancy plasma and sodium thioglycollate might be responsible for the prolonged antidiuretic action of dDAVP in vivo.     

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Norharman inhibition of [3H]diazepam binding in mouse brain

Norharman competitively inhibits specific binding of [³H]-diazepam in mouse brain homogenates. $\text{In vivo}$ this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of $\text{in vivo}$ concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.     

In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus

The binding sites for [3H]corticosterone (3HB) and [3H]aldosterone (3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a "pure glucocorticoid' RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)