Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

Effects of carcinogenic halogenated aliphatic hydrocarbons on [3H]thymidine incorporation into various organs of the mouse. A comparison between 1,2-dibromoethane and 1,2-dichloroethane

The effects of 1,2-dibromoethane (DBE) and 1,2-dichloroethane (DCE) on the incorporation of [3H]thymidine into DNA were evaluated in various tissues of mice. The compounds were given intraperitoneally 24 h before sacrifice in an equimolar dose (293 mumoles/kg body weight). 2 h before the animals were killed, 0.5 mu Ci [3H]thymidine/g body weight was injected intraperitoneally. Both agents inhibited the [3H]thymidine incorporation in the forestomach, a site for their carcinogenic action. Whereas DBE also suppressed the [3H]thymidine incorporation in the nasal mucosa, the thymus, and the "glandular stomach", DCE was inhibitory only in the kidney. The observed difference in the effect of DBE and DCE on the thymus had its counterpart in a DBE-induced decrease of acid-insoluble radioactivity, demonstrated with whole-body autoradiography. The results indicate that in vivo screening of [3H]thymidine incorporation into various organs of an intact experimental animal is a sensitive technique for comparing cyto- and/or genotoxic effects of chemicals with a similar chemical structure.     

Uptake of orotate and thymidine by normal and regenerating rat livers

1. At 1h after operation livers from partially hepatectomized rats showed a 60-100% increase in the capacity to concentrate (3)H radioactivity from orotate, thymidine or uridine with respect to the radioactivity in plasma. Uptake of [(3)H]cytidine into liver was unaffected, as was entry of any precursor studied into any tissue other than liver. 2. This increase in intracellular radioactivity was detectable 10min after operation with both orotate and thymidine. With orotate the augmentation had disappeared by 3 days, but with thymidine it was still evident 8 days after partial hepatectomy, when [(3)H]thymidine incorporation into DNA was no longer increased. Competition studies established that orotate was not entering the liver by the same mechanism as thymidine. 3. In the soluble fraction of the liver all the (3)H radioactivity from orotate was present as uridine nucleotides. Thymidine was not phosphorylated, and was believed to be catabolized.     

Cytoplasmic DNA in rat corpora lutein cells : Effect of prolactin on [H]thymidine incorporation

Biochemical fractionation studies of homogenates of massively luteinized ovaries showed that DNA could be isolated from mitochondrial and microsomal fractions of this tissue and that prolactin administration enhanced [³H]thymidine incorporation into mitochondrial DNA in vivo. These findings were confirmed by autoradiographic analysis of tissue sections at the light and electron microscopic levels. Further support for the existence of microsomal DNA in situ was provided by the autoradiographic detection of acid-insoluble grains from [³H]thymidine over the cytoplasm of differentiating corpora lutein cells in the control and experimental groups. A significant effect on [³H]thymidine incorporation into microsomal DNA by prolactin could not be demonstrated in this experimental system.     

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.     

Dynamics of extracellular DNA in the marine environment

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Dynamics of extracellular DNA in the marine environment.

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Thymidine Metabolism and the Measurement of the Rate of DNA Synthesis in Carrot Suspension Cultures: EVIDENCE FOR A DEGRADATION PATHWAY FOR THYMIDINE

The kinetics of [³H]thymidine incorporation into the DNA of carrot suspension cultures were investigated. At a thymidine concentration of 0.1 micromolar, incorporation into DNA is not quantitative but ceases after only 14% of the thymidine has been incorporated. Thymidine incorporation into DNA is resumed following addition of a second aliquot of thymidine, which is consistent with substrate depletion. In vivo tracer experiments indicate that this may be due to a catabolic route for converting thymidine to β-aminoisobutyric acid. Bearing these observations in mind, conditions for determining the rate of DNA synthesis using [³H]thymidine incorporation have been investigated. It is concluded that by increasing the thymidine concentration to 10 micromolar the assay period may be increased, by reducing the influence of the degradative pathway, and that cell density and incubation time are critical factors in establishing a valid measure of the rate of DNA synthesis using this method.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

The rate of deoxyribonucleic acid synthesis by cultured Chinese-hamster ovary cells. An application of isotope-dilution analysis.

The rate of DNA synthesis is exponentially growing cells was determined by isotopedilution analysis of the incorporation of [me-3H]thymidine. Thymidine concentrations greater than 7 micrometer were used so that the rate-limiting step governing incorporation would be at the level of DNA polymerase rather than at the level of thymidine kinase [Sjostrom & Forsdyke (1974) Biochem. J. 138, 253-262]. In early exponential phase the rate determined by isotope-dilution analysis closely correlated with the rates calculated either from growth curves or from known cell-cycle parameters. However, in late-exponential phase the rate calculated from the growth curve was less than that determined by isotope-dilution analysis. We conclude that, under certain conditions, the pool-corrected rate of incorporation of [me-3H]thymidine, as determined by isotope-dilution analysis, can accurately reflect the rate of DNA synthesis. Discrepancies between the observed rate of DNA synthesis and increase in cell number could reflect an exponential degeneration of post-S-phase cells.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.     

In vivo inhibition of enterocyte metabolism by delta 9-tetrahydrocannabinol

Mice were given 10 to 100 mg/kg by stomach tube of delta 9-tetrahydrocannabinol (THC) in a single dose or for 4 consecutive days. [3H]Thymidine or [3H]glucosamine was given 3 or 24 hr before sacrifice. Enterocytes were isolated, and the incorporation of radioactivity into the acid insoluble fraction was measured. THC significantly inhibits in a dose-related fashion (from 10 to 90%) in vivo enterocyte metabolism. This inhibition is found in all enterocytes whatever their position in the intestinal tract; it is also independent of the state of differentiation of enterocytes. After a single ingestion of THC, crypt cells which synthesize DNA incorporate 37 to 45% less thymidine, and villus cells, which synthesize important amounts of glycoproteins, incorporate 15 to 39% less glucosamine. After 4 days of THC administration, the inhibition of thymidine incorporation is even more significant (up to 88%).     

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [3H]thymidine or [3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [3H]dCyd is used instead of [3H]dThd as the labelled deoxynucleoside.     

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Effects of cancer chemotherapeutic agents on testicular DNA synthesis in the rat. Evaluation of a short-term test for studies of the genetic toxicity of chemicals and drugs in vivo.

Changes in the rate of testicular DNA synthesis in the rat were studied at various times after single doses of 12 cancer chemotherapeutic agents. The animals were given intravenous injections of [14C]thymidine and [3H]thymidine 24 and 3 h, resp., before they were killed. By combining measurements of the free serum radioactivity and the testicular incorporation of the differentially labelled precursor, different response patterns were obtained for agents with different modes of action. The DNA-damaging agents cyclophosphamide, chlorambucil, thio-TEPA, busulphan, CCNU and procarbacine, after some delay, caused a decrease of testicular thymidine incorporation and a corresponding increase of free serum radioactivity. The non-DNA-damaging agents 5-fluorouracil, methotrexate, hydroxyurea and cytosine arabinoside had a rapid effect on testicular thymidine incorporation and produced diverse response patterns different from that of the DNA-damaging agents. Actinomycin D and also vinblastine caused changes in testicular thymidine incorporation and showed response patterns different from those of the other agents. These results show that simple measurements of testicular DNA synthesis may provide useful information for the evaluation of genotoxic effects of chemical compounds and may help one to distinguish between DNA-damaging agents and metabolic inhibitors of DNA synthesis.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

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.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.