Rebound of rat carrageenin granuloma following cessation of anti-inflammatory steroid therapy

Glucocorticoid therapy induced rapid involution of chronic granulomatous inflammation in rats by subcutaneous injection of carrageenin. Hydrocortisone acetate injected into the granuloma pouch at doses higher than 3 mg/kg/day for 3 days caused maximum involution. After withdrawal of the corticoid therapy, rebound of the granulomatous inflammation took place resulting in rapid recovery of the wet weight and total content of tissue DNA and non-collagen proteins. A dose of 3 mg/kg/day was optimal for observing this rebound phenomenon. In order to investigate metabolic aspects of the rebound phenomenon minced granuloma was incubated in vitro with [³H] thymidine or [³H] proline. The rate of incorporation of the labeled precursor into non-collagen protein was elevated near to the normal level by 24 hr after the interruption of the corticoid treatment. A second step in the course of the recovery was a rapid increase in the incorporation of labeled thymidine into DNA which was attained by 48 hr after the last injection of the corticoid. The rate of recovery of the total amount of non-collagen protein, however, was rather slow compared with that of DNA which reached the control level 3 days after the withdrawal of the corticoid therapy. The total non-collagen protein of the granuloma reached almost complete recovery 1 day later. These results suggest that the synthesis of some fractions of the granuloma proteins which involve proteins essential for DNA synthesis was activated before the reactivation of the synthesis of DNA and some other proteins. Recovery of collagen synthesis was not complete until 4 days after the cessation of the corticoid treatment. Consequently, the total amount of collagen was still lower than that of the control on the last day of the experiment.     

The effect of external deoxyribonucleosides on deoxyribonucleoside triphosphate concentrations in human lymphocytes.

The effects of deoxyribonucleosides on the intracellular levels of deoxyribonucleoside triphosphates (dNTP) and on the rate of labelled thymidine incorporated into DNA of human phytohaemag-glutinin-stimulated lymphocytes have been studied. Thymidine (10^?2–10^?6M) expanded the dTTP and reduced dATP and dCTP levels. Deoxycytidine (10^?3M) expanded the dCTP level and caused inhibition of [³H] thymidine incorporation into DNA but had no detectable effect on the other dNTP concentrations. Deoxyadenosine (10^?3 M) expanded the dATP level, and reduced the other dNTP levels and deoxyguanosine (10^?4M) expanded the dGTP level and reduced the dCTP level; both inhibited [³H] thymidine incorporation into DNA. The sensitivity of these cells to the addition of deoxynucleosides to their culture medium indicates that the plasma and tissue levels of nucleosides may profoundly influence DNA synthesis by human cells in vivo.     

Effect of the administration of antirheumatic drugs on experimental granuloma in rat

Subcutaneous granulomas were induced in rats by viscose cellulose-sponge and allowed to develop for 7 or 21 days. Before sacrificing the rats were treated for 5 days with antirheumatic drugs: sodium acetylsalicylate 300 mg/kg per day, prednisolone 1 and 10 mg/kg per day, indomethacin 0.1 and 10 mg/kg per day or phenylbutazone 100 mg/kg/day. The contents of DNA and especially of RNA in the granulomas were decreased, and the incorporation of ³H-cytidine into DNA in vitro was suppressed. The amounts of total nitrogen and of hydroxyproline (and consequently of collagen) were decreased. The incorporation of ³H-proline into proteins of granuloma slices was also decreased, except after treatment with the moderate dose of indomethacin, which apparently increased the incorporation of proline into collagenous proteins. This paradoxical effect of indomethacin is discussed. The content of total hexosamine was also decreased but that of uronic acids was increased. It is suggested that by multiple mechanisms the antirheumatic drugs retard the development of granulation tissue, but not the synthesis of acid mucopolysaccharides. At the proliferative phase the granulomas are more sensitive to the drugs than at the mature phase, and the incorporation rates are more affected than the total amounts of nitrogenous components.     

A biochemical study of the cotton pellet granuloma in the rat: Effects of dexamethasone and indomethacin

The subcutaneous implantation of a cotton pellet into a rat results in the formation of a granuloma at the site of the implant. The early events comprise an accumulation of fluid and proteinaceous material together with an infiltration of neutrophils. The granuloma formed by day 7 is characterized by the formation of a vascularized fibrous capsule containing fibroblasts and infiltrating mononuclear cells which are rich in $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ (NAG). Granuloma development was quantitated by dry weight measurements, and its cellular content was measured by assaying activity of NAG and total nucleic acid content. Nucleic acid determinations showed that cell infiltration into the granuloma took place at a virtually constant rate over a 7-day period. In contrast, the NAG activity did not change significantly until after day 5 when a large increase in the amount of enzyme extractable from the granuloma was seen. Systemic treatment of the animal with dexamethasone or indomethacin resulted in an inhibition of granuloma weight gain, NAG activity and nucleic acid levels. The data suggest that the two drugs acted during the early phase of granuloma development at the level of cell infiltration. Both drugs given on days 0–3 alone suppressed granuloma formation, whereas treatment on days 4–7 was without effect.     

Toxicity of mercuric chloride in cultures of neurons and nonneuronal cells derived from embryonic chick sympathetic ganglia

The effects of mercuric chloride were studied in vitro using both mixed and highly purified cultures of neurons and nonneuronal (glial) cells. Cultures were prepared from the sympathetic ganglia of 11-day chick embryos and treated with mercuric chloride (0.1 nm to 100 μm) for either 2 or 3 days. Acetyl cholinesterase specific activity, [³H]thymidine incorporation per microgram of protein, and [¹⁴C]leucine incorporation per microgram of protein were quantified, and the following results were obtained. First, all three biochemical measures either were stimulated or were unaffected by exposure of mixed ganglion cell cultures to ≤10 μm mercuric chloride for 2 days. In contrast, exposure to 100 μm mercuric chloride for 2 days depressed incorporation of both leucine and thymidine, but stimulated acetyl cholinesterase specific activity. Second, incorporation of both leucine and thymidine rapidly returned to control levels following removal of the mercuric chloride from the culture medium after 2 days of exposure. In contrast, acetyl cholinesterase specific activity remained elevated. Third, all biochemical measures were either inhibited or unaffected by a 3-day exposure of mixed ganglion cell cultures to any concentration of mercuric chloride. Fourth, sympathetic neurons appeared to be much more sensitive to mercuric chloride than homologous nonneuronal cells. Fifth, most of the stimulatory and inhibitory effects of mercuric chloride appeared to result from direct action of the heavy metal on either the neurons or nonneuronal cells.     

Comparison of the subacute effects of cadmium exposure upon nucleic acid, cyclic adenosine 3',5'-monophosphate and polyamine metabolism in lung and kidney cortex.

The timecourse of the cadmium-inflicted changes in DNA, polyamines, and cyclic AMP has been investigated in lung and renal cortex tissue of rats. In pulmonary tissue, heavy metal administration (2 × 1.0 mg/kg/day) produced an initial depression in the incorporation of [¹⁴C]-thymidine into DNA after 3 days, followed by a subsequent, significant enhancement at 5 days with maximal augmentation occurring after 7 days of cadmium treatment. In contrast the incorporation of labeled thymidine into kidney cortex DNA was decreased at all time periods studied and statistically significant reduction to at least half of the control values was noted at 3, 5 and 7 days. No apparent differences were found between pulmonary and renal DNA concentrations which were decreased at 1 day and elevated after 7 days of cadmium exposure in both tissues. Whereas cadmium significantly lowered lung RNA concentrations after 1, 3, or 5 days, heavy metal treatment failed to produce any significant change in RNA content of the kidney cortex. In general, subacute exposure to cadmium resulted in a significant rise in pulmonary putrescine, spermidine, and spermine after 3 and 5 days, although a significant depression was observed at 7 days in the case of putrescine. Surprisingly, the renal concentrations of putrescine and spermine were elevated in metal-treated animals but there was a statistically significant reduction in spermidine content. As in the case of incorporation of [¹⁴C]-thymidine into DNA, pulmonary cyclic AMP concentrations were depressed initially at 1 day and continuation of cadmium treatment for 3, 5, or 7 days resulted in enhancement of cyclic nucleotide concentration. In contrast, administration of the heavy metal lowered both the incorporation of thymidine into DNA and the concentration of cyclic AMP in renal cortex at all time points examined. The data demonstrate that even though the responsiveness of lung to subacute cadmium exposure differs from that of kidney cortex, the observed alterations in DNA synthesis may be mediated through modulation of cyclic AMP (and possibly polyamine levels) in both tissues.     

Biosynthesis of deoxyribonucleic acid, ribonucleic acid and protein in vivo by neonatal mice after a toxic dose of cyclophosphamide

Swiss-Webster mice received 80 $\text{mg}\text{kg}$ of cyclophosphamide 1 day after birth and were allowed to develop or were given 100 $\text{μCi}\text{kg}$ of [¹⁴C]labeled thymidine, uridine or leucine at various times afterwards. Growth retardation and increased mortality were noted during the postnatal period. DNA synthesis, as measured by [¹⁴C]thymidine incorporation, was inhibited in the liver, brain and carcass for at least 3 days after cyclophosphamide treatment. DNA synthesis was reduced 1 day after treatment to 8.4, 26 and 25 per cent of control in the liver, brain and carcass respectively. RNA synthesis, as measured by [¹⁴C]uridine incorporation, was reduced only in the liver and brain. Liver RNA synthesis was reduced to 36 and 64 per cent of control at 1 and 5 days after cyclophosphamide treatment respectively. Brain RNA synthesis was reduced to 78 and 64 per cent of control 1 and 4 days after treatment. [¹⁴C]leucine incorporation, taken as a measure of protein synthesis, was not affected in a manner which would indicate that drug treatment altered this parameter of differentiation. The data suggested that cyclophosphamide neonatal toxicity may be related to a prolonged inhibition of DNA and RNA synthesis during the first 5 days of life. This observation was correlated with the slow rate of cyclophosphamide activation and excretion in neonatal mice.     

Lymphoid suppression by cis-platinum(II)amines: What are the active agents?

Cisplatin and its various hydrolysis products were tested in vitro for their effects on the incorporation of [³H]thymidine into lymphocytes isolated from thymuses, spleens and stimulated lymph nodes of rats. Neither cisplatin nor the μ-hydroxo-bridged oligomers formed after hydrolysis significantly inhibited thymidine incorporation at pH7.4. However, freshly neutralised cis-diaquodiammineplatinum(II) was a potent inhibitor of thymidine incorporation by all three lymphocyte populations.In other experiments, rats were given either cisplatin or one of its hydrolysis products i.p. Cells isolated 17 hr later from the thymuses of all of these animals incorporated much less [³H]thymidine into DNA in vitro than thymocytes from saline-injected control animals. None of the platinum species significantly affected either [³H]uridine incorporation or the oxidation of [¹⁴C]octanoate to ¹⁴CO₂ by the thymocytes.Evidence for anation of di- and tri-nuclear μ-hydroxo-bridged platinum(II)amines by chloride has been obtained from spectrophotometric analyses and ¹⁹⁵Pt-NMR studies. Thiols also reacted with these platinum complexes at different rates (cis-[(NH₃)2Pt(H₂O₂)]²⁺ > derived oligomers > cisplatin).Various mechanisms for lymphoid suppression by cisplatin and its hydrolysis products are considered. It is proposed that cisplatin and its μ-hydroxo-bridged derivatives owe their lymphotoxic activity primarily to their in vivo transformation to platinum species containing aquo ligands.     

5-Ethyl-2'-deoxyuridine: evidence for incorporation into DNA and evaluation of biological properties in lymphocyte cultures grown under conditions of amethopterine-imposed thymidine deficiency

It was demonstrated that 5-ethyl-2′-deoxyuridine (EtUdR), an analogue of thymidine, can restore cell proliferation in human phytohemagglutinin (PHA)-stimulated lymphocyte cultures grown under condition of DNA synthesis block due to amethopterine induced thymidine deficiency. Under similar conditions, ¹⁴C-EtUdR was found to be incorporated into nuclei of lymphocytes entering the S (DNA synthesis) phase of the cell cycle, following release of a G₁/S cell cycle block by the analogue. The lower efficiency of EtUdR, as compared with thymidine, for reversal of amethopterine imposed DNA synthesis inhibition was shown to be due to the slowdown of the passage of cells through the proliferation cycle, when thymidine was substituted by the analogue. High excess of EtUdR blocks the passage of cells through the cycle, similarly or even more efficiently than thymidine, but does not affect metabolic processes preceding DNA synthesis.No changes in chromosome structure were found in cultures grown in media containing amethopterine and EtUdR, which implies that the analogue does not induce chromosome aberrations at least during the first generation following incorporation into DNA of somatic cells.     

The in vitro effect of trimethoprim on blastogenic transformation of human peripheral blood lymphocytes.

Trimethoprim (TP) at concentrations of 1 × 10^?6 to 1 × 10^?3 M inhibits the incorporation of thymidine, uridine, formate, and leucine by human lymphocytes undergoing blastogenic transformation induced by conconavalin A. TP does not alter cell viability, as measured by trypan blue exclusion, or cell numbers. An effect of TP on dihydrofolate reductase, such as is seen with methotrexate, is insufficient to explain the inhibitory effects of TP since an in vivo estimation of dihydrofolate reductase activity, utilizing deoxyuridine incorporation into deoxyribonucleic acid, is not inhibited. Also the observed inhibition of precursor incorporation is not fully reversed by either folate or folinate. TP did not alter the percentage of cells transforming but reduced the number of autoradiographically-labelled lymphoblasts. Immunosuppression by TP in vivo may be associated with these findings.     

Comparative effects of selected antifolates on transforming human lymphocytes and on established human lymphoblastic cell lines

When 15 nM methotrexate was added to the medium in which human peripheral lymphocytes stimulated with phytohemagglutinin were incubated, it caused a 50 per cent decrease in the maximum number of blasts produced, in the number of cells in mitosis and in the incorporation of [³H]deoxyuridine into DNA. However, [³H]thymidine incorporation into DNA was increased by methotrexate concentrations up to 0.5 mM. When 50 nM methotrexate was present continuously, blast formation, mitosis and deoxyuridine incorporation were virtually abolished, but if this concentration was present only during the induction phase (the first 24 hr), the subsequent effect on blast proliferation was slight. In contrast, 24-hr exposure during the proliferative phase (days 3–5) severely affected blast proliferation. The effects of methotrexate were largely reversible by thymidine, but hypoxanthine or purine nucleosides had no significant effect so that the metabolic block appears to be entirely at thymidylate synthetase under the experimental conditions. The inhibitory effects of ten other antifolates on transforming lymphocytes were determined and, with one exception, their relative effectiveness was found to be as predicted from inhibitory effects on highly purified bovine dihydrofolate reductase. The growth of four established lines of human lymphoblastic cells was inhibited to essentially the same extent by six of the antifolates, and these cells were only slightly less sensitive to the antifolates than were the transforming normal lymphocytes.     

Enhanced liver DNA synthesis in partially hepatectomized rats pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver DNA synthesis was studied in rats after a $\text{1}\text{3}$ hepatectomy. The rats were maintained on a controlled feeding schedule and were treated with 5 μg/kg of TCDD or acetone/corn oil (control). Five days after treatment a $\text{1}\text{3}$ hepatectomy was performed and at designated times thereafter liver DNA synthesis was measured by [³H]thymidine incorporation into DNA. The main finding was that liver DNA synthesis was increased 8- to 10-fold by TCDD over that which was observed in control rats. This increase occurred after a latency period that was appropriate for the regenerative liver DNA synthesis response. Other experiments showed that increased incorporation of thymidine in TCDD-treated rats could be blocked by hydroxyurea, an inhibitor of semiconservative DNA synthesis, and that the increased incorporation was secondary to increased DNA synthesis and not increased thymidine kinase activity. Thus, hepatocytes in TCDD-treated rats respond in a quantitatively different manner than control rats to the same proliferative signal, $\text{1}\text{3}$ hepatectomy. Liver DNA synthesis in nonhepatectomized TCDD-treated rats tended to be greater than control, but the difference was not statistically significant.     

Effect of gold sodium thiomalate on proliferation of human rheumatoid synovial cells and on collagen synthesis in tissue culture

Synovial tissue obtained from patients with rheumatoid arthritis who were undergoing reconstructive joint surgery was used to obtain explant cultures of synovial cells. The experiments described were performed on growing monolayer cultures during the second to fifth passages. Synovial cells were exposed to gold sodium thiomalate (GST) in concentrations equivalent to levels attained in synovial tissues during chrysotherapy (3–50 μg/ml). After 5 days of exposure of cells to 10 or 50 μg/ml of GST, [³H]thymidine incorporation into DNA was inhibited 94 or 99 per cent, respectively. After 10 days of exposure to 10 μg/ml of GST, cell number was decreased 50 per cent, although no change in cell number was found after a 5-day exposure to 100 μg/ml of GST. The total collagen content of the media was decreased in flasks of cells exposed to 50 μg/ml of GST for 15 days, which reflects the decrease in cell numbers observed at this concentration. However, after 15 days of exposure of synovial cells to 12 μg/ml of GST, incorporation of [¹⁴C]proline into total collagen per cell increased 4-fold. This increase in [¹⁴C]proline incorporation occurred predominantly in type I collagen. In these experiments, the percentage of type III collagen containing [¹⁴C]proline and the amount found in media were suppressed 50 per cent by a fifteen day exposure to 3 μg/ml of GST. The ability of GST to increase the relative commitment of these cultures to make type I collagen is dose dependent in the range from 3 to 12 μg/ml. These data indicate that changes in cell proliferation and in the nature (genetic composition) of the extracellular matrix produced are direct effects of GST on the synovial cell in tissue culture and may represent one important mechanism of action of chrysotherapy in the treatment of patients with rheumatoid arthritis.     

Effect of slow and rapid cystometry on in vitro rat urinary bladder DNA synthesis

1. Partial outflow obstruction induces marked changes in detrusor contractile function and morphology. One common finding in all experimental animal models of partial outflow obstruction is a significant increase in bladder mass.2. Previous studies have demonstrated that partial outlet obstruction induces a rapid and substantial increase in [³H]thymidine incorporation into virtually all cellular elements of the bladder.3. The present study was designed to investigate the [³H]thymidine uptake and localization induced by exposure of the in vitro whole rat bladder model to various intravesical pressures and rates of intravesical infusion.4. The results are as follows:(a) There were no differences in DNA concentration between control and other groups.(b) Slow infusion induced a mild increase in DNA synthesis ([³Hthymidine incorporation) at 0.5 ml and a significantly greater level of DNA synthesis at 1.6 ml.(c) [³H]thymidine incorporation was significantly increased by exposure to 7.5 cm H₂O, 15 cm H₂O, and 30 cm H₂O.(d) Exposure to 60 cm H₂O and 90 cm H₂O did not initiate an increase in [³H]thymidine incorporation.(e) Autoradiography showed that all tissue elements (urothelium, connective tissue, smooth muscle) participated in the response.     

The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

Antiviral activity of 5-thiocyanatopyrimidine nucleosides.

The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU¹, 5-NCSdU, 5-NCSaraU and tri-O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri-O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [³H]uridine and [³H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [³H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [³H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.     

The mechanisms of action of three fluorine substituted cytosine analogs: implications for cancer chemotherapy

The mechanisms of action in DNA synthesis of three fluorine substituted cytosine analogs have been studied in human phytohaemagglutinin-stimulated lymphocytes. 5-Fluorocytidine (over a wide range of concentrations) and 5-fluorocytosine (at high concentration) inhibited ³H-deoxyuridine incorporation into DNA without inhibiting ³H-thymidine incorporation. 5-Fluorocytidine caused reduction in the free cell concentration of TTP. In contrast, ara-fluorocytosine markedly inhibited both ³H-deoxyuridine and³H-thymidine incorporation into DNA but did not cause a consistent change in thymidine triphosphate concentration. These results indicate that 5-fluorocytidine, and to a lesser extent, 5-fluorocytosine block de novo TMP synthesis in human cells. It seems likely that they are both metabolized to 5-fluorodeoxyuridinc monophosphate which inhibits the enzyme TMP synthetase. On the other hand, the results suggest that ara-fluorocytosine has an action identical to that of cytosine arabinoside, inhibition of DNA polymerase. It is postulated that cells resistant to cytosine arabinoside which have high cytidine deaminase levels might be selectively sensitive to 5-fluorocytidine and to 5-fluorocytosine.     

Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice

The administration of butylated hydroxytoluene (BHT) to mice results in lung cell damage followed by cellular proliferation which was quantitated by measuring the increase in thymidine incorporation into pulmonary DNA. Administration of SKF 525-A or piperonyl butoxide to mice treated with BHT prevented the increase in thymidine incorporation into pulmonary DNA. This effect was dose dependent, with complete protection from 400 mg/kg BHT achieved with 10 mg/kg SKF 525-A or 400 mg/kg piperonyl butoxide. SKF 525-A and piperonyl butoxide completely prevented the BHT-induced increase in pulmonary DNA synthesis even when given 1–2 hr after BHT and a partial protective effect was evident when they were given 6–12 hr after BHT. Pretreatment of mice with cobaltous chloride diminished the BHT-induced increase in thymidine incorporation into pulmonary DNA. Following the in vivo administration of [¹⁴C]BHT, radioactivity was covalently bound to lung, liver, and kidney macromolecules of both mice, which exhibited BHT-induced lung damage, and rats, which did not. The greatest amount of radioactivity was bound to lung tissue from mice. This binding was prevented by the administration of SKF 525-A and was a linear function of the BHT dose within a range of 50–600 mg/kg. Binding to other tissues from the mouse and all tissues examined in the rat was minimal and unaffected by SKF 525-A. These data suggest that a reactive metabolite of BHT rather than the parent compound produces lung damage in mice.