Circadian ACTH 1-17 effects on murine tritiated thymidine incorporation into DNA of thymus, bone marrow and spleen; also on total splenic RNA and DNA

The importance of administration time along the 24-h scale is shown for a potent corticosteroidogenic adrenocorticotropin analogue, ACTH 1-17 (Synchrodyn 1-17). This molecule affects the incorporation of [3H]TdR into DNA (DNA synthesis) in the thymus, bone marrow and spleen, and total RNA and DNA of spleen in CD2F1 mice, standardized in light alternating with darkness at 12-h intervals. As a function of timing, the same dose of ACTH 1-17 at one time increases, at another time decreases (in each case with statistical significance) and at still another time elicits no response in DNA synthesis or in total RNA and DNA of spleen. Effects upon DNA synthesis are recorded with doses of 0.02 IU/kg body weight. The most marked effect with 20 IU/kg body weight is a decrease of DNA synthesis seen (4 h) after administration of ACTH 1-17 late in the dark span and early in the light span. The effect of ACTH 1-17 on the thymus is more prominent than that on bone marrow and spleen. Time-dependence also characterizes placebo effects by comparison to values in untreated controls. At the cellular level responses to ACTH 1-17 or placebo are characterized by critical interactions of treatment kind with treatment timing as well as interval-to-kill-time. The study documents the need to time-specify, in several ways, responses to ACTH 1-17 and suggests more broadly that 'increases' and 'decreases' may have to be complemented by changes in endpoints of rhythms in all those endocrine studies that involve rhythmic variables and rhythm-dependent effects upon these variables.     

Distribution and changes in properties of ferromagnetic iron particles administered to the animal body

The registration of ESR signals of organs and tissues in the wide range of temperatures permits to study properties and distribution of ferromagnetic particles in animal organisms. High dispersed powder (HDP) of iron (particle dimension--50-100 nm) was administered subcutaneously to mice in the doses 2 and 100 mg/kg weight of animals. One week after the administration HDP was accumulated in the animal organs under study. Two weeks after the treatment of mice with HDP in the dose of 2 mg/kg the ESR signals with g = 2.1 appeared in the animal tissues: liver, spleen, kidney, heart and lungs. Six weeks after the treatment the ESR signals of the studied tissues did not differ from those in control animals.     

Changes in the intensity of DNA synthesis in immunocompetent mouse tissues by peptidoglycans of bacterial origin]

The data are presented on the effect of activation of by peptidoglycans from cell walls of Staphylococcus aureus and Brevibacterium flavum on the DNA synthesis intensity in the mice spleen, thymus and bone marrow in vivo. Maximum of the peptidoglycan influence on the DNA synthesis intensity is observed 12-36 hours after injection. As a rule, higher doses of the studied peptidoglycans (1 mg per one animal) activate the DNA synthesis more efficiently. Peptidoglycan from Br. flavum stimulates the DNA synthesis in all tissues more intensively then the peptidoglycan from S. aureus, which may be a result of its monomers' structure.     

Enhancement of ribonucleic acid synthesis by chromium(III) in mouse liver

The effect of Cr(III) administration on hepatic RNA synthesis in mice was studied. It was found that Cr accumulated in mouse liver. Forty-eight hours after intraperitoneal injection of CrCl3 (0.005-5 mg Cr/kg body weight) approximately 10% of the administered dose per g of tissue remained. The accumulated Cr was still retained 64 days after administration (5 mg Cr/kg) with only a slight decrease. Approximately 20% of the hepatic Cr was detected in the nuclei. By administering CrCl3. RNA synthesis in mouse liver was markedly enhanced without altering the pool size of nucleotides. This enhancement was dose-dependent and statistically significant at doses of 0.05 (p less than 0.05), 0.5 (p less than 0.01), and 5 mg Cr/kg (p less than 0.01), and remained so for at least 16 days after administration of 5 mg Cr/kg. The synthesis of DNA and protein in mouse liver were not significantly changed by CrCl3 administration. On the other hand, Cr(VI) administration did not enhance but rather inhibited RNA synthesis in mouse liver. These results suggest that Cr(III) specifically enhances RNA synthesis in mouse liver.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Recovery response of dividing cells in the thymus of whole-body gamma-irradiated mice.

Mice were irradiated with different doses of gamma-rays 30 min after the administration of 32P-orthophosphate. The dose-response curves determined at 72 hours after exposure showed an inflection point in the total activity present in the DNA in thymus and spleen. In the low dose-range, the dose-response curves have D0 = 55 rad (n = 2-5) for thymus and DO = 95 rad (n = 2-5) for the spleen. Thirty minutes after the administration of 32P-orthophosphate, the dividing cells from thymus were partially synchronized by the administration of 80 mg per kg body-weight hydroxyurea. At different time-intervals, the mice were irradiated with 80 rad, and the total activity of DNA was determined at 72 hours after synchronization. A significant maximum of recovery was found at 5 hours (S phase) after the administration of hydroxyurea. In similar conditions, the dose-response curves corresponding to the G1, S and M phase of the division cycle were also determined. The synchronization of dividing cells induced by hydroxyurea failed in the spleen.     

Acute metabolic acidosis inhibits muscle protein synthesis in rats

In this study, we investigated the effect of acute metabolic acidosis on tissue protein synthesis. Groups of rats were made acidotic with intragastric administration of NH(4)Cl (20 mmol/kg body wt every 12 h for 24 h) or given equimolar amounts of NaCl (controls). Protein synthesis in skeletal muscle and a variety of different tissues, including lymphocytes, was measured after 24 h by injection of l-[(2)H(5)]phenylalanine (150 micromol/100 g body wt, 40 moles percent). Results show that acute acidosis inhibits protein synthesis in skeletal muscle (-29% in gastrocnemius, -23% in plantaris, and -17% in soleus muscles, P < 0.01) but does not affect protein synthesis in heart, liver, gut, kidney, and spleen. Protein synthesis in lymphocytes is also reduced by acidosis (-8%, P < 0.05). In a separate experiment, protein synthesis was also measured in acidotic and control rats by a constant infusion of l-[(2)H(5)]phenylalanine (1 micromol.100 g body wt(-1).h(-1)). The results confirm the earlier findings showing an inhibition of protein synthesis in gastrocnemius (-28%, P < 0.01) and plantaris (-19%, P < 0.01) muscles but no effect on heart and liver by acidosis. Similar results were also observed using a different model of acute metabolic acidosis, in which rats were given a cation exchange resin in the H(+) (acidotic) or the Na(+) (controls) form. In conclusion, this study demonstrates that acute metabolic acidosis for 24 h depresses protein synthesis in skeletal muscle and lymphocytes but does not alter protein synthesis in visceral tissues. Inhibition of muscle protein synthesis might be another mechanism contributing to the loss of muscle tissue observed in acidosis.     

Tissue selectivity of pravastatin sodium, lovastatin and simvastatin. The relationship between inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rat.

Tissue selectivity of pravastatin sodium (pravastatin), lovastatin and simvastatin, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors was examined by measuring inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rats after a single oral administration (25 mg/kg) of these drugs. Regarding tissue drug concentrations, all three drugs were liver selective: concentrations of drugs in the liver were about ten-times higher than those in the spleen and testis. On the other hand, pravastatin was far more liver selective in inhibiting sterol synthesis than two other inhibitors: pravastatin inhibited de novo sterol synthesis in the liver but minimally in the spleen and testis, whereas lovastatin and simvastatin inhibited in all three tissues. Microautoradiographic and in vitro cellular-uptake studies demonstrated that pravastatin remained in the extracellular space in the spleen, whereas the other drugs entered the cell. We conclude that pravastatin exhibits a liver-selective inhibition of sterol synthesis because the agent permeates the cell membrane in the liver, but not in non-hepatic tissues.     

The inhibitory effect of histamine on lymphoid tissue proliferation in mice

Histamine, injected subcutaneously (10 mg/kg), inhibited the DNA synthesis response to a contact-sensitizing agent (picryl chloride) and also had an inhibitory effect on DNA synthesis in untreated mice. The synthesis was measured by 5-[125I]iodo-2'-deoxyuridine incorporation in spleen, lung, liver, and peripheral lymph nodes and the inhibitory effect was marked and consistent in spleen in both sensitized and nonsensitized animals, but was variable in the other tissues. Since histamine is believed to activate suppressor cells, it is suggested that the inhibition of DNA synthesis in picryl chloride-treated mice is due to the activation of those suppressor cells which limit the specific DNA synthesis in response to the contact-sensitizing agent. The inhibition of DNA synthesis in untreated mice could be due to the activation of suppressor cells that control the ongoing immune response to environmental antigens.     

Effect of PYCNOGENOL on the toxicity of heart, bone marrow and immune organs as induced by antitumor drugs.

PYCNOGENOL is a mixture of water-soluble bioflavonoids extracted from the bark of pine trees growing in the southwest coastal region of France. In the present paper the effects of PYCNOGENOL (Pyc) on the toxicity of bone marrow, heart and immune organs induced by anticancer drugs were investigated, in mice. The following results were obtained: 1. Pyc at the orally-administered dose of 200 and 150 mg/kg body wt. markedly prevented the elevation of serum creatine phosphokinase (CPK) activity and the decrease of heart rate in mice treated with doxorubicin (Dox); 2. Pyc at 100 and 150 mg/kg body wt. significantly antagonized the inhibition of DNA synthesis in thymus induced by subcutaneous injection of cyclophosphamide (Cyc); 3. Pyc at 150 and 200 mg/kg body wt. markedly induced increase of erythrocytes and hemoglobin, but had no effect on leukopenia, in Cyc-treated mice; and 4. Pyc has no antagonizing effect on the anticancer activity of Dox and Cyc. All the results suggest that Pyc possesses a protective effect on the cardiotoxicity of Dox and the inhibition of thymus DNA synthesis induced by Cyc in mice.     

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.     

Semisynthetic derivatives of daunorubicin and carminomycin: inhibition of DNA synthesis after intravenous administration to mice

Inhibition of DNA synthesis in the liver, kidneys, spleen and heart of mice after intravenous administration of 0.1 and 0.3 LD50 of semisynthetic derivatives of rubomycin (daunorubicin) and carminomycin was studied. The level of DNA synthesis inhibition was estimated by a decrease in incorporation of (methyl-3H) thymidine. Under the action of 13-trebutoxycarbonyl hydrazone and 14-salicyloiloxy derivatives of rubomycin and carminomycin maximum inhibition of DNA synthesis was reached later while its recovery started earlier as compared to the initial antibiotics.     

Incorporation of 5-lododeoxyuridine into the DNA of mouse embryos: its relation to embryotoxicity.

Pregnant female ICR mice were administered, ip, either a trace (200 muCi/kg) or teratogenic (200 muCi + 300 mg/kg) dose of [6(-3)H] 5-iododeoxyuridine (IdU) on day 10 of gestation. Maternal liver, spleen, intestine, and kidneys, and placentas and embryos were removed at various time intervals after injection, weighed, and homogenized in cold 0.5 m perchloric acid. The half-lives of IdU-derived nucleotides in the acid-soluble fraction ranged from 31-46 min (trace) to 57-131 min (teratogenic) for the tissues analyzed. [3H]IdU was incorporated into the DNA of all mitotically active tissues after both dosages. The presence of the label in iodouracil was demonstrated by thin-layer chromatography of DNA bases extracted from maternal spleen and embryo. Growth of embryos following injection on day 10 resulted in decreased 3H-specific activity in the DNA fraction and concomitant retention of total activity. It is suggested that the previously demonstrated embryotoxicity of IdU is related to its retention at its presumed intracellular site of action.     

Ecdysterone Modulates Antitumor Activity of Cytostatics and Biosynthesis of Macromolecules in Tumor-Bearing Mice

The influence of therapeutic and half doses of cisplatin and adriamycin combination with the anabolic drug ecdysterone (20-hydroecdison) on development of subcutaneously and intraperitoneally transplanted P388 and L1210 leukemia and metastasizing B16 melanoma was studied. Ecdysterone significantly stimulated the chemotherapeutic effect of low doses of the cytostatics: inhibition of tumor growth, mice survival rate, their lifespan, and the antimetastatic activity index were comparable or better than after therapy with high doses of the antitumor drugs. The influence of high and low doses of cisplatin and its low dose in combination with ecdysterone on the dynamics of protein and DNA biosynthesis in the liver, pancreas, thymus, spleen, and adrenals of tumor-bearing mice were also studied. Although the therapeutic effect of 4 mg/kg cisplatin by activated protein biosynthesis and DNA repair is comparable or better than that of its low dose (2 mg/kg) in combination with ecdysterone, in terms of chemotherapy the combination looks preferable since the therapeutic dose of cisplatin is toxic for the intact tissues.     

Modification of tissue distribution of trace elements by cyclosporin in rats

Cyclosporin-induced hypomagnesaemia, as observed in patients, could also be induced in rats by intramuscular administration of the drug (20 mg/kg/d) for 12 d. moreover, cyclosporin administration induced modifications in the concentrations of other elements in tissues, particularly an increase in Mg, Cu, and Zn in the thymus and an increase in Mo in the spleen and Sr in the liver.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

The acute-phase response of the liver in relation to thyroid hormone-induced redox signaling

Recently, we reported that 3,3',5-triiodothyronine (T3) induces the expression of redox-sensitive genes as a nongenomic mechanism of T3 action. In this study, we show that T3 administration to rats (daily doses of 0.1 mg/kg ip for 3 consecutive days) induced a calorigenic response and liver glutathione depletion as an indication of oxidative stress, with higher levels of interleukin (IL)-6 in serum (ELISA) and hepatic STAT3 DNA binding (EMSA), which were maximal at 48-72 h after treatment. Under these conditions, the protein expression of the acute-phase proteins haptoglobin and beta-fibrinogen is significantly augmented, a change that is suppressed by pretreatment with alpha-tocopherol (100 mg/kg ip) or gadolinium chloride (10 mg/kg iv) before T3. It is concluded that T3 administration induces the acute-phase response in rat liver by a redox mechanism triggered at the Kupffer cell level, in association with IL-6 release and activation of the STAT3 cascade, a response that may contribute to reestablishing homeostasis in the liver and extrahepatic tissues exhibiting oxidative stress.     

Effects of pravastatin sodium on mevalonate metabolism in common marmosets

In experimental animals and humans, the concentration of serum mevalonate (MVA), a direct product of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is considered to reflect the activity of whole-body sterol synthesis. The relationship between the concentration of serum MVA and the activity of sterol synthesis in tissues, however, has not been fully clarified. In the present study, we examined MVA metabolism by using pravastatin, a liver-selective inhibitor of HMG-CoA reductase, and common marmosets, a good model animal for studying lipid metabolism. In the time course study, the maximal reduction in the concentration of serum MVA was observed 2 h after a single oral administration of 30 mg/kg pravastatin to common marmosets. We, therefore, examined the relationship between the concentrations of serum and hepatic MVA, and sterol synthesis in some tissues at this time point. Sterol synthesis was determined ex vivo in tissue slices by measuring the incorporation of [14C]acetate into digitonin-precipitable [14C]sterols. Pravastatin at 0.03-30 mg/kg reduced dose-dependently the activity of hepatic sterol synthesis, whereas no significant reduction of sterol synthesis was observed in other tissues such as intestine, kidney, testis and spleen, even with the highest dose (30 mg/kg). The liver-specific inhibition of sterol synthesis caused parallel reductions in the concentrations of both serum and liver MVA. In addition, there were good correlations between the concentration of either serum or hepatic MVA and the activity of hepatic sterol synthesis. These data indicate that the major origin of serum MVA is the liver, and that the concentration of serum MVA reflects the concentration of hepatic MVA and the activity of hepatic sterol synthesis 2 h after a single oral administration of pravastatin in common marmosets.     

Effect of acute administration of mercuric chloride on the disposition of copper, zinc, and iron in the rat

The present study was designed to investigate the effect of mercuric chloride administration on copper, zinc, and iron concentrations in the liver, kidney, lung, heart, spleen, and muscle of rats. The results showed that after dose and time exposure to mercuric chloride, the concentration of mercury in the six tissues was significantly elevated. Data showed that there were no interaction between mercury and tissue iron. There was a considerable elevation of the content of copper in the kidney and liver. The most significant changes in the copper concentration took place in the kidneys. About a twofold increase in the copper content of the kidney was noted after exposure to mercuric chloride (3 mg and 5 mg/kg). Only slight elevations in the copper content occurred in the liver, especially in high dose and longer exposure time. In the remaining organs, the copper content was not changed significantly (p>0.05). The most significant changes in the zinc concentration took place in liver, kidney, lung, and heart (5 mg/kg). Marked changes in kidney zinc concentrations were observed at any of the specified doses. Zinc concentrations were significantly increased in kidney of rats sacrificed 9–48 h after sc injection of HgCl₂ (5 mg/kg); in liver obtained from rats at 18, 24, or 48 h after injection; and in lung after 24 or 48 h of treatment. The heart and spleen zinc concentrations were elevated at 24 and 48 h after injection of HgCl₂ (5 mg/kg), respectively. The results of this study implicate that effects on copper and zinc concentrations of the target tissues of mercury may play an important role in the pathogenesis of acute mercuric chloride intoxication.