Hepatic ornithine decarboxylase activity in the rat as influenced by retinyl acetate and ethanol or tryptophan

Ethanol and tryptophan have been demonstrated earlier to induce a rapid stimulation of hepatic ornithine decarboxylase (ODC) activity in overnight-fasted rats. In this study the effect of the administration of retinyl acetate prior to administering ethanol or tryptophan was investigated. The levels of ODC activity in the livers of control and experimental rats were assayed in vitro by measuring the release of 14CO2 from DL-[1-14C]ornithine. Intraperitoneal administration of retinyl acetate (1 microgram/100 g body wt) 1 hr before tube feeding ethanol (0.75 g as a 50% solution/100 g body wt) or L-tryptophan (30 mg in 3 ml water/100 g body wt) and 3 hr before killing caused an enhanced stimulation of hepatic ODC activity compared to that when each agent was administered alone. In vitro [14C]leucine incorporation into protein using hepatic microsomes of tryptophan-treated rats with or without retinyl acetate was increased in comparison with that of controls while decreases were observed when using microsomes of ethanol-treated rats with or without retinyl acetate. Although retinyl acetate has been reported earlier to inhibit the stimulation of hepatic ODC activity due to a variety of agents, including some agents known as carcinogens or promoters, it did not act in this manner against the acute administration of ethanol or tryptophan.     

Lysosome-vacuolar system reactivity during early cell regeneration.

The activities of five selected acid hydrolases have been measured in early regenerating liver, 24 hr after partial hepatectomy. A comparable significant decline in the specific activity of acid phosphohydrolase, β-glucuronidase, β-glucosidase, and β-galactosidase was found. Less significant changes were noted in N-acetylglucosaminidase. No significant decline in the specific activity of cytochrome c oxidase or lactate dehydrogenase was observed. An apparent resistance to thermal and osmotic activation of lysosomal N-acetyl-β-glucosaminidase was noted in the partially hepatectomized animals at 24 hr. A significant increase in the ratio; free/total activity of β-galactosidase and N-acetylglucosaminidase was noted following $\text{50 μg}\text{100 g}$ body wt glucagon injection in sham and control animals. Glucagon administration to partially hepatectomized animals failed to produce a corresponding increase in the free/total ratio suggesting that regenerating liver is insensitive to the action of large doses of glucagon. Electron microscopy failed to reveal autophagic vacuole formation at 24 hr after partial hepatectomy. Quantitative analysis of organelle populations after partial hepatectomy revealed a significant decline in the number of lysosomes per 100 μm². Small decreases in mitochondria and uricosomes were evident when expressed per unit area but the lysosome/mitochondria index was significantly lowered after partial hepatectomy. These observations suggest a potential decrease in the reactivity of the lysosome-vacuolar apparatus after partial hepatectomy and may contribute evidence suggesting a decrease in the catabolic potential of the cell at a time of increased macromolecular synthesis.     

Circadian rhythms in the rat: Constant darkness,entrainment to T cycles and to skeleton photoperiods

Free running activity and drinking rhythms of male Sprague-Dawley rats were observed in constant darkness (DD) for up to 44 days. The average period of the rhythms ($\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$) was 24.2 hr (±0.12 hr) and the activity time was near one half of the circadian cycle. In the second experiment, rats were entrained to T cycles (T=period) with 2 hr of light per cycle. At T=23 and T=26 about one half of the rats entrained indicating that these periods are near the limits of entrainment. T=23 induced a lasting aftereffect on $\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$ while T=26 affected $\text{τ}{}_{\mathrm{<mtext>dd</mtext>}}$ only briefly. In contrast to some other nocturnal rodents, activity time was not compressed as T neared the limits of entrainment. In the third experiment, rats and hamsters were entrained to 24 hr skeleton photoperiods (two 1 hr light pulses/cycle). Rats phase jumped to the longer subjective night when the interval between the light pulses was reduced to 6 or 5 hr, while most hamsters phase jumped at 3.5 hr. Furthermore, all rats phase jumped by means of delaying transients while most hamsters showed advancing transients. Finally, while skeleton photoperiods compressed activity time in hamsters to 6 hr or less, activity time remained fairly constant in rats. These results demonstrate considerable differences in the organization of the circadian system among commonly studied nocturnal rodents.     

Differential effects of choline administration on liver microsomes of female and male rats

Choline was administered for 3 weeks, twice daily, in the amount of $\text{50}\text{mg}\text{100}\text{g}$ body weight to male and female rats. The content of phosphatidyl cholines (PC) and the enzymatic activities related to the smooth endoplasmic reticulum were increased in the female rats receiving choline but not in males. Such changes were accompanied by an increase in surface of smooth membranes, evaluated by stereological analyses. Both enzymatic activities and values of surface and volume densities of the endoplasmic reticulum were lower in the untreated females than in the corresponding group of males. Morphological electron microscopic findings well agree with the increased biogenesis of smooth surfaced membranes in the hepatocytes of the liver of female rats receiving choline.     

Biochemical effects of three carcinogenic chlorinated methanes in rat liver

Three chlorinated methanes, carbon tetrachloride, chloroform, and methylene chloride, known to cause liver tumors in rodents, were given by oral gavage to adult female rats both 21 h and 4 h before sacrifice. Then hepatic DNA damage, ornithine decarboxylase (ODC), cytochrome P-450, glutathione content, and serum alanine aminotransferase (SGPT) activity assays were performed. Carbon tetrachloride increased rat hepatic ODC activity and decreased cytochrome P-450 content at doses both below and above cytotoxicity (as measured by increased SGPT activity). At 54 and 160 mg/kg, chloroform increased hepatic ODC activity with minimal or no elevation in SGPT activity. At 480 mg/kg chloroform increased hepatic ODC and SGPT activity. A dose of 1,275 mg/kg methylene chloride caused a small, but significant amount of hepatic DNA damage. When these three compounds are compared on either an equimolar or equitoxic (1/5 LD50) basis, their ability to induce hepatic ODC or increase SGPT activity was carbon tetrachloride greater than chloroform greater than methylene chloride. The results of this biochemical study are interpreted with respect to the ability of chemicals to cause hepatic cancer by either genetic or epigenetic mechanisms.     

Effect of cycloheximide on the distribution of alpha-naphthylisothiocyanate in rats

Others have shown that in rats the administration of cycloheximide (2 mg/kg) within 2 hr before or after 300 mg/kg of α-naphthylisothiocyanate (ANIT) blocks the development of hyperbilirubinemia and cholestasis. The effects of cycloheximide on the tissue distribution of ANIT labeled with ¹⁴C or ³H were examined. In cycloheximide-treated animals, less ANIT-derived radioactivity was found in blood, liver, fat and several other tissues at various times after ANIT than in controls. Rats given a mixture of (naphthyl-4-³H) ANIT and (isothiocyanate-¹⁴C) ANIT with a ratio of ${}^6\text{H}{}^{14}\text{C}\text{:6.3}$, excreted in their bile during the first 8 hr after ANIT, more ³H compared to ¹⁴C. In rats treated with cycloheximide, the ${}^3\text{H}{}^{14}\text{C}$ ratio did not change. The data suggest that normal animals excrete an ANIT metabolite in their bile from which the ¹⁴C label in the isothiocyanate moiety has been lost, whereas this does not happen in cycloheximide-treated animals. This provides further evidence that biotransformation of ANIT is necessary for the development of hyperbilirubinemia and cholestasis. Bile might be a convenient starting material for the isolation and identification of ANIT metabolites.     

Glycoprotein processing in mutants of HSV-1 that induce cell fusion

A new biosynthesis of thymidylate, the direct deamination of 5mdCMP to dTMP, in Xanthomonas oryzae infected by phage Xp12 was demonstrated both in vivo and in vitro. In vivo administration of DNA precursors [methyl-¹⁴C]5-methyldeoxycytidine and [methyl-³H]thymidine resulted in thymidine being incorporated only into the thymine of Xp12 DNA, whereas 5-methyldeoxycytidine was incorporated into both the thymine and the 5-methylcytosine equally. This implied that 5-methylcytosine could be directly converted into thymine at a certain level of 5-methylcytosine during Xp12 infection. This possibility was further proved by tracing the ${}^{14}\text{C}{}^3\text{H}$ ratio of double-labeled [methyl-¹⁴C, 6-³H]5-methyldeoxycytidine into the thymine residue of Xp12 DNA. In an in vitro study, an enzyme 5-methyldeoxycytidylate aminohydrolase, responsible for this conversion, was detected in a cell-free system of Xp12-infected cells. The enzyme could not utilize dCMP as substrate and preferred 5mdCMP over 5-methyldeoxycytidine; it seems likely that the conversion occurred at the nucleotide level of 5-methylcytosine.     

Influence of contraceptive and other sex steroids on aortic collagen and elastin

The effects of estradiol, progesterone, the contraceptive steroid combination mestranol-norethynodrel (M-N), and testosterone on aortic connective tissue were studied in ovariectomized and intact female rats. Estradiol and M-N administration to ovariectomized rats resulted in decreased accumulation of aortic collagen and elastin and, after injection of rats with [¹⁴C]proline, a lower specific activity of hydroxyproline in aortic collagen and elastin. The ratio of collagen to elastin ($\text{C}\text{E}$), which is an index of passive stiffness, was relatively low in aortas of all rats receiving female hormones and high in ovariectomized oil-treated and in testosterone-treated rats. Estradiol and M-N reduced $\text{C}\text{E}$ mainly by decreasing the percentage collagen, but progesterone reduced it by increasing the percentage elastin. It is concluded that sex hormones differentially alter aortic collagen and elastin synthesis and degradation and in so doing alter the accumulation and proportions of the two fibers in the vessel wall.     

Glutamate metabolism in ageing rat brain

The metabolism of glutamate, taken as an index of the metabolic state of the brain, was studied in brains of 3-, 12- and 30-month-old rats. Following the injection of a mixture of [³H] acetate and d-[2-¹⁴C] glucose, the brain levels of glutamate and aspartate were decreased in 30-month-old rats when compared with those of 3-month-old rats. No significant age-related differences were found in glutamine levels. Neither the protein levels nor the incorporation of the radioactivity in brain proteins differed among the three age groups, suggesting that there are no age-related differences in protein synthesis. The incorporation of $\text{d-[2-}{}^{14}\text{C}\text{]}$ glucose into aspartate and glutamine, expressed as the respective relative specific activities (RSA: specific activity of amino acid/specific activity of glutamate), did not change with age. Since glucose is the precursor of the large glutamate pool in brain, it can be concluded that no age-related changes occur in the metabolism of glutamate in the large compartment. The incorporation of [³H] acetate into aspartate, expressed as the RSA, did not differ among the age groups. The RSA of ³H-labelled glutamine, however, was significantly decreased 10 minutes after injection of the precursor mixture in brains of 30-month-old rats when compared with those of 3-month-old rats. This difference had disappeared 20 minutes after injection, suggesting a somewhat delayed metabolism of glutamate in the small compartment, for which acetate is a precursor.These results and all the other parameters measured indicate that no large age-related metabolic changes in rat brain occur.     

Thrombosis and atherosclerosis: the organization of pulmonary thromboemboli in the pig. Individual phospholipids, fatty acid composition of lecithin, sphingomyelin, esterified cholesterol, and 3 H-cholesterol specific activity

The organization of pulmonary thromboemboli in 8 to 12-week-old pigs was studied at intervals from 6 hr to 4 weeks. Thrombi were prepared in vitro in Chandler rotating loops.The predominant phospholipids of organizing emboli at all time periods were lecithin (45–57%) and sphingomyelin (15–24%). The content of all phospholipids decreased from 6 hr to 5 days. From 5 days to 4 weeks, there was no statistically significant change in the content of any phospholipid. The predominant fatty acids of organizing emboli were present in the order, 16:0 > 18:1 > 18:0 in lecithin, and 16:0 > 18:0 > 18:1 in both sphingomyelin and esterified cholesterol.Spontaneous aortic fatty streaks and fibrous plaques of 5- to $\text{7}\text{1}\text{2}\text{-}\text{year-old}$ pigs contained greater amounts of lecithin and particularly sphingomyelin than the organizing emboli. The predominant fatty acids of fibrous plaques were present in the order, 18:0 > 18:1 > 16:0 in lecithin, 16:0 > 18:0 > 20:0 in sphingomyelin, and 18:2 > 18:1 > 16:0 in esterified cholesterol.Intravenous injection of ³H-cholesterol in autologous plasma resulted in significant amounts of both labeled free and esterified cholesterol in the emboli at all periods of organization.This study indicates that the organization of pulmonary thromboemboli into fibro-fatty plaques is not associated with phospholipid and fatty acid profiles which evolve toward those of spontaneous atherosclerotic plaques.     

Effect of tryptophan on hepatoma and host liver of rats: Influence after treatment with hypertonic sodium chloride and carbon tetrachloride

Female inbred Buffalo rats bearing intrahepatically transplanted hepatoma 5123 were subjected intraperitoneally to the acute administration of hypertonic NaCl or CCl₄ followed by a tube-feeding of l-tryptophan. The responses in terms of changes in polyribosomal aggregation and protein synthesis (in vitro) of host liver and hepatoma were evaluated. While treatment with hypertonic NaCl or CCl₄ caused disaggregation of polyribosomes and inhibition of protein synthesis in both host liver and hepatoma, the subsequent administration of tryptophan caused some improvement in both parameters in host liver but not in hepatoma. Administration of hypertonic NaCl alone caused a decrease in [¹⁴C]orotate incorporation into poly(A)-mRNA of host liver and hepatoma, whereas administration of tryptophan after hypertonic NaCl caused a significant improvement in host liver alone. Following the tryptophan administration, the activities of nuclear DNA-dependent RNA polymerases I and II, and of nuclear-envelope nucleoside triphosphatase, as well as labeled nuclear RNA release in vitro were slightly elevated in host liver but not in hepatoma. Tryptophan-related compounds, 5-hydroxy-dl-tryptophan, 5-fluorotryptophan, indole, and 3-hydroxyanthranilic acid, when administered in place of l-tryptophan, did not appreciably affect polyribosomal aggregation or protein synthesis in vitro in host liver or hepatoma.     

The effects of cycloheximide, actinomycin D, and ethionine on the biliary excretion of labelled alpha-naphthylisothiocyanate in rats.

It has recently been shown that rats given a mixture of [³H-4-naphthyl] α-naphthylisothiocyanate (ANIT) and [¹⁴C-isothiocyanate] ANIT having a ${}^3\text{H}{}^{14}\text{C}$ ratio equal to 6.3, excreted more ³H than ¹⁴C into bile during the first 8 hours after ANIT administration, the ratio increasing to 7.5. However, in rats pretreated with cycloheximide, the ratio remained similar to that of the ANIT mixture administered (6.3) and data indicated that this inhibitor blocked this increase in ³H. Here we present further data showing that this effect by cycloheximide is dose-dependent, can be observed as late as 16 hours after ANIT administration, and is sensitive to early posttreatment times not to exceed 1 hour after ANIT. Actinomycin D and dl-ethionine also significantly blocked the increase in ${}^3\text{H}{}^{14}\text{C}$ ratio but it was found necessary to give both inhibitors 4 hours before ANIT administration in order to effect the decrease within 8 hours after ANIT administration. If given 30 minutes before ANIT, these inhibitors showed no effect until 9 hours after ANIT administration. These data further support the conclusion that cycloheximide, as well as actinomycin D and dl-ethionine, may act by inhibiting the formation of a toxic moiety of ANIT. The effects of these inhibitors on decreasing the excretion of a ³H moiety of ANIT in bile seem to coincide, on the basis of potency and protection, with their effects against hyperbilirubinemia and cholestasis.     

Preventive effect of a galactoglucomannan (GGM) from Dendrobium huoshanense on selenium-induced liver injury and fibrosis in rats

This study was carried out to investigate the preventive effects of galactoglucomannan (GGM), a homogeneous polysaccharide from Dendrobium huoshanense, on liver injury and fibrosis induced by sodium selenite. Sprague–Dawley rats injected subcutaneously with sodium selenite at the dosage of 3.28 mg kg^?1 b. wt. were set as the model groups. Rats treated with sodium selenite at the dosage of 3.28 mg kg^?1 b. wt. and GGM at 50–200 mg kg^?1 b. wt. were set as the prevention groups. Biochemical and histological analysis showed that GGM significantly ameliorated selenite-induced liver injury and fibrosis in rats. Oral administration of GGM effectively attenuated the toxicity of selenite to liver tissue, which was judged both by the decreased activities of serum hepatic enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and by liver histopathological examination. Meanwhile, GGM also reduced the levels of H₂O₂ and malondialdehyde (MDA), elevated the levels of GSH, restored the fluidity of hepatic plasma membrane, and retained the activities of endogenous enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). The prevention of selenite-induced liver injury and fibrosis by GGM was further supported by the reduced expression of transforming growth factor-β1 (TGF-β1) and type I collagen. These results suggested that GGM may be developed into a novel antifibrotic agent for the prevention of liver injury and fibrosis.     

Ischemia-induced canine myocardial lysosome labilization: The role of endogenous prostaglandins and cyclic nucleotides

We tested the hypothesis that alterations in myocardial prostaglandin levels were associated with ischemia-induced lysosome labilization. To test this hypothesis, endogenous prostaglandin synthesis was restricted by administering indomethacin (INDO). After a thoracotomy in dogs, INDO (5 mg/kg) or vehicle was infused iv and 15 min later myocardial ischemia was induced by occlusion of the left anterior descending (LAD) coronary artery and the results compared to sham-operated animals. Myocardial tissue samples were biopsied 3 hr post-LAD ligation, homogenized, and the lysosome fraction subjected to a mild hypo-osmotic shock. The lysosomes from ischemic tissue were more labile compared to lysosomes from nonischemic tissue and this was associated with a decrease in myocardial $\text{cAMP}\text{cGMP}$ in ischemic tissue. Myocardial $\text{cAMP}\text{cGMP}$ correlated positively with myocardial lysosome stability (P < 0.05) and tissue prostaglandin E and A concentrations correlated negatively with myocardial cAMP (P < 0.05). In an in vitro liver lysosome system, INDO had no effect on enzyme release. These data suggest that ischemia-induced lysosome labilization via an increased prostaglandin release causes a lowered $\text{cAMP}\text{cGMP}$.     

Corticosterone "basal levels" and response to ether anesthesia in rats on a water deprivation regimen.

Male hooded rats were subjected to a 23 $\text{2}\text{3}\text{-}\text{hr}$ water deprivation schedule for 8 weeks. The corticosterone levels of these rats increased between 0800 and 1000 hr if they had always been watered at 1200 hr, but increased between 0600 and 0800 if the daily watering time had been randomized between 0800 and 1200 hr the previous 8 weeks. The corticosterone levels remained high until 1200 hr for both treatments. The results indicate temporal entrainment and a stable, elevated baseline corticosterone concentration. Eight weeks on the deprivation schedule resulted in accelerated corticosterone responses to acute stressors, but the acceleration was minimized when the rats were maintained under continuous ether anesthesia. The procedure of blood sampling under ether anesthesia is valid for water-deprived rats if the sample is drawn within 1.5 min, but it is not valid for up to 3 min as in ad lib rats.     

Mechanism of renal necrosis induced by bromobenzene or chlorobenzene

Treatment of mice or rats with a single intraperitoneal dose of [¹⁴C]-bromobenzene or [¹⁴C]-chlorobenzene produced necrosis of the proximal convoluted renal tubules within 24–48 hr. The development of renal necrosis was associated with the covalent binding of substantial amounts of radiolabeled material to kidney proteins, and autoradiograms revealed that most of the covalently bound material was localized within the necrotic tubular cells. Prior inhibition of [¹⁴C]-bromobenzene metabolism in vivo with piperonyl butoxide blocked both the necrosis and the binding, suggesting that the necrosis and binding are caused by a toxic metabolite. Studies on the metabolism and covalent binding of [¹⁴C]-bromobenzene in hepatic and renal microsomes in vitro were compatible with the interpretation that the renal necrosis was caused by a metabolite formed in the liver and transported by the circulation to binding sites in the renal tubules.     

Antispermatogenic activity of 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid (AF 1312/TS) in rats. I. Trials of single and short-term administrations with study of pharmacologic and toxicologic effects.

A new antispermtogenic agent is described. Following single or short-term administrations, 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid, or $\text{AF 1312}\text{TS}$, produced in rats a long-lasting inhibition of the spermatogenic process. In adult rats the secondary sex organs were not affected, while in young rats some weight decrease of the prostate, seminal vesicles and levator ani was observed. The interstitial tissue of the testes as well as thymus, adrenals and hypophysis were not affected. $\text{AF 1312}\text{TS}$ proved to be devoid of the most common pharmacological activities.