Sequential changes in lipid metabolism and the fatty acid profile in liver lipids during fasting and sepsis.

The sequential changes in lipid metabolism and in the fatty acid profile of liver lipids during fasting and sepsis were studied. Liver and blood specimens were taken from normally fed rats and from nonseptically and septically fasted rats at 5, 24, and 48 hr. Sepsis was induced by injecting live Escherichia coli bacteria intraperitoneally. Sepsis attenuated the fasting-induced increase in beta-hydroxybutyrate and reduced liver and serum triglycerides at 5 hr. There was a percentage decline in the most abundant fatty acids in neutral lipids, namely oleic (18:1w9) and linoleic (18:2w6) acids. This was seen throughout fasting and septic fasting. These results indicate that 18:1w9 and 18:2w6 are used as energy substrates and are oxidized to beta-hydroxybutyrate during fasting and mainly to carbon dioxide during septic fasting. On the contrary, the most abundant fatty acids in phospholipids, stearic (18:0), arachidonic (20:4w6), and docosahexaenoic (22:6w3) acids, accumulated in neutral lipids and in phospholipids throughout fasting. However, during sepsis this accumulation was reduced in neutral lipids and reversed to a level below that in the fed and fasted state in phospholipids. These results indicate that a disturbance in membrane integrity and function induced by septic fasting may have pathophysiological consequences for lipid metabolism and liver function during sepsis.

Comparison of the calcium-antagonistic effects of terodiline, nifedipine and verapamil.

Studies were carried out on the Ca-antagonistic effects of terodiline and its enantiomers on the potassium-stimulated mesenteric and coronary arteries, on the spontaneous myogenic activity and norepinephrine- and acetylcholine-induced contractions of the protal vein and on electrically stimulated papillary muscle. The effects were compared with those of the Ca-antagonists, nifedipine and verapamil. Terodiline is relatively weak as a Ca-antagonist, having IC50-values between 5 X 10(-6) and 2 X 10(-5)M for all the tissues studied. Nifedipine is the most potent Ca-antagonist on vascular smooth muscle (IC50 3-6 X 10(-9) M), but is considerably less potent on the papillary muscle (IC50 10(-7)M). Verapamil is most potent on the papillary muscle (IC50 7 X 10(-8)M and the portal vein (IC50 6 X 10(-8)M, but is 10 times less potent on the mesenteric and coronary arteries (IC50 3-5 X 10(-7)M). Nifedipine is 1000 times and verapamil and (-)-terodiline 10 times more potent on the slow component of the K-induced contraction while (+/-)- and (+)-terodiline are almost as active on the fast as on the slow component of K-induced contractions on the mesenteric artery. Furthermore, (+/-)-and (+)-terodiline are 10 times more potent in antagonizing acetylcholine- and norepinephrine-induced contractions, whereas (-)-terodiline is equally potent and nifedipine and verapamil are 10 times more potent in blocking the myogenic activity of the portal vein. On the Ca2+-nifedipine and verapamil. However, nifedipine and verapamil, but not terodiline, in concentrations which blocked the maximal norepinephrine-induced response in non-depleted muscle, antagonized the contractions induced by norepinephrine together with Ca2+ in the Ca2+ depleted portal vein. These results show that terodiline blocks the uptake of Ca2+ and, in addition, blocks the utilization of some intracellular stores of Ca2+.

Effects of triethyllead chloride on hepatic microsomal N- and C-oxygenation of N,N-dimethylaniline in rats.

Triethyllead chloride was added directly to male rat liver microsomes (0.0, 0.05, 0.25, 0.5, 1.0, and 3.0 mM), or the rats were pretreated with ip injections, once a day for 2 days (0.0, 1.5, and 3.0 mg triethyllead chloride/kg body wt), and microsomes prepared without any further treatment on Day 3. The effects of these treatments on microsomal N-oxygenation and C-oxygenation in vitro were studied by using N-oxide formation and N-demethylation of N,N-dimethylaniline (DMA) as test reactions. Increased microsomal N-oxygenation was obtained by in vivo treatment and decreased microsomal C-oxygenation by in vitro treatment. As a result, either treatment thus gave rise to an increase in the N-oxygenation/C-oxygenation ratio. Conditions favoring accumulation of the N-oxide product may potentiate the tumor-inducing characteristics of carcinogenic aromatic amines.

Effects of dimethylnitrosamine on metabolism of N,N-dimethylaniline by rat liver microsomes. A comparative study of treatment in vivo, in isolated liver perfusion and in the microsomal system.

The effect of dimethylnitrosamine (DMN) on rat liver microsomal detoxication was studied, using the non-carcinogenic aromatic amine N,N-dimethylaniline (dimethylaniline) as substrate. Prior to the preparation of microsomes, the rat liver was exposed to DMN either in vivo (by i.p. injection) or in the isolated liver perfusion system (by addition to the perfusion medium). DMN treatment in vivo (20 mg/kg body wt.) caused a 40% increase in dimethylaniline N-oxygenation and a 30% decrease in dimethylaniline C-oxygenation. When DMN was added to the perfusion medium to a final concentration of 5 or 25 mM, a similar effect was observed. With the 5 mM dose, C-oxygenation was decreased by 20% with a non-significant increase in N-oxygenation. The higher dose caused a 50% increase in N-oxygenation, whereas the decrease in C-oxygenation remained at 20%. When microsomes were incubated with both DMN (5 mM) and dimethylaniline (5 mM) in the system, a small but significant decrease in both N- and C-oxygenation of dimethylaniline was observed. The effect of DMN on the amino acid incorporation into liver and plasma proteins was also studied in the liver perfusion system. The synthesis of both liver and plasma proteins was reduced by DMN.

Increased transcription activity of rat liver chromatin after protein restriction and limited digestion of nuclei with micrococcus nuclease.

Protein restriction has been shown to produce either an enhancement or a reduction of transcription activity in vitro. Conditions for an enhanced transcription activity were investigated. Young male rats were fed a complete diet containing either 20% or 3% casein for 6 days. Body weight changed +7.4 g/day and -0.5 g/day, respectively. Liver wet weights were 6.8 and 3.7 g and the DNA amounts/g were 1.31 and 1.67 mg. Liver nuclei were incubated without or with 1 unit micrococcus nuclease (EC 3.1.4.7) per milligram of nuclear DNA, and chromatin was fractionated into a 2,000 x g, a 102,000 x g pellet and a supernatant fraction. In the livers of rats fed a low protein diet, chromatin-bound RNA polymerase I plus III and II activity/mg of fractional and nuclear DNA and soluble RNA polymerase activity were increased, while heparin-stimulated RNA polymerase II activity remained unchanged. An increased number of chains was synthesized by RNA polymerase I plus III without change in chain length and incorporation rate per chain. The length and incorporation rate per chain increased, while the number of chains synthesized by RNA polymerase II did not. After stimulation by heparin, an increased number of short chains was synthesized at a lower rate of incorporation per chain. In the complex chromating structures the capacity for RNA synthesis was determined by specific enzyme activity, RNA chain number and length.

Reduced transcription activity of rat liver chromatin after protein restriction and selective digestion of nuclei with micrococcus nuclease.

Restriction of protein intake in the diet has been shown to produce a change in transcription activity as determined in vitro. Conditions for a reduced transcription activity were investigated with selective digestion of nuclei with micrococcus nuclease (EC 3.1.4.7). Liver nuclei from young male rats fed a diet containing either 20 or 3% casein for 6 days were digested with 1.3 microgram of micrococcus nuclease protein/mg of nuclear DNA (specific enzyme activity 15,000 or 150 units/mg of protein). Part of the chromatin-bound RNA polymerase activity was transferred from the 2,000 x g to the 102,000 x g pellet. Independent of the type of endonuclease use, the specific activity of chromatin-bound and soluble RNA polymerase I plus III was similar in the two groups of rats. After protein restriction RNA polymerase II activity was significantly diminished in the 2,000 x g pellet, and was unchanged in the 102,000 x g pellet. Heparin-stimulated and soluble RNA polymerase II activities were significantly reduced. Number and length of RNA chains synthetized by chromatin-bound RNA polymerase I plus III remained unchanged by dietary treatment. After a low protein diet, RNA polymerase II in the absence and presence of heparin synthesized an unchanged number of RNA chains with reduced length. A selective digestion of chromatin with micrococcus nuclease is needed to show the reduction in RNA polymerase II activity after protein restriction.

Detoxication disturbances and uncoupling effects in vitro of some chlorinated guaiacols, catechols and benzoquinones.

The effects of chlorinated guaiacols, catechols and benzoquinones - earlier identified in discharges from pulp and paper mills -- on oxidative phosphorylation in mitochondria and on detoxication mechanisms in microsomes have been investigated. The same biological systems have been used in testing outgoing water from a sulphite plant. Trichlorocatechol and tetrachlorocatechol and the corresponding guaiacols increase N oxygenation of N,N-dimethylaniline, while C-oxygenation decreases, indicating disturbances in microsomal detoxication. The substances tested have in general an uncoupling effect on mitochondrial oxidative phosphorylation. However, tetrachloroguaiacol seems to differ in having a specific effect on the succinate dehydrogenase part of the respiratory chain. Extracts from waste water from a sulphite plant have a considerable effect on the mitochondrial system such as to produce an increase in basal respiration and a loss of respiratory control. N- and C-oxygenation and phosphorylation have been extensively used in this and other laboratories for evaluating the toxicity of chemicals. The application on water extract described here is rapid and easily handled and thus provides a valuable complement to other water quality tests.

Isolated liver perfusion--a tool in mutagenicity testing for the evaluation of carcinogens.

An isolated liver perfusion system suitable for the combination with Chinese hamster V79 cells is described. With this system, it is possible to study, with the V79 cells as genetic targets, the mutagenic effect of a chemical after metabolic activation in the intact organ. Those substances commonly used in mutagenicity testing as inducers of drug metabolising enzymes, i.e. Arochlor 1254. Phenobarbital(PB) and 3-Methylcholantrene(3-MC), were studied for their effect in the isolated perfused liver. PB increased the bile flow, which was not significantly affected by the other inducers. Only Arochlor caused a significant increase in the amino acid incorporation into plasma proteins and total liver proteins (expressed per mg liver protein). None of the inducers had an effect on gluconeogenesis from lactate or urea synthesis. All three inducers caused an increase in the level of microsomal P-450 enzymes, the biggest increase being seen after Arochlor-induction (170%), followed by PB(90%) and 3-MC(50%). Arochlor- and PB-induction had a dramatic effect on N- and C-oxygenation of N, N-dimethylaniline: N-oxygenation was decreased by 35% and 40% respectively and C-oxygenation increased by 130% and 140% respectively. The advantages of the isolated perfused liver as an intact metabolising unit is discussed in relation to other mutagenicity assays, in which subcellular fractions are used as the metabolising system.

Ethanol inhibition of vinyl chloride metabolism in isolated rat hepatocytes.

Isolated rat liver cells convert [14C]vinyl chloride into non-volatile metabolites. The metabolism is not increased by in vivo pretreatment with phenobarbital. It is sensitive to inhibition by ethanol, which at a concentration of 4 mM inhibits vinyl chloride metabolism to 50% in hepatocyte suspensions. The metabolic activity is NADPH-dependent and is localized in the microsomal fraction of the liver. The enzyme is also strongly inhibited by tetrahydrofuran, indicating that it could be identical to an ethanol-inducible cytochrome P-450 described in the literature [1].

Influence of cystein deficiency on the inhibition of hepatic microsomal detoxication by methyl mercury in two rat strains.

Two rat strains, Wistar, strain R and Sprague--Dawley, were subjected to cystein deficiency and methyl mercury pretreatment, both separately and in combination, after which the hepatic microsomal N- and C-oxygenation of N,N-dimethylaniline (DMA) was studied. Cystein deficiency caused a reduction in C-oxygenation in strain R microsomes, and this reduction was nearly doubled by methyl mercury pretreatment of the depleted rats. Methyl mercury pretreatment per se of strain R rats on the standard diet gave no effect. By contrast microsomes from cystein deficient SpD rats showed no statistically significant decrease in C-oxygenation, and cystein deficiency did not further enhance the inhibitory effect obtained with methyl mercury pretreatment alone. N-oxygenation was not significantly affected by any treatment of the two strains.

Influence of protein or cystein deficiency on hepatic subcellular distribution of methyl mercury in two rat strains.

The influence of protein deprivation and cystein deficiency on the distribution of methyl mercury between 4 subcellular fractions of liver was studied in 2 rat strains (Wistar, strain R and Sprague-Dawley). Kept on a standard diet, the 2 strains showed a similar distribution pattern, with the highest mercury level found in the cytosol, followed by the mitochondrial, microsomal and nuclei fractions. The protein free diet caused on increase in the total amount of bound mercury in both strains, the greatest increase, being found in livers from strain R rats. The cystein deficient diet, on the other hand, gave rise to diverging results. Whereas the level of mercury bound to the subcellular fractions was increased in livers from strain R rats, it was markedly reduced in livers from Sprague-Dawley rats.

Influence of protein deficiency on the inhibition of hepatic microsomal detoxication by methyl mercury in two rat strains.

N- and C-oxygenation of N,N-dimethylaniline was studied in liver microsomes from 2 rat strains (Wistar, Strain R and Sprague-Dawley) subjected to protein deprivation and methyl mercury pretreatment, separately and in combination. A striking interstrain difference was observed. Strain R microsomes from 2 rat strains (Wistar, Strain R and Sprague-Dawley) subjected to tion, but little effect after methyl mercury pretreatment. With Sprague-Dawley microsomes, C-oxygenation was slightly reduced after both treatments. N-oxygenation was little affected in either strain. Methyl mercury treatment of protein deprived rats strongly inhibited C-oxygenation in microsomes from both strains, with N-oxygenation being unaffected in strain R microsomes whereas markedly reduced in microsomes from Sprague-Dawley.

Subcellular distribution, a factor in risk evaluation of pentachlorophenol.

Pentachlorophenol (PCP) is a potent uncoupler of mitochondrial phosphorylation in vitro and also interferes with microsomal detoxication functions in vitro. This favours flavin mediated oxygenation compared with flavin cytochrome P-450 dependent reactions. Gas chromatographic analysis of subcellular fractions, obtained by zonal centrifugation showed markedly lower PCP concentration in mitochondria and a high accumulation in microsomes compared with cytosol. This increases the likelihood that PCP in vivo causes a malfunction in microsomal detoxication.

Disturbance of microsomal detoxication mechanisms in liver by chlorophenol pesticides.

The pesticide pentachlorophenol known as an uncoupler of mitochondrial oxidative phosphorylation was shown to disturb liver microsomal detoxication functions by a selective inhibition of the terminal oxygenation enzyme P-450. At lower concentrations the flavin moiety of this enzyme chain is not inhibited but rather is stimulated, whereby a qualitative shift in detoxication of aromatic amines from C-oxygenation to N-oxygenation is obtained. The effects were due to the pentachlorophenol itself and not to a metabolite. Similar effects of varying strength were also obtained with other chlorophenol pesticides; 2,4,di-, 2,4,6,-tri and 2,3,4,6-tetrachlorophenol, di- and hexachlorophen, tri- and nonachloro-2-hydroxydiphenyl ethers. The relevance of these findings to the possible synergistic influence of chlorophenols on the carcinogenic effects of polyaromatic amines and hydrocarbons is discussed.

Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro.

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Some 250 compounds, representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects encountered for positive standards. When employing the present cell toxicity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the compounds studied, are not taken into account. The most active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation obsered between functional groups and toxicity permits, within the range of functionalities studied, prediction of the toxicity for a compound of known structure.