Skeletal muscle myopathy caused by Triton WR-1339

After administration (1 g/kg every other day for a total of five injections) of Triton WR-1339, the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles of the chicken underwent distinct pathological modifications. Some of the morphological alterations in the muscles paralleled those seen after administration of chloroquine, increased autophagic vacuole formation in the ALD muscle and swelling of the sarcoplasmic reticulum in the PLD muscle, but other changes were unique to Triton WR-1339. These included loss of myofilaments and whole myofibrils, indentation of the sarcolemma as well as increased numbers of ribosomes in the ALD muscle and swelling of the T-tubular system in the PLD muscle. These results are compared with other lysosome mediated pathologies, as well as with other myopathies.     

Reversal by Triton WR-1339 of ethynyloestradiol-induced hepatic cholesterol esterification

Rats treated with ethynyloestradiol have marked hypolipidaemia: serum cholesterol is decreased to 5%, triacylglycerol to 10% and phospholipid to 70% of control concentrations. Loss of serum cholesterol follows an exponential decay, with a half-life of 1.13±0.09 days. After 4 days of treatment, serum cholesterol concentrations remain relatively constant (ranging from 1 to 20mg/100ml) for at least 30 days. There is a concomitant 20-fold decrease in the d<1.21 fraction of serum proteins and a similar decrease in serum apolipoproteins as measured by sodium dodecyl sulphate/10%-polyacrylamide-gel electrophoresis. The activity of hepatic microsomal acyl-CoA–cholesterol O-acetyltransferase (EC 2.3.1.26) was significantly increased by ethynyloestradiol treatment (P<0.05). This activation caused hepatic cholesteryl esters containing mainly C_18:1 fatty acids to increase linearly as serum cholesterol concentrations decreased (r=0.9675, P<0.001). Triton WR-1339, a non-ionic detergent that inhibits lipoprotein catabolism, was used to estimate hepatic lipid secretion by measuring the increment in serum lipids after its administration. At 15h after Triton WR-1339 administration, serum cholesterol concentrations were increased equally in both control and ethynyloestradiol-treated rats. In contrast, the increment of serum triacylglycerol of treated rats was 40% of that found in control rats, indicating that ethynyloestradiol inhibits hepatic triacylglycerol secretion. Triton WR-1339 inhibited the oestrogen activation of hepatic microsomal acyl-CoA–cholesterol O-acyltransferase and restored hepatic cholesteryl ester concentrations to normal values. These data suggest that ethynyloestradiol and its pharmacological `antagonist' Triton WR-1339 alter hepatic triacylglycerol secretion via a mechanism associated with changes in hepatic cholesterol esterification.     

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Triton WR-1339 on lecithin-cholesterol acyltransferase in the rat

The effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase was measured in rat serum following addition of Triton to the serum in vitro or after intravenous injection of the detergent. The inhibitory effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase when the detergent was added in vitro was dose dependent and appeared to result from a direct action on the enzyme rather than from a physical modification of the substrate by the detergent. The serum half-life ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of Triton WR-1339 injected intravenously in the rat was 23.1 ± 1.0 h. The inhibitory effect of Triton on serum LCAT activity when the detergent was given intravenously was also dose dependent and was reversed when the serum concentration of Triton decreased; under specific conditions, LCAT activity reached values higher than control. This behavior after treatment of the animal may be explained by increased concentration of the enzyme in the plasma, by stimulation of LCAT activity by the very low density lipoprotein or metabolites accumulating in the plasma of rats treated with Triton WR-1339, or by a combination of these factors.     

The influence of Cortisone on the hepatic uptake of Triton WR-1339 in adrenalectomized rats.

The effect of cortisone acetate on the hepatic uptake of Triton WR-1339 was studied in adrenalectomized rats. The hormone was found to retard the uptake of Triton in the liver, and at the same time reduce the plasma clearance of this compound. The inhibitory effect of the hormone on endocytosis was seen in purified preparations of both Kupffer cells and parenchymal cells. Triton renders the liver lysosomes progressively lighter. The change in equilibrium density (in sucrose gradients) was found to occur more rapidly in hormone-treated animals. The possibility that cortisone reduces the number and increases the size of the lysosomes is discussed. Our data indicate that the uptake of Triton in non-parenchymal (Kupffer) cells represents about 20% of the total hepatic uptake.     

Effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase of rat.

The effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200 mg per 100 g body weight. Catalase activity decreased to about 35% of that of the control at 42-48 h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96 h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degradation (kd) decreased to about 50% of the control.     

Ultrastructural studies on the effect of Triton WR-1339 on macrophage-mycobacteria interaction

Mycobacterium bovis (BCG organisms) suspended in saline or a 5% solution of a non-ionic detergent, Triton WR-1339, was injected intraperitoneally into mice. Electron-microscopic observation was carried out on peritoneal exudate cells harvested therefrom. Electron-lucent vacuoles limited by the membrane structure were found in macrophages of the mice injected with BCG suspended in the detergent, but not in polymorphonuclear leukocytes or lymphocytes. Mycobacterial cells were present within such vacuoles. Without the detergent, the ingested mycobacterial cells were in close contact with the phagosomal membrane. Within the electron-lucent vacuoles, however, such close contact was not present. These observations, together with other collateral findings, led us to a view that Triton WR-1339 may inhibit the interaction between mycobacteria and the phagosomal membrane by intervening between them thus making the progress of infection delayed.     

Voluntary food consumption and gastric emptying in rats given intravenous Triton WR-1339

Intravenously administered Triton WR-1339, a nonionic surface active agent, has been used as an endogenous hyperlipemic agent since 1951. We expected Triton to increase food consumption to supply, at least partially, the energy and acetyl groups necessary for producing the hyperlipemic state. In this study, however, we observed that the rats injected intravenously with various dose levels of Triton decreased their voluntary food intake in a dose-related manner. Two other nonionic surface active agents, Tween 20 and Tween 80, given intravenously did not alter food intake. Further studies revealed that Triton WR-1339 administered intravenously 30 min before feeding by stomach tube resulted in a marked delay in the rate of gastric emptying which was also dose related. A delay in gastric emptying has previously been suggested as one mechanism that controls food intake. Tween 20 and Tween 80 did not alter the rate of gastric emptying. We suggest that the mechanism responsible for the decrease in voluntary food consumption in Triton WR-1339 injected rats may be due to the delay of gastric emptying in these animals.     

Effects of the lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, on the synthesis and secretion of lipids by rat hepatocytes

The lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, were incubated with rat hepatocytes. Triton WR-1339 increased the recovery of triacylglycerol in the hepatocytes and incubation medium by 31% and 38%, respectively. Tetrahydrolipstatin decreased the accumulation of newly synthesized, and of total triacylglycerol in the medium. This compound might be useful in determining mechanisms involved in intracellular triacylglycerol metabolism and the secretion of very low density lipoproteins.     

Mitochondrial damage and inhibition of respiration in animal cell cultures treated with Triton WR-1339

Inhibition of cellular respiration by treatment with the nonionic detergent Triton WR-1339 was found to be related to the cytotoxic response of cell to the surfactant. Respiration of sensitive cell lines (AV-3 and HeLa) markedly inhibited by Triton concentrations as low as 125 μgm/ml. Conditionally sensitive lines (BHK-21 and L-929) were affected by 500 μgm/ml while the respiration of insensitive cultures (primary rat and chick embryo cells) was unaffected by this concentration. Macrocyclon, a cyclic analogue of Triton, failed to alter the respiration rate of any of the above cell cultures. The levels of isocitric and succinic dehydrogenases in sensitive and conditionally sensitive cells were depressed within 2 hours after treatment with 500 μgm/ml of Triton was initiated and by 6 hours the activity was only 25% of the untreated controls. Similar results were obtained with mitochondrial preparations from these cells. Enzyme levels in insensitive cells were unaffected by Triton treatment. Mitochondrial damage was the most striking characteristic noted in treated cells examined by electron microscopy. The mitochondria were quite distorted and had lost most of their cristae formation. This mitochondrial damage was seen in all cell types examined although the rate at which it occurred varied. With sensitive cultures, damage was pronounced within 6 hours after the addition of Triton while mitochondria from conditionally sensitive cells were not grossly affected until 48 hours and they appeared to repair the damage following the removal of Triton.     

The origin of lipid accumulated in liver lysosomes after administration of triton WR-1339

The origin of the lipids accumulated in liver lysosomes after administration of Triton WR-1339 was investigated. When Triton WR-1339 was injected into rats, serum triglyceride and cholesterol increased markedly. The highest content of triglyceride was observed in the second-day serum, from which very-low-density lipoprotein (VLDL) was isolated. The VLDL was administered to normal rats, then the light mitochondrial fraction of the liver at 24 h was centrifuged in a sucrose density gradient. The activities of lysosomal enzymes, acid phosphatase, N-acetyl-beta-D-glucosaminidase and acid lipase, were all shifted to less dense fractions as compared with those of normal lysosomes. [3H]Triglyceride-labeled VLDL was injected similarly, and at 12 and 24 h after the administration, the light mitochondrial fraction of the liver was fractionated by sucrose gradient centrifugation. Protein content and radioactivity in the immunoprecipitate with anti-VLDL serum at 12 h showed almost the same distribution as acid phosphatase activity. At 24 h, though acid phosphatase activity, immunoprecipitable protein content and radioactivity were all found in less dense fractions than in the case of normal lysosomes, the former two distributions were significantly different from the latter. The anti-VLDL serum reacted in Ouchterlony tests not only with Triton-induced VLDL and normal VLDL but also with the extract from low-density lysosomes. These results suggest that the lipids accumulated in low-density lysosomes following the administration of Triton WR-1339 were probably derived from the elevated serum VLDL induced by the treatment.     

Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339

Triglyceride (TG), a water-insoluble energy-rich lipid, is secreted by the liver as part of very low density lipoproteins (VLDLs) to supply energy to extrahepatic tissues. Overproduction of VLDL is associated with increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG production. The TG production rate is determined by measuring temporal increases in plasma TG under conditions in which TG hydrolysis by lipoprotein lipase (LPL) is inhibited. The nonionic detergent, Triton WR-1339 (Triton), has commonly been used to inhibit LPL for this purpose. Triton, in addition to inhibition of TG hydrolysis, has properties that have the potential to adversely influence lipoprotein metabolism. Another nonionic detergent, poloxamer 407 (P-407), also inhibits LPL. In these studies, we demonstrate that P-407 is comparable to Triton in the determination of TG production but without the unwanted side effects of Triton.     

The effects of Triton WR-1339, protamine sulfate and heparin on the plasma removal of emulsion models of chylomicrons and remnants in rats

Protein-free lipid emulsions with compositions modelling chylomicrons (chylomicron-like emulsion) or chylomicron remnants (remnant-like emulsion) were injected intra-arterially into nonanesthetized rats. Compared with control untreated rats, treatment with Triton WR-1339, protamine sulfate or heparin strongly modified the plasma removal of triacylglycerols and cholesteryl ester moieties of chylomicron-like emulsions, but had little effect on removal rates of triacylglycerols or cholesteryl esters of remnant-like emulsions. The effects on chylomicron-like removal were similar to those on natural lymph chylomicrons. The relative lack of effects on remnant-like emulsion removal provides additional evidence that remnant-like emulsions are a metabolic model for natural chylomicron remnants.     

The effect of Triton WR-1339 on the subcellular distribution of Trypan Blue and 125I-labelled albumin in rat liver

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured (125)I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 (125)I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.     

Effects of gamma-terpinene on lipid concentrations in serum using Triton WR1339-treated rats

We studied lipid metabolism to evaluate the effects of gamma-terpinene on suppression of increases in serum lipid concentrations using Triton WR1339-treated rats. At 6 hr after Triton WR1339 injection, the total cholesterol and triglyceride concentrations in the gamma-terpinene group underwent statistically significant decreases (18.3 and 30.3%, respectively) compared with those of the Triton-treated group.     

Hypolipidemic effect and antioxidant activity of glycoprotein isolated from Ulmus davidiana Nakai in Triton WR-1339-treated mouse

The glycoprotein isolated from Ulmus davidiana Nakai (UDN) (UDN glycoprotein) has a molecular weight of 116 kDa and consists of 78.65% carbohydrate content and 21.35% protein content. In the present study, we investigated the hypolipidemic effect of UDN glycoprotein on Triton WR-1339-induced mice. With pretreatment with UDN glycoprotein, the triacylglycerol (TAG), total cholesterol and low density lipoprotein-cholesterol (LDL-C) concentrations were significantly reduced, whereas high density lipoprotein-cholesterol (HDL-C) concentration was increased in the plasma of Triton WR-1339-induced mice. With respect to antioxidative activity, UDN glycoprotein significantly decreased the level of thiobarbituric acid reactive substances (TBARS) and improved activities of catalase and glutathione peroxidase (GPx), without an apparent change of superoxide dismutase (SOD) activity. Also UDN glycoprotein significantly increased nitric oxide (NO) production in Triton WR-1339-induced mice. These results indicate that UDN glycoprotein has a hypolipidemic effect, possesses antioxidant activity and has an ability to stimulate NO production. Thus, we speculate that UDN glycoprotein is an example of natural compound that lowers plasma lipid level together with having an antioxidant function in Triton WR-1339-induced mice.