Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

The irreversible binding of¹⁴C from¹⁴CCl₄ to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl₄-induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl₄ is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P₁-450) by CCl₄ is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl₄ activation much more effectively than P₁-450.     

Carbon tetrachloride target lipids in the rat liver microsomes. Effect of cystamine administration on their pattern of labeling by 14CCl4

¹⁴C from ¹⁴CCl₄ irreversibly binds to lipids from the smooth (SER) and rough (RER) endoplasmic reticulum. Most of the label is associated with the phospholipid fraction (> 95%). Prior cystamine administration decreased the extent of that binding but does not change its pattern of distribution. About the half of the label in phospholipids is in the phosphatidylcholine fraction; the other half is distributed similarly among lysophosphatidylcholine, sphingomyelin and phosphatidylethanolamine, while only a very minor fraction is associated with diphosphatidyl glycerol. No differences were found in the pattern of labeling of phospholipids in SER and RER.     

Mechanism of in vivo carbon tetrachloride-induced liver microsomal cytochrome P-450 destruction.

Prior administration of aminotriazole (3-amino-1,2,4-triazole) or pyrazole to rats resulted in a significant prevention of the CCl₄-induced decrease in the liver microsomal P-450 content. In A/J mice the CCl₄ activation and P-450 destruction occurred in absolute absence of lipid peroxidation as determined by uv absorption. The data suggest that P-450 destruction is mainly mediated by direct attack of CCl₄ metabolites rather than by CCl₄-induced lipid peroxidation.     

The occurrence of multiple forms of cytochrome P-450 in hepatic microsomes from untreated rats and mice

The hepatic microsomes of rat and mice were subfractionated by the procedure of Dallner. When a 1.3 M sucrose lower layer was used for the two-step discontinuous gradient, no differences in spectral characteristics were noted between subfractions, though the smooth fractions (SER) had higher oxidative activity towards the substrates tested. When lower layers of 1.05, 1.1 or 1.15 M sucrose were used, the SER isolated contained cytochrome P-450 with significantly different spectral characteristics from that of the rough fraction (RER). The SER cytochrome P-450 had a wavelength maximum in the carbon-monoxide reduced difference spectrum that was significantly lower (ca. 1.0 nm) than that in the RER. In addition, the type I:CO-reduced spectral ratio of these fractions is significantly elevated. These data indicate that liver microsomes from untreated rats and mice contain more than one cytochrome P-450 and that these cytochromes may be located in different parts of the endoplasmic reticulum.     

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.     

CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL : II. Effects of Phenobarbital on Rat Hepatocytes

The changes occurring in rat hepatocytes during a 5 day period of treatment with phenobarbital were determined by morphometric and biochemical methods, particular attention being paid to the endoplasmic reticulum. The hepatocytic cytoplasm played an overwhelming part in the liver hypertrophy, while the hepatocytic nuclei contributed to only a moderate extent. The endoplasmic reticulum accounted for more than half of the increase in cytoplasmic volume. The increase in the volume and number of hepatocytic nuclei in the course of phenobarbital treatment was associated with changes in the ploidy pattern. Until the 2nd day of treatment both the rough-surfaced endoplasmic reticulum (RER) and the smooth-surfaced endoplasmic reticulum (SER) participated in the increase in volume and surface of the whole endoplasmic reticulum (ER). Subsequently, the values for RER fell again to control levels, whereas those for SER continued to increase, with the result that by the 5th day of treatment the SER constituted the dominant cytoplasmic element. The specific volume of mitochondria and microbodies (peroxisomes) remained constant throughout the duration of the experiment, while that of the dense bodies increased. The specific number of mitochondria and microbodies displayed a significant increase, associated with a decrease in their mean volume. The phenobarbital-induced increase in the phospholipid and cytochrome P-450 content of the microsomes, as well as in the activities of microsomal reduced nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase and N-demethylase, was correlated with the morphometric data on the endoplasmic reticulum.     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.     

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.     

The Ultrastructure of Circulating Hemolymph Cells of the Marine Snail Cerithidea californica (Gastropoda: Prosobranchiata)

Two morphologically distinct blood cell-types, the granulocyte and hyalinocyte, are found in the hemolymph circulation of the marine prosobranch Cerithidea californica. Granulocytes, measuring 12.7 µ (9.0–15.0 µ) in diameter, possess well-defined ectoplasmic and endoplasmic regions of the cytoplasm, granules of moderate to heavy electron density, tubular rough endoplasmic reticulum (RER), short vesicles of smooth endoplasmic reticulum (SER), and a large cytoplasm to nucleus ratio. Two morphological variants of this cell-type are distinguished depending upon the presence or absence of dense granules or RER. Hyalinocytes, measuring 5.3 µ (4.0–8.0 µ) in diameter, are distinguished from gran ulocytes by possessing a smaller cytoplasm to nucleus ratio and a general lack of dense cytoplasmic granules and SER.     

Role of lipoperoxidation in aflatoxin production

Summary Lipoperoxidation appears to play a role in inducing aflatoxin biosynthesis. In vitro, synthetic lipoperoxides greatly stimulate aflatoxin production when added to cultures of toxigenic strains of Aspergillus parasiticus or A. flavus. In vivo, the amount of toxin formed in sunflower seeds of different ages inoculated with A. parasiticus is directly related to the peroxide number of their oil content: the higher the peroxide number, the higher the aflatoxin production. In cultures of A. parasiticus carbon tetrachloride (CCl₄) greatly stimulates aflatoxin biosynthesis. This effect might be due to the peroxidation of lipids of the endoplasmic reticulum of Aspergillus by the highly reactive CCl _. ³ radicals formed by interaction with the NADPH-cytochrome P-450 system.     

Morphometric analysis of hepatocytes from rats subjected to compound 48/80-induced anaphylactic shock

Morphometric and biochemical techniques were used to analyze hepatic glycogen, endoplasmic reticulum, and mitochondrial matrix granules in rats treated with compound 48/80 to induce an anaphylactic-like state of shock. Thirty minutes after insult there was a significant decrease in glycogen and mitochondrial matrix granules, an increase in rough endoplasmic reticulum (RER), and no change in smooth endoplasmic reticulum (SER). Less glycogen in experimental rats substantiated a previously described glycogenolytic response to compound 48/80. The decrease in matrix granules implies a loss and/or shift in intramitochondrial calcium as occurs in epinephrine-induced glycogenolysis in the rat. Since other glycogenolytic agents, e.g. glucagon, and starvation stimulate an increase in SER presumably from RER, the present morphological data suggest the increase in RER may precede proliferation of SER from RER.     

Targeting of rough endoplasmic reticulum membrane proteins and ribosomes in invertebrate neurons

The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins.     

The Characterization of Tubulin in CNS Membrane Fractions

Abstract— Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and a plasma membrane (PM) fraction enriched in synaptic membranes were isolated from rat forebrain. The proteins in these membrane fractions were analyzed by two-dimensional gel electrophoresis (2DGE) in the isoelectric range of 5.1 to 6.0 by a modification of the O'Farrell procedure. Proteins were detected by Coomassie Brilliant Blue staining of the electrophoretograms. The results of these analyses were compared with 2DGE analysis of cytosol proteins, with particular attention given to tubulin subunits and actin. The RER contained one major protein (53K 5.4) in the β-tubulin region with a molecular weight of 53,000 and an isoelectric point of 5.4. The SER contained at least two major proteins in the β-tubulin region; one with a migration identical to 53K 5.4 and other proteins with slightly higher apparent molecular weights and more acidic isoelectric points (54K, 5.4 to 5.3), identical to cytoplasmic β-tubulin. The PM fraction also contained multiple overlapping proteins (54K, 5.4 to 5.3) in the β-tubulin area and a trace amount of the 53K 5.4 protein. The proteins in the β-tubulin region were removed from the 2DGE electrophoretogram and digested by Staphylococcus aureus V8 protease, and the peptides separated on one-dimensional polyacrylamide gels. The peptide patterns of 53K 5.4 protein from RER and SER were almost identical and differed significantly from the cytoplasmic β-tubulin pattern; however, the peptide maps of the PM and SER β-tubulin region were identical to the cytoplasmic β-tubulin. The 2DGE analysis of RER did not contain proteins in the region of cytoplasmic α-tubulin. SER and PM contained proteins in the α-tubulin region with a similar, but not identical, peptide analysis to cytoplasmic α-tubulin. Significant amounts of actin were detected in 2DGE analysis of SER and PM, and the peptide analysis of the actin was identical to the cytoplasmic actin analysis. The RER fraction contained only trace amounts of actin. The cytosol and all membrane fractions contained a protein (68K 5.6) found among microtubule-associated proteins, as judged by molecular weight and isoelectric point. Several proteins present in all membrane fractions (61K 5.1 and 58K 5.1) bound to concanavalin A agarose.     

Protective Effect of Cornuside against Carbon Tetrachloride-Induced Acute Hepatic Injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl₄)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl₄. Sixteen h after CCl₄ treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl₄-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl₄ treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl₄-treated rats. Our data indicate that cornuside protects the liver from CCl₄-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Incorporation of radioactive- -aminolevulinic acid into microsomal cytochrome P 450 : selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl₄ to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P₄₅₀ within 1 hr. The amount of cytochrome b₅ and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl₄ administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b₅ and NADPH cytochrome c reductase. By using ³H-δ-aminolevulinic acid to label microsomal cytochrome P₄₅₀ heme, the effect of AIA and CCl₄ on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P₄₅₀ heme accumulate in the liver after AIA or CCl₄ treatment.     

Effect of halomethanes on intracellular calcium distribution in hepatocytes

Exposure of isolated rat hepatocytes to hepatotoxic halomethanes results in a 40–60% decrease in intracellular Ca²⁺ content. The order of halomethane potency (CBrCl₃ CCl₄ CHCl₃) suggests that this effect requires halomethane metabolism by the hepatic mixed function oxidase system. Although the Ca²⁺ sequestering ability of the endoplasmic reticulum is destroyed by CBrCl₃ and CCl₄, it appears that much of the Ca²⁺ lost from the cell is mitochondrial in origin. Paradoxically, saturating concentrations of CCl₄ cause a marked increase in cell Ca²⁺. CCl₄ also causes an acute increase in cytoplasmic free Ca²⁺ (from about 60 nM to about 90 nM), but this effect does not appear to require CCl₄ metabolism and is probably a result of direct action of CCl₄ on the plasma membrane.