Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane- bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.     

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol⁻¹ min⁻¹ 100 g⁻¹ body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features This revised version was published online in November 2006 with corrections to the Cover Date.     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol^?1 min^?1 100 g^?1 body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features     

Induction of cytochrome P-450 in cultured rat hepatocytes. The heterogeneous localization of specific isoenzymes using immunocytochemistry.

Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of 'induced' cells. However, whereas maximal concentrations of beta-naphthoflavone resulted in virtually all cells containing induced levels of MC1b, a maximal concentration of phenobarbitone resulted in only 30% of the cells containing induced levels of PB3a/PB3b. These results are discussed in relation to the heterogeneous distribution and induction of cytochrome P-450 in the intact liver.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Deprivation and repletion of androgen in vivo modifies triacylglycerol synthesis by rat hepatocytes

Given the same quantity of fatty acid, livers from male rats esterify less fatty acid and secrete less triacylglycerol in very-low-density lipoprotein than do livers from female animals. To elucidate the role of testosterone in maintenance of this male pattern, conversion of [1-14C]oleic acid into triacylglycerol was assessed in vitro by rat hepatocytes (male) following gonadectomy and replacement with testosterone. Following castration, incorporation of fatty acid into triacylglycerol was increased. In contrast, esterification of exogenous fatty acid into phospholipid, cholesteryl esters, and diacylglycerol was unchanged. Treatment with testosterone (75 micrograms/day) reduced incorporation of exogenous fatty acid into triacylglycerol. Higher doses of testosterone (200 or 100 micrograms/day) modified the effect, such that inhibition was observed only at low oleate (0.5 mM) concentrations. At higher substrate concentrations (1.0-2.0 mM) the inhibitory effect was no longer observed. Further, a similar dose-dependent effect of testosterone was observed following in vivo treatment of castrate females with testosterone. These data support the concept of a regulatory role of testosterone in hepatic triacylglycerol synthesis. These findings also demonstrate a biphasic effect of testosterone, an effect that is dependent not only upon the dose of testosterone administered, but also on the concentration of fatty acid to which the hepatocyte is exposed in vitro.     

Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.     

An in vivo role of bone morphogenetic protein-6 in aldosterone production by rat adrenal gland

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex. We previously reported the presence of a functional BMP system including BMP-6 in human adrenocortical cells. BMP-6 contributes to Ang II-induced aldosterone production by activating Smad signaling, in which endogenous BMP-6 action is negatively controlled by Ang II in vitro. In the present study, we examined the in vivo role of BMP-6 in regulation of aldosterone by neutralizing endogenous BMP-6 in rats treated with immunization against BMP-6. Three-week-old male rats were actively immunized with rat mature BMP-6 antigen conjugated with keyhole limpet hemocyanin (KLH). The immunization treatment had no effect on bilateral adrenal weight or its ratio to body weight. Urinary aldosterone excretion was time-dependently increased during the 8-week observation period in the control group. Of note, the level of urinary aldosterone excretion in BMP-6-KLH-immunized rats was significantly reduced compared to that in the control group, suggesting that endogenous BMP-6 contributes to the induction of aldosterone production in vivo. Moreover, the level of urinary aldosterone/creatinine after 8-week treatment was significantly lowered by treatment with BMP-6-KLH. In contrast, with chronic Ang II treatment, urinary aldosterone and creatinine-corrected values at 8 weeks were not significantly different between the two groups, suggesting that the effects of BMP-6-KLH were impaired under the condition of chronic treatment with Ang II. The mRNA levels of Cyp11b2, but not those of Star, P450scc and 3βhsd2, were significantly decreased in adrenal tissues isolated from BMP-6-KLH-immunized rats after 8-week treatment. Furthermore, the ratio of plasma aldosterone level to corticosterone was significantly decreased by immunization with BMP-6-KLH. Collectively, the results indicate that endogenous BMP-6 is functionally linked to aldosterone synthesis by the zona glomerulosa in the adrenal cortex in vivo.     

Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS

Lipopolysaccharide (LPS) induces hepatocellular down-regulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2). Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis. Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver. In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase. In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes. Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2. Furthermore, we found a strong induction of Mrp5 mRNA. Likewise, Mrp6 mRNA was up-regulated, however Mrp6 protein expression was not significantly altered. It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPS-induced loss of Mrp2 in the perivenous area. Induction of pericentral Mrp3 and up-regulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.     

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.     

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [(3)H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(-) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(-/-) knockout mice, and Bcrp1(-/-) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(-/-) mice or in Bcrp1(-/-) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(-) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(-) rats and functional DMP uptake into isolated TR(-) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(-) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(-) rats expelled phalloidin back into blood circulation.     

Determination of immunoreactive melatonin in the colon of the rat by immunocytochemistry

Melatonin immunofluorescence in the rat colon was measured with a cadmium sulfide photometer in conjunction with an immunohistological double antibody procedure. The method proved to be very reliable with 5-10% intra- and interassay reliabilities. In addition, manipulation of the primary antibody concentration, the incubation time, and the nature of the first antibody produced predicted changes in fluorescence intensity. Treatment with p-chlorophenylalanine led to a marked reduction of melatonin-specific fluorescence.     

Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

Two monoclonal antibodies (ADM-1-11 and 79-31 mAbs) were raised against daunomycin (DM) conjugated to bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide. The monoclonal antibodies (mAbs) specifically detected DM as well as its analogs doxorubicin and epirubicin, but did not react with other anticancer antibiotics, including pepleomycin, mitomycin C, and actinomycin D. The mAbs reacted strongly with glutaraldehyde-conjugated DM in an enzyme linked immunosorbent assay (ELISA) used as a model system for immunocytochemistry as well as in appropriately pretreated sections of tissues from animals injected with DM. No staining occurred in tissues from uninjected animals. In order to perform DM ICC a number of tissue treatment conditions critical to the detection of low molecular weight substances were employed. Uptake of DM was studied in rats after a single i.v. or i.p. administration of the drug. In the heart, accumulation of DM occurred in nuclei and in the cytoplasm. In the kidney, DM immunoreactivity accumulated in all segments of the nephron except for the proximal tubules. Since the proximal tubules are known to be where a variety of transport systems including P-glycoprotein (Pgp) and organic anion-transporting polypeptides (OATPs) in drug interactions occur, the absence of DM accumulation in these segments may reflect a transport phenomenon depending upon such transporters. The availability of methods to study sites of accumulation of DM offers possibilities for understanding toxic side effects of this drug on the heart and kidney. Moreover, the immunocytochemical methodology developed may prove useful for the localization of other low molecular weight drugs that can be fixed in situ by glutaraldehyde.     

DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal

The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 mg/ml glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg/kg body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 mg/kg glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.     

Inhibition of diacylglycerol acyltransferase by 2-bromooctanoate in cultured rat hepatocytes

Triacylglycerol synthesis in cultured rat hepatocytes was inhibited by 2-bromooctanoate with a concomitant accumulation of diacylglycerols. 2-Bromooctanoate inhibition could be ascribed to its thioesterification by medium chain fatty acyl-CoA synthase (Raaka, B.M., and Lowenstein, J.M. (1979) J. Biol. Chem. 254, 6755-6762) with 2-bromooctanoyl-CoA acting as a competitive inhibitor of diacylglycerol acyltransferase. The Ki of 2-bromooctanoyl-CoA was 1.5 microM compared with a Km of 25 microM for the palmitoyl-CoA substrate. Diacylglycerol esterification was also inhibited by C12-C16 2-bromo fatty acids. However, inhibition of triacylglycerol synthesis by long chain 2-bromo fatty acids was accompanied by decreased overall neutral lipid synthesis as a result of inhibition of the long chain fatty acyl-CoA synthase. Since 2-bromooctanoate was a poor inhibitor of the long chain fatty acyl-CoA synthase, it appears to function selectively as an inhibitor of diacylglycerol acyltransferase in cultured rat hepatocytes.