Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.     

A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin

A direct fluorometric procedure for the continuous determination of cytochrome P-450-dependent mixed function oxidases, using 3-cyano-7-ethoxycoumarin substrate, is described. The reaction product, 3-cyano-7-hydroxycoumarin, is fluorescent at neutral pH values (excitation and emission wavelength maxima: 408 and 450 nm, respectively). Using hepatic microsomal preparations from control rats, the enzyme(s) had an apparent Km of 16 microM. Vmax values (0.5 nmol/min/mg protein) were induced 6- and 21-fold by pretreatment of rats with phenobarbitone and about 50- to 100-fold more sensitive than the ethoxyresorufin deethylase assay. Reaction rates using 3-cyano-7-pentoxycoumarin as substrate were generally much lower than with the ethoxy analog. 3-Cyano-7-ethoxycoumarin can also be used as a substrate to measure mixed function oxidases in isolated hepatocytes. However, 3-cyano-7-hydroxycoumarin shows a time- and concentration-dependent loss of fluorescence when incubated with such cells. This causes an approximately 5% underestimate of the true reaction rates.     

Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice

Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.     

Preferential activation of 6-aminochrysene and 2-aminoanthracene to mutagenic moieties by different forms of cytochrome P450 in hepatic 9000 X g supernatants from the rat

6-Aminochrysene and 2-aminoanthracene were activated to metabolites which were mutagenic to Salmonella typhimurium TA98 by hepatocytes or hepatic 9000 X g supernatants (S9s) from control or xenobiotic-treated rats. Hepatocytes from Aroclor-1254-treated rats were more efficient than hepatocytes from untreated rats at activating these aromatic amines. When plate-incorporation and liquid-incubation bacterial mutagenesis assays were performed in the presence of limiting amounts of rat hepatic S9, 2-aminoanthracene was activated to a greater extent in both cases, as judged by his+ revertant formation, by 3-methylcholanthrene-induced hepatic S9 than by phenobarbital-induced or control S9s. In contrast, 6-aminochrysene was activated more efficiently by phenobarbital-induced S9 than by 3-methylcholanthrene-induced or control S9s. This unexpected finding was confirmed employing polyclonal antibodies directed against specific forms of rat cytochrome P450. Thus, when employing Aroclor-1254-induced S9 as a source of metabolic activation, antibody directed against cytochrome P450IA1 inhibited the activation of 2-aminoanthracene but not of 6-aminochrysene. In contrast, antibody directed against cytochrome P450IIB1 inhibited the activation of 6-aminochrysene but not of 2-aminoanthracene. These results suggest that under conditions in which the amounts of S9 added are rate-limiting, the two aromatic amines are preferentially activated by different induced forms of cytochrome P-450.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Identification and quantification of a messenger ribonucleic acid induced by polynuclear aromatic hydrocarbons--using a cloned human cytochrome P-450 gene

We have isolated four overlapping human genomic clones associated with the polynuclear aromatic hydrocarbon-induced form of cytochrome P-450. The form of P-450 most closely associated with polynuclear aromatic hydrocarbons induction has been defined as P1-450. These four overlapping genomic clones span a total of 31.0 X 10(3) base pairs in length with the coding sequence lying in the center of these clones. Translation in vitro of 3-methylcholanthrene-induced mRNA, selected with the human P1-450 genomic clone, detect a protein with Mr 52000, which is immunoprecipitable by the anti-(mouse P1-450) antibody. The isolated human P1-450 genomic clone hybridizes to 3-methylcholanthrene-induced mRNA from monkey liver, benzanthracene and 3-methylcholanthrene-treated human mammary tumor cells (MCF-7), but not to isosafrole-treated human cells. Upon treatment with polynuclear aromatic hydrocarbons there is a positive correlation between induced arylhydrocarbon hydroxylase (flavoprotein-linked monoxygenase) activity and the amount of mRNA that hybridizes to the isolated human genomic clone for P1-450. The size of mRNA, induced from human cells and monkey liver by polynuclear aromatic hydrocarbons, is around 3.3 X 10(3) base pairs, which is the same as the larger of two mRNA induced by polynuclear aromatic hydrocarbons in the inbred strain of mouse (C57BL/6N). Our data also showed that the isolated DNA clone can detect a mRNA size of 3.3 X 10(3) base pairs from phytohemagglutinin-activated, benzanthracene-treated human lymphocytes. Densitometer scanning indicated the presence of a 3.6-fold variation (highest-lowest) in the levels of lymphocyte P1-450 mRNA contents among six individuals studied.     

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins)

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent K_m-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes—the ethyl, propyl and butyl ethers—but the magnitudes of the spectra differed in the order 3MC- > PB- > control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold).The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.     

Metabolism of benzo[a]pyrene: Effect of 3-methylcholanthrene pretreatment on metabolism by microsomes from lungs of genetically “responsive” and ”nonresponsive” mice

The metabolism of [¹⁴C]benzo[a]pyrene by microsomes from the lungs of normal and 3-methylcholanthrene-treated DBA/2J, C57BL/6J, and A/HeJ mouse strains was quantitatively analyzed by high-pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with lung microsomes than with liver microsomes in all three strains. The ratio of epoxide hydrase to monooxygenase activity in mouse lung was shown to be considerably higher than in mouse liver. Benzo[a]pyrene metabolism by control lung microsomes showed some strain differences. C57BL/6J and A/HeJ mice formed twice as much dihydrodiols as a percentage of total metabolism compared to DBA/2J mice. DBA/2J mice produced somewhat less phenol 2 fraction and considerably more quinone 1 and 2 fractions than the other two mouse strains as a percentage of total metabolism. Treatment of C57BL/6J and DBA/2J mice with 3-methylcholanthrene resulted in a 20-fold increase in the metabolism of benzo[a]pyrene, while A/HeJ mice were induced more than 50-fold. The profiles of metabolites from the 3-methylcholanthrene-induced animals were nearly identical in all three mouse strains.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

The level and activity of molecular forms of monooxygenase P-450b and P-450c during their separate and consecutive induction in the liver

Studies with monospecific antibodies to individual forms of monooxygenases P-450b (phenobarbital-induced) and P-450c (3-methylcholanthrene-induced) by immunochemical, kinetic and spectral methods revealed differences in the dynamics of xenobiotic-induced changes in the content and monooxygenase activity of the total microsomal CO-binding hemoprotein and of its molecular forms. Correction was made in the estimation of the benzphetamine-demethylase and benzpyrene-hydroxylase activities based on the content of specific forms of P-450b and P-450c. Since consecutive injection of phenobarbital and 3-methylcholanthrene (or vice versa) is accompanied by selective induction of the corresponding isoforms and the redistribution of the relative content of P-450b and P-450c in the total pool of cytochrome P-450 in rat liver microsomes, a conclusion was drawn that these molecular forms are induced by different populations of hepatocytes. This conclusion was confirmed by data from immunohistochemical analysis.     

Immunolocalization of mitochondrial 3-hydroxy-3-methylgutaryl CoA synthase in rat liver

We report the preparation of specific polyclonal antibodies raised against two synthetic peptides deduced from the cDNA sequence for the rat liver mitochondrial 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) synthase gene. Immunoelectron microscopy using these antibodies on hepatic cryoultrathin sections confirms the mitochondrial localization of this protein in hepatocytes. Immunofluorescence microscopy on frozen sections of adult rat liver revealed fluorescence inside all hepatocytes, with no evidence of zonation, indicating that ketogenesis may not be limited to specific regions of rat liver but is extended to all hepatocytes. © 1995 Wiley-Liss, Inc.     

Acute suppression of albumin synthesis in systemic inflammatory disease: an individually graded response of rat hepatocytes.

The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.     

Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats.

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

Development of binuclearity and DNA-polyploidization in the growing mouse liver

Summary Suspensions of intact liver cells were prepared from 36 male NMRI mice of different age after perfusion of the liver with ice-cold calcium- and magnesium-free phosphate buffer (CMF). The suspensions of the isolated hepatocytes were smeared on slides, fixed, hydrolized and stained by fluorescent acriflavine-Schiff-Feulgen reaction. The number of nuclei per cell was determined in a phase-contrast microscope. Quantitative fluorescent cytophotometric measurements of nuclear Feulgen-DNA were performed on individual nuclei. At the age of 0.5 month, 55% of the hepatocytes were found to be mononuclear, 45% binuclear. In the animal groups aged 1 month, 1.5 months, 3 months, 6 months and 12 months, the percentage of binuclear hepatocytes remained constant at about 80%. Very few liver cells with 3 or 4 nuclei were detected. Feulgen-DNA-measurements revealed a predominance of 2c and 4c nuclei at ages 1 month and 1.5 months with logarithmic increase of 8c nuclei and a decrease of the 2c nuclei. From 1.5 months on 16c nuclei were found, which never exceeded 8%. When total DNA-ploidy of the hepatocytes was calculated similar kinetics at a higher ploidy level were observed. 2c hepatocytes existed in small percentages at very young ages up to 1.5 months, but were also occasionally found in older animals. With increasing age the number of 16c hepatocytes increased logarithmically with a concomitant decrease of the 4c hepatocytes. The percentage of 8c liver cells remained more or less constant. Few hepatocytes with a 32c total DNA content were found in mice aged 3 months and older. In one-year-old mice the mean DNA-ploidy was calculated to be 5.8c per liver nucleus and 10.0c per whole hepatocyte.Supported by Deutsche Forschungsgemeinschaft, Grant No Bo 395/5     

Severe hypoalphalipoproteinemia in mice expressing human hepatic lipase deficient in binding to heparan sulfate proteoglycan

Unlike human hepatic lipase (hHL) that is mainly cell surface-anchored via binding to heparan sulfate proteoglycans (HSPG), mouse HL (mHL) has a low affinity to HSPG and thus is largely blood-borne. The reduced HSPG binding of mHL is attributable to the C-terminal amino acids. To determine the functions of HSPG binding of hHL in vivo, we created adenovirus vectors encoding hHL or a chimeric protein (designated hHLmt) in which the C-terminal HSPG-binding sequences were replaced with the corresponding mouse sequences. Injecting hHLmt-expressing virus into C57BL/6J mice (1.8 x 10(10) virus particles/mouse) resulted in a 3-fold increase in pre-heparin HL activity, whereas infection with an identical dose of hHL virus did not change pre-heparin HL activity. In hHLmt-expressing mice, the concentration of total cholesterol and phospholipids was inversely related to the hHL activity in pre-heparin plasma in a dose- and time-dependent manner, and the decrease was mainly attributable to high density lipoproteins (HDL) cholesterol and HDL phospholipids. The expression of hHL exhibited no change in plasma total cholesterol or phospholipid levels as compared with control mice infected with luciferase or injected with saline. The reduced HDL lipids in the hHLmt-expressing mice were accompanied by markedly decreased plasma and hepatic apolipoprotein (apo) A-I. In primary hepatocytes isolated from hHLmt-expressing mice, the concentration of cell-associated and secreted apoA-I was decreased by 2-3-fold as compared with hepatocytes isolated from control mice, whereas the levels of apoB and apoE were unaltered. Infection of primary hepatocytes with hHLmt virus ex vivo also resulted in reduced apoA-I secretion but had no effect on cell-associated apoA-I. These results suggest that expression of HSPG binding-deficient hHL has a profound HDL-lowering effect.     

Tracker Dyes to Probe Mitochondrial Autophagy (Mitophagy) in Rat Hepatocytes

Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes co-loaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), alsodecreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.     

Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes

Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.     

Short term treatment with St. John's wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse

This investigation was designed to determine whether St. John's wort (SJW)(435 mg/kg/d), a readily available antidepressant, or its purported active constituents hypericin (1 mg/kg/d) and hyperforin (10 mg/kg/d) were able to induce various hepatic cytochrome P450 (CYP450) isoforms. SJW, hypericin and hyperforin were administered to male Swiss Webster mice for four consecutive days and hepatic microsomes were prepared on day 5. None of the three treatments resulted in a statistical change in total hepatic CYP450 (SJW treated 0.95 +/- 0.09 nmol/mg vs control 1.09 +/- 0.14 nmol/mg). Furthermore, the catalytic activities of CYP1A2. CYP2E1 and CYP3A were unchanged from control following all three treatments as determined by ethoxyresorufin O-deethylation, p-nitrophenol hydroxylation and erythromycin N-demethylation respectively. Additionally, western immunoblotting demonstrated that there was no significant change in the polypeptide levels of any of the three isoforms. These results indicate that four days of treatment with moderate to high doses of SJW, hyperforin or hypericin fails to induce these CYP450 isoforms in the male Swiss Webster mouse.