Liver Subcellular Fractions Research Articles

Stability of cytochrome P4501A-associated enzyme activity in cryogenically stored teleost liver samples

Induction of cytochrome P4501A (CYP1A), more specifically the associated enzyme activity aryl hydrocarbon hydroxylase (AHH), has been shown to be one of the most sensitive measures of exposure of vertebrate animals to a range of organic chemical contaminants. However, in order to reliably use this biochemical method for analyzing archived samples, the stability of the enzyme activity in storage must be ascertained. Stability of AHH activity was determined for both tissue sections and subcellular fractions of liver collected from English sole ( Pleuronectes vetulus), and held in cryogenic storage for up to 24 months. Our overall recommendations for sample collection, handling, storage, and assay are given.

Bioactivation and inactivation of aflatoxin B 1 by human, mouse and rat liver preparations: Effect on SCE in human mononuclear leucocytes

The purpose of this study was to investigate the use of human and animal subcellular liver fractions in an in vitro evaluation of carcinogenic risk. The bioactivation and bioinactivation of the known genotoxic carcinogen aflatoxin B 1 by human, mouse and rat liver preparations was investigated using the SCE assay in human lymphocytes as a genotoxic endpoint. There was a 10-fold variation in SCE response (1.1–11.6 SCE/Cell) in human mononuclear leucocytes (MNLs) after aflatoxin B 1 was activated by human liver microsomes ( n=6). Activation correlated with the CYP1A2 phenotype of livers ( r=0.8; p<0.05), but there was no correlation with either GST M1 genotype or epoxide hydrolase phenotype. Mouse liver microsomes activated aflatoxin B 1 to a greater extent [(1 μM) 12.8±2.51 SCE/Cell] than either rat [(10 μM) 12.0±3.84 SCE/Cell] or human (L25) [(10 μM) 8.8±2.00 SCE/Cell] liver microsomes. The addition of mouse liver cytosol and reduced glutathione (GSH) significantly ( p<0.001) reduced aflatoxin B 1-dependent genotoxicity, whereas the addition of either human or rat cytosol (+GSH) was without effect. These data indicate that species variation in both bioactivation and bioinactivation can exist. Therefore there is a necessity for careful selection of activation systems from species whose biochemical profile reflects that of man.

Denitrification of the genotoxicant 2-nitropropane : relationship to its mechanism of toxicity

1. The stabilities of the industrial chemical and constituent of cigarette smoke 2- nitropropane (2-NP) and its aci tautomer propane 2-nitronate (P2N) towards hepatic enzymes and proteins such as serum proteins and oxyhaemoglobin were investigated in vitro in biological (hepatocytes and subcellular liver fractions) and model systems (serum proteins, oxyhaemoglobin, methylene blue). 2. Denitrification of 2-NP, P2N and 2-deutero 2-nitropropane (2H-NP) occurred in murine hepatocytes significantly faster than in rat cells.For 2-NP the rates were 1271 ± 167 versus 820 ± 125 pmol nitrite × min−1 × 106 cells−1. 3. A similar observation was made in microsomes, where 2-NP denitrification was 1460 ± 110 (mouse) versus 480 ± 80 pmol nitrite × min−1 × mg protein−1 (rat). 4. The major NO2−-forming activity was found to be localized in the microsomal fraction. 5. Conversionof 2-NP into P2N, either chemically or enzymatically,was a prerequisite for rapid denitrification. 6. Serum proteins and oxyhaemoglobin proved to be capable of denitrifying P2N (198 ± 24 pmol nitrite × min−1 × mg protein−1 and 7·1 ± 1·0 nmol nitrite × min−1 × nmol HbO2−1 respectively), but were much less active towards 2-NP (24 ± 2 pmol nitrite × min−1 × mg protein−1 and none respectively). 7. Methylene blue decomposed 2-NP and P2N at rates of 11 ± 3 and 192 ± 4 pmol nitrite × min−1 × nmol methylene blue−1 respectively. The dye also enhanced NO2− formation from P2N and 2-NP in the presence of hepatocytes or serum proteins, with a concomitant enhancement of both 2-NP and P2N toxicity. 8. The results presented report species differences in the denitrification rate of 2-NP and highlight the crucial nitro-aci tautomerism of 2-NP as a pivotal determinant of 2-NP toxicity.

On the effects of thia fatty acid analogues on hydrolases involved in the degradation of m etabolisable and non-m etabolisable acyl-CoA esters

1. We investigated the nature and roles of various xenobiotic acyl-CoA hydrolases in liver subcellular fractions from rat treated with sulphur-substituted (thia) fatty acids. To contribute to our understanding of factors influencing enzymes involved in the degradation of activated fatty acids, the effects on these activities of the oppositely acting thia fatty acid analogues, the peroxisome proliferating 3-thia fatty acids (tetradecylthioacetic acid and 3- dithiacarboxylic acid), which are blocked for β-oxidation, and a non-peroxisomeproliferating 4-thia fatty acid (tetradecylthiopropionic acid), which undergoes one cycle of β-oxidation, were studied. 2. The hepatic subcellular distributions of palmitoyl-CoA, tetradecylthioacetyl-CoA and tetradecylthiopropionyl-CoA hydrolase activities were similar to each other in the control and 3-thia fatty acid-treated rat. In control animals, most of these hydrolases were located in the microsomal fraction, but after treatment with the 3-thia fatty acids, the specific activities of the mitochondrial, peroxisomal, and cytosolic palmitoyl-CoA, tetradecylthioacetyl-CoA, and tetradecylthiopropionyl-CoA hydrolase activities were significantly increased. This increase in activity was seen mostly for the enzymes using tetradecylthiopropionyl-CoA and tetradecylthioacetyl-CoA as substrates. The increased mitochondrial activities for these two substrates were seen already after 1 day of treatment, whereas the peroxisomal activities increased after 3 days. No stimulation was seen after treatment with the 4-thia fatty acid analogue, tetradecylthiopropionic acid, but a decrease in peroxisomal hydrolase activities for all three substrates was observed. 3. The cellular distributions of clofibroyl-CoA, POCA-CoA, and sebacoyl-CoA hydrolase activities were different from those of the 'long-chain acyl-CoA' hydrolases mentioned above both in the normal and 3-thia fatty acid treated rat. This group of hydrolases was found in the mitochondrial, peroxisomal, and cytosolic fractions. 3-Thia fatty acid treatment increased the activities of clofibroyl-CoA and sebacoyl-CoA hydrolases in all three fractions. Clofibroyl-CoA and sebacoyl-CoA hydrolase activities were increased after 1 day of treatment. Only the cytosolic POCA-CoA hydrolase was stimulated after 3- thia fatty acid treatment after only 1 day of treatment, whereas treatment with the 4-thia fatty acid led to an increase of enzyme activity in the mitochondrial and peroxisomal fractions. 4. Based on the subcellular distributions and specific activities, we suggest that several enzymes exist which may act as regulators of intracellular acyl-CoA levels.

Modulatory role of GTP-binding proteins in the endogenous ADP-ribosylation of cytosolic proteins.

The endogenous ADP-ribosylation of cytosolic proteins and the pattern of NAD degradation were analyzed in subcellular fractions of rat liver in order to investigate the modulation of these reactions by GTP-binding (G) proteins. We could show that intracellular membranes from rat liver have a guanine nucleotide- and divalent cation-dependent pyrophosphatase activity able to rapidly degrade NAD to AMP. This enzymatic activity was investigated by two different approaches: the degradation of [32P]-NAD in the presence of intracellular membranes and the mono-ADP-ribosylation of cytosolic proteins. Divalent cations, preferentially Zn2+ and Mn2+, were required for the pyrophosphatase activity, since in the presence of the Zn2+ chelator TPEN (N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine) or EDTA, the NAD degradation was inhibited by about 50%. Accordingly, in the presence of TPEN the endogenous ADP-ribosylation of cytosolic proteins was enhanced, whereas Zn2+ caused a significant inhibition of this reaction. GDP beta S was able to strongly activate the mono-ADP-ribosylation of cytosolic proteins. This effect was abolished by GTP gamma S, suggesting that a G protein, or rather one of the subunits of a heterotrimeric G protein, is involved in the modulation of the pyrophosphatase and consequently, of endogenous ADP-ribosylation. We propose that a regulatory pathway involving a heterotrimeric G protein modulates enzymes affecting the NAD turnover and availability of NAD for endogenous mADPRTs.

Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA- and ADP-ribosylation factor (ARF)-regulated isoenzymes

Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the highes: specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.

Characterization of p92, karyopherin beta, co-purified with N-acetylglucosamine-bearing nucleoporins from rat liver nuclear envelopes.

A 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin beta, which was found recently in the cytosolic fraction. It was shown using anti-p92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.

Biotransformation of the Fungicide Chlorthalonil by Glutathione Conjugation

The biotransformation of the nephrotoxic fungicide chlorthalonil (2,4,5,6-tetrachloroisophthalonitril) has been studied in the rat and in rat liver subcellular fractions. In rat liver cytosol, chlorthalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in the presence of glutathione in a reaction catalysed by glutathione S-transferases. 4-(Glutathion-S-yl)-2,5,6-trichloroisiphthalonitril was observed as an intermedite in the glutathione-dependent biotransformation of chlorthalonil. In the bile of rats dosed with chlorthalonil (0.18 mmol/kg, orally) 4,6-bis (glutathion-S-yl)-2,5-dichloroisophthalonitril was recovered as a minor metabolite. In rats, chlorthalonil was transformed to 4,6-bis (N-acetyl-cystein-S-yl)-2,5-dichloroisophthalonitril. This metabolite was excreted in small amounts in the urine of rats dosed orally with chlorthalonil (0.66 and 2.64 mmol/kg). In all experiments, a major part of the dose was recovered as unchanged chlorthalonil in feces. Oral administration of chlorthalonil (0.66 and 2.64 mmol/kg) resulted in a slight increase in the urinary excretion of gamma-glutamyltranspeptidase, indicative of nephrotoxicity. Other parameters indicative for renal proximal tubular damage (urinary glucose and protein excretion) were not changed by acute chlorthalonil administration. Very minor renal lesions were observed in chlorthalonil-dosed animals by histopathological examination.

Purification and properties of rat D-3-hydroxyacyl-CoA dehydratase: D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

We have previously purified two D-3-hydroxyacyl-CoA dehydratase preparations from human liver. One preparation contained a 77-kDa polypeptide and smaller polypeptides, and the other was a homodimer of a 46-kDa polypeptide. Three different purified rat peroxisomal D-3-hydroxyacyl-CoA dehydratase preparations have been reported. Therefore, rat enzyme was purified in this study to confirm the enzyme structure. Two preparations with similar molecular structures to the human enzyme preparations were obtained, and these were similar to each other in immunochemical and catalytic properties. It was suggested that the native enzyme was a homodimer of the 77-kDa polypeptide, and this enzyme was modified to a homodimer of the 46-kDa polypeptide, because conversion of the 77-kDa polypeptide to smaller polypeptides including the 46-kDa polypeptide was clearly observed during purification. Rat liver subcellular fractionation study indicates that this enzyme is located in peroxisomes. The enzyme preparation containing the 77-kDa polypeptide catalyzed the D-3-hydroxyacyl-CoA dehydrogenase reaction as well as the dehydratase reaction. Thus, it is proposed that this enzyme is D-3-hydroxyacyl-CoA dehydratase/ D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

Increases in tissue levels of ubiquinone in association with peroxisome proliferation

Rats were treated with various peroxisome proliferators and concomitant changes in ubiquinone levels were monitored. In addition to clofibrate and di(2-ethylhexyl)phthalate, acetylsalicylic acid, 2-ethylhexanoic acid, thyroxine and dehydroepiandrosterone were used as proliferators. Administration of these compounds increased the contents of ubiquinone in liver and, to some extent, in kidney and muscle. No change in corresponding values for heart or brain were observed. The treatments did not influence cholesterol levels, but increased the amounts of dolichol in the liver to various extents. Treatment of rats with the catalase inhibitor aminotriazole increased the ubiquinone levels in kidney, heart and muscle but not in liver. Comparison of peroxisomal fatty acid β-oxidation with ubiquinone amounts in liver homogenates after treatment with a number of peroxisome proliferators demonstrated a direct correlation between these two parameters. Subcellular fractionation of liver after peroxisome proliferation revealed that the ubiquinone level was increased in mitochondria and lysosomes which are the main compartments for this lipid, but an increase was also observed in both peroxisomes and microsomes. The increase in hepatic ubiquinone after treatment with various types of proliferators was related to the decrease in blood cholesterol level. These results show that the volume of the peroxisomal compartment and the ubiquinone content in animal tissues are interrelated.

Contribution of lysosomes to the subcellular distribution of basic drugs in the rat liver.

We examined the subcellular distribution of the basic drugs, chlorpromazine (CPZ), imipramine (IMP) and biperiden (BP), in rat liver, and evaluated the contribution of lysosome (Lys) to their intracellular distribution in comparison with that of mitochondria (Mit). In an in vivo distribution, the concentrations of CPZ, IMP and BP in the liver subcellular fractions were determined. In an in vitro study, uptake of [3H]IMP into Lys and Mit fractions was determined in the presence or absence of several agents. The distribution of these drugs 10 min after administration was quite similar. However, the relative specific contents (the drug concentration per protein of each fraction divided by that of the total homogenate) in Lys were 7.3, 9.6 and 4.2, respectively for CPZ, IMP and BP, whereas those in the other organella were only 0.4 approximately 1.7. In an in vitro uptake study, the dose response of IMP uptake into Lys was biphasic, while that into Mit fractions was monophasic. The binding of IMP to the high affinity sites of Lys was pH dependent and disappeared in 50 mM NH4Cl or 50 microM CPZ, both of which increased the intralysosomal pH, the low affinity sites were not affected by these drugs. The results indicated that Lys has the highest affinity for the basic drugs in the liver and that its contribution to their subcellular distribution depends on the intralysosomal pH, which is also affected by these drugs. The importance of these effects may become significant in combination therapy using various basic drugs.

Biotransformation of the Fungicide Chlorthalonil by Glutathione Conjugation

The biotransformation of the nephrotoxic fungicide chlorthalonil (2,4,5,6-tetrachloroisophthalonitril) has been studied in the rat and in rat liver subcellular fractions. In rat liver cytosol, chlorthalonil was rapidly transformed to 4,6-bis(glutathion-S-yl)-2,5-dichloroisophthalonitril in the presence of glutathione in a reaction catalysed by glutathione S-transferases. 4-(Glutathion-S-yl)-2,5,6-trichloroisiphthalonitril was observed as an intermedite in the glutathione-dependent biotransformation of chlorthalonil. In the bile of rats dosed with chlorthalonil (0.18 mmol/kg, orally) 4,6-bis (glutathion-S-yl)-2,5-dichloroisophthalonitril was recovered as a minor metabolite. In rats, chlorthalonil was transformed to 4,6-bis (N-acetyl-cystein-S-yl)-2,5-dichloroisophthalonitril. This metabolite was excreted in small amounts in the urine of rats dosed orally with chlorthalonil (0.66 and 2.64 mmol/kg). In all experiments, a major part of the dose was recovered as unchanged chlorthalonil in feces. Oral administration of chlorthalonil (0.66 and 2.64 mmol/kg) resulted in a slight increase in the urinary excretion of gamma-glutamyltranspeptidase, indicative of nephrotoxicity. Other parameters indicative for renal proximal tubular damage (urinary glucose and protein excretion) were not changed by acute chlorthalonil administration. Very minor renal lesions were observed in chlorthalonil-dosed animals by histopathological examination.

Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection.

Potentially serious idiosyncratic reactions associated with sulfamethoxazole (SMX) include systemic hypersensitivity reactions and hepatotoxicity. Covalent binding of SMX to proteins subsequent to its N-hydroxylation to form N4-hydroxysulfamethoxazole (SMX-HA) is thought to be involved in the pathogenesis of these reactions. A polyclonal antibody was elicited in rabbits against a SMX--keyhole limpet hemocyanin conjugate that recognized covalent protein adducts of SMX in microsomal protein and was used to characterize the covalent binding of SMX and its putative reactive metabolites to hepatic protein in vivo and in vitro. In vitro covalent binding of SMX to rat and human liver microsomal protein was NADPH-dependent, while binding of SMX-HA was not dependent on NADPH. SMX and SMX-HA produced similar patterns of covalent binding, with major protein targets in the region of 150, 100 (two bands), 70 (two bands), and 45-55 kDa. The pattern of covalent binding to human and rat liver microsomal protein was similar. Binding of SMX-HA was completely eliminated by GSH or by addition of cytosolic fractions and acetylcoenzyme A. The acetoxy metabolite of SMX also led to covalent binding, but it was primarily attributable to the formation of SMX-HA from acetoxySMX. In vivo exposure of rats to SMX did not result in detectable covalent binding by the methods employed. When rat liver slices were incubated with 2 mM SMX or 500 microM SMX-HA, no toxicity was observed and yet covalent binding of SMX-HA to 130, 100, 70, and 55 kDa proteins could be detected. These results confirm that covalent binding of SMX occurs via the formation of SMX-HA and that covalent binding of SMX-HA in vitro results from its conversion to the more reactive nitroso metabolite. Acetylation of SMX-HA protected against its covalent binding. Further studies are required to determine how this in vitro covalent binding relates to in vivo covalent binding in humans and to either direct or immune-mediated cytotoxicity in SMX idiosyncratic drug reactions.

Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon o-quinones produced by dihydrodiol dehydrogenase

Eight polycyclic aromatic hydrocarbon (PAH) o-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH o-quinones were potent cytotoxins yielding IC 50 values for cell survival in the range 1–30 μM. PAH o-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained o-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[ a]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained o-quinones (e.g., benz[ a]-anthracene-3,4-dione and 5-methylchrysene-1,2-dione) which reduced cell survival but had no effect on cell viability; and group III contained o-quinones (e.g., benzo[ a]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that o-quinones underwent preferential enzymatic one-electron redox-cycling and produced Superoxide anion radical (O 2 ∸) and/or o-semiquinone anion or alternant radicals. o-Quinones that reduced cell viability produced O 2 ∸ and caused the most total free radical formation, while those that reduced cell survival produced o-semiquinone anion or alternant radicals only. PAH o-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of o-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH o-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or o-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH o-quinones to intercalate and covalently modify DNA.

Epoxidation of plasmalogens: source for long-chain alpha-hydroxyaldehydes in subcellular fractions of bovine liver.

1. Masked long-chain alpha-hydroxyaldehydes were trapped in all subcellular fractions of bovine liver by application of pentafluorbenzyloxime derivatization [van Kuijk, Thomas, Stephens and Dratz (1986) Biochem. Biophys. Res. Commun. 139, 144-149] and quantified via GLC/MS using characteristic ion traces. 2. The chain-length profile of long-chain 2-hydroxyalkanales clearly indicates their relationship to plasmalogens as precursor molecules. 3. The previously postulated existence of alpha-acyloxyplasmalogens as precursor molecules of masked long-chain alpha-hydroxyaldehydes in bovine tissue lipids [Lutz and Spiteller (1991) Liebigs Ann. Chem. 1991, 563-567] was excluded. 4. The constant oxidation rate of plasmalogens in all subcellular fractions provides conclusive evidence for a non-enzymic plasmalogen epoxidation process (probably via hydroperoxy radicals). 5. The high reactivity of alpha-hydroxyaldehydes sheds some doubt on the postulation that plasmalogens protect mammalian cells against oxidative stress as postulated previously [Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11590-11596; Morand, Zoeller and Raetz (1988) J. Biol. Chem. 263, 11597-11606].

Rat Liver Subcellular Folate Distribution Shows Association of Formyltetrahydropteroylpentaglutamates with Mitochondria and Methyltetrahydropteroylhexaglutamates with Cytoplasm

The ternary complex method for the determination of folylpolyglutamates was combined with procedures for interconverting folate derivatives to measure 28 different folate derivatives in the subcellular fractions of rat liver. Folates in the homogenate showed a typical distribution with nearly equal quantities of penta- and hexaglutamates and pteridine derivatives in decreasing order as follows: 1) methyl substituted folates [5-methyltetrahydropteroylglutamates], 2) unsubstituted folates [tetrahydropteroylglutamates + 5,10-methylenetetrahydropteroylglutamates], 3) formyl substituted folates [5-formyltetrahydropteroylglutamates + 10-formyltetrahydropteroylglutamates + 5,10-methenyltetrahydropteroylglutamates], and 4) oxidized folates [dihydropteroylglutamates]. In the homogenate the methyl substituted folates exhibited a higher hexa:pentaglutamate ratio than did the other pteridine derivatives. As the fractionation proceeded toward purer subcellular components, the methyl substituted folates were found almost exclusively in the soluble fraction, and this fraction also contained the higher hexa:pentaglutamate ratio characteristic of the methyl substituted folates. The plasma membrane, the microsomal and the nuclear fractions did not contain appreciable folate. The mitochondrial fraction contained primarily formyl substituted and unsubstituted folates, and these folates exhibited the lower hexa:pentaglutamate ratios. These data support the hypothesis that folate-dependent one-carbon metabolism is compartmentalized in the eukaryotic cell.

Comparative metabolism of 2-acetylaminofluorene by rainbow trout and rat liver subcellular fractions

In order to assess the cause of the resistance of rainbow trout ( Oncorhynchus mykiss), Shasta strain, to the hepatocarcinogenic effects of 2-acetylaminofluorene (AAF), we have investigated the metabolism of this model chemical by subcellular fractions from trout liver. Treatment of trout with 3-methylcholanthrene (3-MC) enhanced the rate of metabolism of AAF by liver microsomes. The major AAF metabolites produced by trout liver microsomes at saturating substrate concentration (34.3 and 19.5 μM AAF for control and 3-MC-induced microsomes, respectively) were 7-hydroxy-AAF and 5-hydroxy-AAF which represented 75 and 21% of total AAF metabolites, respectively for control microsomes, and 79 and 18% of total metabolites, respectively for induced microsomes. With both control and 3-MC-induced microsomes, N-hydroxy-AAF (the proximate carcinogen of AAF) was a minor metabolite accounting for less than 1% of total AAF metabolites. At a low substrate concentration (0.18 μM AAF), 7-hydroxy-AAF formation accounted for 95 and 91% of total metabolites for control and 3-MC induced microsomes, respectively, and a slight relative increase in N-hydroxy-AAF production (1.5–2.5%) was observed. Liver microsomes from untreated male Sprague-Dawley rats (a species susceptible to hepatocarcinogenesis by AAF) formed N-hydroxy-AAF in greater proportions (14-fold) and ring-hydroxylated metabolites in smaller proportions compared to trout liver microsomes. The activities of cytosolic N-hydroxy-AAF sulfotransferase and acyltransferase in trout liver (24 and 17 pmol/mg h −1, respectively) were found to be substantially lower than those reported for Sprague-Dawley rats. The low activity of hepatic enzymes implicated in the metabolic activation of AAF (AAF-N-hydroxylase, N-hydroxy-AAF-sulfotransferase, and N-hydroxy-AAF acyltransferase) coupled with a relatively high capacity of the trout liver for ring-hydroxylation (detoxification) of AAF may explain, in part, the resistance of rainbow trout to the hepatocarcinogenic action of AAF.

Intracellular Sorting of Lysosomal β-Glucuronidase Is Altered Due to Administaration of Dibutyl Phosphate1

Organophosphate compounds are known to cause a selective increase of beta-glucuronidase activity in rat serum. Previous data suggested that increase of serum beta-glucuronidase activity was well correlated with decrease of that activity in rat liver microsomal fraction, thereby, suggesting a role of the microsomal enzyme in mediating the organophosphate effect. To investigate further the intracellular sorting pathway of beta-glucuronidase in dibutyl phosphate-treated rats, liver subcellular fractions were prepared at 12 or 48 h after in vivo administration of [3H]leucine and it was established that microsomal beta-glucuronidase was the origin of the increased serum enzyme. To characterize the intracellular secretory pathway of beta-glucuronidase in dibutyl phosphate-treated rats, Golgi subfractions were isolated and a time course study was carried out. At 30 min after administration of dibutyl phosphate, specific activity of beta-glucuronidase in GF-2 (Golgi intermediate fraction) and GF-3 (Golgi heavy fraction) was significantly increased to the maximum. Furthermore, colchicine pretreatment of rats caused a delay of the peak of specific activity for 30 min in GF-2 and GF-3, and accumulation of enzyme activity in Golgi subfractions was observed. Colchicine pretreatment also had an inhibitory effect on release of beta-glucuronidase into serum until 30 min after dibutyl phosphate injection. The electrophoretic pattern of microsomal beta-glucuronidase on polyacrylamide gel was found to show two major bands of microsomal enzyme type and lysosomal enzyme type in dibutyl phosphate-treated rats. Taken together, these findings indicate that microsomal beta-glucuronidase follows the intracellular secretory pathway and is secreted into serum via Golgi complex in response to dibutyl phosphate.

Seasonal changes in hepatocyte ultrastructure correlated with the cyclic synthesis of secretory proteins in the winter flounder (Pleuronectes americanus)

Liver tissue was sampled from flounder (Pleuronectes americanus) throughout the year with the intention of documenting changes in the ultrastructure coincident with the production and secretion of antifreeze proteins. In the winter, hepatocytes are dedicated to the production of these proteins and, in the female, also reproductive proteins. In both sexes, liver cells in the summer contain abundant lipid and glycogen stores. In the female, there is a conspicuous hepatocyte transformation from a fat-filled cell in the summer to one with well-developed rough endoplasmic reticulum in the winter. Large amounts of rough endoplasmic reticulum (11.2 mg/gm) were recovered after subcellular fractionation of female wintertime liver. The increased appearance of secretory organelles and the high number of nucleolar profiles observed in winter animals is consistent with the elevated demand for protein secretion and synthesis in both sexes. The fractional volumes occupied by lipid droplets and mitochondria were different when comparisons were made between sex and season. Females contained a greater volume of lipid than did males, and summer animals contained more lipid than those in winter.

Highly efficient establishment of immortalized cells from adult human liver

Human liver tissue slices, homogenates, organ cultures, subcellular fractions and primary hepatocyte cultures represent valuable tools for analysis of metabolism, toxicity and efficacy of xenobiotics. However, the limited access to human tissue and the high test variability with different tissue isolates underline the significance of having standardized and defined human liver cell models. This report describes the establishment of replicative cultures of human liver epithelial cells and their immortalization in serum-free medium. Highly efficient immortalization is achieved by the infection of primary liver cells with a retroviral recombinant carrying the simian virus 40 (SV40) large T antigen gene. These cultures have an indefinite life-span, are non-tumorigenic and express differentiation markers and metabolic functions of normal primary hepatocytes. Therefore, these cells are useful for various pharmaco-toxicological applications.