Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide.

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Different appearance of hepatic collagenase and lysosomal enzymes in recovery of experimental hepatic fibrosis.

1. Both activities of hepatic collagenase and lysosomal enzymes (acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase) have been observed in the recovery from experimental hepatic fibrosis in rats treated with carbon tetrachloride for 6 to 20 weeks, and compared with the disappearance of newly formed collagen fibers in the recovery process. 2. In the process of experimental hepatic fibrosis, collagenase activity reached maximum on sethe accumulation of collagen fibers in reversible hepatic fibrosis, but decreased to the same level as that of non-treated rat liver in cirrhotic stage. In the reocvery from reversible hepatic fibrosis, collagenase activity reached maximum on second day after the discontinuation of carbon tetrachloride, and decreased to the same extent of that of non-treated rat liver on seventh day. 3. Lysosomal enzyme activity was parallel to the activity of hepatic collagenase and to the accumulation of collagen fibers in the process of hepatic fibrosis. In the recovery stage, lysosomal enzyme activity in mesenchymal cells within the septa increased markedly on second day after the discontinuation of toxic agent but turned to the same level of that of non-treated rat liver seven days later, which was consistent with the appearance and disappearance of collagenase activity. On the other hand the appearance of lysosomal enzymes activities in Kupffer cells and hepatocytes was different from that of collagenase activity. That is lysosomal enzyme activity in Kupffer cells decreased in early days but increased five days later, and the enzyme activity in hepatocytes markedly decreased but gradually recovered to normal level seven days later. 4. The appearance of collagenase was observed at the beginning of the recovery stage. It indicates that mammalian collagenase initiates the collagen degradation and lysosomal enzymes might have a role in the subsequent degradation of collagen.     

Ontogenic changes in metabolism and transport of glutathione in the rat

Changes in the level of glutathione (GSH), the turnover rate, and gamma-glutamyltransferase (GGT) activity were examined in newborn, weanling, and adult male Wistar rats, the objective being to elucidate the mechanisms which control the hepatic GSH level during maturation as well as under conditions of different degrees of protein ingestion. The hepatic GGT activity in the newborn rats was high at birth, decreased within a few days to 1 to 2% of the initial level, and remained unchanged thereafter, when these rats were fed a normal diet after 3 weeks of age. In contrast, the hepatic GSH level increased 3-4-fold while total GGT activity in the kidney increased 6-8-fold. When weanling rats were fed a low protein diet (containing 10% soy protein) for 3 weeks, the hepatic GSH level decreased markedly while the GGT activity increased 5-6-fold. The turnover rate of hepatic GSH also increased, as determined by the use of buthionine sulfoximine, a specific inhibitor of GSH synthesis; a value of 2.1 h was obtained in comparison with 3.5 h for that of rats fed the normal laboratory chow (CRF-1). On the other hand, feeding adult rats on the low protein diet resulted in a marked decrease in hepatic GSH level with no effect on either hepatic or renal GGT activity. These results together with other observations may suggest that GSH translocated out of liver cells in the newborn rats is degraded mainly by these cells, while the tripeptide secreted by hepatocytes of adult rats is metabolized predominantly in extrahepatic tissues, such as the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Metabolism of hydralazine by rat hepatocytes

The metabolism of hydralazine and its pyruvic acid hydrazone was studied in a medium containing isolated adult rat hepatocytes. Hydralazine undergoes conversion to 3-methyltriazolophthalazine, while hydralazine pyruvic acid hydrazone is not metabolized in rat hepatocytes.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

Background

PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms.

Methodology/Principal Findings

Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo.

Conclusions/Significance

Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.     

The enzymatic reaction of chlorotrifluoroethylene with glutathione

The nephrotoxic gas chlorotrifluoroethylene is a substrate for glutathione S-transferase activity in rat hepatic cytosolic and microsomal fractions. The rates of reaction, determined by measuring glutathione disappearance, were 5–15 or 35–70 nmol/min/mg of cytosolic or microsomal protein, respectively. Glutathione disappearance was completely abolished by heat-denaturing the subcellular fractions. A product of the cytosolcatalyzed reaction between chlorotrifluoroethylene and glutathione was isolated and shown by amino acid analysis and ¹H- and ¹⁹F-NMR to be S-(2-chloro-1,1,2-trifluoroethyl)glutathione. This appears to be the first demonstration of a glutathione S-transferase-catalyzed addition reaction with a halogenated olefin, and this reaction may be of toxicological significance.     

Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis.

It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 mM), a potent inhibitor of the transamination reaction, it was shown that there exists an "insulin-sensitive" pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of alpha-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Metabolism of monohydroxybenzo(a)pyrenes by rat liver microsomes and mammalian cells in culture.

The 3-, 6-, and 9-monohydroxybenzo(a)pyrenes are metabolized by microsomes from rat liver in vitro. The metabolism of 3-hydroxybenzo(a)pyrene requires the presence of NADPH and is inhibited by carbon monoxide, suggesting that the reaction is mediated by a microsomal mixed-function oxygenase. The metabolic activity can be induced by in vivo treatment with 3-methylcholanthrene. 7,8-Benzoflavone strongly inhibits the induced activity but has little effect on the constitutive enzyme. The inducibility and inhibition characteristics, as well as the metabolic rate of the conversion of 3-hydroxybenzo(a)pyrene, closely resemble those of the oxidative metabolism of benzo(a)pyrene. The microsomal NADPH-dependent metabolism of [3H]3-hydroxybenzo(a)pyrene leads to the formation of a number of products of which a major fraction cochromatographs with the 3,6-quinone of benzo(a)pyrene. In mammalian cell cultures 3-hydroxybenzo(a)pyrene is converted by a mechanism different from that in hepatic microsomes. The disappearance of the phenol in cultures of hamster embryo cells is independent of the action of inducers or inhibitors of the aryl hydrocarbon hydroxylases and also occurs in the mouse L-cell line, A9, which lacks detectable aryl hydrocarbon hydroxylase activity. In A9 cells, [3H]3-hydroxybenzo(a)pyrene is largely converted to water soluble derivatives.     

Protective mechanism of sodium molybdate against the acute toxicity of cadmium in rats. II. Prevention of cytoplasmic acidification

In order to clarify the protective mechanism of sodium molybdate against the acute toxicity of cadmium chloride in rat, the effect of in vivo sodium molybdate pretreatment on the cytotoxic action of cadmium in isolated hepatocytes was studied. The cytosolic pH of hepatocytes isolated from untreated rats immediately decreased with incubation in either neutral Hank's balanced salt solution (HBS), pH 7.4, containing 5 µM cadmium chloride minimum or acidic HBS (pH 7.1, 6.8, 6.5, and 6.2). The presence of 5 µM cadmium in HBS adjusted to pH 7.1 aggravated cytosalic acidification induced by the acidic medium alone. Cell viability of hepatocytes incubated in HBS at pH 6.2 was significantly reduced as compared to that of control cells in HBS at pH 7.4, but the presence of cadmium in the acidic HBS had no aggravating action against such a toxic action of the acidic medium although cellular uptake of the metal in the medium increased, as compared to that in HBS at pH 7.4. Molybdenum pretreatment alleviated cytoplasmic acidification induced by the treatment with HBS at pH 7.4 or 7.1 containing cadmium or by extracellular acid load wothout cadmium. This pretreatment also prevented the loss of cell viability induced by the treatment with HBS at pH 6.2 but could not attenuate that when cadmium was present in the medium.These facts suggest that molybdenum pretreatment alleviated the acute toxicity of cadmium in rat by preventing cytoplasmic acidification caused by the harmful metal.     

Allogeneic hepatic cell suspension in the treatment of liver insufficiency

A study was made of a possibility of using isolated hepatocytes for the treatment of diseases of the liver in animals. The optimal dose of the cell suspension in intravascular, intraperitoneal, intrapleural and subcutaneous administration was determined; a reaction of the experimental animals to this biological substrate was studied. The efficacy of the isolated hepatocytes in the treatment of hepatic insufficiency is demonstrated; also a comparative assessment of the mentioned methods of the cell suspension administration is given.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.     

Microbial transformations of styrene and [14C] styrene in soil and enrichment cultures.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.