Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Inhibitory effect of salicylate on 2,4-dinitrophenol and diethyl maleate in isolated rat intestinal epithelial cells

Studies on the mechanism of chemically induced intestinal epithelial injury were carried out using isolated, rat small intestinal epithelial cells. Compounds such as 2,4-dinitrophenol (DNP) and diethyl maleate (DEM), caused NADH loss, an increase in cytosolic Ca2+ concentration and protein thiol loss. Further, these compounds accelerated cell aggregation and decreased cell viability. Calmodulin antagonists inhibited protein thiol loss induced by either of the compound, inhibited cell aggregation and prolonged cell viability, but did not influence NADH loss. It has been reported that the calmodulin-binding protein may regulate cytoskeletal activity. Therefore, the inhibition of protein thiol loss by calmodulin antagonist may be due to a dissociation of calmodulin-binding proteins from cytoskeletal elements. Salicylate also inhibited protein thiol loss induced by DNP and DEM, and inhibited cell aggregation. However, salicylate may have a direct effect in reducing the cytosolic free Ca2+ concentration by complexation and subsequent facilitated release of Ca2+ from cells. Further, in the present study, the induction of cell aggregation may be caused by the appearance of specific sites on the cell membrane surface to which arsenazo III could adsorb, since adsorption of arsenazo III to the isolated epithelial cells seemed to correlate with increased cell aggregation.     

Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents

The transport activity for cystine and glutamate in cultured human diploid fibroblasts is enhanced in response to diethyl maleate treatment. The enhancement is time- and dose-related, with a lag of about 3 h, and maximum enhancement (approximately 3-fold increase in the rate of uptake) is attained after 1 to 2 days of incubation of the cells with 0.1 mM diethyl maleate. The enhancement of the transport activity is accompanied by an increase in the Vmax and little change in the Km, and it requires RNA and protein synthesis. Other electrophilic agents, such as cyclohex-2-en-1-one, ethacrynic acid, 1,2-epoxy-3-(p-nitro-phenoxy)propane, and sulfobromophthalein, similarly enhance the transport activity. These electrophiles are known as agents that interact with glutathione. For example, diethyl maleate at high concentrations, i.e. 1 mM, depletes intracellular glutathione and injures the cells. However, at relatively low concentrations diethyl maleate and other electrophilic compounds do cause increases in the intracellular levels of glutathione which we attribute to the enhanced uptake of cystine. It is suggested that the transport system for cystine and glutamate is involved in a protective mechanism of cells against an electrophilic attack.     

Tissue-specificity of apoptosis in hepatoma-derived cell lines

Apoptosis is known to play a critical role in development and homeostasis in metazoans. Although apoptotic responses vary widely among cell types, the underlying mechanisms responsible for these differences are not known. In order to understand the molecular basis for these differences, we have studied a cell culture model comparing hepatoma cells to dedifferentiated cell lines derived from them. We recently reported evidence suggesting that a common regulatory locus affects both liver-specific function and sensitivity to lipopolysaccharide (LPS)-mediated apoptosis. Here, we show that dedifferentiated hepatoma cells undergo apoptosis in response to multiple compounds, including sorbitol (to induce hyperosmotic shock), TNF alpha and the microtubule damaging agent vinblastin. In contrast, the hepatoma parental cells fail to undergo apoptosis in response to any of the compounds tested. Further analysis of LPS-mediated cell death found that antioxidants N-acetylcysteine and alpha-tocopherol partially prevented apoptosis. Lastly, evidence is presented showing that LPS-mediated cell death of the hepatoma variant cell lines is caspase-dependent. These results suggest that pathways dictating hepatic phenotype also affect general cellular survival mechanisms in response to multiple agents. The dedifferentiated cells provide a model to examine the influence of cell-type specific expression on apoptotic signaling.     

Biosynthesis of functionally active heparin cofactor II by a human hepatoma-derived cell line

Human plasma heparin cofactor II (HCII) inhibits thrombin by rapidly forming a stable, equimolar complex in the presence of heparin or dermatan sulfate. Cultured human hepatoma-derived cells (PLC/PRF-5) secreted (approximately equal to 200 ng/ml in 3 days) a protein of MW - 72 kD that was immunoisolated and immunoblotted with anti-HCII, co-migrated on SDS-PAGE with human plasma HCII, and formed covalent complexes with thrombin (MW - 101 kD) in the presence but not absence of heparin or dermatan sulfate; these complexes co-migrated with those obtained by incubating thrombin with human plasma under the same conditions. HCII was not detectable (less than 0.13 ng/ml) in post-culture medium from cultured human umbilical vein endothelial cells or human foreskin fibroblasts.     

Steroid synthesis in rat brain cell cultures

Primary cultures derived from neonatal rat forebrains were established and cultured for several weeks. They grow entirely as glial cultures composed of oligodendrocytes and astrocytes. Glial cells undergo maturation and differentiation in culture. This was shown by measuring the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a specific marker for expression of oligodendrocyte differentiation. CNPase activity increased from days 10-21 of culture. Both cell types were characterized by indirect immunofluorescence staining using monoclonal antibodies to galactocerebroside (Gal C) and myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) for astrocytes. Using the above criteria, we measured about 60% oligodendrocytes and 40% astrocytes after 3 weeks of culture. Oligodendrocytes, expressing Gal C and MBP, were highly immunoreactive to monospecific polyclonal antibodies to the cytochrome P-450scc, enzyme involved in the synthesis of pregnenolone from cholesterol. After incubation of glial cultures with [3H]mevalonolactone in the presence of mevinoline and trilostane, biosynthesis of [3H]cholesterol, [3H]pregnenolone (P) and [3H]pregn-5-ene-3 beta, 20 alpha-diol (20-OHP) was demonstrated. Steroid biosynthesis was related to oligodendroglial differentiation, as the initial and rapid rate of increase in CNPase activity was found to occur at the same time as the onset of steroid synthesis. Both reached a maximum at 3 weeks of culture and remained stable for several weeks. Steroid synthesis was increased by dibutyryl cAMP (0.2 mM), as well as by dexamethasone (10 nM). When aminoglutethimide, a potent inhibitor of cytochrome P-450scc, was added during the incubation of cells with [3H]mevalonolactone, [3H]cholesterol accumulated in the cells. After the release of aminoglutethimide blockade, [3H]20-OHP was the major steroid produced and released in the culture medium. The demonstration of de novo steroid biosynthesis and of the cholesterol side-chain cleavage cytochrome P-450 in normal rat glial cells brings additional support to the concept of "neurosteroids".     

Studies of alpha-protein in human cell cultures

alpha-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium A3. AGF is effective at less than 0.4 microgram per ml. By using the procedures described in the text, it is possible to culture HeLa cells in very simple media such as Eagle's basal medium. The properties of AGF are such that it may be adsorbed on glass or plastic flasks. Glass flasks treated with AGF retain full activity after washing with acetone, and treatment with ethyl ether and chemically defined medium. Adsorbed AGF is destroyed by trypsin. AGF can detoxify protamines, polylysines or histones. It will reverse the aggregation response induced by adding complexes composed of carboxymethylcellulose (CMC) and basic proteins. The results support the contention that highly adsorptive AGF functions at the cell surface and is capable of modifying the response of the cell to its environment.     

Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein ("VLDL"), low density lipoprotein ("LDL"), and high density lipoprotein ("HDL"), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B "LDL", compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B "LDL" possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B "VLDL" particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. "HDL" harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 "HDL." "HDL" from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoA-II without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized "HDL" not found in HepG2 medium. NPLC "HDL" had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that "HDL" harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The "HDL" apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutathione-dependent cytotoxicity of the chloroacetanilide herbicides alachlor, metolachlor, and propachlor in rat and human hepatoma-derived cultured cells

Alachlor, metolachlor, and propachlor are widely used chloroacetanilide herbicides. Their cytotoxicity in rat (Fa32) and human (Hep G2) hepatoma-derived cells was investigated, in connection with their influence on the endogenous glutathione (GSH) content, on the xenobiotic-metabolizing phase I enzymes 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD), and phase II glutathione transferase (GST). The cytotoxicity was measured by the neutral red uptake inhibition assay. The following toxicity range was observed in both cell lines : propachlor>alachlor>metolachlor. When the endogenous GSH content was reduced by pretreatment of the cells with L-buthionine (S,R)-sulfoximine, the cytotoxicity of the herbicides increased strongly in both cell lines. EROD and PROD activities were dose-dependently increased to different degrees in Fa32, as was EROD in Hep G2, but no PROD activity was observed in these cells. The GSH content was not altered after 1 h treatment, and was approximately doubled after 24 h. GST activity was increased in Fa32 cells but not in Hep G2. A comparable cytotoxicity was observed for the investigated chloroacetanilides in both the rat and the human cell lines. Different interactions with xenobiotic-metabolizing phase I and II enzymes were observed, and GSH showed a protective effect against the acetanilides in both cell lines.     

d-glucose uptake in human liver cell cultures

Summary The kinetic parameters ford-glucose uptake were studied in human liver cell cultures under strictly defined experimental conditions. Using a wide concentration range (0.005 to 30 mmol/l), the kinetic data obtained suggested strongly thatd-glucose in human liver cell cultures can be transported by two separate systems. For the high-affinity system, the apparentK _m was 0.645±0.21 mmol/l and the V_max, 12.49±3.74 nmol/mg protein per min. For the low-affinity system, the apparentK _m was 6.91±0.58 mmol/l and the V_max, 79.90±5.27 nmol/mg protein per min. At a concentration of 2.1×10⁻⁷ mol/l, cytochalasin B preferentially inhibited the high-affinityd-glucose site or transport system. The time course ofd-glucose uptake, studied in two cell lines from patients with hereditary fructose intolerance, was significantly higher than for the control lines. This work was supported by Grant I.N.S.E.R.M. CRL 77-5-210-4.     

The use of cell cultures in human genetics

Advice on rearing locusts is followed by suggestions for simple investigations, needing only inexpensive equipment, which will help students to understand the circumstances in which locusts become a major threat to food supplies in many parts of the Tropics.