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Biomedical subjects

H Greim

Publications and source records attributed to H Greim.

At least 109 records · Page 6Linked to original sources
PubMed1980

Effects of pentachlorophenol and 2,4,6-trichlorophenol on the disposition of sulfobromophthalein and respiration of isolated liver cells.

The effect of pentachlorophenol (PCP) and 2,4,6-tricholorphenol (2,4,6-T) on the disposition of the hepatodiagnostic dye, sulfobromophthalein (BSP) has been studied in isolated liver cells. PCP (4-6 microM) as well as 2,4,6-T (50-100 microM) interferes with the disposition of BSP. The main effect apparently occurs at the secretion step as both drugs severely impair the release of the glutathione conjugate of BSP into the medium. As a consequence, BSP and its conjugate accumulate in the cell. High doses of PCP did not increase the release of lactate dehydrogenase from the hepatocytes. Concentrations of the two phenols which interfere with the secretion of BSP also completely uncouple the oxidative phosphorylation of hepatocellular mitochondria. The dysfunction of liver cells described here may therefore be explained by the effect of PCP and 2,4,6-T on the energy production of the cells. The higher toxicity of PCP as compared to 2,4,6-T observed in our system corresponds well with the higher LD50 of the latter compound.

Animals↗
PubMed1980

Mutagenicity testing with Salmonella microsome test.

This in vitro mutagenicity test system comprises five different strains of S. typhimurium as target cells with the rat liver S-9 fraction and appropriate co-factors for metabolic activation of the chemical tested. The bacterial tester strains detect both mutations induced by base pair substitutions and intercalation (frame shift mutations). Usually 10(8)--10(9) cells of an overnight culture or an exponentially growing culture are incubated for 2-3 days with a mixture of S-9, co-factors, soft agar and the chemical on histidine-deficient agar. The S-9 fraction is obtained from the livers of rats pretreated with 500 mg/kg chlorinated biphenyls (Clophen A-50, Aroclor 1254) to obtain high metabolic activity. For reproducibility it is essential to standardize metabolic activity and protein content of the S-9 and to use three different concentrations thereof in the test system. Since solvents inhibit metabolic activation of the chemicals they must not exceed 4% of the final 2.6 ml incubate. Several independent studies have shown that between 85 and 93% of chemical carcinogens are mutagens in the test. Regarding extrapolation to man one has to consider that the test is preferentially adapted for metabolic activation of the chemicals, whereas inactivation processes are absent or are less active than in vivo. Thus, the test provides qualitative rather than quantitative information on mutagenic effects of a chemical.

Animals↗
PubMed1980

Glutathione and glutathione S-transferases in the Salmonella mammalian-microsome mutagenicity test.

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH. The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.

Animals↗
PubMed1980

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450.

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.

Aldrin↗
PubMed1978

Methylmercury chloride induces learning deficits in prenatally treated rats.

Methylmercury chloride (MMC) was given to pregnant rats on the 6th, 7th, 8th, and 9th day after conception in doses of 0.05 and 2.0 mg/kg/day. The female offspring of these animals were tested 90 days after birth for learning ability using operant conditioning procedures. The rats were kept at 90% of their normal body weight and trained in a lever-box to press a bar in order to obtain a food pellet. Significant differences in the acquisition speed became apparent when the ratio of bar presses to reward was increased in a classical contingency of differential reinforcement of high rates even at MMC-doses of 4 X 0.05 mg/kg. These differences were not found in the general motility level nor in motor coordination.

Animals↗
PubMed1978

Bile acid conjugation in the chimpanzee: effective sulfation of lithocholic acid.

To characterize the hepatic biotransformation in the chimpanzee of the primary bile acid chenodeoxycholic acid (chenic) and its major bacterial metabolite lithocholic acid (lithocholic) a mixture of trace amounts of 14C-lithocholic and 3H-chenic was injected intravenously into two animals with a bile fistula; the chemical form of radioactivity appearing in bile was inferred using thin layer chromatography. About 80% of chenic, and 70% of lithocholic was recovered in 90 min. Chenic was completely conjugated in bile, appearing predominantly as chenyltaurine (52%) and chenylglycine (37%). An unidentified conjugate (about 11%) was also found. Lithocholic was excreted completely as taurine and glycine conjugates, but the majority (63%) of conjugates was sulfated. Sulfation increased progressively with time, and lithocholylglycine was sulfated more than lithocholyltaurine. We conclude that the chimpanzee is similar to man in that the secondary bile acid lithocholic is efficiently sulfated. The chimpanzee thus differs from the baboon and rhesus monkey which sulfate lithocholic poorly. However, the chimpanzee differs from man and is similar to the baboon and rhesus monkey in showing preferential conjugation of bile acids with taurine. The results imply that hepatotoxicity caused by chenic, which is well documented in the rhesus monkey and baboon and has been related to defective lithocholic sulfation, should not occur in the chimpanzee.

Animals↗
PubMed1977

Mutagenicity and chromosomal aberrations as an analytical tool for in vitro detection of mammalian enzyme-mediated formation of reactive metabolites.

1. Incubation of trichloroethylene, 1,1-dichloroethylene, vinylchloride, tetra-chlorocyclopentadiene, the nitroso derivatives of the pesticides Carbaryl, Prometryn, and Dodin in the presence of metabolically active mouse liver microsomes and bacteria as target cells were mutagenic, whereas tetrachloroethylene, 1,2 cis- and transdichloroethylene, hexachlorocyclopentadiene, carbontetrachloride, chloroform, halothane, trichlorofluoromethane and styrene were not activated to mutagenic species. 2. In a similar in vitro test system using freshly isolated human lymphocytes as target cells dimethylnitrosamine induced chromosomal aberrations. 3. It is concluded from the experiments that submammalian or mammalian in vitro cell systems with metabolically active liver microsomes are not only suitable to screen for chemical mutagens but to demonstrate formation of reactive intermediates, which are short lived and cannot be detected by chemical procedure.

Animals↗
PubMed1977

Taurolithocholate inhibits taurocholate uptake by isolated hepatocytes at low concentrations.

The cholestatic bile acid taurolithocholate inhibits taurocholate uptake by isolated liver cells non-competitively. Inhibition is instantaneous and inversely related to the cell number in the incubate. The Ki amounts to 7 micron in the presence of 2 mg cellular protein per ml. Secretion of taurocholate by isolated liver cells is not affected by taurolithocholate up to a concentration of 50 micron. This indicates a difference between the carrier for taurocholate uptake and the carrier for taurocholate secretion. Inhibition of bile acid uptake of liver cells may be involved in the pathogenesis of lithocholate-induced cholestasis.

Animals↗
PubMed1977

Metabolic activation of haloalkanes and tests in vitro for mutagenicity.

1. During incubation of 14CCl4, 14CHCl3, [14C]halothane, or 14CCl3F with liver microsomes and NADPH, considerable radioactivity is bound irreversibly to endoplasmic protein and lipid. However, no 14C was detected in the ribosomal RNA. 2. None of the four haloalkanes studied induced mutations after incubation with liver microsomes and the bacterial tester strains S. typhimurium TA 1535 and TA 1538. 3. Very low or no activity was associated with soluble protein or RNA added to incubation mixtures of the four haloalkanes with liver microsomes.

Animals↗
PubMed1977

Current concepts in intrahepatic cholestasis.

The investigations on experimental intrahepatic cholestasis of the last 10 years suggest that any of several different functional alterations may lead to cholestasis. In most studies very high doses of cholestatic compounds have been used. The relevance of these studies to clinical syndroms of cholestasis which usually are observed at much lower doses, is unclear. One target of cholestatic compounds may be the lipid phase of several cell structures such as the sinusoidal and canalicular membrane, the endoplasmic reticulum and the mitochondria. By their capacity to interact with the lipid layer and proteins of membranes these compounds impair specific cellular functions: the activity of carrier proteins, the activity of the hydroxylating system and the energy supply. Another target may be the binding proteins in the cytoplasma and possibly the microfilaments. One may suggest that other factors such as interference with regulatory processes in the cell may be of interest in the future. So far the primary event of drug-induced intrahepatic cholestasis is still unknown and it is not even clear whether the increased bile acid levels are cause or consequence of the cholestatic condition.

Animals↗
PubMed1976

Excretion of taurocholate from isolated hepatocytes.

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux. Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 muM. Ouabain increased the rate of efflux. The observations indicate that secretory functions are maintained in isolated liver cells.

Animals↗
PubMed1976

Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1.

Environmental chemicals including pesticides carrying secondary and tertiary amino groups are suggested to be a health hazard to man since potentially carcinogenic nitroso compounds may be formed in the presence of nitrite at low pH values resembling conditions in the human stomach. Nitrosation of the isopropylamino-triazine Prometryne, the n-dodecyl guanidine Dodine and the N-methylcarbamate carbaryl was investigated in the presence of HCl and acetic acid at pH 1 and excess sodium nitrite for 4 h at 37 degrees C. The reaction products were extracted with CCl4 and were analyzed qualitatively and quantitatively by infrared spectroscopy, nuclear-resonance spectrometry, GC/mass spectrometry and by spectrophotometry. All compounds investigated formed N-nitroso derivatives in the following yields: carbaryl 67%, Dodine 12% and Prometryne 14%. The N-nitroso derivatives per se were not or only slightly mutagenic to Escherichia coli K12 or Salmonella typhimurium TA 1538. However, significantly increased mutation frequencies were seen after metabolic activation by mouse-liver microsomes. These results add to the observations that among environmental chemicals not only those containing methyl- or ethyl-substituted amino groups form potentially carcinogenic nitroso derivatives but also those with iso-propylamino groups as well as alkyl-substituted guanidine derivatives.

Carbaryl↗
PubMed1976

Human lymphocytes as target cells in a metabolizing test system in vitro for detecting potential mutagens.

A metabolizing test system is presented that comprises mouse liver microsomes for metabolic activation of the test compound, NADPH as co-factors, dimethylnitrosamine (DMN) as model mutagen and human peripheral lymphocytes as target cells. Lymphocytes were cultured in RPMI 1640 medium in the presence of phytohemagglutinin for 48 h before being used in the system. Incubation of the lymphocytes for 45 min in the metabolically active system reduced thymidine incorporation by 50%, whereas in the metabolically inactive system without NADPH thymidine incorporation was reduced by 15%. The latter reduction was due to the cytotoxic effects of the mouse liver microsomes and of DMN. When the lymphocytes were cultured for another 24 h after exposure to the metabolizing system and DMN, significant increased formation of chromosomal aberrations such as gaps were observed, cross-overs and breaks being less frequent.

Cells, Cultured↗