In vitro effect of fenugreek extracts on intestinal sodium-dependent glucose uptake and hepatic glycogen phosphorylase A.

Fenugreek (Trigonella foenum-graecum L. seed) is a food with traditional medicinal use in diabetes. Beneficial effects have been demonstrated in diabetic animals and both insulin-dependent and non-insulin-dependent diabetic subjects. Effects of a lipid extract A, crude ethanolic extract B, further sub-fractions of B (saponin-free C, saponin D and sapogenin E) and a gum fibre fraction F on intestinal sodium-dependent glucose uptake were investigated in vitro using rabbit intestinal brush border membrane vesicles. All fractions except A inhibited glucose-uptake at 0.33 and/or 3.3 mg/mL (p < 0.001). Greatest inhibition was observed with fractions D and E. Diosgenin and trigonelline (compounds reported in fenugreek) also inhibited glucose-uptake (IC50 values approximately 3 mg/ml, equivalent to 8 mM and 19 mM respectively) but did not account for the activity of the crude extracts. Fenugreek extracts had no effect on basal levels of glycogen phosphorylase a (HGPa) activity in rat hepatocyte suspensions. However fractions C and E caused a marginal but statistically significant inhibition (18.9 and 15.1% respectively, p < 0.05) of glucagon induction of this enzyme suggesting a glucagon-antagonist effect. Diosgenin (1.65 mg/ml; 4 mM) inhibited glucagon-induced HGPa activity by 20% (p < 0.05), and was more effective than trigonelline (non significant inhibition of 9.4% at 1.65 mg/ml, 10 mM).

SB-203207 and SB-203208, two novel isoleucyl tRNA synthetase inhibitors from a Streptomyces sp. II. Structure determination.

Two novel isoleucyl tRNA synthetase inhibitors, SB-203207 and SB-203208 have been isolated from a Streptomyces sp. and found to be structurally related to altemicidin. Structures of SB-203207 and SB-203208 have been deduced by a combination of spectroscopic techniques, derivatisation, hydrolysis studies and found to be 4-(aminocarbonyl)-7-[[(2-amino-3-methylpentanoyl)aminosul phonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-hydroxy-2-methyl-1H-2- pyrindine-7-carboxylic acid (1) and 4-(aminocarbonyl)-7-[[(2-amino-3-methyl pentanoyl)-aminosulphonyl]acetamido]-2,4a,5,6,7,7a-hexahydro-6-(2- amino-3-phenylbutanoyl oxy)-2-methyl-1H-2-pyrindine-7-carboxylic acid (2), respectively.

The inhibition of human cytomegalovirus (hCMV) protease by hydroxylamine derivatives.

Aryl hydroxylamine derivatives have been synthesised that are some of the most potent inhibitors of hCMV protease prepared to date (IC50 14-60 nM). Mass spectrometry studies indicate that oxazinone derived hydroxylamines inhibit the enzyme by acylation of Ser132 whereas non-oxazinone derived hydroxylamines appear to inhibit via formation of a sulfinanilide at Cys138.

Maintenance of steroid metabolism and hormone responsiveness in cryopreserved dog, monkey and human hepatocytes.

The efficient and effective use of hepatocytes from larger species and rare human material requires a reliable storage method for cells not needed on the day of preparation. Cryopreservation would seem to be the only viable alternative. In this study the suitability of a published cryopreservation technique on dog, monkey and human hepatocytes has been examined and the cells were tested for functionality directly after thawing and subsequent to culture using steroid metabolism and hormone responsiveness of glycogen phosphorylase a. Monkey and human hepatocytes appear to survive the freezing and thawing process better than dog cells-the latter losing the ability to respond to adrenergic stimuli and their ability to maintain steroid metabolism in culture. Although monkey and human cells do preserve their steroid metabolising capacity after freeze/thawing, there is not the significant increase in enzyme activity seen during culturing freshly isolated cells. It would appear, therefore, that some damage has occurred to the cells during the freeze/thaw process. As previously noted, Williams' medium E is superior to Ham's F-10 in maintaining enzyme activities in culture. It is suggested that cryopreservation is the way forward for the development of stockpiles of viable hepatocytes for biomedical and toxicological research and development but that further modifications to the process are still necessary to optimise the maintenance of liver-specific functions in the thawed cells.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Inhibition of metallo-beta-lactamases by a series of mercaptoacetic acid thiol ester derivatives.

A series of mercaptoacetic acid thiol esters have been identified as metallo-beta-lactamase inhibitors. Electrospray mass spectrometry (ESMS) has shown that irreversible inhibition of the Bacillus cereus II metallo-beta-lactamase by SB214751, SB214752, and SB213079 was concomitant with a 90-Da increase in mass of the enzyme. Tryptic digestion of the B. cereus II inhibited with SB214751 illustrated that the peptide fragment, containing the only cysteine of the enzyme, had undergone a mass increment of 90 Da. It was further demonstrated that B. cereus II hydrolyzed this type of compound across the thiol ester bond to yield mercaptoacetic acid. Mercaptoacetic acid is the only molecular fragment common to SB214751, SB214752, and SB213079, and free mercaptoacetic acid does not bind covalently to B. cereus II. Therefore, it is concluded that these compounds inhibit B. cereus II by the mechanism-based delivery of mercaptoacetic acid, forming a disulfide linkage with the active sites cysteine (predicted mass shift = +90 Da) under the aerobic conditions of the assay. The different thiol esters examined had a broad range of potencies against the metallo-beta-lactamases tested. For example SB214751, SB214752, and SB213079 all had 50% inhibitory concentrations of < 10 and > 1,000 microM for the Stenotrophomonas maltophilia L-1 and Bacteroides fragilis CfiA enzymes, respectively. SB216968 was particularly active against the Aeromonas hydrophila CphA metallo-beta-lactamase and was found to be an uncompetitive inhibitor of this enzyme (Ki = 3.9 microM), whereas it exhibited irreversible inhibition of the L-1 enzyme. These observations with this series of compounds have revealed subtle differences between the active sites of different metallo-beta-lactamases. Finally, a novel application for isothermal titration calorimetry for assessing the zinc chelating activity of candidate inhibitors is also presented.

Isolation and culture of human hepatocytes.

The liver performs a wide range of physiologically important functions, including the synthesis and secretion of albumin, fibrinogen, and other plasma proteins; the synthesis of cholesterol and bile acids, and the metabolism of drugs, steroids, and amino acids. The liver has a central role in energy metabolism as the major store of glycogen, as the site of gluconeogenesis, and in the synthesis of fatty acids and triglycerides. The liver is, therefore, a vital organ, but it is difficult to study specific liver functions in vivo owing to interfering influences from other organs, e g., the kidney, gut, and lungs, which metabolize drugs and the muscle involvement in glucose homeostasis. An isolated liver preparation seems necessary, and the isolated human hepatocyte appears to be a suitable experimental model for the study of liver-specific functions.

beta-Lactamase ragged ends detected by electrospray mass spectrometry correlates poorly with multiple banding on isoelectric focusing.

Purified preparations of TEM-2, P99, Bacillus cereus I and B. cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry. The ES mass spectra of the B. cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends). The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends. All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends. Multiple banding patterns were seen with each beta-lactamase preparation. Although these may correlate with the presence of ragged ends in the two B. cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry. In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends. Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples. This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.

Induction of rat hepatic glucocorticoid-inducible cytochrome P450 3A by metyrapone.

The bipyridyl compound metyrapone is a potent inhibitor of cytochromes P450, a gene superfamily of haemoproteins involved in the metabolism of many xenobiotics as well as endogenous compounds such as steroid hormones. Administration of metyrapone to male rats induces the expression of the cytochrome P450 sub-family 3A (CYP3A). In order to determine whether metyrapone was causing the induction of CYP3A by blocking endogenous glucocorticoid metabolism, CYP3A levels were examined in rat hepatocytes cultured in serum-free medium supplemented with hydrocortisone 21-hemisuccinate plus or minus metyrapone. Western blotting indicated that metyrapone alone induces CYP3A and that hydrocortisone 21-hemisuccinate is ineffective. However, hydrocortisone 21-hemisuccinate enhanced the levels of CYP3A induced by metyrapone. In contrast, glucocorticoid-inducible tyrosine aminotransferase (TAT) activity was unaffected by metyrapone but metyrapone enhanced the levels induced by hydrocortisone 21-hemisuccinate. An examination of the metabolism of hydrocortisone by rat hepatocytes in vitro indicated that metyrapone perturbed the catabolism of hydrocortisone under conditions which give rise to an enhancement of hydrocortisone 21-hemisuccinate and hydrocortisone-dependent TAT induction. However, evidence is presented to suggest that such a perturbation of hydrocortisone metabolism could not account for the glucocorticoid potency amplifying property of metyrapone. Thus the induction of CYP3A and the enhancement of glucocorticoid-mediated TAT induction appears not to be associated with any perturbation in glucocorticoid metabolism but with some other as yet undefined mechanism(s).

Problems in using isolated and cultured hepatocytes for xenobiotic metabolism/metabolism-based toxicity testing-Solutions?

Isolated and cultured hepatocytes would seem to be an ideal model for the study of hepatic xenobiotic metabolism and metabolism-based hepatotoxicity. However, a serious drawback with this system of testing is the loss of xenobiotic-metabolizing capacity in a reaction-dependent manner when the cells are kept in culture for any extended period. Many ways of modifying culture methods to overcome this problem have been investigated, for example the use of different culture media, culture media additions (e.g. hormones, serum, minerals, vitamins, and xenobiotics such as metyrapone and dimethyl sulfoxide), culturing on a substratum of components of the extracellular matrix or on microcarriers, and co-culturing with other hepatic or with non-hepatic cells. However, these methods have achieved only limited success, with only some enzyme activities being preserved or many activities being preserved to only a limited degree. Furthermore, the variations between the methods used in hepatocyte isolation and culture preclude interlaboratory correlations of results. It is likely that a combination of the methods that have been investigated will yield a workable and reliable culture method to maintain xenobiotic metabolizing capacity in isolated hepatocytes, and co-ordination of research in this area should be promoted to achieve this outcome.

Changes in adenosine sensitivity in the hippocampus of rats with streptozotocin-induced diabetes.

1. Hippocampal slices have been used to assess the sensitivity of the CNS to adenosine and gamma-aminobutyric acid (GABA) in diabetes. The effects of adenosine, 2-chloroadenosine, GABA, muscimol and baclofen were studied on orthodromic synaptic potentials recorded in the CA1 region of slices taken from normal rats or animals made diabetic by the injection of streptozotocin. 2. In diabetic animals the sensitivity to adenosine was increased 4 fold compared with normal rats. The potency of 2-chloroadenosine was unchanged. 3. The nucleoside transport inhibitor, hydroxynitrobenzylthioinosine (HNBTI), increased the potency of adenosine in slices from normal rats but not in slices from diabetic rats. 4. No change was observed in the potency of GABA or muscimol, although a small but significant decrease was detected in the EC50 value for baclofen. 5. Treatment of diabetic animals with insulin restored the potency of adenosine to control levels. 6. It is concluded that the diabetic state is accompanied by substantial changes of adenosine sensitivity due to the loss of nucleoside uptake processes. Secondary neurochemical changes following from this in human diabetic patients may contribute to the reported behavioural changes.

Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics.

Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.

The interaction of oral hypoglycaemic drugs with insulin on steroid metabolism in hepatocytes isolated from control and diabetic male rats.

The effects of the oral hypoglycaemic drugs, phenformin and tolbutamide, and insulin, alone and in combination, on steroid metabolism in hepatocytes isolated from control and streptozotocin-diabetic male rats has been studied. Both phenformin and tolbutamide mimic the action of insulin in stimulating hepatic steroid metabolism in a dose-dependent manner in control cells. Unlike insulin, however, both drugs give a similar effect in cells derived from diabetic animals although to a lesser extent. Both drugs can partially restore the effect of insulin in cells derived from diabetic animals. Biguanides and sulphonylureas, therefore, have a direct effect on liver cells to mimic insulin action and can still have an effect under conditions where insulin is inactive. Both types of oral hypoglycaemics can also affect insulin-insensitive cells isolated from diabetic rat liver to restore to a certain extent their response to insulin.

The role of microsomal phospholipids and their fatty acid composition in the control of hepatic metabolism of lignocaine.

1. Sex differences exist in the metabolism of lignocaine by the rat liver. Microsomal phospholipids have been implicated in the control of these sex differences. Induction of diabetes in the male rat abolishes these sex differences. The difference in drug metabolism between the male and female rat is, thus, the same as that between the control and diabetic male rat. 2. By using reconstitution of delipidated male microsomal proteins with male-, female- and diabetic-derived phospholipids as well as synthetic phospholipids, it should be possible to delineate the role of phospholipids in the control of drug metabolism. 3. Female- and diabetes-derived phospholipids decrease the activity of the male-specific lignocaine N-deethylase specifically by between 35 and 52%. 4. Analysis of the phospholipid classes and fatty acid content of the various fractions indicated that stearic acid content was increased and arachidonic acid content decreased in both female- and diabetic-derived lipids as compared to control males. Linoleic acid content was decreased in female- but increased in diabetic-derived lipids as compared to control males. Subsequent correlation to N-deethylase activity, however, rules out all but the arachidonic acid content of the phospholipids as a controlling factor of lignocaine metabolism. 5. Use of synthetic phosphatidylcholine (PC) species for reconstitution indicates that diarachidonyl-PC is the most efficient at activating the N-deethylase and indicates that the degree of unsaturation of the fatty acyl side-chains of PC is of major importance in the regulation of this enzyme activity. 6. The presence of unsaturated fatty acids, and especially arachidonic acid, in the phospholipids is, thus, a major controlling influence on the specific activation of lignocaine N-deethylase in the rat liver.