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Biomedical subjects

G Powis

Publications and source records attributed to G Powis.

At least 127 records · Page 7Linked to original sources
PubMed1990

Expression of glutathione S-transferases and cytochrome P450 in normal and tumor breast tissue.

The level of expression of glutathione S-transferases (GSTs) and cytochrome P450s in breast tissue are potentially important determinants in both the susceptibility of this tissue to the mutagenic effects of chemical carcinogens and in the response of breast tumors to chemotherapy. In this study we have investigated the expression of these proteins in 41 tumor and surrounding normal breast tissue samples by measurement of substrate metabolism. Western blot analysis and immunohistochemistry. In addition, we have quantitated the concentration of alpha, mu and pi class GST subunits using radioimmunoassay. All three classes of GST were expressed in breast tissue. The pi and mu class enzymes preponderate. Both the polymorphic mu class GST as well as a further form, present in all individuals, were found in high concentration. The polymorphic mu class GST was expressed in approximately 50% of the samples, which is consistent with the frequency of this polymorphism in the population and therefore does not appear to be a factor in susceptibility to this disease. Interestingly, although levels of the alpha class GST were very low, in two tumor samples extremely high levels of the B1B1 subunit were detected. Immunohistochemical studies showed significant variability in the localization of the pi class of GST between normal epithelial cells, infiltrating plasma cells and tumor cells, and in some samples GST pi appeared to be almost absent from the tumor tissue. No direct, or inverse correlation was found between GST pi concentration determined by radioimmunoassay and estrogen receptor levels. However, when studied by immunohistochemistry estrogen receptor negative tumors did tend to have higher GST pi content. The only cytochrome P450 detectable by Western blot analysis was a member of the P450IIC gene family. This was apparently distinct from the P450IIC proteins expressed in the liver and was detected in normal and tumor tissues to a similar extent.

Adult↗
PubMed1990

Glutathione S-transferase and glutathione peroxidase expression in normal and tumour human tissues.

Glutathione S-transferases play a central role in drug detoxification and have been implicated in the sensitivity of tumour cells to anticancer drugs. In this study, glutathione S-transferase (GST) isozyme expression in normal and tumour tissue from human lung, colon, stomach, breast, kidney and liver tissue has been quantified using sensitive and subunit specific radioimmunoassays (RIA), together with Western blot analysis and measurement of substrate metabolism. Glutathione S-transferase pi was the predominant GST in the majority of the tumours examined. The concentration of this enzyme was increased significantly in tumour tissue relative to normal lung, colon, and stomach tissue. A strong correlation was observed (r = 0.77, P less than 0.01) between GST activity and GST pi levels in those tumour samples. The concentrations of the alpha class GST, the predominant isoenzymes in normal stomach, kidney and liver, decreased dramatically in tumour tissue from these organs. Western blot analysis revealed the presence of novel polypeptides that cross-reacted with antisera raised against alpha and mu class GST. Our data demonstrates that although GST pi is the predominant GST isoenzyme in many tumours, significant levels of the other GST subunits are also present and collectively can represent a significant proportion of the GST content. Therefore the properties of all the GST isoenzymes need consideration when assessing the role of these proteins in drug resistance. Selenium-dependent glutathione peroxidase, an enzyme activity also implicated in the mode of action of certain antitumour agents, was also studied and shown to be the predominant glutathione-dependent peroxidase in all tumours except the hepatoma.

Blotting, Western↗
PubMed1990

The effects of volatile anesthetics on Ca++ mobilization in rat hepatocytes.

This study provides direct evidence that in hepatocytes, intracellular Ca++ is released from internal stores by halothane, enflurane, and isoflurane. Hepatocytes isolated from rat livers were used fresh or treated with saponin and then incubated in 45Ca++ media. The uptake of 45Ca++ by hepatocytes was maximal following 13-16 min of incubation (untreated or saponin-treated) and the effects of various agents on the release of 45Ca++ was studied following maximal loading. The agents used included halothane, enflurane, isoflurane, and several putative intracellular second messengers. The anesthetics, to various degrees, all stimulated a significant release of 45Ca++ from internal stores at concentrations that were at or less than clinical concentrations. The release of intracellular 45Ca++ by each of the anesthetic agents was dose-dependent with halothane and enflurane being equally potent at concentrations equivalent to 1 MAC exposure. The halothane-induced release was only somewhat suppressed by preincubation in either 2 mM LaCL3 or 10 microM dantrolene, both suggested Ca++ channel blockers. Transient increases in intracellular Ca++ regulates a number of enzyme systems, including glycogenolysis, while prolonged elevation in Ca++ concentrations have been implicated in the mechanism of hepatotoxicity.

Anesthetics↗
PubMed1990

Inhibition of thioredoxin reductase (E.C. 1.6.4.5.) by antitumor quinones.

Thioredoxin reductase (TR) is a widely distributed flavoenzyme that provides reduced thioredoxin, a dithiol hydrogen donor for protein disulfide reduction and for the reduction of ribonucleotides to deoxyribonucleotides, the first unique step of DNA synthesis. Antitumor quinones were found to exhibit time- and concentration-dependent inhibition of purified rat liver TR that requires the presence of NADPH. Diaziquone initially shows competitive inhibition of the enzyme with 5,5'-dithiobis 2-nitrobenzoic acid as substrate with a Ki of 7.5 microM, which becomes non-competitive after 1 hour incubation with NADPH with a Ki of 0.5 microM. Doxorubicin shows non-competitive inhibition both initially and after 1 hr incubation with NADPH, with Ki values of 10 microM and 0.5 microM, respectively. Electron spin resonance spectroscopy showed the formation of semiquinone free radicals by TR incubated under anaerobic conditions with doxorubicin or diaziquone and NADPH. Redox cycling and formation of oxygen radicals does not play a major role in the inhibition of TR by antitumor quinones as shown by the minor effect on inhibition of removing O2, and the lack of effect of superoxide dismutase and catalase. Diaziquone causes time- and concentration-dependent inhibition of TR activity in intact A204 human rhabdomyosarcoma cells that is associated with growth inhibition. The results suggest that inhibition of TR by antitumor quinones could contribute to their growth inhibitory properties.

Animals↗
PubMed1990

Doxorubicin blocks the increase in intracellular Ca++, part of a second messenger system in N1E-115 murine neuroblastoma cells.

Exposure of differentiated N1E-115 murine neuroblastoma cells, microinjected with the Ca(++)-sensitive photoprotein aequorin, to doxorubicin for 1 hr, but not for 2 min, produced a reversible block of the rise in intracellular free Ca++ [( Ca++]i) produced by histamine. The resting level of [Ca++]i was increased from 0.23 to 1.22 microM (P less than 0.05) by 10(-4) M histamine. After exposure to 10(-6) M doxorubicin for 1 hr, histamine increased [Ca++]i to only 0.34 microM (P less than 0.05 compared to the histamine alone value). Doxorubicin exposure for 1 hr completely blocked the increase in inositol trisphosphate caused by histamine. There was no block by doxorubicin of the release of intracellular Ca++ after microinjection of the cells with inositol 1,4,5-trisphosphate. Based on the results from studies with differentiated N1E-115 neuroblastoma cells doxorubicin may: 1) block the histamine-induced rise in [Ca++]i by decreasing synthesis of inositol polyphosphates, 2) block plasma membrane Ca++ channels that allow entry of extracellular Ca++ in response to histamine and/or 3) prevent recovery of histamine receptors after desensitization.

Aequorin↗
PubMed1989

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels.

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Toxicity of intrathecally administered cytotoxic drugs and their antitumor activity against an intrathecal Walker 256 carcinosarcoma model for meningeal carcinomatosis in the rat.

Meningeal carcinomatosis in humans is refractory to most attempts at therapy and has a grave prognosis. As part of a search for new agents to treat meningeal carcinomatosis, the toxicity of a series of antitumor agents administered through an implanted catheter directly into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 200-g rats has been examined. The highest non-toxic dose (HNTD) of the agents producing no signs of histological damage, motor dysfunction, or neurobehavioral changes was bleomycin (80 micrograms), cytarabine (640 micrograms), dacarbazine (1 microgram), doxorubicin (20 micrograms), 5-fluorouracil (150 micrograms), methotrexate (1000 micrograms), mitomycin C (10 micrograms), and triethylene phosphoramide (800 micrograms). No toxicity was observed at the highest dose that could be administered because of limited solubility of 1,3-bis(2-chloroethyl)-1-nitrosourea (100 micrograms), or diaziquone (70 micrograms). The rat model gave reasonable prediction of the toxicity of antitumor agents that have been administered intra-CSF to humans but with a tendency to underpredict toxicity. The antitumor activity of the agents administered intra-CSF at the HNTD against a Walker 256 carcinosarcoma model for meningeal carcinomatosis in the rat was examined. Diaziquone, doxorubicin, methotrexate, and mitomycin C gave a 25% or greater increase in lifespan and diaziquone and mitomycin C gave some long-term survivors. BCNU, bleomycin and 5-fluorouracil gave less than a 25% increase in lifespan. When mitomycin C was administered intra-CSF at the HNTD and diaziquone at the limit of solubility daily for 4 days there was no toxicity and an increase in survival of Walker 256 carcinosarcoma tumored animals compared to animals administered a single daily dose. Intrathecal diaziquone was more active against meningeal Walker 256 carcinosarcoma than i.v. diaziquone. Intravenous methotrexate had no activity against meningeal Walker 256 carcinosarcoma. We conclude that intra-CSF administration of some anticancer agents such as diaziquone and mitomycin C at doses that do not produce toxicity can significantly increase the survival of rats with experimental meningeal carcinomatosis.

Animals↗
PubMed1989

Preclinical pharmacologic studies of the new antitumor agent carmethizole (NSC-602668) in the mouse and beagle dog.

The chemical breakdown of carmethizole [1-methyl-2-methylthio-4,5-bis-(hydroxymethyl)imidazole-4',5'- bis(N-methylcarbamate)hydrochloride] and its pharmacokinetics in the mouse and beagle dog were studied. Carmethizole was relatively unstable in aqueous media, having a half-life of less than or equal to 1 h in 0.9% sodium chloride, human whole blood, human plasma, and dog urine at 37 degrees C. Its major breakdown product in 0.9% sodium chloride and pH 5.0 sodium phosphate buffer was carmethizole diol. When carmethizole was added to pH 7.0 or pH 9.0 sodium phosphate buffer, the major breakdown product was carmethizole diol-4'-monophosphate. Carmethizole reacted directly with glutathione at pH 8.0, forming a glutathione adduct of carmethizole monocarbamate. Elimination of the drug from the plasma of the beagle dog following i.v. bolus doses of 22.4 and 4.3 mg/kg was biphasic. At these doses the terminal half-life was 39 and 46 min, respectively, and the respective total body clearance was 4.6 and 7.7 ml/min per kg. The 22.4 mg/kg dose was lethal to the beagle dog by day 4. Elimination of carmethizole from the plasma of mice following an i.v. bolus dose of 115 mg/kg was monoexponential, with a half-life of 11.6 min and a total body plasma clearance of 43.6 ml/min per kg. When the drug was infused at 230 mg/kg over 8 h into mice, the total body clearance was 40.8 ml/min per kg. Following the i.v. bolus administration of carmethizole to mice, 30% of the total dose was excreted in urine over 3 h as carmethizole diol, 10%, as carmethizole diol-sulfate, 3.4%, as carmethizole 4'-monocarbamate, and 2.4%, as unchanged drug.

Animals↗
PubMed1989

Isolation, identification and biological activity of a phyllanthoside metabolite produced in vitro by mouse plasma.

The antitumor agent phyllanthoside is rapidly metabolized in vitro by mouse plasma. This metabolite has now been isolated from mouse plasma and its structural properties and cytotoxicity characterized. The isolated metabolite was estimated to be greater than 98% pure by HPLC analysis. Mass spectral analysis (fast atom bombardment and tandem mass spectrometry) indicated that the metabolite was the aglycone of phyllanthoside that resulted from the cleavage of the ester bond linking the aglycone and the disaccharide moieties of phyllanthoside. This identification was based on identical collision-induced dissociation spectra of both phyllanthoside and the metabolite. The aglycone was not formed by mouse plasma that had been boiled, filtered to remove proteins, or treated with 1.0 mM diisopropyl fluorophosphate. These results suggest that aglycone formation occurs as a result of plasma esterase activity. Michaelis-Menten constants, Vmax and Km, for conversion of phyllanthoside to the aglycone at 22 degrees C were estimated to be 1.1 mmol/ml plasma/min and 2.0 mM, respectively. Concentrations of phyllanthoside and metabolite required to inhibit cell-colony formation by human A204 rhabdomyosarcoma in vitro were 0.47 nM and 24 microM, respectively. The toxicity of phyllanthoside, and perhaps its efficacy as an antitumor agent in mice, may depend on its rate of conversion to the aglycone.

Animals↗
PubMed1989

Model systems for detecting the hepatic toxicity of pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides.

Indicine N-oxide (INO) is a pyrrolizidine alkaloid (PA) with antitumor activity in animals and humans. Prior studies showed that despite the known hepatic toxicity of the PAs, INO did not produce hepatic toxicity in animals but caused unpredictable lethal hepatic toxicity in humans. In this study we have attempted to find a model system for predicting the hepatotoxic potential of antitumor PAs. Primary cultures of rat hepatocytes showed toxicity only with the most hepatotoxic PAs such as lasiocarpine, but did not detect toxicity with other PAs. Subchronic intraperitoneal administration of PAs to weanling rats and adult mice produced, in surviving animals, hepatic megalocytosis and centrilobular necrosis with heliotrine (H) and 9-O-(R(-)-2-(4'-chlorophenyl)-2-hydroxybutyryl)retronecine N-oxide (RC1NO) but only megalocytosis with INO. Thus, despite previous reports, weanling rats offered no advantage over adult mice for detecting significant hepatic toxicity with PAs. Phenobarbital pretreatment of the mice did not increase the hepatic toxicity of any of the PAs. Subchronic oral administration of PAs to adult mice produced hepatic megalocytosis and centrilobular necrosis in surviving animals with H and RC1NO and megalocytosis with INO. Animals that died acutely following oral administration of INO showed hepatic centrilobular necrosis. Administration of several courses of INO intravenously to dogs produced histological evidence of centrilobular hemorrhagic necrosis. It is concluded that there is no single animal model that will predict hepatic toxicity of the type seen in humans with the antitumor PAs. A combination of studies using adult mice and dogs and lethal doses of the PAs offers the best way of detecting potential hepatic toxicity.

Administration, Oral↗
PubMed1989

Studies of chemical toxicity to fresh and cryopreserved rat hepatocytes.

Isolated hepatocytes are useful for studying the metabolism and mechanisms of hepatic toxicity of foreign chemicals. A problem with using human hepatocytes is the limited and irregular availability of normal human liver. Cryopreservation could provide a useful way of storing hepatocytes until they are needed. As a preliminary step to using human hepatocytes we have compared the toxic response to chemical toxicants of primary cultures of fresh rat hepatocytes and rat hepatocytes cryopreserved as previously described (G. Powis, K. S. Santone, D. C. Melder, L. Thomas, D. J. Moore, and T. J. Wilke, 1987. Drug Metab. Dispos. 15, 826). After 24 hr in culture the cryopreserved hepatocytes had a plating efficiency 75% that of noncryopreserved hepatocytes. The cultured cryopreserved hepatocytes showed a small increase in spontaneous lactate dehydrogenase release compared to that of cultured noncryopreserved hepatocytes. A similar toxic chemical-induced increase in lactate dehydrogenase release occurred in the cultured cryopreserved as in the noncryopreserved hepatocytes. The 50% effective concentrations (EC50) for lactate dehydrogenase release (+/- SE, n = 3 preparations) from cultured cryopreserved and noncryopreserved hepatocytes for chlorpromazine were 235 +/- 20 and 215 +/- 30 microM, for cadmium chloride 200 +/- 5 and 272 +/- 23 microM, and for menadione (2-methyl-1,4-naphthoquinone) 24 +/- 7 and 44 +/- 8 microM, respectively. The EC50 values for intracellular glutathione depletion in cultured cryopreserved and noncryopreserved hepatocytes were for chlorpromazine 200 +/- 8 and 235 +/- 8 microM, for cadmium chloride 242 +/- 19 and 213 +/- 7 microM, and for menadione 22 +/- 2 and 21 +/- 3 microM, respectively. The results show that cryopreservation offers a practical way of storing rat hepatocytes for studies of chemical toxicity.

Animals↗
PubMed1989

Free radical formation by antitumor quinones.

Quinones are among the most frequently used drugs to treat human cancer. All of the antitumor quinones can undergo reversible enzymatic reduction and oxidation, and form semiquinone and oxygen radicals. For several antitumor quinones enzymatic reduction also leads to formation of alkylating species but whether this involves reduction to the semiquinone or the hydroquinone is not always clear. The antitumor activity of quinones is frequently linked to DNA damage caused by alkylating species or oxygen radicals. Some other effects of the antitumor quinones, such as cardiotoxicity and skin toxicity, may also be related to oxygen radical formation. The evidence for a relationship between radical formation and the biological activity of the antitumor quinones is evaluated.

Alkylating Agents↗
PubMed1989

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions.

1. The oxidative metabolism of 14C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by h.p.l.c. employing continuous radioactivity monitoring of the column effluent. 2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per mg protein, 2-pyridone (2PO) at 207 pmol/min per mg and 4-pyridone (4PO) at 154 pmol/min per mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites. 3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate. 4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism. 5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per mg protein, 2PO at 17 pmol/min per mg and 4PO at 61 pmol/min per mg. 6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.

Animals↗
PubMed1989

High molecular weight dextran sulfate inhibits intracellular Ca2+ release and decreases growth factor-induced increases in intracellular free Ca2+ in Swiss 3T3 fibroblasts.

High molecular weight (500 kDa) dextran sulfate (DXS) inhibited the release of Ca2+ induced by myoinositol 1,4,5-trisphosphate from non-mitochondrial stores of saponin-permeabilized Swiss 3T3 fibroblasts with an IC50 of 20 micrograms/mL. Low molecular weight (5 kDa) DXS did not have this effect. DXS was more inhibitory than heparin, which in the same system had an IC50 of 62 micrograms/mL. DXS also produced a small inhibition of Ca2+ release by arachidonic acid and GTP but did not affect Ca2+ release by 4-bromo A23187 or halothane. The transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in intact Swiss 3T3 cells caused by platelet-derived growth factor was completely inhibited by 100 micrograms/mL of DXS, but DXS had no effect on the [Ca2+]i increase caused by bradykinin or vasopressin. The specific binding of platelet-derived growth factor, but not of bradykinin or vasopressin, to Swiss 3T3 fibroblasts was decreased by DXS. The effect of DXS in decreasing growth-factor mediated increases in [Ca2+]i may be mediated by an effect on the binding of growth factor to its receptor. An effect of DXS on the intracellular release of Ca2+ by second messengers to decrease changes in [Ca2+]i, however, cannot be ruled out.

Arachidonic Acid↗
PubMed1988

An increase in intracellular free Ca2+ associated with serum-free growth stimulation of Swiss 3T3 fibroblasts by epidermal growth factor in the presence of bradykinin.

Bradykinin gave a biphasic increase in intracellular free Ca2+ concentration ([Ca2+]i) in serum-deprived Swiss 3T3 fibroblasts loaded with the photoprotein aequorin. Epidermal growth factor (EGF) alone did not increase [Ca2+]i, but when added after bradykinin there was an increase in [Ca2+]i. The EGF-dependent increase in [Ca2+]i was maximal at 3 min and disappeared with a half-life of 6 min after bradykinin. Removing Ca2+ from the external medium did not abolish either the bradykinin or the EGF-induced [Ca2+]i responses. Although prostaglandins E2 and F2 alpha also gave [Ca2+]i responses and permitted an EGF-dependent [Ca2+]i response, the effect of bradykinin did not appear to be mediated by prostaglandins since it was not blocked by indomethacin. Vasopressin and phorbol 12-myristate 13-acetate both gave a [Ca2+]i response but did not facilitate a [Ca2+]i response by EGF. Bradykinin or EGF alone did not increase DNA synthesis in growth-arrested Swiss 3T3 fibroblasts, but EGF added together with, or after, bradykinin increased DNA synthesis. The effect disappeared with a half-life of 180 min after the addition of bradykinin. It is concluded that stimulation of receptor protein tyrosine kinase is unlikely, by itself, to explain the increase in DNA synthesis produced by EGF. The observed increase in [Ca2+]i caused by EGF after bradykinin probably reflects the interaction of intracellular second messenger pathways leading to facilitation of DNA synthesis.

Aequorin↗