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Biomedical subjects

D F Lewis

Publications and source records attributed to D F Lewis.

At least 163 records · Page 9Linked to original sources
PubMed1987

Spectral and kinetic studies of the interaction of imidazole anti-fungal agents with microsomal cytochromes P-450.

1. The imidazole antifungal agents, ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of the phenobarbital-induced cytochromes P-450 and the 3-methylcholanthrene-induced cytochromes P-448-dependent rat hepatic microsomal mixed-function oxidases. 2. All three drugs were more potent inhibitors of the phenobarbital-induced O-deethylation of ethoxycoumarin than of the 3-methylcholanthrene-induced activity indicating selective inhibition of the phenobarbital-induced cytochromes P-450. In both types of microsomes ketoconazole was always the weakest inhibitor. 3. All three compounds elicited type II spectral interactions with both types of microsomes, and had similar Ks values. Miconazole and clotrimazole, and to a lesser extent ketoconazole, also interacted with the substrate binding sites of both phenobarbital-induced cytochromes P-450 and to a lesser extent with the 3-methylcholanthrene-induced cytochrome P-448. 4. It is concluded that at least part of the inhibitory effect of these antifungal agents may reflect competitive inhibition at the substrate binding site.

Animals↗
PubMed1987

Molecular orbital calculations and quantitative structure-activity relationships for some polyaromatic hydrocarbons.

1. Correlations between biological activity and electronic structure for 7 polyaromatic hydrocarbons are reported. 2. Molecular orbital (MO) calculations by the MINDO/3 method indicate that the hydrophobic parameter, log P (the logarithm of the octanol/water partition coefficient) exhibits a parallelism with total electrophilic superdelocalizability, and a similar relationship is shown with protein binding. 3. Inhibition of dimethylnitrosamine (DMN) demethylase activity is linearly related to total nucleophilic superdelocalizability, and other correlations are shown with mutagenicity and benzo[a]pyrene hydroxylase (AHH) activity.

Aryl Hydrocarbon Hydroxylases↗
PubMed1986

Molecular dimensions of the substrate binding site of cytochrome P-448.

The molecular geometries of specific substrates, inhibitors and inducers of cytochrome P-448 activity were determined using computer-graphic techniques for use in defining the molecular dimensions of the substrate binding site of this enzyme. Specific substrates of cytochrome P-448 are essentially planar molecules characterised by a small depth and a large area/depth ratio. In contrast, compounds that do not serve as substrates of cytochrome P-448 are bulky, non-planar molecules characterised by small area/depth ratios and greater flexibility in molecular conformation. Specific inhibitors of cytochrome P-448 whose effect is mediated through interaction with the haem still meet the dimensional criteria for substrates indicating that they must also interact with the substrate binding-site, which is probably located in proximity to the haem. Inducers of cytochrome P-448 activity exhibit similar molecular geometries to the substrates from which it may be inferred that the cytosolic receptor associated with the induction of cytochrome P-448 activity is structurally related to the active site of the cytochrome.

Animals↗
PubMed1986

Structure activity studies on the induction of peroxisomal enzyme activities by a series of phthalate monoesters in primary rat hepatocyte cultures.

A series of 9 phthalate monoesters were cultured with primary rat hepatocytes for 70 h and their effect on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) determined. Marked quantitative differences in the induction of enzyme activity were observed with both alkyl chain length and position of side chain substitution affecting compound potency. With all 9 compounds a good correlation was observed between electronic structural parameters obtained by molecular orbital calculations and biological activity in the cell culture system. These results demonstrate a relationship between chemical structure and biological activity for a series of phthalate monoesters and indicate the potential usefulness of primary hepatocyte cultures to screen compounds for peroxisome proliferation.

Acyl Coenzyme A↗
PubMed1986

The interaction of a homologous series of hydrocarbons with hepatic cytochrome P-450. Molecular orbital-derived electronic and structural parameters influencing the haemoprotein spin state.

The spectral interaction of a homologous series of alkyl-substituted benzenes and related compounds with purified mammalian cytochrome P-450 has been investigated. Each of the 10 hydrocarbons produced a Type I spectral change, indicative of a low to high spin transition of the haem iron of cytochrome P-450. The extent of perturbation of the cytochrome P-450 spin equilibrium varied for each compound and was used to quantify the spin shifts of the haemoprotein and consequently the substrate-bound spin equilibrium constant, K2. Molecular orbital calculations were utilised to determine the electronic structural parameters of the 10 hydrocarbons investigated, including the electrophilic and nucleophilic super-delocalizabilities summed over all atoms (sigma SE and sigma SN, respectively), the sum of the absolute values of net atomic charge (sigma QT) and the energy levels of both the highest occupied and lowest unoccupied molecular orbitals (E (HOMO) and E (LUMO) respectively). Multiple regression analyses were then utilized to generate quantitative structure-activity relationships between the above structural parameters and the substrate-bound spin equilibrium constant, K2. Good correlations were observed between sigma SE, sigma SN and sigma QT, indicating the importance of hydrophobicity and steric factors in the perturbation of the haemoprotein spin equilibrium. In addition, the electron-accepting potential of the hydrocarbons was an important structural feature and exhibited better correlations with K2 than the electron donating parameter. Taken collectively, our data show the importance of the hydrophobic and charge transfer characteristics of hydrocarbon substrates in dictating the position of the cytochrome P-450 spin equilibrium, and as such, provides a rational molecular explanation based on sound chemical principles for the differential interaction of hydrocarbons with cytochrome P-450.

Animals↗
PubMed1984

Caffeine inhibition of aflatoxin synthesis: probable site of action.

Aflatoxin production by pregrown cultures of Aspergillus parasiticus was completely inhibited by incorporation of 2 mg of caffeine per ml into the medium. This was accompanied by a decrease in glucose utilization and an inhibition of oxygen uptake and carbon dioxide evolution. Enzyme analyses indicated no significant differences in specific activities on glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphofructokinase, fructose 1,6-diphosphatase, pyruvate kinase, or malate dehydrogenase. Glucose uptake kinetics indicated a linear dose-related inhibition of glucose uptake. It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins.

Aflatoxins↗
PubMed1984

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes.

Aflatoxins↗
PubMed1975

Mitochondrial enzyme pathways and their possible role during curing.

The velocity of oxidation of exogenous ferrocytochrome c by nitrite under anaerobic conditions in the presence of skeletal muscle mitochondria is dependent upon pH over at least the range 5.6-6.7, increasing markedly as the pH is lowered. A product of the reaction is the complex formed between nitric oxide and ferricytochrome c. At levels up to 20 mM, nitrite inhibits aerobic cytochrome oxidase action; at higher concentrations, however, a partial resuscitation of the oxidation of ferrocytochrome c occurs, the enhancement of reaction velocity being considerably greater at pH 6.0 than at 6.5. Mitochondrial respiration is also inhibited by nitrite but no similar resurgence was, however, observed and thus the oxidation of ferrocytochrome c by high levels of nitrite is considered to be a direct non-enzymic action. Under anaerobic conditions, the rate of increase of the velocity constant of the oxidation of ferrocytochrome c with nitrite concentration in the presence of muscle mitochondria similarly decreased with rise of pH over the same range. The permeability of the muscle mitochondrion to nitrire has been demonstrated by swelling studies and by the rapid conversion of endogenous ferrocytochrome a3 into its nitrosyl-derivative. Over longer periods of anaerobic incubations of mitochondria with nitrite, oxidation of endogenous cytochromes occurs with the formation of nitrosylferricytochrome c. Above a nitrite concentration of 0.3 mM, the mitochondrial enzyme system probably involved is increasingly inhibited but by a concentration of 30 mM a direct non-enzymic oxidation has intervened. Commercial vacuum packed bacons were examined by electron microscopy. Mitochondria were clearly recognisable although they contained fewer cristae than those observed in fresh meat.

Electron Transport Complex IV↗
PubMedDate not supplied

The use of computers in the safety evaluation of drugs and other chemicals.

The toxicity and carcinogenicity of drugs and other chemicals is, in most cases, mediated by highly reactive intermediates which are generated following metabolism catalysed by the enzymic apparatus of the exposed organisms. These reactive intermediates readily interact covalently with vital cellular components to provoke toxicity and carcinogenicity. The ubiquitous cytochrome P450-dependent mixed-function oxidases are the most important enzyme system in the activation of chemicals. This enzyme system comprises a number of families, each of which contains one or more subfamilies. The CYPIA and CYP2E subfamilies are the most closely associated with the production of reactive intermediates and, consequently, the manifestation of toxicity and carcinogenicity. A computer based molecular structure procedure (COMPACT) has been developed which, via a calculation of the molecular and electronic structure of the chemical, determines whether the chemical will interact with either of these two cytochrome P450 subfamilies and hence be metabolised to form reactive intermediates that manifest toxicity. As the basal levels of these two subfamilies are generally low, the ability of a chemical to induce them selectively, on repeated administration, is an important determinant of its toxic and carcinogenic potential. This inductive capability may be determined in short-term studies (ENACT) using only a small number of animals. Thus the combination of COMPACT and ENACT provides a rapid and inexpensive means for the preliminary screening of chemicals for toxicity and carcinogenicity before undertaking the long-term and expensive rodent lifetime bioassays.

Animals↗
PubMedDate not supplied

Hypoxia-induced increase in soluble Flt-1 production correlates with enhanced oxidative stress in trophoblast cells from the human placenta.

OBJECTIVE: Placental trophoblast cells (TCs) produce soluble Flt-1 (sFlt-1). Hypoxia induces placental oxidative stress and modulates trophoblast function. The aim of this study was to investigate whether hypoxia mediates TC sFlt-1 production and whether increased sFlt-1 production correlates with increased oxidative stress in placental TCs. METHODS: Placentas were obtained immediately after delivery from normal pregnant women (n = 8). Placental TCs were isolated by Dispase digestion of villous tissue and purified by Percoll gradient centrifugation. Isolated TCs were cultured under normoxia (21% O2: 5% CO2/95% air) and hypoxia (2% O2/5% CO2/93% N2) conditions for 3 days in vitro. TC productions of sFlt-1, VEGF, and PlGF were measured by enzyme-linked immunosorbent assay (ELISA). Lipid peroxide production and superoxide dismutase (CuZn-SOD) levels were evaluated. Messenger RNA expressions of Flt-1, VEGF and PlGF were determined by RT-PCR. Messenger RNA expressions for superoxide dismutase (CuZn-SOD) and heme oxygenase-1 (HO-1) were also determined. Data are expressed as mean +/- SE. A p level less than 0.05 was considered statistically different. RESULTS: Our results show that sFlt-1 production was significantly increased by TCs cultured under hypoxia condition that correlates with increased lipid peroxide production. We also found that under hypoxia condition: (1) the ratio of PlGF/VEGF production was reversed; (2) the ratio of lipid peroxides to superoxide dismutase production was increased. The increased mRNA expressions for Flt-1 and VEGF and the decreased mRNA expression for PlGF in TCs were consistent with the protein productions under hypoxia condition. CONCLUSION: We concluded that upregulation of sFlt-1 and unbalanced PlGF/VEGF production associated with increased oxidative stress are consequences of hypoxia in placental TCs. Our results suggest that placental TCs are major sources of sFlt-1 and VEGF levels in the maternal circulation in women with preeclampsia.

Cell Hypoxia↗
PubMedDate not supplied

Protease chymotrypsin mediates the endothelial expression of P- and E-selectin, but not ICAM and VCAM, induced by placental trophoblasts from pre-eclamptic pregnancies.

OBJECTIVES: Soluble endothelial-cell adhesion molecules (ICAM, VCAM and PECAM) are markers of endothelial activation, and are elevated in the maternal circulation during pregnancy and even further increased in pregnancies complicated by pre-eclampsia (PE). To identify possible sources of endothelial activators during pregnancy, we addressed whether factors released from placental trophoblast cells (TCs) activate endothelial cells (ECs) to enhance adhesion molecule expression on ECs. We also examined whether proteases released by placental cells induce the endothelial cell surface molecule expression in PE. METHODS: Confluent ECs were co-cultured with placental TCs derived from normal (n=9) or PE (n=8) pregnancies or with placental conditioned media (CM) derived from PE placental cultures (n=7). ICAM, VCAM, P-selectin and E-selectin were quantified using an enzyme-linked immunosorbent assay (ELISA). The protease inhibitors alpha(2)-macroglobulin (alpha(2)M), thrombin inhibitor (TI) and chymotrypsin inhibitor (CI) were tested in the co-culture system. mRNAs for ICAM, VCAM, P-selectin and E-selectin were determined by RNase protection assay (RPA). NF-kappaB activity in ECs was also determined. RESULTS: (1) ICAM and VCAM expression was significantly increased on ECs co-cultured with both normal-TCs and PE-TCs, compared to control ECs (P<0.01). ICAM and VCAM expression in ECs co-cultured with normal-TCs did not differ from ECs co-cultured with PE-TCs. (2) E-selectin expression was increased on ECs co-cultured with normal-TCs (P<0.05) and further increased in ECs co-cultured with PE-TCs (P<0.01). (3) P-selectin expression was increased on ECs co-cultured with PE-TCs, but not ECs co-cultured with normal-TCs compared to control ECs (P<0.05). (4) alpha(2)M and TI did not alter the ICAM, VCAM, P-selectin and E-selectin expression on ECs induced by PE-CM. (5) CI blocked the upregulation of P-selectin and E-selectin (P<0.05), but not ICAM and VCAM expression, in ECs cultured with PE-CM. (6) Changes in mRNA for ICAM, VCAM, P-selectin and E-selectin paralleled the increases in protein expression on ECs cultured with PE-CM. (7) NF-kappaB activity was also increased in cells challenged with PE-CM. CONCLUSIONS: (1) Factor(s) released from both normal-TCs and PE-TCs promote ICAM and VCAM expression on ECs. (2) Factor(s) released from PE-TCs significantly increase EC P-selectin and E-selectin expression. (3) CI blocks the upregulation of P-selectin and E-selectin on ECs induced by factors released from PE placental cells, suggesting that chymotrypsin is responsible for the increased endothelial expression of P-selectin and E-selectin in pre-eclampsia.

Cell Adhesion Molecules↗
PubMedDate not supplied

Expression of platelet-derived growth factor-A mRNA in human placenta: effect of magnesium infusion in pre-eclampsia.

The expression of platelet-derived growth factor-A (PDGF-A) mRNA was examined in the cotyledons of normal human placentae and those from patients with pre-eclampsia. These patients exhibited pre-delivery blood pressure of 154+/-4/99+/-4 mmHg (mean+/-SEM) and met the criteria established for pre-eclampsia. During labour they received MgSO4 infusion for various time intervals (4-25 h). The PDGF-A message was quantitated to beta-actin by the solution hybridization nuclease protection assay. Since the two groups differed in two parameters (pre-eclampsia and MgSO4 treatment), the direct comparison was not feasible. An analysis of covariance revealed a significant difference in the message between the pre-eclamptic and control groups (P<0.01); the gestational age was not a significant covariate for either group but the time on MgSO4 in pre-eclampsia group was significant (P<0.002). A linear regression analysis of PDGF-A mRNA values for the pre-eclamptic group showed a time-dependent downregulation of the message by MgSO4 (P<0.01, r=- 0.796). These results show a uniform expression of PDGF-A mRNA in cotyledons of normal human placenta between 35 and 40 weeks of gestation. Furthermore, MgSO4 has an inhibitory effect on the expression of this message which may have aside from its anticonvulsive action beneficial effect on the function of pre-eclamptic placenta.

Actins↗
PubMedDate not supplied

Activation of endothelial cells in preeclampsia: increased neutrophil-endothelial adhesion correlates with up-regulation of adhesion molecule P-selectin in human umbilical vein endothelial cells isolated from preeclampsia.

OBJECTIVE: Increased endothelial activation has been suggested to be important in the pathophysiology for preeclampsia. Our objective was to examine whether in preeclampsia neutrophil adherence to endothelial cells is increased and whether endothelial cell-surface adhesion molecule expression is up-regulated. METHODS: Endothelial cells were isolated from normal (n = 10) and preeclamptic (n = 9) human umbilical veins (HUVECs). Neutrophils were isolated from normal, healthy, nonpregnant female volunteers. Freshly isolated neutrophils were labeled with 51Cr, and labeled neutrophils were coincubated with confluent normal and preeclamptic endothelial monolayers. Adhesion assays were then performed. To determine whether in preeclampsia endothelial cellular-surface adhesion molecules are responsible for increased neutrophil-endothelial adhesion, cellular adhesion molecule expression of P-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and E-selectin were examined by an enzyme-linked binding assay. Furthermore, adhesion assays were also performed on HUVECs pretreated with antibodies against P-selectin, ICAM-1, VCAM-1, and E-selectin. RESULTS: Neutrophil adhesion to the HUVECs from preeclamptic pregnancies was significantly increased compared with neutrophil adhesion to the HUVECs from normal pregnancies (P < .01). Expression of cellular-surface adhesion molecule of P-selectin was significantly higher (P < .01) and ICAM-1 was significantly lower (P < .05) in HUVECs isolated from preeclampsia than from normal controls, whereas there was no difference for VCAM-1 and E-selectin expression between HUVECs from normal and preeclamptic pregnancies. No differences were found for neutrophil-endothelial adhesion on normal HUVECs pretreated with anti-P-selectin, anti-ICAM-1, anti-VCAM-1, and anti-E-selectin compared with the untreated cells. However, pretreatment of preeclampsia HUVECs with anti-P-selectin, anti-ICAM-1, anti-VCAM-1, and anti-E-selectin completely or partially blocked the neutrophil-endothelial adhesion compared to the untreated cells. CONCLUSION: There is a significant increase in neutrophil adhesion to HUVECs that are isolated from preeclamptic pregnancies compared with normal controls. This increase appears to be a result of up-regulation of the cell-surface adhesion molecule P-selectin. Elevated P-selectin expression may play a significant role in neutrophil-endothelial hyperadhesiveness and contribute to vascular complications associated with preeclampsia.

Antibodies↗
PubMedDate not supplied

Increased neutrophil-endothelial adhesion induced by placental factors is mediated by platelet-activating factor in preeclampsia.

OBJECTIVE: Endothelial cell activation or dysfunction and neutrophil-endothelial cell adhesion have been suggested to be important in the pathophysiology of preeclampsia. However, the mechanisms that underlie the alteration of endothelial cell function in preeclampsia are unknown. Placenta from preeclamptic pregnancies produces mediators and autacoids, which may be released into the maternal circulation and modulate endothelial function. In this study, the effect of placental factor(s) on neutrophil-endothelial adhesion and the possible role of platelet-activating factor (PAF) in mediating the response have been examined. METHODS: Endothelial cells were isolated from human umbilical veins (HUVECs) from normal pregnancies. Confluent primary passage HUVECs were exposed to conditioned medium derived from normal and preeclamptic placental tissue cultures, with unconditioned medium as a control. Placental-conditioned medium was prepared by incubation of placental whole villous tissue in Dulbecco's Modified Eagle's Medium (DMEM) for 48 hours. Neutrophil-endothelial adhesion assays were performed to evaluate placental factors in mediating neutrophil-endothelial adhesion, and a PAF-3H scintillation proximity assay (SPA) system was used to determine endothelial PAF production. The PAF-receptor antagonist WEB 2086 was used to block placental factor-mediated increased neutrophil-endothelial adhesion induced by conditioned medium derived from preeclamptic placenta. RESULTS: Neutrophils were significantly more adherent to HUVECs treated with conditioned medium from preeclamptic placentas (28.44 +/- 2.47%) than to HUVECs treated with conditioned medium from normal placentas (18.95 +/- 1.57%) or with unconditioned medium (14.60 +/- 1.29%, P < .01). Also, HUVECs exposed to preeclamptic placental-conditioned medium produced more PAF than the cells exposed to normal conditioned medium and unconditioned medium, 416.18 +/- 17.14 pg/1 x 10(7) cells versus 330.90 +/- 35.70 and 296.43 +/- 44.40 pg/1 x 10(7) cells, P < .05, respectively. The PAF receptor antagonist WEB 2086 completely blocked increased neutrophil-endothelial adhesion induced by preeclamptic placental-conditioned medium (13.24 +/- 0.81% versus 31.31 +/- 4.75%, P < .01). CONCLUSION: In preeclampsia, the placenta releases one or more factors promoting neutrophil-endothelial adhesion. The increased neutrophil-endothelial adhesion thereby induced is a PAF-mediated event. It is suggested that if preeclamptic placentas release toxic factors into the maternal circulation in vivo, these factors may contribute to the altered vascular endothelial cell function in preeclampsia.

Azepines↗
PubMedDate not supplied

The mutagenicity and interactions of 2- and 4-(acetylamino)fluorene with cytochrome P450 and the aromatic hydrocarbon receptor may explain the difference in their carcinogenic potency.

A study has been undertaken to identify the properties of 2-(acetylamino)fluorene which render it carcinogenic, in contrast to the 4-isomer, the carcinogenic potential of which is, at best, very weak. Compared to the 4-isomer, 2-(acetylamino)fluorene was a more potent inducer of cytochrome P450 1A (P450 1A) activity, as exemplified by the O-deethylation of ethoxyresorufin (P450 1A1), the metabolic activation of Glu-P-1 (P450 1A2), and immunoblot analysis. These findings were consistent with the relative affinity of these compounds for the cytosolic aromatic hydrocarbon receptor, determined by the displacement of tetrachlorodibenzo-p-dioxin. In the presence of Aroclor 1254-induced hepatic microsomal and cytosolic preparations 2-(acetylamino)-fluorene elicited a potent mutagenic response in the Ames test whereas the 4-isomer, in both cases, elicited a weak mutagenic response. Molecular orbital calculations of the relative energetics of the nitrenium ions revealed that the ion of the 2-isomer was more stable than the nitrenium ion of 4-(acetylamino)fluorene. Control hepatic microsomal and cytosolic fractions could activate 2-(acetylamino)fluorene to mutagens in the Ames test. Pretreatment of animals with 2-(acetylamino)fluorene enhanced both the microsome- and cytosol-mediated activation. In contrast, microsomal and cytosolic fractions from control animals could not activate 4-(acetylamino)fluorene, but following pretreatment with the compound itself, a weak mutagenic response in the presence of microsomes, but not of cytosol, was evident.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗