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Biomedical subjects

D Lucas

Publications and source records attributed to D Lucas.

At least 55 records · Page 3Linked to original sources
PubMed1997

Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1.

4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

Animals↗
PubMed1997

Adrenomedullary secretion of DOPA, catecholamines, catechol metabolites, and neuropeptides.

Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine >> norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol > or = DOPA >> [Met]enkephalin >> neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 microM carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca2+-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.

Adrenal Medulla↗
PubMed1996

Determination of cytochrome P450 2E1 activity in microsomes by thin-layer chromatography using [2-14C]chlorzoxazone.

A thin-layer chromatographic assay was developed as an alternative method for the determination of cytochrome P450 2E1 (CYP2E1) in microsomes using [2-14C]chlorzoxazone. After incubation of microsomes with 0.125 microCi/mmol chlorzoxazone, chlorzoxazone and its single metabolite, 6-hydroxychlorzoxazone, were extracted using chloroform-2-propanol (85:15, v/v) and chromatographed on silica gel 60 F254 plates with acetone-hexane (45:55, v/v) as solvent . The plates were then exposed to X-ray film for 2 days to localize the radiolabelled chlorzoxazone and 6-hydroxychlorzoxazone. The metabolite and substrate regions were scraped and counted in a liquid scintillation analyzer. This method is sensitive enough to determine constitutive and induced CYP2E1 activities in liver or kidney microsomes. The precision of the method was similar to that of the HPLC method. The correlation coefficient between both methods was found to be 0.97 (n = 21). Therefore, the TLC method constitutes a valuable tool for the determination of chlorzoxazone metabolism in microsomes.

Animals↗
PubMed1996

Cytochromes P4502E1 and P4501A1 genotypes and susceptibility to cirrhosis or upper aerodigestive tract cancer in alcoholic caucasians.

Genetic polymorphisms of various cytochromes P450 have recently been described and could be implicated in the individual susceptibility of alcoholics to ethanol-related diseases. Rsal and Dral polymorphisms of CYP2E1 and Mspl polymorphism of CYP1A1 were studied in 260 controls and 511 alcoholic patients, without any clinical symptoms (n = 202) or with various ethanol-related diseases (n = 309), such as liver cirrhosis (n = 110), esophageal cancer (n = 62), upper aerodigestive tract cancer (n = 96), and other miscellaneous diseases (n = 41). Frequencies of the mutated alleles were found to be 2.5% (Rsal), 7.9% (Dral), and 8.7% (Mspl) in controls; 4%, 14.1%, and 12% in alcoholics without clinical symptoms; and 3.1%, 12.5%, and 11.2% in alcoholics with ethanol-related diseases. The only significant difference was found in the Dral polymorphism, whose frequency was enhanced in alcoholics with (p < 0.05) or without ethanol-related diseases (p < 0.01) when compared with controls. No differences were found between alcoholics without clinical symptoms and alcoholics with cirrhosis, esophageal cancer, or upper aerodigestive tract cancer. However, in liver cirrhosis and in ethanol-related cancers, the rare Dral-C allele was three times less frequent in patients under the age of 45 than in older patients, suggesting a protective role for this allele. In conclusion, our data indicate that the aforementioned mutations do not play a critical role in the development of cirrhosis, esophageal cancer, or upper aerodigestive tract cancers in Caucasians.

Adult↗
PubMed1996

Contributions of liver and kidneys to glycerol production and utilization in the dog.

The classical concept holds that liver and kidneys are the main sinks of glycerol released by adipose tissue. However, rates of glycerol appearance (Ra) exceed the rate of glycerol delivery to liver and kidneys. We measured the hepatic and renal contributions to glycerol production and utilization in anesthetized dogs that were fasted either overnight or for 24 h after 3 days on a carbohydrate-free diet. Dogs were infused with [2H5]glycerol, and the concentration and 2H enrichment of glycerol were measured across liver and kidney. After a baseline period, either norepinephrine or glucose plus insulin was infused to alter the rate of glycerol production. Our study shows that the production of glycerol by liver and kidneys amounted to 4-9% and 4-7% of the Ra of glycerol, respectively. Uptake of glycerol by liver and kidneys amounted to 26-30 and 10-19% of the Ra of glycerol, respectively. Thus, contrary to the classical concept, the bulk of glycerol utilization occurs in nonhepatic, nonrenal tissues that have very low glycerol kinase activity per gram.

Animals↗
PubMed1996

Managed competition with prefunding: the solution for long-term care?

Managed competition with prefunding could be a useful approach to long-term care. One version, described here, has two main components: First, people would be required to save over their working lives to cover a portion of the expected cost of a minimum required level of long-term-care insurance. Second, people would begin purchasing long-term-care insurance around the time of retirement from one of a number of competing insurers, under a system of managed competition. Potentially large social gains would arise from improving insurance arrangements, distributing long-term-care costs more equitably across and within generations, and eliminating many of the distortions inherent in the current Medicaid program.

Humans↗
PubMed1995

Interaction between two probes used for phenotyping cytochromes P4501A2 (caffeine) and P4502E1 (chlorzoxazone) in humans.

The first steps in the metabolism of caffeine and chlorzoxazone are primarily catalysed by CYP1A2 and CYP2E1, respectively. Accordingly, these compounds have been developed as metabolic probes for non-invasive phenotyping of these two P450s. Their specificities, however, have been shown to overlap. In this study, 140 mg of caffeine and 500 mg of chlorzoxazone were administered alone or together in 16 healthy subjects under standardized conditions. The metabolites of these two probes were measured in the blood and also in the urine for caffeine. CYP1A2 activity was determined either by the paraxanthine/caffeine ratio in the blood or by the usual caffeine metabolic ratio in the urine. The CYP2E1 activity was determined by the 6-OH-chlorzoxazone/chlorzoxazone ratio in blood. CYP1A2 activities measured in blood and urine were highly significantly correlated. CYP2E1 activity was not modified when chlorzoxazone was given together with caffeine. In contrast, an inhibition of CYP1A2 by chlorzoxazone was demonstrated by a 16% decrease in the caffeine metabolic ratio in urine when both caffeine and chlorzoxazone were given together. Under the same conditions, the paraxanthine/caffeine ratio in plasma also decreased by about 20%. These results were confirmed in vitro by the incubation of 1 mM caffeine with human hepatic liver microsomes in the presence of 0.4 mM chlorzoxazone. The overall metabolism of caffeine decreased by 38% compared to controls incubated without chlorzoxazone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Cytochrome P450 2E1 genotype and chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects.

Susceptibility to cancer or ethanol-related liver diseases may be associated with a large variability in cytochrome P450 2E1 activity. This variability may be of genetic origin or reflect environmental factors. To test the role of genetics, the phenotype and genotype of this enzyme were determined in 42 non-alcoholic and 74 alcoholic patients hospitalized for detoxification treatment. Chlorzoxazone metabolism was used to assess CYP2E1 phenotype. Restriction length fragment polymorphisms with Rsa I or Pst I, and Dra I endonucleases were used to determine the two mutant alleles, Pst I/Rsa I-c2 and Dra I-C. A significant gender difference in basal CYP2E1 activity was observed in non-smoking controls (p < 0.05) but not in alcoholics or smokers. Subjects heterozygous for the C or c2 mutated allele did not show any difference in CYP2E1 activity at the basal level, compared with the wild type homozygotes. Conversely, patients with the mutated genotype appeared less inducible than the others after ethanol induction (p < 0.01).

Adult↗
PubMed1995

Decrease in cytochrome P4502E1 as assessed by the rate of chlorzoxazone hydroxylation in alcoholics during the withdrawal phase.

To evaluate cytochrome P4502E1 (CYP2E1) induction in alcoholics, the ratio of the concentrations of 6-hydroxychlorzoxazone (6-OH-CHZ) and chlorzoxazone (CHZ) was measured in blood 2 hr after CHZ ingestion using a HPLC method. This ratio was determined in controls and in alcoholic patients after 1, 2, 3, 4, 5, 8, and 21 days withdrawal. It was found to be 0.34 +/- 0.03 in 30 controls and 1.05 +/- 0.14 in 41 alcoholic patients within 2 days following ethanol withdrawal. This ratio decreased rapidly during withdrawal as attested by the short half-life of CYP2E1, which was found to be 2.5 days. Patients tested for CHZ metabolism after 8 or 21 days alcohol abstinence displayed the same ratio as controls [0.35 +/- 0.03 (n = 28) and 0.31 +/- 0.03 (n = 34), respectively]. No correlation was observed between gamma-glutamyltransferase, carbohydrate-deficient transferrin values, the amount of alcohol consumed/day, and the 6-OH-CHZ/CHZ ratio. There was no influence of smoking on the rate of CHZ hydroxylation, because smokers displayed the same ratio as nonsmokers [0.33 +/- 0.025 (n = 62) and 0.33 +/- 0.02 (n = 30), respectively]. The CHZ hydroxylation ratio seems to be a good reflection of the hepatic and extrahepatic CYP2E1 activity in humans.

Adult↗
PubMed1995

Model of extreme hypoglycemia in dogs made ketotic with (R,S)-1,3-butanediol acetoacetate esters.

The rationale behind this study is that controlled starvation of poorly differentiated (anaplastic) fast-growing tumor cells, but not host cells, might be possible in vivo. The energy metabolism of anaplastic tumor cells, but not host cells, is largely dependent on carbohydrate metabolism at all times. Therefore depleting plasma of carbohydrate fuels could place these tumor cells at a significant metabolic disadvantage. Hence an animal model was developed in which all cells would be required to oxidize fatty acids, ketoacids, and/or 1,3-butanediol to satisfy their energy needs. To achieve this aim, one would need ketosis, severe hypoglycemia, and low lactatemia. Anesthetized normal dogs were infused with somatostatin and a mixture of (R,S)-1,3-butanediol monoacetoacetate and (R,S)-1,3-butanediol diacetoacetate; these latter compounds are nonionized precursors of ketoacids. They were infused at 90% of the dog's caloric requirement. After establishment of a moderate ketosis (2-3 mM) over < 100 min, a severe degree of hypoglycemia (close to 0.5 mM) without rebound and without hyperlactatemia was induced by infusing insulin and dichloroacetate. Tracer kinetic measurements showed 1) a 20% decrease in the rate of appearance of glucose, 2) 50 and 62% increases in glycerol and nonesterified fatty acid rates of appearance, reflecting stimulation of lipolysis, and 3) no change in the rate of glutamine appearance. We suggest that this model may prove useful for selectively starving those cancer cells that are unable to utilize fat-derived fuels while preserving nutrient supply to vital organs.

3-Hydroxybutyric Acid↗
PubMed1995

Cytochrome P-450 2E1 in rat liver, kidney and lung microsomes after chronic administration of ethanol either orally or by inhalation.

In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contents and activities were tested in liver, kidney and lung from Wistar rats after the following treatments (1) oral administration of a 10% ethanol solution for 4 weeks; (2) pair fed controls; (3) oral administration of a 5% acetone solution for 1 week; (4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activity was measured using chlorzoxazone as substrate and CYP2E1 content was measured using Western blot analysis. In addition, the cellular distribution of CYP2E1 was studied in liver, lung and kidney by immunohistochemistry. Basal liver CYP2E1 was 10-20 times lower in lung and kidney than in liver. Inhalation was clearly the most efficient way of inducing CYP2E1, probably due to the continuous and high alcohol exposure. Among the organs tested, lung appeared to be the tissue least sensitive to induction even after ethanol inhalation, suggesting the absence of local induction. After ethanol intoxication, immunostaining was increased in the centrilobular region of the liver, in the alveolar cells of the lung and in the proximal convoluted tube of the kidney. The CYP2E1 activities decreased to control values in the three tissues tested, within 24 h after cessation of intoxication.

Administration, Inhalation↗
PubMed1994

Assessment of cytochrome P4502E1 induction in alcoholic patients by chlorzoxazone pharmacokinetics.

Chlorzoxazone is mainly metabolized to 6-hydroxychloroxazone (6-OHchlorzoxazone) by the ethanol-inducible cytochrome P450 2E1 (CYP2E1). To evaluate the impact of ethanol consumption on the enzyme induction, the pharmacokinetics of chlorozoxazone and 6-OHchlorzoxazone were studied in alcoholic and control subjects. Fifteen alcoholic male inpatients (all smokers, daily intake 333 +/- 191 g of absolute ethanol) and 20 healthy male volunteers (10 smokers and 10 non-smokers, weekly intake < 100 g of absolute ethanol) participated in this study. Following a 12 hr fasting period, each subject was orally administered 500 mg of chlorzoxazone. Venous blood and urine samples were collected over a 10 hr period. Areas under the curve of plasma concentration versus time (AUC) of chlorzoxazone and 6-OHchlorzoxazone was calculated. The total plasma clearance of chlorzoxazone was measured as the dose/AUC ratio. The mean total plasma clearance was not different between smoker and non-smoker controls but it was enhanced by 73% in alcoholic patients. These results indicate a negligible and non-significant effect of cigarette smoking in controls but an increased metabolism of chlorzoxazone in alcoholic patients (P < 0.05). This increase was corroborated by the 2-fold enhancement of the 6-OHchlorzoxazone/chlorzoxazone AUC ratio, compared to controls. A good correlation was found between this AUC ratio and the 6-OHchlorzoxazone/chlorzoxazone concentration ratio at t = 2 hr in patients and in controls (r = 0.88 and 0.85, respectively, P < 0.01). The concentration ratio increased by 150% in alcoholic patients and decreased by 65% in the seven alcoholics tested after 7 days of alcohol abstinence. It is therefore concluded that the 6-OHchlorzoxazone/chlorzoxazone concentration ratio at t = 2 hr could constitute a simple and non-traumatic marker of CYP2E1 induction.

Adult↗
PubMed1994

RU 58,668, a new pure antiestrogen inducing a regression of human mammary carcinoma implanted in nude mice.

RU 58,668, a new steroidal antiestrogen, has been synthesized. Its in vitro and in vivo pharmacological activities have been compared to those of tamoxifen and ICI 182,780. In vitro, it displayed affinities for human and murine estrogen receptors equivalent to those of 4-hydroxy-tamoxifen, along with moderate affinities for progestin and glucocorticoid receptors. RU 58,668 proved to be a potent antiproliferative agent on MCF-7 cells stimulated by estradiol, or by exogenous or endogenous growth factors (IC50, 0.01 nM). It also inhibited the growth of the insulin-stimulated T47D cell line. In vivo, RU 58,668 displayed a total anti-uterotrophic activity in mice or rats without exhibiting any agonistic effect. Moreover, RU 58,668 was the only antiestrogenic compound tested so far to be able to induce a long term regression of human mammary MCF-7 tumors implanted in nude mice, suggesting its potential use for the treatment of advanced breast cancer.

Animals↗
PubMed1994

Acetaldehyde exposure causes growth inhibition in a Chinese hamster ovary cell line that expresses alcohol dehydrogenase.

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.

Acetaldehyde↗
PubMed1994

Splenic reimplantation does not affect outcome in chronic canine model.

The effects of septic insult were compared in a canine model of splenic reimplantation. Sequential changes in hematologic, hepatic, and immunologic function were monitored biweekly in 18 dogs during 10 months after splenectomy, splenectomy with reimplantation, or sham operation. There was no significant difference in these measures between the two groups. At the end of the 10-month period, spleen scans with technetium (99Tc) labeled, heat-damaged RBCs were obtained on the reimplanted dogs. 99Tc scanning revealed no active splenic implants at 10 months. All dogs were then infected with intravenous Type III pneumococcus for 9 consecutive days. There were no measurable hematologic, hepatic, or immunologic differences between groups before or after the septic insult. These animals were then sacrificed for histologic analysis of the splenic reimplants. Reimplant histology showed active germinal centers, but the surrounding pulp was fibrotic and lymphocyte-depleted. Splenic reimplantation in this canine model yields no apparent benefit.

Analysis of Variance↗