The analysis of current and future cosmological surveys of Type Ia supernovae (SNe Ia) at high redshift depends on the accuratephotometric classification of the SN events detected. Generating ...realistic simulations of photometric SN surveys constitutes anessential step for training and testing photometric classification algorithms, and for correcting biases introduced by selectioneffects and contamination arising from core-collapse SNe in the photometric SN Ia samples. We use published SN time-seriesspectrophotometric templates, rates, luminosity functions, and empirical relationships between SNe and their host galaxies toconstruct a framework for simulating photometric SN surveys. We present this framework in the context of the Dark EnergySurvey (DES) 5-yr photometric SN sample, comparing our simulations of DES with the observed DES transient populations.We demonstrate excellent agreement in many distributions, including Hubble residuals, between our simulations and data.We estimate the core collapse fraction expected in the DES SN sample after selection requirements are applied and beforephotometric classification. After testing different modelling choices and astrophysical assumptions underlying our simulation,we find that the predicted contamination varies from 7.2 to 11.7 per cent, with an average of 8.8 per cent and an r.m.s. of 1.1 percent. Our simulations are the first to reproduce the observed photometric SN and host galaxy properties in high-redshift surveyswithout fine-tuning the input parameters. The simulation methods presented here will be a critical component of the cosmologyanalysis of the DES photometric SN Ia sample: correcting for biases arising from contamination, and evaluating the associatedsystematic uncertainty.
In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. ...Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with 1-14C-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorbenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J.T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R), 15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R), 12(S)-EET; P450 2C9 is 69.4% selective for the 11(S), 12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.
The propensity of centrilobular liver damage to develop in alcohol abusers after exposure to various hepatotoxins, including ethanol itself, has been linked to the induction by ethanol of P‐4502E1, a ...microsomal P‐450 enzyme that bioactivates these agents to reactive metabolites. Whereas long‐term ethanol consumption elicits a marked increase in hepatic P‐4502E1 content, the molecular mechanism by which ethanol produces this effect is the subject of controversy in animals, and it has not been elucidated in human beings. Possible mechanisms include increased enzyme synthesis stemming from elevated 2E1 messenger RNA levels, enhanced translation of preexisting messenger RNA or stabilization of P‐4502E1 protein. To determine which, if any, of these mechanisms underlies P‐4502E1 induction in human beings, we examined the effects of ethanol intake on the hepatic intralobular distribution of P‐4502E1 messenger RNA and the corresponding protein. Liver sections derived from needle biopsy specimens were obtained from five recently drinking alcoholics (last drink no more than 36 hr before) and eight control subjects (five abstaining alcoholics last drink 96 hr or more before and three nondrinkers). In situ hybridization of these liver sections with a human P‐4502E1 complementary DNA probe was used to localize P‐4502E1 messenger RNA transcripts. Quantitative image analysis of hybridized sections from control subjects revealed that P‐4502E1 transcript content in perivenular (zone 3) hepatocytes was significantly higher (p < 0.05) than in midzonal (zone 2) and periportal (zone 1) cells (18.3 ± 1, 9.5 ± 2 and 3.1 ± 2 arbitrary density units, respectively; mean ± S.E.M.). In recent drinkers, acinar regions containing P‐4502E1 transcripts were elevated 2.9‐fold compared with those in controls (32.8% ± 7% vs. 11.2% ± 2%; p < 0.01), with this messenger RNA increase occurring mainly in perivenular cells (29.6 ± 3 vs. 18.3 ± 1 units; p < 0.01). P‐4502E1 protein distribution, assessed by the immunohistochemical staining of liver sections with P‐4502E1 antibodies, was found to be analogous to that of the messenger RNA in control subjects (the level in perivenular cells was greater than that in midzonal cells, which was greater than that in periportal cells), whereas recent drinkers exhibited marked elevations in enzyme content in both perivenular and midzonal hepatocytes. Moreover, cellular levels of P‐4502E1 protein and messenger RNA were significantly correlated (rs = 0.79; p < 0.001) in all patients. Our results indicate that the induction by ethanol of P‐4502E1 protein in human liver tissue is associated with, and appears to stem from, a corresponding increase of P‐4502E1 messenger RNA. This ethanol‐mediated enhancement of P‐4502E1 enzyme levels mediated through the encoding messenger RNA, a process occurring primarily in perivenular hepatocytes, may explain why these cells are preferentially damaged after exposure to P‐4502E1–activated hepatotoxins. (HEPATOLOGY 1993;17:236–245.)
We introduce a novel digital lock-in detection technique for simultaneously measuring the amplitude and phase of multiple amplitude-modulated signals. Using particular modulation and sampling ...constraints and averaging filters, we achieve optimal noise reduction and discrimination between sources of different modulation frequencies. Furthermore, it is shown that the digital lock-in technique can be performed as a simple matrix multiplication, which considerably reduces the computation time. The digital lock-in algorithm is described and analyzed under certain sampling and modulation conditions, and results are shown for both numerical and experimental data.
The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on ...these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of gamma-glutamyltransferase, gamma-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.
Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, includingS-mephen-ytoin, omeprazole, warfarin, tolbutamide, and ...nonsteroidal anti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast orEscherichia colihave been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidizeS-mephenytoin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabolizedS-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0–6.4 vs 2.4–4.3 nmol hydroxytolbutamide formed/min/nmol P450). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km= 650 μM,Vmax= 3.71 min−1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km= 178–407 μM,Vmax= 2.95–7.08 min−1). Polyclonal CYP2C19 antibodies markedly decreasedS-mephenytoin 4′-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9. Moreover, excellent correlations were found between immunoreactive CYP2C19 content,S-mephenytoin 4′-hydroxylase activity (r= 0.912;P< 0.001), and omeprazole 5-hydroxylase activity (r= 0.906;P< 0.001) in liver samples from 13–17 different subjects. A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r= 0.664;P< 0.02) but not with CYP2C19 content (r= 0.393;P= 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88.5 ± 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 ± 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 ± 12 nmol/mg). Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant ofS-mephenytoin 4′-hydroxylation and low-Kmomeprazole 5-hydroxylation in human liver. Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof.
The mechanisms responsible for ethanol-mediated teratogenesis have not been resolved. However, possible etiologies include the local formation of the teratogen acetaldehyde or oxygen radicals by ...fetal ethanol-oxidizing enzymes. As alcohol dehydrogenases are expressed at very low concentrations in human embryonic tissues, the ethanol-inducible P450 enzyme, CYP2E1, could be the sole catalyst of fetal ethanol oxidation. With this in mind, we examined the expression of this P450 in liver samples from fetuses ranging in gestational age from 16 to 24 weeks. Immunoblot analysis of fetal liver microsomes revealed the presence of a protein immunoreactive with CYP2E1 antibodies that exhibited a slightly lower molecular weight than that found in adult liver samples. Embryonic CYP2E1 expression was further confirmed by the reverse transcriptase reaction with RNA from a 19-week gestational fetal liver used as template. Catalytic capabilities of human fetal microsomes were assessed by measurement of the rate of ethanol oxidation to acetaldehyde, which were 12-27% of those exhibited by adult liver microsomes. Immunoinhibition studies with CYP2E1 antibodies revealed that the corresponding antigen was the major catalyst of this reaction in both fetal and adult tissues. We then assessed whether embryonic CYP2E1 was, like the adult enzyme, inducible by xenobiotics. Treatment of primary fetal hepatocyte cultures with either ethanol or clofibrate demonstrated a 2-fold increase in CYP2E1 levels compared with untreated cells. Collectively, our results indicate that CYP2E1 is present in human fetal liver, that the enzyme is functionally similar to CYP2E1 from adults, and that fetal hepatocyte CYP2E1 is inducible in culture by xenobiotics, including ethanol. Because fetal CYP2E1 mediates ethanol metabolism, the enzyme may play a pivotal role in the local production of acetaldehyde and free radicals, both of which have potential deleterious effects on the developing fetus.
Carbohydrate‐deficient transferrin (CDT) is now considered to be the most sensitive and specific biological marker of alcohol abuse. However, the mechanism by which chronic alcohol consumption causes ...an elevation of CDT levels in serum is still not understood. Therefore, we fed eight pairs of male rats a nutritionally adequate liquid diet containing either alcohol (36% of energy) or isocaloric dextrose (control) for 4 weeks, after which blood and liver samples were obtained. Serum CDT content in alcohol‐treated rats increased by 45% (P < .05) as assessed with an isoelectric focusing/Western blot method (IEF/WB) thereby mimicking the clinical condition. In particulate fractions (plasma membranes) prepared from the liver samples, the activity of sialidase was increased 1.62‐fold (P < .05) in ethanol‐fed animals compared with their corresponding controls. In contrast, in rats fed ethanol, the activities of sialyltransferase (ST), galactosyltransferase (GT), and N‐acetylglu‐cosamine transferase (N‐AGT), which are glycosyltransferases involved in transferrin carbohydrate side chain synthesis, were diminished by 24% and 40% (P < .05), 23% and 51% (P < .05, .001), and 20% and 26% (P < .05) in total liver homogenates and Golgi fraction (GF) 1, respectively, when expressed as units/100 g body weight. These enzymes were also significantly less active in hepatic GFs 2 and 3. The depression of the transferase activities in ethanol‐fed rats appeared to be due, at least in part, to enzyme inactivation by acetaldehyde, whereas ethanol itself was without effect. Similar results were obtained in humans; five alcohol abusers were found to exhibit a 23% decrease in hepatic sialyltransferase and a 41% increase in sialidase activities, respectively, when compared with three nondrinking subjects. In conclusion, the increased activity of sialidase in liver particulate fractions, combined with the decrease in transferrin glycosyltransferases in GFs, mediated by ethanol (or acetaldehyde derived from ethanol) most likely accounts for the enhanced serum CDT in response to chronic ethanol consumption in rats as well as in humans. (Hepatology 1995; 22:1462–1468).
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