Activation of p53 as a causal step for atherosclerosis induced by polycyclic aromatic hydrocarbons

AbstractThis study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells

Biomonitoring of Urinary Cotinine Concentrations Associated with Plasma Levels of Nicotine Metabolites after Daily Cigarette Smoking in a Male Japanese Population

Human biomonitoring of plasma and urinary levels of nicotine, cotinine, and 3′-hydroxycotinine was conducted after daily cigarette smoking in a population of 92 male Japanese smokers with a mean age of 37 years who had smoked an average of 23 cigarettes per day for 16 years. Members of the population were genotyped for the nicotine-metabolizing enzyme cytochrome P450 2A6 (CYP2A6). The mean levels of nicotine, the levels of its metabolites cotinine and 3′-hydroxycotinine, and the sum of these three levels in subjects one hour after smoking the first cigarette on the sampling day were 20.1, 158, 27.7, and 198 ng/mL in plasma and 846, 1,020, 1,010, and 2,870 ng/mL in urine under daily smoking conditions. Plasma levels of 3′-hydroxycotinine and urinary levels of nicotine and 3′-hydroxycotinine were dependent on the CYP2A6 phenotype group, which was estimated from the CYP2A6 genotypes of the subjects, including those with whole gene deletion. Plasma cotinine levels were significantly correlated with the number of cigarettes smoked on the day before sampling (r = 0.71), the average number of cigarettes smoked daily (r = 0.58), and the Brinkman index (daily cigarettes × years, r = 0.48) under the present conditions. The sum of nicotine, cotinine, and 3′-hydroxycotinine concentrations in plasma showed a similar relationship to that of the plasma cotinine levels. Urinary concentrations of cotinine and the sum of nicotine metabolite concentrations also showed significant correlations with the plasma levels and the previous day’s and average cigarette consumption. The numbers of cigarettes smoked per day by two subjects with self-reported light smoking habits were predicted by measuring the urinary cotinine concentrations and using linear regression equations derived from above-mentioned data. These results indicate that biomonitoring of the urinary cotinine concentration is a good, easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers independent of genetic polymorphism of CYP2A6

Determination of Serum Spermidine by High-Performance Liquid Chromatography after Fluorescence Derivatization with Orthophthalaldehyde

Repor

Selective Solubilization of Microsomal Electron-Transfer Proteins with Alkylglucoside

A series of alkylglucosides (AGs) was examined for the solubilization of four microsomal electron-transfer proteins (cytochrome P450 (P450), cytochrome b5 (b5), NADPH-cytochrome P450 reductase (fp2), and NADH-cytochrome b5 reductase (fp1)) in rat liver microsomes. Among the four proteins, b5 and fp2 were completely solubilized, and thus, almost recovered in the supernatant fraction after centrifugation, while P450 and fp1 were in the pellet. In particular, the solubilization yield of P450 was only about 10%. With a high ratio of alkylglucoside to membrane, along with a low ionic strength, a greater selectivity for b5 and fp2 could be obtained. Such high selectivity was not observed by the use of sugar ester, bile salt, and poly(oxyethylene) alkylphenyl ether types of surfactants. After re-solubilization of the pellet with sodium cholate, the supernatant fraction contained P450 free from b5 and the final recovery of P450 was about 40%. Thus, this two-step solubilization provides a simple method for the partial purification of P450 in microsomes

Polymer-Induced Phase Separation in Aqueous Micellar Solutions of Alkylglucosides for Protein Extraction

Aqueous micellar solutions of alkylglucosides were separated into two phases at 0°C upon addition of polyethylene glycol 6000 (PEG) or Dextran T-500 (Dextran). One was an aqueous phase in which hydrophilic proteins, cytochrome c and peroxidase (horseradish), were retained, and the other was a surfactant-rich phase into which hydrophobic membrane proteins, bacteriorhodopsin and cytochrome b5, were extracted. A combination of octyl-β-D-thioglucoside (OTG) (or nonyl-β-D-glucoside (NG)) with PEG (or Dextran) was the best choice for extraction of hydrophobic proteins. Extraction yields (50-90%) and concentration factors (7-30) of the hydrophobic proteins were dependent on the types of nonionic surfactants and water-soluble polymers. Solubilization and phase separation in processing cell membranes could be made in a single step at 0°C. Hence the present method would be useful for the purification of thermolabile proteins

Comparative Cytochrome P450-1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 expressions in human larynx tissue analysed at mRNA level

WOS: 000242307900001PubMed ID: 16894644The metabolic activation of numerous exogenous and endogenous chemicals is catalysed by cytochrome P450 enzymes (CYPs). The aim of this study was to analyse the expression of the individual forms of CYP at the mRNA level in human larynx and quantitatively to compare their expressions in human liver, the main in organ of CYP expression. Individual forms of CYP mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CYPs -1A1, -1A2, -2A6,-2B6,-2C, -2D6, -2E1, -3A3/4 ,-3A5, -3A7 and 4B1. An RNA competitor of known copy number, covering the primer sequences necessary to amplify the entire object CYPs within a single molecule, was used as reference. This study reports a consistent detection of mRNAs for the CYPs -1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 in the human larynx tissue. The data indicate that the human larynx highly resembles the lung tissue in CYP content, as a comparable subset of CYP mRNAs was detected in the larynx previously reported for human lung with the exception of CYP1A2. The results are discussed in quantitative ratios of the detected CYP mRNAs in relation to the hepatic CYP expression. Copyright (c) 2006 John Wiley & Sons, Ltd

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population.

The reduced capacity of flavin-containing monooxygenase 3 (FMO3) to N-oxidize trimethylamine (TMA) is believed to cause a metabolic disorder. The aim of this study was to investigate the inter-individual variations of FMO3. Genomic DNA of case subjects that showed only 10–20% of FMO3 metabolic capacity among self-reported trimethylaminuria Japanese volunteers was sequenced. Functional analysis of recombinant FMO3 proteins was also performed. One homozygote for a novel single nucleotide substitution causing a stop codon at Arg500 was observed. The biological parents of this Proband A were heterozygous and showed >90% TMA N-oxygenation metabolic capacity. Another Proband B had the Arg500Stop and Cys197Stop codons. The TMA N-oxygenation metabolic capacities of the father and brother of this Proband B were apparently observed by possessing Arg205Cys mutant that coded for decreased TMA N-oxygenase. Recombinant Arg500Stop FMO3 cDNA expressed in Escherichia coli membranes and a series of highly purified truncation mutants at different positions of the C-terminus of FMO3 showed no detectable functional activity toward typical FMO3 substrates. The results suggest that individuals homozygous for either of the nonsense mutations, Arg500Stop and/or Cys197Stop alleles, in the FMO3 gene can possess abnormal TMA N-oxygenation