Comparison of the mutations induced by p-benzoquinone, a benzene metabolite, in human and mouse cells

Benzene is one of the chemicals widely contaminating the environment. Benzene is suggested to be a human leukemogen. When benzene is absorbed in the human body, it is metabolized firstly in the liver and subsequently in the bone marrow where it provokes initiation of leukemia. In the present study, we analyzed mutations induced by p-benzoquinone (p-BQ), a benzene metabolite, in human cells using a shuttle vector plasmid pMY189, and compared frequencies, types and spectra of the mutations with those of the mutations previously revealed in mouse cells using a similar plasmid pNY200. We found that p-BQ induces mutations in human and mouse cells at similar frequencies but with different types of mutagenesis. The proportion of tandem base mutations was significantly lower in human cells than in mouse cells. Most base substitutions were induced in G:C base pairs in both human and mouse cells. However, the proportion of G:C→C :G transversion is significantly higher in human cells. These findings indicate that the p-BQ-induced DNA damage in human and mouse cells is processed in a different manner, and that extrapolation of mice findings on experimental benzene carcinogenesis to human cancer risk assessment should be conducted carefully.     

Application of the high-performance liquid chromatographic method for separation, purification and characterisation of p-bromophenylacetylurea and its metabolites

This study presents a HPLC method for the separation and purification of p-bromophenylacetylurea (BPAU) and its metabolites. The method effectively separated and purified BPAU and its metabolites. Three metabolites of BPAU, M1, M2 and M3 were characterised by mass spectroscopy and nuclear magnetic resonance. They are named as N′-hydroxy-p-bromophenylacetylurea, 4-(4-bromophenyl)-3-oxapyrrolidine-2,5-dione and N′-methyl-p-bromophenylacetylurea, respectively. The major metabolic pathways of BPAU were proposed. The establishment of the HPLC method and characterisation of BPAU metabolites make it possible for further pharmacokinetic studies to explore the mechanism of BPAU-induced delayed neuropathy.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart^® (125 mm×4 mm I.D.) Nucleosil 100-5 μm C₁₈ AB cartridge column, using a gradient elution of MeOH–buffer pH 3.9 1:99→15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate–citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H₃PO₄ 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2±2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (Cl_R) and systemic (Cl_S) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney Cl_R/Cl_S calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73±0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87±0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.     

Monoclonal antibodies to p-coumarate

p-Coumaric acid is one of the predominant phenolic acids acylating the cell walls of grasses; p-coumarates are mainly esterified by lignins and arabinoxylans. Here we describe the production and characterisation of two monoclonal antibodies against p-coumarates.The 5-O-pCou-Ara(1 → 4)Xyl was chemically synthesized and conjugated to a carrier protein. Two interesting antibodies were obtained, hereinafter named INRA-COU1 and INRA-COU2. The specificity of these monoclonal antibodies has been evaluated using competitive-inhibition assays with different oligosaccharides and phenolic compounds. INRA-COU1, recognized free p-coumaric acid or p-coumarate esters. INRA-COU1 did not react with any of the other hydroxycinnamic acids and related compounds found in plants. INRA-COU2, only recognizes esterified p-coumarate. These antibodies were used to study the localization of p-coumarates in the cell walls of grasses. Immunocytochemical analyses indicated noticeable amounts of p-coumarate in the cell walls of the aleurone layer of wheat grain, in the epiderm of cereal straw, and in the exoderm of wheat root.The use of these antibodies will contribute to a better understanding of the organisation and developmental dynamics of cell walls in Graminaceae.     

Determination of fluoxetine and its metabolite norfluoxetine in serum and brain areas using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method using only 0.1 ml of serum or homogenate from brain areas has been developed for the determination of fluoxetine (FLU) and its metabolite, norfluoxetine (N-FLU), with ultraviolet detection at 227 nm. The small volume of sample required in this method allows studies in small animals, such as mouse. The method provides recoveries of up to 90% for both compounds. Acceptable coefficients of variation were found for both within-run and day-to-day assays. The limit of detection was 5.0 ng/ml. No interferences were found with tricyclic antidepressant drugs and benzodiazepines, which allows this method to be used in clinical studies. Pharmacokinetic parameters for the two compounds are reported in mouse serum, frontal cortex and caudate nucleus. We also report the values of FLU and N-FLU in serum from humans who were treated once daily with 20 mg of FLU, obtained after 1, 14 and 28 days of treatment.     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.     

Production of Gastrodin Through Biotransformation of p-hydroxybenzaldehyde Using Cell Suspension Cultures of Datura tatula L.

The conversion of exogenous p-hydroxybenzaldehyde into p-hydroxy-methyl-phenol-β-D-glucoside (gastrodin) was studied using cell suspension cultures of Datura tatula L. The chemical structure of the synthesized gastrodin was identified on the basis of spectral analysis and chemical evidence. The procedure of conversion of p-hydroxybenzaldehyde into gastrodin by D. tatula L. cell suspension cultures was established. The synthesized gastrodin (II) was isolated from the ferment liquor and identified by spectral analysis. Simultaneously, the p-hydroxybenzyl alcohol (I) that was converted through biotransformation of p-hydroxybenzaldehyde by cell suspension cultures of D. tatula L. was also isolated and identified. The efficiency of glucosylation of p-hydroxybenzaldehyde was remarkably enhanced by the addition of salicylic acid (0.1 mg/L) and the maintenance of low pressure (0.001 MPa) in a 25-L airlift loop bioreactor. The biotransformation of exogenous p-hydroxybenzaldehyde to gastrodin using cell suspension cultures of D. tatula L. is a promising approach.     

Interactions of D-cellobiose with p-toluenesulfonic acid in aqueous solution: a C NMR study

The effects of adding D₂SO₄, and p-toluenesulfonic acid-d to D-cellobiose dissolved in D₂O were investigated at 23 °C by plotting ¹³C NMR chemical shift changes (Δδ) against the acid to D-cellobiose molar ratio. ¹³C Chemical shifts of all 18 carbon signals from α and β anomers of D-cellobiose showed gradual decreases due to increasing acidity in aqueous D₂SO₄ medium. The C-1 of the α anomer showed a slightly higher response to increasing D⁺ concentration in the surrounding. In the aqueous p-toluenesulfonic acid-d medium, C-6′ and C-4′ carbons of both α, and β anomeric forms of D-cellobiose are significantly affected by increasing the sulfonic acid concentrations, and this may be due to a 1:1 interaction of p-toluenesulfonic acid-d with the C-6′, C-4′ region of the cellobiose molecule.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

p-香豆酸对西洋参的化感作用及生理机制

西洋参(Panax quinquefolium L.)栽培中存在严重的连作障碍现象,前期发现p-香豆酸在以滤纸片为基质的条件下,能够显著抑制西洋参胚根的生长。为了明确p-香豆酸在土壤基质中对种胚的化感活性以及对成株西洋参生长的作用及生理机制,以自然土壤为基质,观察p-香豆酸作用后种胚的生长情况;采用室内水培试验,观察p-香豆酸作用下2年生西洋参种根从出苗至结果期的生长及部分生理指标的变化。种胚生长实验在土壤中分别添加0.0024、0.012、0.06、0.3、1.5、7.5 mg/g的p-香豆酸,处理7 d后测定西洋参种胚的胚根长和胚芽长。水培试验中全营养液中分别添加0.012 mg/mL、0.06 mg/mL、0.3 mg/mL p-香豆酸,处理后每隔5 d测定植株叶片展开情况、株高、冠幅等生长指标;于展叶期(10 d)、现蕾期(20 d)、结果期(30 d)测定地上部分及新生须根的生物量,同时测定新生须根苯丙氨酸解氨酶(PAL)活力;叶片完全展开后测定植株净光合速率(Pn)、表观电子传递速率(ETR)和最大光化学效率(Fv/Fm)等光合特性参数。结果表明,土壤中添加0.0024-7.5 mg/g p-香豆酸西洋参胚根长度降低28.52%-100%,胚芽长度降低1.09%-100%,并呈现一定的剂量抑制效应。实验浓度内的p-香豆酸可显著抑制西洋参植株地上部分生长,推迟展叶期;结果期地上部生物量比对照降低17.17%-54.55%(P < 0.05,Dunnett-t test);同时,叶片的Pn、ETR受到抑制(P < 0.05),但Fv/Fm不变;对须根的影响主要表现为0.06 mg/mL p-香豆酸处理组在展叶期PAL酶活力提高69.05%,之后PAL活力和生物量均比对照下降,浓度增加至0.3 mg/mL时整个培养期内PAL酶活力和生物量均低于对照。由此推论,根系环境中的p-香豆酸在自然土壤中对西洋参种胚具有显著抑制其生长的化感作用;对成株西洋参的作用主要为抑制地上部分生长,并通过降低成株西洋参叶片光合能力,从而表现出明显的化感作用,0.06 mg/mL p-香豆酸诱导须根PAL酶活力先升高再降低并最终降低生物量的结果也表明p-香豆酸是西洋参根系生长的胁迫因素。结果证实p-香豆酸对西洋参种胚和成株的生长均具有自毒作用,其抑制生长的生理机制在于抑制叶片的光合作用。     

Simultaneous determination of fluoxetine and its metabolite p-trifluoromethylphenol in human liver microsomes using a gas chromatographic-electron-capture detection procedure

An gas chromatography-electron-capture detection method has been developed for simultaneous determination of fluoxetine and p-trifluoromethylphenol (TFMP), an O-dealkylated metabolite of fluoxetine in human liver microsomes. Prior to the analysis, aliquots of alkalinized microsomal mixture were extracted with ethyl acetate solvent containing acetonitrile (10%, v/v) and the derivatizing reagent, pentafluorobenzenesulfonyl chloride (0.1%, v/v). The organ phase was retained and taken to dryness, the residue was reconstituted in methanol, and the aliquot of extracts was injected directly into a gas chromatograph equipped with an electron-capture detector. 2,4-Dichlorophenol was added to the initial incubation mixture and carried through the procedure as the internal standard. The method provided the mean recoveries of up to 103% for fluoxetine and 104% for TFMP. Acceptable relative standard deviations were found for both within-run and day-to-day assays. The practical limit of detection (signal-to-noise ratio=3) was 1.62 ng/ml for TFMP and 6.92 ng/ml for fluoxetine in human liver microsomes, and the limit of quantitation was 8.1 pg for TFMP and 34.6 pg for fluoxetine. The assay is rapid and sensitive and has been applied successfully to simultaneous quantification of fluoxetine and TFMP in human liver microsomes with different CYP2C19 genotypes.     

Determination of a new blood glucose-lowering agent in biological fluids by capillary gas chromatography using electron-capture detection

A gas chromatographic method for the determination of 5-(4-chlorophenyl)-2-[(4-methylphenyl)sulphonyl]-4-pentynoic acid (I) in plasma (serum) and urine has been developed. After an extraction process, the cleaned-up organic extract was derivatized with diazomethane at ambient temperature. Results are evaluated from peak-height ratios with respect to the appropriate internal standard. The detection limit following extraction of a 1-ml plasma sample is about 20 ng/ml.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Determination of an arylacetamide antiarrhythmic in rat blood and tissues using reversed-phase high-performance liquid chromatography

A method was developed for quantification of (+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b- ]thiophene-4-acetamide (compound I), an antiarrhythmic drug, in rat whole blood, heart, brain, liver and skeletal muscle. Blood and tissue samples were homogenized and purified by chemical extraction. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography (HPLC) coupled with UV detection (215 nm). Drug recoveries from the extraction procedure ranged from 77 to 90%. Within- and between-day reproducibility of peak area (coefficient of variation) ranged from 1.1 to 15.7%. The detection limit was 80–200 ng/ml (in a 500-μl extracted solution) depending on the type of biological sample. This method was used in a pharmacokinetic study of compound I disposition in rats after a bolus intravenous dose of 3.1 mg/kg.     

Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen–phosphorus detection (GC–NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179°C at 4°C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10 000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within ±15%.     

Hydroxyurea,a natural metabolite and an intermediate in d-cycloserine biosynthesis in streptomyces garyphalus

It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [¹⁴C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-¹⁴C]-arginine and l-[ureido-¹⁴C]-citrulline. The ¹⁵N of H₂NCO¹⁵NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the ¹⁵N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [¹⁵N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine.     

Determination of sialic acids in biological fluids using reversed-phase ion-pair high-performance liquid chromatography

A simple, rapid and sensitive reversed-phase ion-pair high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in biological fluids is described. Determination of N-acetylneuraminic acid released by acidic hydrolysis, in serum, urine and saliva, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in urine, without hydrolysis, was accomplished by injecting the sample without derivatization, into the chromatograph. Measurements were carried out isocratically within 6 min using a C₁₈ column and a mobile phase of aqueous solution of triisopropanolamine, as ion-pair reagent, 60 mM, pH 3.5 at room temperature with UV absorbance detection. The present method is reported for the first time for the determination of sialic acids in biological fluids. Recoveries in serum, urine and saliva ranged from 90 to 102% and the limits of detection were 60 nM and 20 nM for the two sialic acids, respectively. The method has been applied to normal and pathological sera from patients with breast, stomach, colon, ovarian and cervix cancers, to normal urine and urine from patient with sialuria and to normal saliva.