Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species

We previously reported that benzo[a]pyrene (BaP) and UVA radiation synergistically induced oxidative DNA damage via 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vitro. The present study shows that microsomal BaP metabolites and UVA radiation potently enhance 8-OHdG formation in calf thymus DNA about 3-fold over the parent compound BaP. Utilization of various reactive oxygen species scavengers revealed that singlet oxygen and superoxide radical anion were involved in the 8-OHdG formation induced by microsomal BaP metabolites and UVA. Two specific BaP metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/-) (anti) (BPDE) and BaP-7,8-dione, were further tested for synergism with UVA. BaP-7,8-dione showed an effect on 8-OHdG formation induced by UVA radiation that was similar to that of the parent BaP, whereas BPDE exhibited significantly higher induction of 8-OHdG than BaP. At as low as 0.5 microM, BPDE plus UVA radiation substantially increased 8-OHdG levels about 25-fold over the parent BaP. BPDE increased the formation of 8-OHdG levels in both BPDE concentration- and UVA dose-dependent manners. Additionally, singlet oxygen was found to play a major role in 8-OHdG induction by BPDE and UVA. These results suggest that BaP metabolites such as BPDE synergize with UVA radiation to produce ROS, which in turn induce DNA damage.     

Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2-5.0ng/microL, and limits of detection (LOD) were lower than 0.01ng/microL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4+/-21.3% to 92.4+/-3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC-MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.     

Metabolism of and DNA adduct formation by benzo[alpha]pyrene in human skin epithelial cells in vitro pretreated with cytochrome P450 modulators

This study was designed to investigate the effects of four compounds that are shown to influence the cytochrome P450 system, on the metabolism of and DNA adduct formation by benzo[alpha]pyrene (BaP) in human skin epithelial cells in culture. Radiolabeled BaP was used in the metabolism studies, and the levels of metabolites in the ethylacetate extracts of the intracellular and extracellular fractions were determined by HPLC. Among the various metabolites detected BaP-7,8-diol was the only one that was an intermediate on the activation pathway of BaP to the ultimate carcinogen, BPDE I. Both BHA and 7,8-BF pretreatment significantly decreased intracellular production of BaP-7,8-diol compared to cultures treated with only radiolabeled BaP. MeBHA pretreatment greatly increased intracellular BaP-7,8-diol formation compared to BaP treated controls, while disulfiram pretreatment had no effect on the intracellular concentration. Cultures pretreated with BHA, 7,8-BF or disulfiram formed 30-40% less BPDE I-dG adducts than nonpretreated cultures, while cultures pretreated with MeBHA exhibited approximately 200% increase in the BPDE I-dG adduct formation. Thus, BHA and 7,8-BF act similarly in reducing BaP activation and adduct formation. Alternatively, MeBHA increased BaP activation and adduct formation in human keratinocyte cultures in vitro. Disulfiram pretreatment did not reduce BaP-7,8-diol formation, but decreased BPDE I-dG adducts. These studies indicate that modulators of the P450 system act in different fashions at the level of production of an oxygenated procarcinogen metabolite, altering the amount of specific carcinogen-dG adducts that lead to the expression of a transformed phenotype.     

Southern flounder hepatic and intestinal metabolism and DNA binding of benzo[a]pyrene (BaP) metabolites following dietary administration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene

The lipid peroxidation (as malondialdehyde, MDA), activities of superoxide dismutase (SOD) and catalase (CAT), and benzo[a]pyrene (BaP) metabolites were investigated in sera and erythrocytes of male Sprague-Dawley rats treated with BaP (20 mg per rat). MDA levels were significantly increased in sera (16.98+/-3.29 nmol/ml serum, P<0.05) 12 h after BaP treatment and persisted up to 96 h (13.80+/-1. 65 nmol/ml serum, P<0.05), but no significant change in NIDA levels was observed in erythrocytes. SOD and CAT activities were significantly increased in erythrocytes shortly after BaP exposure, and they were slightly decreased in sera, indicating an inverse correlation between lipid peroxidation and antioxidant enzyme activity. BaP and BaP-quinones (BaP-1,6-quinone and BaP-3,6-quinone) were measured in sera during the study period. A rapid increase of unmetabolized BaP was observed in sera (41.27+/-4.14 pmol/ml serum) 3 h after BaP treatment, reaching a peak at 6 h (48.56+/-4.62 pmol/ml serum) followed by a sharp decrease. Formation of the BaP-1, 6-quinone and BaP-3,6-quinone started in sera 3 h after BaP treatment, reached a peak at 24 h (7.23+/-1.02 pmol/ml serum) and 12 h (9.20+/-0.98 pmol/ml serum), respectively, and then decreased gradually. The time-dependent pattern of serum lipid peroxidation and the level of erythrocyte antioxidant enzymes were shown to be related to the concentrations of the BaP-quinone metabolites. These results suggest that BaP treatment, probably via the formation of BaP-quinones, oxidatively altered lipids and antioxidant enzymes in the blood, and might be associated with BaP-related vascular toxicity including carcinogenesis.     

Transitory metabolic disruption and cytotoxicity elicited by benzo[a]pyrene in two cell lines from rainbow trout liver

Two cell lines, RTL-W1 and R1, from rainbow trout liver were used to investigate the effects of benzo[A]pyrene (BaP). BaP induced a catalytic measure of CYP1A, 7-ethoxyresorufin-O-deethylase (EROD) activity, in the rainbow trout liver cell line RTL-W1 but not in R1. Geldanamycin inhibited EROD induction by BaP. Potential BaP metabolites, BaP-7,8-dihydrodiol (BDP) and 6,12-BaP quinone (BQ) also induced EROD activity in RTL-W1. Very low BaP concentrations slightly stimulated cell proliferation in both cell lines. Higher BaP concentrations caused cytotoxicity in RTL-W1 but not in R1. Cytotoxicity was detected in a cell viability assay with 5-carboxyfluorescein diacetate acetoxymethyl ester, and as a decline in cell number. In both cell lines, BaP exposure impaired the reduction of the redox dye, alamar Blue (AB). After BaP removal, AB reduction recovered. Similar results were observed with BQ. As AB monitors metabolic activity, this novel phenomenon was termed transitory metabolic disruption. This decline in AB readings that was caused by BaP was ameliorated in RTL-W1 by alpha-naphthoflavone and geldanamycin, which suggests a role for CYP1A, and in R1 by indomethacin, which suggests involvement of prostaglandin-H-synthase. The significance of the response to BaP that is detected with AB and whether other PAHs cause it will be interesting future questions.     

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.     

Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection.

We developed a method to determine dihydrofolate reductase (DHFR) activity at pH 7.4 (37 degrees C) by monitoring its product, tetrahydrofolate (H(4)folate), using HPLC with electrochemical detection. After the assay mixture was deproteinized by 0.5 M perchloric acid, the H(4)folate concentration was measured. Using sodium ascorbate at 20 mM, H(4)folate was stable in our assay system. The enzyme activity was also stable. The detection limit of this method was less than 1 nM of H(4)folate in the enzyme assay system, which was 1/100 lower than those for the NADPH-spectrophotometric assay, which is commonly used for analysis of DHFR activity. This value of 1 nM allowed us to control the conversion from dihydrofolate (H(2)folate) to H(4)folate less than 10% of initial substrate concentrations during assay, when we used a concentration around K(m) values reported for DHFR from various sources. The rate of reduction showed a linearity at concentrations around the K(m). The reduction rate must be evaluated exactly around the K(m), in order to obtain an accurate profile of Michaelis-Menten kinetics. This assay method has a sensitivity high enough to determine the reduction rate at H(2)folate concentrations around K(m). In addition, the assay procedure is very simple. Therefore, our method may be useful for studying DHFR.     

Rapid screening method for simultaneous determination of four polycyclic aromatic hydrocarbons in water samples by derivative non-linear variable-angle synchronous fluorescence spectrometry.

Derivative non-linear variable-angle synchronous fluorescence spectroscopy (D-NLVASFS) has been developed to improve the selectivity of the fluorescence measurement without loss of sensitivity. We report a simple screening approach for the rapid and simultaneous determination of 1,2.5,6-dibenzoanthracene (DBA), 2,3-benzofluorene (BF), pyrene (Pyr) and 3,4-benzopyrene (BaP) in a mixture by using first-derivative NLVASFS. The method is efficient, fast and straightforward, and the measurement was simply based on a single spectrum via a single spectral scanning of a sample. The linear ranges 0.005-0.30, 0.02-0.30, 0.02-0.40 and 0.005-1.0 microg/mL and the detection limits 0.08, 1.14, 1.64 and 0.12 ng/mL are obtained for DBA, BF, Pyr and BaP, respectively. The method was applied to water samples spiked with the four polycyclic aromatic hydrocarbons (PAHs), with mean recoveries of 103.0% (SD 5.9%) for DBA, 96.4% (SD 4.2%) for BF, 96.2% (SD 4.0%) for Pyr and 98.2% (SD 3.0%) for BaP.     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder.

1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudopleuronectes americanus. 2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP. 3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP. 4. Both metabolites and BaP formed DNA adducts in liver.     

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits,vegetables, and flavonoids

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).     

High-performance liquid chromatographic method for the determination of histamine and 1-methylhistamine in biological buffers

A method for the determination of histamine and its catabolite 1-methylhistamine (1-MH) was developed, using HPLC with fluorescence detection. Derivatization of both compounds occurred on-column with o-phthaldialdehyde dissolved in an alkaline borate buffer, followed by separation on a reversed phase C18 column. Histamine and 1-MH could be detected with comparable sensitivity (limit of quantification, 50 nM). The method was proven suitable to investigate catabolism of histamine by epithelia of pig colon. The method should be useful in research on histamine metabolism.     

Binding of 3-hydroxybenzo[a]pyrene to bovine hemoglobin and albumin

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.     

Simultaneous determination of eight beta-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.     

Effects of saline-alkaline stress on benzo[a]pyrene biotransformation and ligninolytic enzyme expression by <Emphasis Type="Italic">Bjerkandera adusta</Emphasis> SM46

Benzo[a]pyrene (BaP) accumulates in marine organisms and contaminated coastal areas. The biotreatment of waste water using saline-alkaline-tolerant white rot fungi (WRF) represents a promising method for removing BaP under saline-alkaline conditions based on WRF’s ability to produce ligninolytic enzymes. In a pre-screening for degradation of polycyclic aromatic hydrocarbons of 82 fungal strains using Remazol brilliant blue R, Bjerkandera adusta SM46 exhibited the highest tolerance to saline-alkaline stress. Moreover, a B. adusta culture grown in BaP-containing liquid medium exhibited resistance to salinities up to 20 g l^?1. These conditions did not inhibit fungal growth or the expression of manganese peroxidase (MnP) or lignin peroxidase (LiP). The degradation rate also became higher as salinity increased to 20 g l^?1. Fungal growth and enzyme expression were inhibited at a salinity of 35 g l^?1. These inhibitory effects directly decreased the degradation rate (>24 %). The presence of MnSO₄ as an inducer improved the degradation rate and enzyme expression. MnP and LiP activity also increased by seven- and fivefold, respectively. SM46 degraded BaP (38–89 % over 30 days) in an acidic environment (pH 4.5) and under saline-alkaline stress conditions (pH 8.2). Investigating the metabolites produced revealed BaP-1,6-dione as the main product, indicating the important role of ligninolytic enzymes in initializing BaP cleavage. The other metabolites detected, naphthalene acetic acid, hydroxybenzoic acid, benzoic acid, and catechol, may have been ring fission products. The wide range of activities observed suggests that B. adusta SM46 is a potential agent for biodegrading BaP under saline conditions.     

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High‐Performance Liquid Chromatography with Fluorescence Detection after pre‐Column Derivatisation

Introduction

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.

Objective

To develop a sensitive and selective analytical method for tanshinones by high‐performance liquid chromatography (HPLC) with fluorescence detection after pre‐column derivatisation.

Methodology

The proposed method depends on derivatisation reaction of tanshinones with 4‐carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.

Results

The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.

Conclusion

The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o‐quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.     

Determination of urinary adenosine using resonance light scattering of gold nanoparticles modified structure-switching aptamer

A novel sensitive method has been developed for the detection of adenosine (AD) in human urine by using enhanced resonance light scattering (RLS). This method is based on the specific recognition and signal amplification of adenosine aptamer (Apt) coupled with gold nanoparticles (GNPs) via G-quartet-induced nanoparticle assembly, which was fabricated by triggering a structure switching of the 3′ terminus G-rich sequence and aptamer duplex. RLS signal linearly correlated with the concentration of adenosine over the range of 6-115 nM. The limit of detection (LOD) for adenosine is 1.8 nM with relative standard deviations (RSD) of 2.90-4.80% (n = 6). The present method has been successfully applied to determination of adenosine in real human urine, and the obtained results were in good agreement with those obtained by the HPLC method. Our investigation shows that the combination of the excellent selectivity of aptamer with the high sensitivity of the RLS technique could provide a promising potential for aptamer-based small molecule detection, and be beneficial in extending the application of RLS.     

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.     

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder

• 1.1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudo pleuronectes americanus.
• 2.2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP.
• 3.3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP.
• 4.4. Both metabolites and BaP formed DNA adducts in liver.