Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P-postlabeling.

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity - fluorescence labeling of nucleotides followed by CE-LIF detection and (32)P-postlabeling. DNA of Hydra was digested either to deoxyribonucleoside-5'-monophosphates or deoxyribonucleoside-3'-monophosphates selectively conjugated with the fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE-LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5-methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the (32)P-postlabeling method.

Genomic N(6)-methyladenine determination by MEKC with LIF.

2'-Deoxy-N(6)-methyladenosine (N(6)mdA) is frequently found in prokaryotic and unicellular eukaryotic genomes. Although methylated bases represent only a minor fraction of the genome, they, however, exhibit strong biological effects. Here, we report a fast and sensitive method for the quantification of global adenine methylation in DNA. The method is based on a recently developed procedure consisting of fluorescence labeling of deoxyribonucleotides with BODIPY FL EDA and analysis by CE with LIF. An oligodeoxyribonucleotide site specifically modified with N(6)mdA was used for peak assignment, to establish separation conditions and to determine the LOD. The method yielded a LOD for N(6)mdA of 280 pM (1.4 amol), which is equivalent to ∼1 N(6)mdA per 10(4) normal nucleotides (0.01%) using 1 µg of DNA as the matrix. After calibration with completely dam methylated λ DNA, the assay was applied to the analysis of various DNAs.

St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary endothelial cells (PBCEC) and freshly isolated brain capillaries from pig were used as in vitro/ex vivo blood-brain barrier model. SJW modulation of P-glycoprotein function was studied in PBCEC using a calcein-AM uptake assay and in isolated pig brain capillaries using the fluorescent cyclosporine A derivative NBD-CSA and confocal microscopy. RESULTS: SJW extract and the constituents hyperforin, hypericin, and quercetin decreased P-glycoprotein transport activity in a dose- and time-dependent manner. SJW extract and hyperforin directly inhibited P-glycoprotein activity, whereas hypericin and quercetin modulated transporter function through a mechanism involving protein kinase C. Quercetin at high concentrations decreased P-glycoprotein transport activity, but increased transporter function at low concentrations. This increase in P-glycoprotein activity was likely due to trafficking and membrane insertion of vesicles containing transporter protein. CONCLUSIONS: Our findings provide new insights into the short-term interaction of SJW with P-glycoprotein at the blood-brain barrier. They are of potential relevance given the wide use of SJW as OTC medication and the importance P-glycoprotein has for CNS therapy.

Accelerated (32)P-HPLC for bulky DNA adducts.

Many compounds can react with DNA forming covalent modifications, so-called DNA adducts, which can influence crucial biological processes. DNA adducts from various DNA-damaging agents can act both as biomarkers and as a measurement of the actual damage of the genome. Therefore, they are of value in determining exposed or sensitive individuals or populations and in identifying agents that can induce DNA damage. A common method to measure DNA adducts is through DNA hydrolysis, adduct enrichment, (32)P-post-labelling and chromatographic separation. High-performance liquid chromatography (HPLC) with online radioactivity detection, the (32)P-HPLC (direct injection of the (32)P-labelled mixture into the HPLC with online (32)P-detection) method, gives both high sensitivity and good resolution of complex mixtures of DNA adducts. One limitation with this method is the capacity when dealing with large numbers of samples. The aim of this study was therefore to increase the analytical capacity by reducing analysis time of the (32)P-HPLC method. A change of HPLC columns to low backpressure columns and adaptation of elution conditions enabled a reduction in time per analysis from 100 to 20 min. This did not affect sensitivity but lowered chromatographic resolution, although bulky DNA adducts were still well resolved from other DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is required, this can be achieved by gradient modifications, which increase the time required per analysis to 30 min. The accelerated (32)P-HPLC increases the capacity in number of samples 3- to 5-fold, depending on resolution requirements, without any negative effect on sensitivity in both in vitro and in vivo samples.

Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts.

We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 µM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.

RNA analysis by MEKC with LIF detection.

We have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-O-methyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.

Formation and persistence of DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in target and non-target organs after intratracheal instillation in rats.

Sprague-Dawley rats were treated by intratracheal instillation with a single dose of 0.2 mg/kg body wt of 3-nitrobenzanthrone (3-NBA), and whole blood, lungs, pancreases, kidneys, urinary bladders, hearts, small intestines and livers were removed at various times after administration. At five posttreatment times (2 days, 2, 10, 20 and 36 weeks), DNA adducts were analysed in each tissue by (32)P-postlabelling to study their long-term persistence. 3-NBA-derived DNA adducts consisting of the same adduct pattern were observed in all tissues from animals killed between 2 days and 36 weeks and between 2 days and 20 weeks in blood. DNA isolated from whole blood contained the same 3-NBA-specific adduct pattern as that found in tissues. Although total adduct levels in the blood were much lower than those found in the lung, the target organ of 3-NBA tumourigenicity, they were related (20-25%, R(2) = 0.98) to the levels found in lung. In all organs, total adduct levels decreased over time to 20-30% of the initial levels till the latest time point (36 weeks) and showed a biphasic profile, with a rapid loss during the first 2 weeks followed by a much slower decline that reached a stable plateau at 20 weeks after treatment. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in target and non-target organs of the rat. The correlation between DNA adducts in lung and blood suggests that persistent 3-NBA-DNA adducts in the blood may be useful biomarkers for human respiratory exposure to 3-NBA.

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats.

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.