Quantitative determination of aristolochic acid-derived DNA adducts in rats using 32P-postlabeling/polyacrylamide gel electrophoresis analysis.

Aristolochic acids (AA) are nephrotoxic and carcinogenic nitroaromatic compounds produced by the Aristolochiaceae family of plants. Ingestion of these phytotoxins by humans results in a syndrome known as AA nephropathy, characterized by renal tubulointerstitial fibrosis and upper urothelial cancer. After activation by cellular enzymes, AA I and II react with DNA to form covalent adducts and as such represent potential biomarkers for studies of AA toxicity. Using site-specifically modified oligodeoxynucleotides as standards, we have developed a method for quantifying 7-(deoxyadenosin-N(6)-yl) aristolactam-DNA or 7-(deoxyguanosin-N(2)-yl) aristolactam-DNA adducts in tissues of Wistar rats using an assay in which (32)P-postlabeling techniques are coupled with nondenaturing polyacrylamide gel electrophoresis. The limit of detection with this technique is five adducts in 10(9) nucleotides for a 5-microg DNA sample. In contrast to previous reports, we find that the levels of AA adducts in renal tissues of Wistar rats treated p.o. with AA for 1 week with 5 mg/kg/day of AA I or AA II were much higher than that in the forestomach. Highest adduct levels were observed in rats treated with AA II, suggesting that this compound may be more genotoxic than AA I. Treatment of rats with aristolactam I, an end-product of AA I metabolism, resulted in a much lower level of adduction. This study establishes the feasibility of using AA-DNA adducts as intermediate biomarkers of exposure in studies of AA nephropathy and its associated urothelial cancer.     

血清样本双向电泳实验方法学研究

目的提高血清双向电泳实验的分辨率,建立理想的血清双向电泳实验方法。方法对血清双向电泳(two-diensional gel electrophoresis,2-DE)中血清标本的处理方法以及硝酸银染色方法等条件和技术方法进行了调整和优化。结果经过优化后,建立了重复性较好的血清2-DE实验方法 ,血清样本中蛋白质斑点的分辨率得到了明显的提高,蛋白质斑点明显增多,且此方法能够兼容后续生物质谱分析。结论通过优化和验证,建立了较实用的血清2-DE实验方案,对血清样本的蛋白质组学分析具有很好的技术参考价值,为进一步分析、寻找和鉴定药物干预或与疾病相关的血清蛋白质奠定了实验基础。     

Detection of DNA Adducts by 32P-Postlabeling and Multifraction Contact-Transfer Thin-Layer Chromatography

³²P-Postlabeling is a sensitive method for detecting DNA adducts.Large bulky adducts, particularly from polycyclic aromatic compounds,are readily detected using this technique. Detection of smallmodifications, such as methylations, has often required specificadditional enrichment procedures prior to ³²P-postlabeling.We report the use of a single analytical procedure that candetect DNA adducts of a wide range of sizes and hydrophobicities(exemplified by adducts produced with methyl methanesulfonate,diepoxybutane, styrene oxide, or benzo[a]- pyrene). This ³²P-postlabeling/thin-layerchromatography procedure is particularly useful when examiningthe potential of novel compounds or their metabolites to formDNA adducts.     

32P-Postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: a comparative study of labeling efficiencies.

32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by (32)P-postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3', 5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T(4) polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 microM(-)(1) min(-)(1)) was approximately 4-fold lower than that (5. 4 microM(-)(1) min(-)(1)) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 microM(-)(1) min(-)(1)) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2, 4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that (32)P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2, 4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical (32)P-postlabeling assays. Since the high specific activity of carrier-free [gamma-(32)P]ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting (32)P-postlabeling analyses of DNA adducts.     

Two-dimensional polyacrylamide gel electrophoresis of hymenoptera venom and venom sac extracts

Hymenoptera venom and venom sac extracts were studied by two-dimensional polyacrylamide gel electrophoresis using non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate electrophoresis in the second dimension. Pure Apis mellifera (honeybee) venom collected by electrical stimulation was resolved into five major and more than 20 minor components. Polistes (paper wasp), Vespula (yellow jacket), Dolichovespula (aerial hornet) and Vespa (old world hornet) venom sac extracts contained more than 40 components each. These results illustrate the complexity of the mixtures in current use for immunotherapy for stinging insect hypersensitivity. The degrees of similarity observed between the various species of Polistes and Vespula studied correlate with the phylogenetic classifications of these species.     

Comparative study of hormonal preparations by isoelectric focusing and polyacrylamide gel electrophoresis

The authors studied the purity of the hormonal drugs by electrophoresis in polyacrylamide gel and isoelectrofocusing. Identified fractions found in insulin, glucagon, andecaline and in other preparations. Established that isoelectrofocusing has a higher separation power as compared with disc electrophoresis in polyacrylamide gel.     

Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.     

Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry

Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n− 1)-phosphate with chain length from 2 to 8 bases. Alkylated and non-alkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O⁶- and N²-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable. Received: 16 January 1997 / Accepted: 17 March 1997     

Formation of DNA adducts from oil-derived products analyzed by 32P-HPLC

The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by 32P-postlabeling and 32P-high-performance liquid chromatography (32p-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250 degrees C and a peak around a distillation point of approximately 400 degrees C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16 substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The 32P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.     

Synthesis, characterization, and 32p-postlabeling analysis of DNA adducts derived from the environmental contaminant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. 3-NBA forms DNA adducts in rodent tissues that arise principally through reduction to N-hydroxy-3-aminobenzanthrone (N-OH-ABA), esterification to its acetate or sulfate ester, and reaction of this activated ester with DNA. We detected 3-NBA-derived DNA adducts in rodent tissues by (32)P-postlabeling and generated them chemically by acid-catalyzed reaction of N-OH-ABA with DNA, but their structural identification has not yet been reported. We have now prepared 3-NBA-derived adducts by reaction of a possible reactive metabolite, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA), with purine nucleosides and nucleotides, characterized them, and have shown that they are present in DNA treated with this 3-NBA derivative. Three of these adducts have been characterized as the C-C adduct N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone, the C-N adduct N-acetyl-N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone, and an unusual 3-acetylaminobenzanthrone adduct of deoxyadenosine, which involves a double linkage between adenine and benzanthrone (N1 to C1, N(6) to C11b), creating a five-membered imidazo type ring system. According to IUPAC fused ring conventions, we propose the following systematic name for this adduct: (9'-(2' '-deoxyribofuranosyl))purino[6',1':2,3]imidazo[5,4-p](1,11b-dihydro-(N-acetyl-3-amino))benzanthrone. The 3'-phosphates of these novel adducts could be 5'-postlabeled using [gamma-(32)P]ATP, although the efficiency of labeling was found to be low (less than 20%). However, none of these adducts could be detected in DNA from 3-NBA-treated rats by (32)P-postlabeling. Two of these synthetic adducts were treated with alkali to generate nonacetylated adducts, and these were also shown by HPLC to differ from those adducts found in rat DNA. Therefore, a different approach to the synthesis of authentic standards is needed for the structural characterization of 3-NBA-derived DNA adducts formed in vivo.     

酸性天然凝胶电泳法研究HIV进入抑制剂ADS-J1的作用机制

目的ADS-J1是通过虚拟筛选从20000个化合物中获得的靶向HIVgp41的小分子HIV进入抑制剂。该研究探讨ADS-J1与gp41的结合位点和作用机制。方法采用酸性天然聚丙烯酰胺凝胶电泳技术(AN-PAGE),研究ADS-J1与衍生于gp41不同功能区的多肽的结合。结果此前采用的天然聚丙烯酰胺凝胶电泳(N-PAGE)等方法,由于不能显示衍生于gp41的N-端多肽,未能确定ADS-J1的作用位点。该研究建立的AN-PAGE技术,能直接显示N-端多肽的条带,证实ADS-J1能与gp41的N-端螺旋重复序列(NHR)复合螺旋核中的深穴结合,从而抑制gp41六螺旋束结构的形成,而且深穴中第574位的赖氨酸残基(K574)与ADS-J1的抑制作用密切相关。结论ADS-J1通过与gp41 NHR靶穴中的K574结合,抑制gp41六螺旋束结构的形成,从而抑制HIV进入靶细胞。此外,该研究建立的AN-PAGE技术,为研究靶向Ⅰ型包膜病毒的病毒进入抑制剂的作用机制提供了一个简便有效的实验方法。     

Gradient polyacrylamide gel electrophoresis for determination of molecular weights of heparin preparations and low-molecular-weight heparin derivatives.

The M(r) values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of M(r) determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the M(r) values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average M(r), weight-average M(r), and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the M(r) values of heparin and LMW heparin.     

Characterization of the protein content of a meningococcal outer membrane vesicle vaccine by polyacrylamide gel electrophoresis and mass spectrometry

The development and evaluation of outer membrane vesicles as vaccines against meningococcal disease has been carried out for more than two decades. Although such vaccines have limitations and are not widely licensed, they continue to be used to disrupt clonal outbreaks caused by group B meningococci and a wealth of information is now available from large-scale clinical studies. One dimensional polyacrylamide gel electrophoresis and semi-quantitative measurement of the major proteins is one method used to evaluate and control these products. However, it is often difficult to determine exactly which bands on a one dimensional gel correspond to the key antigens whose presence must be demonstrated for control and lot release. We have therefore carried out mass spectrometric analyses of outer membrane vesicle vaccine samples to definitively identify the bands containing seven key antigens: Omp85, FetA, PorA, PorB, RmpM, OpcA and NspA. An additional 33 proteins present in the vaccine were also identified and this information will be useful both for future quality control and for the interpretation of data from vaccine trials.     

High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation

A new and simple gel electrophoretic method is described which enables the protein and polypeptide components of bee venom to be resolved on a single gel. The electrophoretic method allows octapeptides to be resolved and species as small as decapeptides can be detected at high sensitivity using the Coomassie blue staining method without prior fixation. This has been achieved by replacing acetic acid by propionic acid in acid/urea polyacrylamide gels and by controlling the amount of TEMED catalyst for the polymerisation of high concentration gels in order to obtain a low effective pore size. We demonstrated the value of ethanol precipitation as a rapid and efficient desalting the fractionation technique and propose that it could be used in combination with gel filtration to purify many of the peptides to homogeneity.     

Measurement of Nuclear DNA Modification by 32P-Postlabeling in the Kidneys of Male and Female Fischer 344 Rats after Multiple Gavage Doses of Hydroquinone

Oral administration of hydroquinone (HQ) to male Fischer 344(F344) rats results in dose-related kidney toxicity beginningwith mild enzymuria by 1 week, significant cell proliferationby 6 weeks, and nephropathy and an increase in the incidenceof renal tubule adenomas after 2 years. Female F344 rats, B6C3F1mice, Sprague-Dawley rats, dogs, and humans are resistant tothe renal toxicity of HQ associated with repeated exposure.To determine the potential of HQ to induce covalent DNA adductsin the kidney, male and female F344 rats were given 0, 2.5,25, or 50 mg/kg HQ by gavage for 6 weeks, and nuclear DNA isolatedfrom kidneys was analyzed by the ³²P-postlabeling assay. At50 mg/kg, males, but not females, showed an increase in therate of excretion of N-acetyl-ß-D-glucosaminidase,indicative of proximal tubular damage. Analysis of nuclear DNApreparations by the postlabeling assay showed that HQ does notproduce covalent DNA adducts in the kidneys of male and femalerats. The assay's lower limit of detection is 1 adduct in 10⁹to 10¹⁰ DNA nucleotides. No treatment-related increases in backgroundradioactivity levels on the chromatograms were seen at locationscorresponding to the major in vitro adducts of HQ and p-benzoquinone.HQ treatment, however, resulted in the reduction of the levelsof certain endogenous adducts (I-compounds), the biologicalsignificance of which is unknown. The results indicate thatHQ does not produce covalent DNA adducts in the kidneys of maleand female rats after repeated oral administration at nephrotoxicdose levels, and suggest a nongenotoxic etiology of benign tumorsin the kidneys of male F344 rats treated with HQ.