Identification of guanine and adenine adducts in DNA alkylated by dibromodulcitol in vitro and in vivo

DNA reacted with dibromodulcitol in neutral solution yielded 3- and 7-alkyl substituted purines after hydrolysis at neutral pH-value at 37°C. The alkylated products were identified by mass spectrometry and by comparison of their UV absorption spectra and chromatographic properties on thin-layer chromatography (TLC) and various columns with those of the corresponding galactitylpurine derivatives obtained by synthetic route from alkylation of the appropriate nucleic bases or nucleosides. The labelled alkylpurines occurring in DNA of Yoshida tumour cells treated with [³H]dibromodulcitol in vivo were also indentified by co-chromatography of labelled DNA hydrolysate with synthetic 3- and 7-alkyl substituted purines. On the basis of the same chromatographic behaviour 3-(1-deoxy-3,6-anhydrogalactit-1-yl)adenine, 7-(1-deoxygalactit-1-yl)guanine, 7-(1-deoxy-3,6-anhydrogalactit-1-yl)guanine and 1,6-di(guanin-7-yl)-1,6-dideoxygalactitol were identified as main alkylated products in tumor cell DNA after in vivo treatment with dibromodulcitol.     

Transfer of polycyclic aromatic hydrocarbons between model membranes: relation to carcinogenicity

Polynuclear aromatic hydrocarbons (PAH), some of which are potent carcinogens, are common environmental pollutants. The transport processes for these hydrophobic compounds into cells and between intracellular membranes are diverse and are not well understood. A common mechanism of transport is by spontaneous desorption and transfer through the aqueous phase. From the partitioning parameters, we have inferred that the rate limiting step involves solvation of the transfer species in the interfacial water at the phospholipid surface. Transfer of 10 PAH (pyrene, 3,4-benzophenanthrene, triphenylene, chrysene, 1,2-benzanthracene, 1,1'-binaphthyl, 9-phenylanthracene, 2,2'-binaphthyl, m-tetraphenyl and 1,3,5-triphenylbenzene) out of phosphatidylcholine vesicles has been examined. Our results show that the molecular volume of the PAH is a rate-determining factor. Moreover, high performance liquid chromatography (HPLC) data confirms the hypothesis that the rate of transfer is correlated with the size of the molecule and with the partitioning of the molecule between a polar and hydrocarbon phase. The kinetics and characteristics of the spontaneous transfer of carcinogens are likely to have a major impact on the competitive processes of PAH metabolism within cells.     

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.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.     

牛生长激素结构不均一性的研究

本文报导用常规方法分离纯化的牛生长激素,在还原性SDS-11％聚丙烯酰胺凝胶电泳中呈分子量很接近的两条主带(22KD,21.5KD)。用单克隆抗体和多克隆抗体亲和层析技术分析了不同分子量形式的牛生长激素的转化,结果表明:牛生长激素可能在pH5.5条件下转化为21.5KD分子,在pH8.3条件下则转化18KD分子。这几种形式的牛生长激素均保留与抗体的结合力,但亲和力不尽相同,如在亲和层析的洗脱性质上存在差异。已检验分离并部分纯化了18KD分子以备作进一步的研究。     

化学诱导的B95-8细胞中TK酶的分离和性质研究

巴豆油和正丁酸钠(nSB)诱导Raji和B95-8细胞株生成胸腺嘧啶核苷激酶(TK),其粗提液,经DEAE—纤维素柱层析,可分成两个性质不同的TK活性峰—峰Ⅰ和峰Ⅱ:(1)峰Ⅰ是穿过峰,峰Ⅱ为洗脱峰,在120mMol/L从K_2HPO_4缓冲液时洗脱下来;(2)峰Ⅱ含量在病毒生产性细胞B95-8中高于非生产性的Raji细胞;(3)B95-8细胞经联合诱导48小时后,峰Ⅱ比活性最高;(4)TTP对峰Ⅰ和峰Ⅱ的抑制效应不同,两峰利用GTP能力也不同;(5)PAGE结果表明:峰Ⅰ的Rm值为0.044,峰Ⅱ呈现两条带,Rm值分别为0.015和0.276;(6)峰Ⅰ的Km值为0.86μMol/L,峰ⅡKm值为0.29μMol/L。根据以上的结果,我们认为:峰Ⅰ是细胞TK(C-TK),而峰Ⅱ具有许多疱疹病毒TK的特性,因此,峰Ⅱ是EB病毒相关TK(EBV-TK)。     

Principles and applications of luminescence spectrometry coupled to liquid chromatographic techniques

An overview is presented of the physicochemical basis of luminescence, and its application to the detection of chemicals (drugs, biomedically important compounds, environmentally active substances) in liquid chromatographic systems.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.