DNA adducts and cell cycle

Summary Cell cycle-dependent differences of transformation sensitivity may be due to alterations in the formation of ultimate electrophilic carcinogens during the cell cycle, preferential primary adduct formation during specific phases of the cell cycle, e.g. binding to single stranded DNA at the replication fork, base-mispairing and mutation of transformation-related genes replicating at critical phases of DNA synthesis, or cell cycle-related differences in the repair of DNA adducts. Some recent data on these subjects are summarized, mainly in context of cell cycle-dependent transformation sensitivity of regenerating rat liver.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986Original work was supported by Deutsche Forschungsgemeinschaft and Dr.-Mildred-Scheel-Stiftung für Krebsforschung     

Characterization of covalent Adriamycin-DNA adducts

Adriamycin is a popular antineoplastic agent whose ability to form covalent adducts with DNA has been correlated to cellular apoptosis (programmed cell death) in tumor models. We have isolated and purified this adduct formed under oxido-reductive (Fenton) conditions in Tris buffer. We show by homo- and heteronuclear NMR spectroscopy that the covalent Adriamycin-DNA adduct is structurally equivalent to that resulting from direct reaction with formaldehyde. Covalent linkage of the drug to one of the DNA strands confers remarkable stability to the duplex, indicated by a 162-fold reduction in the rate of strand displacement compared with the complex with noncovalently bound drug. Glyceraldehyde also engenders covalent Adriamycin-DNA complexes, providing a possible relevant biological context for in vivo adduct formation.     

Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease.

RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini. Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor. However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme. This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation. We tested these alternatives by fabricating partially synthetic test substrates for RNase M5. Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA. The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates. Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule. We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site. The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds.     

DNA adducts of halogenated hydrocarbons

Summary Although formation of DNA adducts has been postulated for several halomethanes, no chemical identification of such adducts has been performed so far. There is, however, evidence that methyl chloride does not act biologically as a DNA methylating agent. 1,2-Dichloroethane and 1,2-dibromoethane are activated through conjugation with glutathione. There is some evidence for formation on an N-7 adduct of guanine which carries an ethyl-S-cysteinyl moiety.Extensive work has been published on adducts of vinyl chloride, both in vitro and in vivo. The major DNA adduct is 7-(2-oxoethyl)guanine; a minor adduct appears to be N²,3-ethenoguanine. Other etheno adducts, i.e., 1,N⁶-ethenoadenine and 3,N⁴-ethenocytosine, are readily formed with DNA, vinyl chloride, and a metabolizing system in vitro and with RNA in vivo, but are usually not detected as DNA adducts in vivo.The data on DNA alkylation by vinyl chloride (and vinyl bromide) metabolites are compared with those of structurally related compounds (acrylonitrile, vinyl acetate, vinyl carbamate).Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986     

Role of stress-activated MAP kinase P38 in cisplatin- and DTT-induced apoptosis of the esophageal carcinoma cell line Eca109

AIM: To study the role of P38 kinase in esophageal cancer cell apoptosis induced by genotoxin, cisplatin and the unfolded protein response (UPR) inducer, dithiothreitol (DTT). METHODS: Esophageal carcinoma cell line Eca109 was cultured in RPMI 1640 medium to 70% confluency and treated with either cisplatin, DTT, or cisplatin plus DTT in the presence or absence of P38 inhibitor, SB203580. The untreated cells served as the control. The esophageal carcinoma cell apoptosis was detected by agarose gel DNA ladder analysis and quantified by flow cytometry. The P38 phosphorylation was detected by immunohis-tochemistry using antibodies specific to phosphorylated P38 protein. RESULTS: (1) Both cisplatin and DTT induced apoptosis in the esophageal cancer cell line Eca109 as shown by DNA ladder formation; (2) As detected by antibodies specific for the phosphorylated P38 protein (p-P38), both cisplatin and DTT treatments activated the stress-activated enzyme, MAP kinase P38. The number of positive cells was about 50% for the treatment groups, comparing to that of 10% for untreated group. DTT treatment, but not cisplatin treatment, induces nuclear localization of p-P38; (3) As measured by flow cytometry, inhibition of P38 activity by SB203580 blocks DTT- and cisplatin-induced apoptosis. The rates for DTT, cisplatin, and DTT plus cisplatin-induced apoptosis were 16.8%, 17.1%, and 21.4%, respectively. Addition of the SB compound during the incubation reduced the apoptotic rate to about 7.6% for all the treatment groups, suggesting that P38 activation is essential for cisplatin- and DTT-induced apoptosis in Eca109 cells. CONCLUSION: (1) Both DTT and cisplatin were able to induce apoptosis in esophageal cancer cell line Eca109; (2) P38 MAP kinase is essential for DTT- and cisplatin- induced apoptosis in Eca109 cells; (3) P38 activation may be the common signaling component relaying the multiple upstream signaling events to the downstream cell death program.     

Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

Background  

Acetaminophen-cysteine adducts (APAP-CYS) are a specific biomarker of acetaminophen exposure. APAP-CYS concentrations have been described in the setting of acute overdose, and a concentration >1.1 nmol/ml has been suggested as a marker of hepatic injury from acetaminophen overdose in patients with an ALT >1000 IU/L. However, the concentrations of APAP-CYS during therapeutic dosing, in cases of acetaminophen toxicity from repeated dosing and in cases of hepatic injury from non-acetaminophen hepatotoxins have not been well characterized. The objective of this study is to describe APAP-CYS concentrations in these clinical settings as well as to further characterize the concentrations observed following acetaminophen overdose.     

New [1,2,4]triazolo[4,3-c]quinazoline enhances cisplatin- and temozolomide-induced growth inhibition and apoptosis in HL-60 cells

The purpose of this study was to investigate the therapeutic potential of a newly synthesized [1,2,4]triazolo[4,3-c]quinazoline (NTCHMTQ) alone and in combination with two anticancer drugs (cisplatin and temozolomide) against HL-60 leukemia cell line. The IC50 value of NTCHMTQ toward HL-60 cells was 19.7 microM. No apoptosis and cell cycle changes were observed in cells treated with 5 microM NTCHMTQ alone. Combination of non-toxic concentrations of NTCHMTQ (1-5 microM) with cisplatin or temozolomide sensitized HL-60 cells to these two drugs and significantly enhanced their efficacies, that is illustrated by combination indexes, sub-G0 cell fraction, apoptotic DNA fragmentation and caspase-3 activity. The results suggest that combined therapy of non-toxic concentrations of NTCHMTQ with chemotherapeutics may provide synergistic regimen for treatment of leukemia. However, further in vitro and in vivo experimental drug-cell and drug-drug studies are warranted.     

DNA adducts and DNA damage by antineoplastic and carcinogenic N-nitrosocompounds

Summary Mechanisms of DNA adduct formation by antineoplastic 2-chloroethyl-N-nitrosoureas (CNUs) and of DNA damage induced by these compounds as well as by carcinogenic 2-hydroxyalkylnitrosamines are discussed. CNUs are monofunctional and bifunctional alkylating agents that form, in a quantitatively minor reaction, DNA-DNA crosslinks (XL). In vitro, by far the most abundant alkylation products of DNA are those resulting from 2-hydroxyethylation. The reaction sequence responsible for 2-hydroxyethylation comprises intermediate oxazolidine ring closure followed by generation of 2-hydroxyethylnitrosourea and ethylene oxide. Oxadiazolium intermediates have not been found to play a role. In contrast to the in vitro experiments, in vivo 2-hydroxyethyl adducts are formed to a much lesser extent und 2-chloroethyl adducts are predominant in rat kidney DNA. 2-Hydroxyethylation of phosphate groups introduces extreme instability into the sugar-phosphate backbone since the resulting phosphotriester rapidly breaks down through a dioxaphospholane ring intermediate. Measurements of DNA XL in target tumor tissue and in bone marrow provides a sensitive tool for evaluation of hormone-linked cytotoxic agents.The potent environmental carcinogen N-nitrosodiethanolamine (NDELA) has been found to be activated in the rat liver by a two-step metabolic transformation sequence involving alcohol dehydrogenase and, subsequently, sulfotransferase. Evidence for this mechanism is provided by measuring DNA single strand breaks in rat liver DNA and by studying the effect of various enzyme inhibitors on the extent of DNA damage induced in vivo by NDELA and its metabolites.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28-March 1, 1986     

慢性淋巴细胞性甲状腺炎伴甲亢的识别和处理

慢性淋巴细胞性甲状腺炎伴甲亢,又称桥本甲状腺毒症(Hashitoxicosis),俗称桥本甲亢,属自身免疫性甲状腺炎(ATD)的一种,而ATD又囊括了甲状腺的多种炎症改变。因此,桥本甲状腺毒症的识别实为多种伴血清甲状腺激素水平升高的甲状腺炎性改变之间的鉴别诊断。桥本甲状腺毒症通常发生于慢性淋巴细胞性甲状腺炎(桥本病)的初期,占慢性淋巴细胞性甲状腺炎不足5%。可因与甲状腺相关的自身抗体与抗原反应时所致的甲状腺滤泡上皮受炎性破坏有关,也可因存在有兴奋性促甲状腺激素受体抗体(TRAb),刺激尚未受到自身免疫性炎症破坏的腺体组织,使甲状腺激…     

Characterization of adducts of ethanol metabolites with cytochrome c

Cytochrome c (cyt c) is found in the mitochondria of all mammalian cells where hydrogen peroxide is produced as a byproduct of the electron transport chain. In the presence of peroxide cyt c generates a ferryl heme and radicals at Tyr residues (Barr et al., 1996). These radicals can be transferred to Trp residues within the protein or to Tyr- and Trp-containing peptides (Deterding et al., 1998). We report that addition of ethanol to this system of cyt c plus peroxide results in replacement of the Tyr/Trp radicals by 1-hydroxyethyl radicals (HER), and covalent binding of up to 10 mol of ethanol per mol of cyt c. In the absence of exogenously added peroxide, ethanol incorporation to cyt c is attained also with a reconstituted system of the ethanol-inducible cytochrome P-4502E1 isozyme. Comparative studies with myoglobin and apomyoglobin suggest that the heme is necessary for ethanol adduction of the protein to occur. Structural analysis by mass spectrometry of the tryptic digestion fractions of adducted cyt c is consistent with several peptides bearing one-to-three acetaldehyde moieties on Lys residues, and three distinct Tyr/Trp-containing peptides: P[28-53], P[56-73], P[73-91] carrying one-to-two HER. The x-ray crystallographic structure of cyt c shows that the Tyr/Trp residues in the adducted peptides are in close proximity to the heme. In conclusion, our data show that ethanol metabolites alkylate cyt c under oxidative stress and point to HER-Tyr/Trp adducts as plausible markers of alcoholism.     

DNA adducts of medicinal drugs: some selected examples

Summary A few selected medicinal drugs, diamminedichloroplatinum (II) componds, mitomycin C, psoralens, and diethylstilbestrol are briefly reviewed with respect to the formation and biological significance of their DNA adducts. Different types of adducts, e.g., DNA intrastrand crosslinks, DNA interstrand crosslinks, or monoadducts appear to represent the critical DNA lesions of the different drugs, accounting for cytotoxicity and carcinogenicity. Thus, these examples serve to illustrate the complexity of DNA adduct formation and its biological sequelae.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986     

吸烟、DNA加合物与慢性阻塞性肺疾病的关系

目的探讨DNA加合物(DNA-BPDE)水平与吸烟及DNA-BPDE水平与慢性阻塞性肺疾病(COPD)的关系.方法应用竞争性酶联免疫吸附测定(ELISA)方法,检测5组SD大鼠在不同被动吸烟时间下DNA-BPDE的水平.检测68名健康人(包括吸烟与不吸烟),88例吸烟COPD患者和87名健康吸烟者DNA-BPDE水平,以及后两者各20名外周血淋巴细胞在香烟代谢产物二氢二醇环氧苯并[a]芘(BPDE)刺激前、后DNA-BPDE水平.应用流式细胞仪检测淋巴细胞的凋亡.结果(1)吸烟与DNA-BPDE的关系①大鼠的DNA-BPDE水平随吸烟天数的增加而增加(r=0.90);②68名健康人中,DNA-BPDE水平随吸烟指数的增加而呈增加的趋势(r=0.67).(2)DNA-BPDE水平与COPD的关系①COPD与健康吸烟者的DNA-BPDE水平(/107核苷酸)分别为91±23和67±15,两者比较差异有显著性(P＜0.01);② 20名COPD患者淋巴细胞DNA-BPDE水平在BPDE刺激前、后分别为94.5±24.1和106.1±27.3,而健康吸烟者淋巴细胞DNA-BPDE水平在BPDE刺激前、后分别为68.3±15.8和72.5±16.7;BPDE刺激前、后DNA-BPDE和DNA-BPDE的增加值两者比较差异均有显著性(P＜0.01).(3)COPD与健康吸烟者淋巴细胞在相同浓度BPDE刺激下,COPD组T细胞和B细胞的凋亡率为18.5%±3.1%和5.8%±0.6%,而健康吸烟组分别为9.5%±1.8%和5.2%±0.7%.COPD组凋亡细胞数明显增高,且以T淋巴细胞凋亡为主,两组的T淋巴细胞凋亡率比较差异有显著性(P＜0.01).结论吸烟可致DNA-BPDE的形成,COPD患者DNA-BPDE水平增高及导致细胞的DNA损伤与凋亡可能与COPD的发病有关.     

Recognition and treatment of cardiogenic shock

Cardiogenic shock has long been a difficult problem for clinicians. The most common cause is left ventricular pump failure after myocardial infarction, but other important causes include mechanical complications of infarction, right ventricular dysfunction, prolonged cardiopulmonary bypass, valvular disease, and cardiomyopathy. Cardiogenic shock is the leading cause of in-hospital death after myocardial infarction. Despite advances in management of heart failure and acute myocardial infarction, clinical outcomes had remained frustratingly poor, with reported mortality rates ranging from 50 to 80%. Recently, however, survival rates have been improving. Improved understanding of the pathophysiology of cardiogenic shock has led to renewed emphasis on the notion that stunned or hibernating myocardium may recover function with hemodynamic support and restoration of flow. This concept has underscored the importance of expeditious initiation of supportive measures to maintain blood pressure and cardiac output, including both medications and intraaortic balloon counterpulsation. Finally, the theory that coronary revascularization would be beneficial by reversing the vicious cycle in which ischemia causes myocardial dysfunction, which in turn worsens ischemia, which had been supported by an extensive body of observational and registry studies, has now been strongly buttressed by the results of two randomized, controlled trials, both of which show improved mortality with early revascularization for cardiogenic shock in the setting of acute infarction.     

Recognition and treatment of juvenile-onset spondyloarthritis

PURPOSE OF REVIEW: The purpose of this review is to discuss the classification, diagnosis and management of juvenile-onset spondyloarthritis. RECENT FINDINGS: There have been changes in the classification criteria for juvenile-onset spondyloarthritis and magnetic resonance imaging has allowed for earlier detection of disease. Additionally, tumor necrosis factor-alpha blockers have been shown to be effective in the treatment of ankylosing spondylitis. There is evidence to suggest that early treatment may lead to better response. A high percentage of patients with enthesitis-related arthritis progress to develop ankylosing spondylitis within 10 years after presentation. Patients with juvenile-onset ankylosing spondylitis appear to have poorer functional outcomes. SUMMARY: Juvenile-onset spondyloarthritis has variable clinical features that may lead to significant impairments. Improved classification criteria exist, but better techniques that are more sensitive are needed to diagnose disease earlier. New therapies appear to improve outcomes, but randomized controlled trials are needed in this population of patients.