Thiols oxidation and covalent binding of BSA by cyclolignanic quinones are enhanced by the magnesium cation

A novel cyclolignanic quinone, 7-acetyl-3',4'-didemethoxy-3',4'-dioxopodophyllotoxin (CLQ), inhibits topoisomerase II (TOPO II) activity. The extent of this inhibition was greater than that produced by the etoposide quinone (EQ) or etoposide. Glutathione (GSH) reduces EQ and CLQ to their corresponding semiquinones under anaerobic conditions. The latter were detected by EPR spectroscopy in the presence of MgCl₂ but not in its absence. Semiquinone EPR spectra change with quinone/GSH mol ratio, suggesting covalent binding of GSH to the quinones. Quinone-GSH covalent adducts were isolated and identified by ESI-MS. These orthoquinones also react with nucleophilic groups from BSA to bind covalently under anaerobic conditions. BSA thiol consumption and covalent binding by these quinones are enhanced by MgCl₂. Complex formation between the parent quinones and Mg⁺² was also observed. Density functional calculations predict the observed blue-shifts in the absorption spectra peaks and large decreases in the partial negative charge of electrophilic carbons at the quinone ring when the quinones are complexed to Mg⁺². These observations suggest a possible role of Mg⁺² chelation by these quinones in increasing TOPO II thiol and/or amino/imino reactivity with these orthoquinones.     

Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

 The stearoyl-acyl carrier protein Δ⁹ desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O₂–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O₂ to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k _app≈3.5 s^–1 in 0.7 M NaN₃, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η¹–terminal structure at pH 7.8 (¹⁴N₃ ^– asymmetric stretch at 2073 cm^–1, resolved into two bands with ¹⁵N¹⁴N₂ ^–) and a μ-1,3 bridging structure at pH<7 (¹⁴N₃ ^– asymmetric stretch at 2100 cm^–1, shifted as a single band with ¹⁵N¹⁴N₂ ^–). Both adducts also exhibit an Fe–N₃ stretching mode at ≈380 cm^–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries. Received: 23 August 1996 / Accepted: 4 October 1996     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

A conundrum in molecular toxicology: Molecular and biological changes during neoplastic transformation of human cells

The process of multistage carcinogenesis lends itself to the concept that the effects of carcinogens are mediated through dose-related, multi-hit, linear changes. Multiplein vitro model systems have been developed that are designed to examine the cellular changes associated with the progression of cells through the different stages in the process; however, these systems may have inherent limitations due to the cell lines used for these studies, the manner of assessing the effects of the carcinogens, and the subsequent growth and differentiation of the exposed cells. Each of these variables results in increasing levels of uncertainty relative to the correlation of the events with the actual process of human tumor development. Therefore, the prediction of the ultimate effect of any carcinogen is difficult. Moreover, relationships between individual biological endpoints resulting from carcinogen treatment appear at best to be approximations. The presence of an activated carcinogen inside the cell can give rise to multiple outcomes, only some of which may be critical events. For example, site-specific modification of the 12th and 13th codons of H-ras is different than that in the adjacent 14th and 15th codons. It is interesting to speculate what effect these differences might have on a biological outcome, e.g., transformation to anchorage-independent growth. The use of different model systems to examine the effects of activated carcinogens also creates additional problems. Comparisons ofin vitro transformed cells with similar cells isolated from human tumors indicate that the culture environment appears to influence the expression of a particular phenotype, in that human tumor cells in culture express many of the same parameters as those found in cells transformed with carcinogensin vitro. If the process of transformation is linear, then less aggressive phenotypes should progress to a more aggressive transformed stage. However, in carcinogen-transformed human cells, the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fial to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cellsin vitro.Abbreviations AIG anchorage-independent growth - B[a]P benzo[a]pyrene - BPDE-I benzo[a]pyrene diol epoxide I - I-NP 1-nitrosopyrene - PCR polymerase chain reaction - PDL population doubling(s)     

Molecular dynamics simulations of excimer forming (+)-anti-BPDE-DNA adducts in aqueous solution

The chemical carcinogen (+)-anti BPDE preferentially binds covalently to the guanine base in the minor groove of DNA. Fluorescence spectroscopic studies have shown that the BPDE molecules bound to DNA can interact in their photo-excited state giving strong excimer fluorescence when bound to poly(dGdC) · poly(dGdC). It was suggested that the formation of such excited state complexes is most probable when the two (+)-anti-BPDE bind to guanines of adjacent base pairs on the two different strands of the DNA. In the present work a model for such an excimer forming DNA-BPDE double adduct system has been constructed and shown to be stable over a 300 ps molecular dynamics simulation in a water box. The model is a d(CG)₃ · d(CG)₃ molecule with two BPDE molecules bound to the guanines at the 4th position on each strand, located in the minor groove and each oriented towards the 5 end of the modified strand, respectively. The results of 300 ps MD simulation show that the two BPDE chromophores exhibited on the average a relative geometry favourable for excimer formation. The local structure at the adduct position was considerably distorted and the helix axis was bent. The modified bases were found to be paired through a stable single non-Watson Crick type of hydrogen bond. Correspondence to: A. Gräslund     

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109–121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117–123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.     

DNA adducts in humans after exposure to methylating agents

Human exposure to methylating agents appears to be widespread, as indicated by the frequent occurrence of methylated DNA adducts in human DNA. The high incidence of methylated DNA adducts even in humans thought not to have suffered extensive exposure to environmental methylating agents implies that chemicals of endogenous origin, probably N-nitroso compounds such as the strongly carcinogenic N-nitrosodimethylamine (NDMA), may be primarily responsible for their formation and raises the question of the carcinogenic risks associated with such exposure. In addition to accumulation of DNA damage, other factors (such as induced cell proliferation) appear to be important in determining the probability of induction of mutation or cancer by NDMA, implying that high to low dose risk extrapolations should not be based on the assumption of dose- or even adduct-linearity. Comparative studies of the accumulation and repair of methylated adducts in humans and animals treated with methylating cytostatic drugs do not reveal significant species differences. Based on this and the dosimetry of adduct accumulation in rats chronically exposed to very low doses of NDMA, it is suggested that the exposure needed to account for the levels of adducts found in human DNA may be of the order of hundreds of micrograms NDMA (or equivalent) per day, a level of exposure which may well represent a significant carcinogenic hazard for man.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

In vivo effect of L-ascorbic acid on benzo(α)pyrene metabolite-DNA adduct formation in rat liver

Pretreatment of male Wistar rats with L-ascorbic acid results in a decrease in thein vivo covalent binding of benzo(a)pyrene to hepatic nuclear DNA.In vitro formation of this adduct is also found to be low in liver slices and in liver nuclei of pretreated rats. No inhibition of the adduct formation is, however, observed when benzo (a) pyrene and exogenous DNA are incubated with liver microsomes isolated from ascorbic acid treated rats.It appears that the presence of ascorbate in the cellular or subcellular environment is essential for its inhibitory action.