Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Conjugation of carcinogens by 6 class glutathione S-transferases : mechanisms and relevance to variations in human risk

Conjugation of chemicals with glutathione (GSH) can lead to decreased or increased toxicity. A genetic deficiency in the GSH S-transferase μ class gene M1 has been hypothesized to lead to greater risk of lung cancer in smokers. Recently a gene deletion polymorphism involving the human θ enzyme T1 has been described; the enzyme is present in erythrocytes and can be readily assayed. A rat θ class enzyme, 5-5, has structural and catalytic similarity and the protein was expressed in the Salmonella typhimurium tester strain TA1535. Expression of the cDNA vector increased the mutagenicity of ethylene dibromide and several methylene dihalides. Mutations resulting from the known GSH S-transferase substrate 1,2-epoxy-3-(4′nitrophenoxy)propane were decreased in the presence of the transferase. Expression of transferase 5-5 increased mutations when 1,2,3,4-diepoxybutane (butadiene diepoxide), 4-bromo-1,2-epoxybutane, or 1,3-dichloracetone were added. The latter compound is a model for the putative 1,2-dibromo-3-chloropropane oxidation product 1-bromo-3-chloroacetone. These genotoxicity and genotyping assays may be of use in further studies of the roles of GSH S-transferase θ enzymes in bioactivation and detoxication and any changes in risk due to polymorphism.

Carcinogenicity and genotoxicity of ethylene oxide : new aspects and recent advances

Long-term inhalation studies in rodents have presented unequivocal evidence of experimental carcinogenicity of ethylene oxide, based on the formation of malignant tumors at multiple sites. However, despite a considerable body of epidemiological data only limited evidence has been obtained of its carcinogenicity in humans. Ethylene oxide is not only an important exogenous toxicant, but it is also formed from ethylene as a biological precursor. Ethylene is a normal body constituent; its endogenous formation is evidenced by exhalation in rats and in humans. Consequently, ethylene oxide must also be regarded as a physiological compound. The most abundant DNA adduct of ethylene oxide is 7-(2-hydroxyethyl)guanine (HOEtG). Open questions are the nature and role of tissue-specific factors in ethylene oxide carcinogenesis and the physiological and quantitative role of DNA repair mechanisms. The detection of remarkable individual differences in the susceptibility of humans has promoted research into genetic factors that influence the metabolism of ethylene oxide. With this background it appears that current PBPK models for trans-species extrapolation of ethylene oxide toxicity need to be refined further. For a cancer risk assessment at low levels of DNA damage, exposure-related adducts must be discussed in relation to background DNA damage as well as to inter- and intraindividual variability. In rats, subacute ethylene oxide exposures on the order of 1 ppm (1.83 mg/m3) cause DNA adduct levels (HOEtG) of the same magnitude as produced by endogenous ethylene oxide. Based on very recent studies the endogenous background levels of HOEtG in DNA of humans are comparable to those that are produced in rodents by repetitive exogenous ethylene oxide exposures of about 10 ppm (18.3 mg/m3). Experimentally, ethylene oxide has revealed only weak mutagenic effects in vivo, which are confined to higher doses. It has been concluded that long-term human occupational exposure to low airborne concentrations to ethylene oxide, at or below current occupational exposure limits of 1 ppm (1.83 mg/m3), would not produce unacceptable increased genotoxic risks. However, critical questions remain that need further discussions relating to the coherence of animal and human data of experimental data in vitro vs. in vivo and to species-specific dynamics of DNA lesions.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Fred’s Flow (Canada) and Murphy Well (Australia) : thick komatiitic lava flows with contrasting compositions, emplacement mechanisms and water contents

Two Archaean komatiitic flows, Fred’s Flow in Canada and the Murphy Well Flow in Australia, have similar thicknesses (120 and 160 m) but very different compositions and internal structures. Their contrasting differentiation profiles are keys to determine the cooling and crystallization mechanisms that operated during the eruption of Archaean ultramafic lavas. Fred’s Flow is the type example of a thick komatiitic basalt flow. It is strongly differentiated and consists of a succession of layers with contrasting textures and compositions. The layering is readily explained by the accumulation of olivine and pyroxene in a lower cumulate layer and by evolution of the liquid composition during downward growth of spinifex-textured rocks within the upper crust. The magmas that erupted to form Fred’s Flow had variable compositions, ranging from 12 to 20 wt% MgO, and phenocryst contents from 0 to 20 vol%. The flow was emplaced by two pulses. A first ~20-m-thick pulse was followed by another more voluminous but less magnesian pulse that inflated the flow to its present 120 m thickness. Following the second pulse, the flow crystallized in a closed system and differentiated into cumulates containing 30–38 wt% MgO and a residual gabbroic layer with only 6 wt% MgO. The Murphy Well Flow, in contrast, has a remarkably uniform composition throughout. It comprises a 20-m-thick upper layer of fine-grained dendritic olivine and 2–5 vol% amygdales, a 110–120 m intermediate layer of olivine porphyry and a 20–30 m basal layer of olivine orthocumulate. Throughout the flow, MgO contents vary little, from only 30 to 33 wt%, except for the slightly more magnesian basal layer (38–40 wt%). The uniform composition of the flow and dendritic olivine habits in the upper 20 m point to rapid cooling of a highly magnesian liquid with a composition like that of the bulk of the flow. Under equilibrium conditions, this liquid should have crystallized olivine with the composition Fo94.9, but the most magnesian composition measured by electron microprobe in samples from the flow is Fo92.9. To explain these features, we propose that the parental liquid contained around 32 wt% MgO and 3 wt% H2O. This liquid degassed during the eruption, creating a supercooled liquid that solidified quickly and crystallized olivine with non-equilibrium textures and compositions.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

Numerical investigation of deformation and failure mechanisms of osteopontin-hydroxyapatite interfaces and mineralized collagen fibril arrays

This thesis is a comprehensive study of deformation and failure mechanisms in bone at nano- and micro-scale levels. It explores the mechanical behaviour of osteopontin-hydroxyapatite interfaces and mineralized collagen fibril arrays, through atomistic molecular dynamics and finite element simulations. This thesis shows some main factors contributing to the excellent material properties of bone and provides some guidelines for development of new artificial biological materials and medical implants.

Nonprofit Service Delivery for Government : Accountability Relationships and Mechanisms

When government purchases social services under contract from a nonprofit organisation, a clear accountability relationship is created. The NPO must give an account for the use of the funds and achievement of outcomes to the funder. This paper explores how accountability is enacted in two different types of funding relationships in Queensland. Support is found for the argument that different relationships have different approaches to accountability.

Structural damage detection method using frequency response functions

Structural damage detection using measured dynamic data for pattern recognition is a promising approach. These pattern recognition techniques utilize artificial neural networks and genetic algorithm to match pattern features. In this study, an artificial neural network–based damage detection method using frequency response functions is presented, which can effectively detect nonlinear damages for a given level of excitation. The main objective of this article is to present a feasible method for structural vibration–based health monitoring, which reduces the dimension of the initial frequency response function data and transforms it into new damage indices and employs artificial neural network method for detecting different levels of nonlinearity using recognized damage patterns from the proposed algorithm. Experimental data of the three-story bookshelf structure at Los Alamos National Laboratory are used to validate the proposed method. Results showed that the levels of nonlinear damages can be identified precisely by the developed artificial neural networks. Moreover, it is identified that artificial neural networks trained with summation frequency response functions give higher precise damage detection results compared to the accuracy of artificial neural networks trained with individual frequency response functions. The proposed method is therefore a promising tool for structural assessment in a real structure because it shows reliable results with experimental data for nonlinear damage detection which renders the frequency response function–based method convenient for structural health monitoring.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA

We report that tumor cells devoid of their mitochondrial genome (mtDNA) show delayed tumor growth and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory supercomplexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.