Endogenous Metabolic Processes Research Articles

Abstract C52: Induction of homologous recombination by DNA nicks

Abstract Homologous recombination (HR) is a major mechanism of DNA repair, such that mutations in HR genes are associated with tumor susceptibility. We have used a recently adapted system for genome engineering to study HR mechanisms in mammalian cells. Clustered regularly-interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems that specifically cleave nucleic acids of invading pathogens. The effector phase of CRISPR system in Staphylococcus pyogenes consists of two main components, the endonuclease Cas9 and RNA strands that dictate the recognition sequence cleaved by Cas9. The activity of Cas9 resides in 2 nucleolytic domains, HNH-like and RuvC1-like, such that mutating the active site of either of these domains turns Cas9 into a DNA nicking enzyme. Here we applied the commonly used DR-GFP HR reporter to investigate the repair of both DNA double-strand breaks (DSBs) and nicks induced by Cas9 in human cells. We show that a DSB, introduced by wild-type (wt) Cas9 and a guide RNA (gRNA) directed to the I-SceI site of the DR-GFP reporter, induces HR at levels similar to the I-SceI endonuclease, demonstrating the robustness of the system and confirming the potential of the CRISPR system as a genome engineering tool. Cas9 with mutations in either of the nucleolytic domains also stimulates HR, albeit at much lower levels, suggesting that a nick can also be repaired by HR. Stimulation of HR by DNA nicks is not reduced by PARP inhibition and has the same genetic requirements as DSB-induced HR. Given that SSBs are induced by endogenous DNA metabolic processes as well as by many anti-cancer treatments, our results may advance the understanding of mechanisms responsible for repair of these abundant DNA lesions. Citation Format: Lianne E.M. Vriend, Przemek M. Krawczyk, Maria Jasin. Induction of homologous recombination by DNA nicks. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C52.

DNA damage in lens epithelium of cataract patientsin vivoandex vivo

DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.

Diagnostic Value of Exhaled Carbon Monoxide as an Early Marker of Exacerbation in Children with Chronic Lung Diseases

Chronic airways infection and inflammation are leading causes of morbidity and mortality in chronic lung diseases (CLD). Pulmonary exacerbations are major causes of morbidity in CLD. Exhaled carbon monoxide (eCO) is a product of endogenous metabolic processes whose presence in exhaled breath is considered an index of inflammatory processes. Objective. To evaluate carbon monoxide (eCO) as inflammatory marker for early detection of acute exacerbation in CLD. Methods. Case control study included 40 children with CLD (twenty in exacerbation, group I and twenty in quiescent period, group II) recruited from the Chest Clinic, Children's Hospital, Ain Shams University. Twenty apparently healthy children were included as controls (group III). Results. Patients' mean age was 9.98 ± 3.29 years: 24 (60%) males and 16 (40%) females. The mean eCO level among patients during exacerbation was 5.35 ± 1.35 (ppm) compared to 2.65 ± 0.49 (ppm) in quiescent stage and 1.30 ± 0.47 (ppm) in controls. eCO cutoff value discriminating cases and control was 1.5 (ppm) (sensitivity; 100% and specificity 70%) and cutoff value discriminating group I from group II was 3 (ppm) (sensitivity: 100% and specificity: 100%). Conclusion. Exhaled CO can be considered a noninvasive early marker of acute exacerbation of CLD.

Assessing stable isotope dynamics of diapausing Calanus finmarchicus and C. hyperboreus during the overwintering period: a laboratory experiment

This study aimed at describing changes in the stable isotopic composition of late copepodite stage V (CV) subarctic marine copepods (Calanus finmarchicus and C. hyperboreus) during overwintering non-feeding periods. Diapausing stage CVs sampled in deep waters of the Lower St. Lawrence Estuary (Québec, Canada) in late-September 2009 were monitored for 4 months under controlled laboratory conditions. CVs and newly moulted adults were analyzed for δ13C and δ15N signatures as well as lipid, carbon and nitrogen content. Lipids were extracted in half of the samples to compare δ13C of individuals with and without lipids and to evaluate the accuracy of mass balance correction models for δ13C under lipid influence. Lipid content generally decreased with time for both species, which was reflected in an increase of δ13C values of CVs but a constant δ13C in newly moulted adults. Accordingly, lipid extraction resulted in an increase of δ13C in CVs and adults. The mean δ13C signature of lipid-extracted individuals remained constant through the time for CVs of both species and for C. finmarchicus adults. δ15N signatures of individuals increased after lipid extraction, but this did not result in a constant value over time, suggesting that several endogenous metabolic processes affected nitrogen isotopic content. The accuracy of the mass balance model differed between species and stages, suggesting that lipid extraction should always be performed prior to applying mathematical corrections.

EVALUATION OF THE PRESENT INFORMATION ON THE MECHANISMS LEADING TO FLOWER BUD INDUCTION, EVOCATION AND DIFFERENTIATION IN OLEA EUROPAEA

Environmental factors such as suitable light and thermal conditions are essential for the reproductive development of olive buds. However, the tree and its buds have to be in a suitable responsive physiological state in order to respond. Furthermore sufficient well developed buds from the previous season with a good nutritive balance, capable to differentiate are required the tree. In the present paper, we are demonstrating the changes in the nutritional state of the tree developing in ON and OFF years affecting the responsiveness of the buds and level of vegetative growth to the environmental conditions. After a heavy crop a nutrient dilution occurred and vegetative growth is inhibited. Thus, a following growing season is required to regain a good nutrient balance for generating vegetative growth with sufficient buds responsive to environmental differentiation stimuli for the initiation of the next ON year. INTRODUCTION The lack of regularity in reproductive organ development leading in many cases to severe alternate bearing is one of the major economical drawbacks of today's olive industry. Although a considerable amount of work has been devoted during the second half of the last century to this problem, our understanding of the metabolism involved and ability to control alternate bearing is still limited. The potential to induce flower bud formation is governed both by the endogenous metabolic processes and the environment, mainly the thermal conditions. It is presently quite clear that both the late spring and winter temperatures play a significant role in controlling the differentiation of reproductive buds. However, neither the required summer nor the low winter temperature regimes are clearly defined. Even the nature of the responses to temperature is still disputed among the various workers in the field. Laboratory experiments with cuttings indicated that lateral bud can develop vegetatively or reproductively according to the thermal conditions during fall and winter. Thus, the genetic potential to initiate flower bud induction and differentiation developing shoots is environmentally dependent. Reproductive buds were reported to show some changes in their RNA already at the end of the summer, however, these buds will develop reproductively only if suitable winter conditions will prevail. The present available data indicate that neither summer-fall nor mid-winter seasons could individually be responsible for flower bud initiation. The developing fruits the tree have a major effect controlling shoot growth as well as the metabolism leading to reproductive development in the buds. The protein composition in leaves of fruiting and non-fruiting trees differs, with specific ones developing in the leaves of each group (Lavee and Avidan, 1994). Furthermore, a conjugated polyphenol, chlorogenic acid, related to the cinnamic acid pass-way is accumulating in leaves of fruiting trees and specifically inhibits the winter differentiation of the buds (Lavee, 1989). The messenger from the developing fruits to change the leaves' metabolism is believed to be a specific gibberellin. While harvest time is effecting bud differentiation fruit load effects has in addition also a major effect shoot growth. The lack of shoot growth a heavily fruit bearing olive tree is directly related to fruiting alternation as no potential buds for differentiation are developing. This lack of growth occurs although the level of carbohydrates in the leaves was found similar in on and off years. The aim of this work was to study the situation of the olive tree and its fluctuation during and after a high

Inflammation and autoimmunity: risk assessment and predictive approaches through the analytical armamentarium

The absence of a clear understanding of human chronic-disease, especially the absence of a clear pathogenesis, has hindered implementation of targeted predictive and preventive medicine. But the emerging sciences of metagenomics and proteomics have now clarified a mechanism whereby not just one or two discrete pathogens can drive the disease process, but where a very large number of viral, fungal and bacterial species, accumulating in the human body over the course of a lifetime, progressively weaken the human innate immune system. Homo sapiens is not a sterile compartment, but a super-organism harboring thousands of species of microbes in its cells and tissues. Those microbes which have evolved to persist inside nucleated and phagocytic cells can engender both genomic and proteomic interference within the host cell metabolism, precipitating the disease processes. Key components of this microbiota persist by reducing expression of, and by, the VDR Nuclear Receptor, which is at the heart of the human innate immune system. Only in Homo sapiens is the VDR responsible for expression of Cathelicidin, TLR2, TACO and a number of other endogenous anti-microbials. Animals have different antimicrobial defenses, and thus cannot properly model human chronic disease. Pathogens already known to persist by dysregulating the human VDR include EBV, Mycobacteria, Borrelia, Aspergillus and HIV. Antimicrobial therapy is difficult, as the diseased VDR cannot be simply restored by endogenous metabolic processes, as it can in healthy individuals. Nor can antibiotics function in-vivo in the absence of a competent innate immune system. We have shown that the drug Olmesartan can be retargeted to function as a VDR agonist, allowing innate immunity to recognize the pathogens, and reverse the disease process. Recovery is very slow, as care must be exercised to ensure the resulting cytokine-storm does not become life-threatening. Over the last decade, we have implemented a worldwide clinical collaboration to demonstrate ‘proof of concept’ efficacy in hundreds of subjects with serious chronic disorders, ranging from schizophrenia and manic depression, through ALS and MS, to sarcoidosis and arthritis. It seems certain that the current epidemic of chronic disease can not only be controlled, it can be reversed by implementation of preventative measures aimed at minimizing the accumulation of pathogenic organisms into the human microbiome.

Volatile Disease Biomarkers in Breath: A Critique

Hundreds of volatile organic compounds (VOCs) are released from human body fluids. Some of them are produced by endogenous metabolic processes in and on the body, and others are derived from the environment. Expressions of some endogenous VOCs can be affected by pathophysiological changes, and several disease-specific volatile biomarkers have been identified and used as diagnostic aids. Monitoring volatile disease markers is attractive since the procedure can be performed in a noninvasive manner with little or no exposure to biohazardous body fluids. Although many VOCs have been claimed as potential biomarkers, only a few compounds have been consistently demonstrated and approved for clinical applications. This is mainly because (1) many of the putative markers are present in the environment as well as in the body and their levels in the environment are often higher than those in the body, (2) there are a large individual variation in the concentrations of biomarkers within diseased and/or healthy subjects, and (3) the origin and biosynthetic pathway of the claimed biomarkers have been frequently neglected. Unfortunately, these aspects have often been ignored in many studies. Here, we review a number of publications that have identified volatile disease biomarkers in breath, argue that many of these have not demonstrated to actually underlie the differences in volatile profiles between diseased patients and healthy subjects, speculate on the reasons for this lack of success, and suggest potential approaches that may provide a better chance of identifying disease biomarkers.

Replication of Damaged Genomes

Cellular DNA is continuously assaulted by chemical and physical agents that arise from both endogenous metabolic processes as well as exogenous insults. Commonly encountered environmental agents include polyaromatic hydrocarbons, polycyclic aromatic amines, the ultraviolet component of sunlight, and ionizing radiation, among many others. Although the kinds of damages and the mechanisms involved in their interaction with DNA vary widely, genotoxic agents alter the structure of DNA in ways that may result in permanent alterations in the DNA sequence or in cell death. To avoid these consequences, cells have evolved countermeasures to reduce the biological consequences of DNA damage. These mechanisms are highly conserved and are present in all eukaryotic cells. In general, cellular responses include the detection of damage, signal transduction to halt cell cycle progression, and the recruitment of repair mechanisms that are tailored to the specific kind of damage. If replication-blocking damage remains when cells enter S-phase, then tolerance mechanisms in the form of complex recombination mechanisms or translesion DNA synthesis using accessory DNA polymerases exist. These mechanisms complete the replication of damaged genomes and suppress cytotoxicity, but at the potential cost of mutagenesis and genomic instability. This review focuses on error-prone mechanisms, including a discussion of the Y-family of DNA polymerases, current concepts of DNA polymerase switching mechanisms, and their relevance to cancer and cancer prevention.

Antioxidant Efficacy on Human Skin in vivo Investigated by UVA-Induced Chemiluminescence Decay Analysis via Induced Chemiluminescence of Human Skin

Background/Aims: Exogenous factors (e.g. physical: UV irradiation; or chemical: hydrogen peroxide) and endogenous metabolic processes (e.g. cellular respiration, oxidative burst, etc.) generate oxidative stress in living tissues which are in balance with enzymatic antioxidative systems and ingested antioxidants under normal conditions. These complex biological reactions are accompanied by chemiluminescence (ultraweak photon emission). However, knowledge about the chemiluminescence decay characteristics of human skin and the modulatory influence of topically applied antioxidants is still scarce. Methods: Using ICL-S (induced chemiluminescence of human skin), a highly sensitive in vivo method, the decay characteristics of UVA-induced photon emission caused by different UVA doses were investigated in detail. In addition, modulatory properties of topical antioxidant pretreatment were examined for 2 weeks. Results: UVA-induced chemiluminescence signals were generally characterized by two distinct decay phases: an initial burst (0–5 s), contributing approximately 80% of the complete signal with an inverse dose-response relationship (UVA dose vs. chemiluminescence intensity), followed by a second decay phase (delayed chemiluminescence, 5–200 s) showing a direct correlation. Antioxidant pretreatment caused a reduction in signal intensity of approximately 50%, which was calculated by signal integration and confirmed using the modulation of the intersection point of decay curves resulting from irradiation with different UVA doses at constant intensity with and without treatment. Conclusion: In addition to the established UVA filter testing (independent from UVB filter content) on human skin in vivo, ICL-S is also a valuable tool for the efficacy testing of topically applied antioxidants under in vivo conditions in humans. The first rapid, but short, decay phase not only provides approximately 80% of the complete chemiluminescence signal, but is also essential for the investigation of antioxidant-mediated effects. Chemiluminescence signal modulations induced by UVA intensity reduction (e.g. UV filters in daily care products) can be clearly distinguished from antioxidant-mediated signal modulations. The probe head dimensions permit comprehensive in vivo testing in humans on practically every skin area (e.g. arms, legs, back, abdomen and face).

Abstract 1834: Sex disparities in cancer mortality

Abstract INTRODUCTION: Previous research has noted that cancer mortality rates are higher for males than females, but no extensive analysis of this has been conducted. We wanted to study the extent of sex disparities in cancer mortality and if these disparities persist after adjustment for characteristics of the person and cancer. METHODS: We extracted U.S. vital statistics mortality data and cancer incidence and survival data from the SEER*Stat system for 36 cancers and undertook age-adjusted mortality rates and survival analyses. For the age-adjusted mortality rate analyses we extracted mortality count, person-years, and mortality rate per 100,000 for each cancer, stratified by sex and age for the period 1977-2006. Male-to-female mortality rate ratios (MRRs) were calculated. For each cancer, we plotted sex-specific mortality rates and male-to-female MRRs. For the survival analyses, we extracted patient id, age and date of diagnosis, sex, primary site, stage, grade, survival time, vital status and cause of death. Relative hazards for the 5-year period following diagnosis were estimated from Cox proportional hazard models adjusted for age, stage, grade, and year of diagnosis. RESULTS: For the vast majority of cancers, age-adjusted mortality rates were higher among males than females; the 5 cancers with the highest male-to-female MRR were lip (5.51, 95% confidence interval (CI):5.05-6.03), larynx (5.37, 95%CI:5.29-5.45), hypopharynx (4.47, 95%CI:4.30-4.65), esophagus (4.08, 95%CI:4.05-4.11) and urinary bladder (3.36, 95%CI:3.34-3.39). Only 3 cancers had a higher mortality rate among females than males: peritoneum, omentum and mesentery (MRR=0.39, 95%CI:0.38-0.41); gallbladder (0.56, 95%CI:0.55-0.57); and anus, anal canal and anorectum (0.78, 95%CI:0.75-0.81). The survival analyses indicated that males had a significantly increased risk of death, relative to females, for the cancers: anus, anal canal and anorectum (hazard ratio: 1.21, 95%CI:1.03-1.44); colon and rectum (1.08, 95%CI:1.07-1.09); floor of mouth (1.29, 95%CI:1.04-1.59); larynx (1.09, 95%CI:1.01-1.18); liver and intrahepatic bile duct (1.20, 95%CI:1.14-1.26); lung and bronchus (1.19, 95%CI:1.17-1.20); pancreas (1.06, 95%CI:1.03-1.08); small intestine (1.16, 95%CI:1.04-1.29); and soft tissue including heart (1.12, 95%CI:1.03-1.21). Females had higher mortality risks for the cancers tongue (0.87, 95%CI:0.81-0.94) and urinary bladder (0.83, 95%CI:0.81-0.86). DISCUSSION: Male cancer mortality rates were higher than equivalent female rates for the majority of cancers studied. Moreover, males also had higher risk of cancer mortality following diagnosis, relative to females, for many cancers even after adjustment for age of diagnosis, stage, grade and year of diagnosis. These observations indicate that there are sex differences in exposure and/or endogenous metabolic processes which modify risk of both cancer incidence and cancer mortality following diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1834.

Quantification of γH2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming gammaH2AX(1). Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)(2). Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB(2,3). This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning approximately 2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete gammaH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy(2). The loss of gammaH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary(4-8). The disappearence of gammaH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C(5,6). Further, removal of gammaH2AX by redistribution involving histone exchange with H2A.Z has been implicated(7,8). Importantly, the quantitative analysis of gammaH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of gammaH2AX foci in gamma-irradiated adherent human keratinocytes(9).

Toxic Alcohol Ingestions

The ingestion of toxic alcohols, specifically ethylene glycol (EG) and methanol, provides unique therapeutic challenges for emergency personnel. If untreated, these agents can result in significant morbidity and mortality. The toxicity of EG and methanol is dependent on endogenous metabolic processes rather than the actual parent compound ingested. These metabolites lead to the characteristic metabolic acidosis and cellular dysfunction typically seen with toxic alcohol ingestions. Fortunately, several options exist for the treatment of these ingestions, which, if implemented in the appropriate time period, can significantly decrease associated morbidity and mortality. The intent of this review is to discuss relevant management issues associated with EG and methanol intoxication in the emergency department.

Impact of diets and nutrients/drugs on early epigenetic programming

Specific, often unbalanced diets are used to circumvent the metabolic defects of patients with monogenic inborn errors of metabolism. Human epidemiological studies and appropriately designed dietary interventions in animal models have provided considerable evidence to suggest that nutritional imbalance and metabolic disturbances, during critical time windows of developmental programming, may have a persistent effect on the health of the child and later in adulthood. Thus patients with monogenic inborn errors of metabolism may also suffer additional types of alterations due to the lack or excess of key nutrients. Interactions of nutrients with the epigenetic machinery lead to epigenetic changes associated with chromatin remodelling and regulation of gene expression that underlie the developmental programming of pathological consequences in adulthood. Today, with the explosion of new technologies, we can explore on a large scale the effects of nutrients on the level of expression of thousands of expressed genes (nutritional genomics and epigenomics), the corresponding protein products and their posttranslationally modified derivatives (proteomics), and the host of metabolites (metabolomics) generated from endogenous metabolic processes or exogenous dietary nutrients and can establish the relationship between these biological entities and diet, health or disease. The combination of these various lines of research on epigenetic programming processes should highlight new strategies for the prevention and treatment of inborn errors of metabolism.

Repair of Genome Destabilizing Lesions

Living organisms are constantly exposed to detrimental agents both from the environment (e.g. ionizing radiation, ultraviolet light, natural and synthetic chemicals) and from endogenous metabolic processes (e.g. oxidative and hydrolytic reactions), resulting in modifications of proteins, lipids and DNA. Proteins and lipids are degraded and resynthesized, but the DNA is replicated only during cell division, when DNA damage may result in mutation fixation. Thus the DNA damage generated has the potential to lead to carcinogenesis, cell death, or other genetic disorders in the absence of efficient error-free repair. Because modifications in DNA sequence or structure may be incompatible with its essential role in preservation and transmission of genetic information from generation to generation, exquisitely sensitive DNA repair pathways have evolved to maintain genomic stability and cell viability. This review focuses on the repair and processing of genome destabilizing lesions and helical distortions that differ significantly from the canonical B-form DNA in mammalian cells. In particular, we discuss the introduction and processing of site-specific lesions in mammalian cells with an emphasis on psoralen interstrand crosslinks.

Recruitment for a pilot case control study of oxidative DNA damage and breast cancer risk.

Oxidative DNA damage (ODD) can result from numerous endogenous metabolic processes as well as from exposure to environmental and dietary oxidants. One important type of ODD that may have a role in carcinogenesis is the formation of hydroxylated DNA bases. Our major purpose was to determine the potential for subject accrual for a multisite case-control study of ODD and breast cancer risk within a large urban university medical center. We examined the levels of a hydroxylated thymine residue, 5-hydroxymethyl-2'-deoxyuridine in DNA obtained from the peripheral blood of 26 women with breast cancer and an age-matched group of 29 control women without breast cancer. The isolated DNA was analyzed for levels of 5-hydroxymethyl-2'-deoxyuridine by gas chromatography with mass spectral detection. Our recruitment methods resulted in a relatively high yield of eligible cases (72%) and a lower yield of controls (46%). We evaluated the dose-response relationship of ODD level to breast cancer risk, using quartiles of ODD. The covariate-adjusted odds ratio of breast cancer exceeded 2.0 for women in the highest quartile of ODD (compared with the lowest quartile), although this result was not statistically significant. ODD levels were significantly more variable among African-American controls (SD = 224.1) than among white controls (SD = 57.5), p < 0.001. Overall, these results suggest a possible slight increase in breast cancer risk among women in the highest ODD quartile, after adjusting for race, menopausal status, and family history of breast cancer.

Hydroperoxide-induced DNA damage and mutations

Hydroperoxides (ROOH) are believed to play an important role in the generation of free radical damage in biology. Hydrogen peroxide (R=H) is produced by endogenous metabolic and catabolic processes in cells, while alkyl hydroperoxides (R=lipid, protein, DNA) are produced by free radical chain reactions involving molecular oxygen (autooxidation). The role of metal ions in generating DNA damage from hydroperoxides has long been recognized, and several distinct, biologically relevant mechanisms have been identified. Identification of the mechanistic pathways is important since it will largely determine the types of free radicals generated, which will largely determine the spectrum of DNA damage produced. Some mechanistic aspects of the reactions of low valent transition metal ions with ROOH and their role in mutagenesis are reviewed with a perspective on their possible role in the biological generation of DNA damage. A survey of hydroperoxide-induced mutagenesis studies is also presented. In vitro footprinting of DNA damage induced by hydroperoxides provides relevant information on sequence context dependent reactivity, and is valuable for the interpretation of mutation spectra since it represents the damage pattern prior to cellular repair. Efforts in this area are also reviewed.

Metabonomics: Metabolic processes studied by NMR spectroscopy of biofluids

NMR spectroscopy of biofluids provides a wealth of information on endogenous metabolic processes in an organism. Details of various types of biofluid and the types of NMR experiment which are useful are given. The features of biofluid NMR spectra are described and practical details of spectral acquisition are also presented. However, spectra are very complex and many resonances have not been assigned. Therefore, in order to focus on significant differences between a set of spectra from control organisms and from abnormals (e.g., humans with diseases or animals in toxic situations), recourse is made to pattern recognition or chemometric methods. This is exemplifed using NMR spectra of a number of different biofluids such as urine, blood plasma, and cerebrospinal fluid. This approach is encapsulated in concept of metabonomics, a subject which can be regarded as complementary to studies of genome (genomics) and the proteins in an organism (proteomics). Metabonomics is defined as the quantitative measurement of multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.

Oxidative DNA damage processing in nuclear and mitochondrial DNA

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, to examine the repair of different types of oxidative lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage.

Oxidative DNA damage processing and changes with aging

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Identification and characterisation of a functional peroxidoxin from Leishmania major

Leishmania spp. encounter damaging oxygen metabolites from endogenous metabolic processes as well as from exogenous sources, such as inside the gut of the sandfly vector and within host macrophages. The recently described peroxidoxin protein family form part of a novel pathway for metabolising hydrogen peroxide that, in trypanosomatids, links peroxide reduction to NADPH oxidation via trypanothione. Here we report the cloning and characterisation of the Leishmania major peroxidoxin gene, tryparedoxin peroxidase ( TryP). TryP is a multi-copy gene arranged in a complex tandem array located on the size polymorphic homologues of chromosome 15. Northern analysis showed that TryP expresses a single 1.6 kb mRNA throughout promastigote development. TryP encodes a 22-kDa protein with two conserved cysteine-containing domains that defines it as a 2-Cys peroxidoxin. Purified recombinant TryP protein catabolised hydrogen peroxide in the presence of the tryparedoxin homologue from Crithidia fasciculata ( Cf-TryX), trypanothione, trypanothione reductase and NADPH. The demonstration that L. major utilises a three-protein peroxidase system confirms that this is a mechanism of protection against oxidative damage in this parasite.