Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts.

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead to the discovery of new targets and thus be of great importance for the development of preventive therapies. Because of this, we developed a real-time base excision repair assay with a specially designed double-stranded reporter oligonucleotides to measure the activity of hOGG1 in lysates of isolated mitochondria. This system presented here differs from the classical assays, in which an endpoint determination is performed via a denaturing acrylamide gel, by the possibility to measure the hOGG1 activity in real-time. In addition, to determine the activity of each enzymatic step (N-glycosylase and AP-lyase activity) of this bifunctional enzyme, a melting curve analysis can also be performed. After isolation of mitochondria from human fibroblasts using various centrifugation steps, they are lysed and then incubated with specially designed reporter oligonucleotides. The subsequent measurement of hOGG1 activity is performed in a conventional real-time PCR system.

Plasmapheresis for Rescue in Severe Encephalopathy and Multiorgan Failure from Fulminant Influenza (H3N2) Infection.

We are presenting a case of 4-years-old previously healthy male with coma, severe acute hepatitis and multiorgan failure in presence of Influenza infection. Literature review highlighted an immune-mediated pathophysiology for such presentations so the child underwent a trial of plasmapheresis which resulted in a rapid clinical improvement and child was discharge in his baseline neurologic status by day 14.

The activity of the DNA repair enzyme hOGG1 can be directly modulated by ubiquinol.

The DNA of human cells suffers about 1.000-100.000 oxidative lesions per day. One of the most common defects in this category is represented by 7,8-dihydro-8-oxoguanine. There are numerous exogenous effects on DNA that induce the intracellular generation of 7, 8-dihydro-8-oxoguanine. Therefore, a quantitatively sufficient repair of all occurring oxidative damaged guanine bases is often only partially feasible, especially in advanced age. Inadequate removal of these damages can subsequently lead to mutations and thus to serious diseases. All these aspects represent a dangerous situation for an organism. However, it is suspected that the amount of the 8-oxoguanine DNA glycosylase can be actively regulated on the level of gene expression by the redox-active properties of ubiquinol and thus its protein expression can be controlled. Using an real-time base excision repair assay including a melting curve analysis, the activity of the human 8-oxoguanine DNA glycosylase 1 was measured under the influence of ubiquinol. It was possible to observe a concentration-dependent increase in the activity of the 8-oxoguanine DNA glycosylase 1 under the influence of ubiquinol for the first time, both on purified and commercially acquired enzyme as well as on enzyme isolated from mitochondria of human fibroblasts. An increase in activity of this enzyme based on a change in cellular redox state caused by ubiquinol could not be confirmed. In addition, an increased gene expression of 8-oxoguanine-DNA glycosylase 1 under ubiquinol could not be observed. However, there was a change in bifunctionality in favor of an increased N-glycosylase activity and a direct interaction between ubiquinol and 8-oxoguanine DNA glycosylase 1. We suggest that ubiquinol contributes to the dissolution of a human 8-oxoguanine DNA glycosylase 1 end-product complex that forms after cutting into the sugar-phosphate backbone of the DNA with the resulting unsaturated 3'-phospho-α, ß-aldehyde end and thereby inhibits further enzymatic steps.

A New Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies.

Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result, e.g., in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital-basal-side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.

Age-Dependent Loss of Mitochondrial Function in Epithelial Tissue Can Be Reversed by Coenzyme Q10.

The process of aging is characterized by the increase of age-associated disorders as well as severe diseases. Due to their role in the oxidative phosphorylation and thus the production of ATP which is crucial for many cellular processes, one reason for this could be found in the mitochondria. The accumulation of reactive oxygen species damaged mitochondrial DNA and proteins can induce mitochondrial dysfunction within the electron transport chain. According to the "mitochondrial theory of aging," understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general, but the measurement of mitochondrial respiration in skin cells from cell cultures cannot completely reflect the real situation in skin. Here, we describe a new method to measure the mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using a XF24 extracellular flux analyzer to evaluate the effect of coenzyme Q10. We observed a decrease in mitochondrial respiration and ATP production with donor age corresponding to the "mitochondrial theory of aging." For the first time ex vivo in human epidermis, we could show also a regeneration of mitochondrial respiratory parameters if the reduced form of coenzyme Q10, ubiquinol, was administered. In conclusion, an age-related decrease in mitochondrial respiration and ATP production was confirmed. Likewise, an increase in the respiratory parameters by the addition of coenzyme Q10 could also be shown. The fact that there is a significant effect of administered coenzyme Q10 on the respiratory parameters leads to the assumption that this is mainly caused by an increase in the electron transport chain. This method offers the possibility of testing age-dependent effects of various substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.

PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity.

Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks.

Fluid Overload and Kidney Injury Score: A Multidimensional Real-Time Assessment of Renal Disease Burden in the Critically Ill Patient.

OBJECTIVE: Interruptive acute kidney injury alerts are reported to decrease acute kidney injury-related mortality in adults. Critically ill children have multiple acute kidney injury risk factors; although recognition has improved due to standardized definitions, subtle changes in serum creatinine make acute kidney injury recognition challenging. Age and body habitus variability prevent a uniform maximum threshold of creatinine. Exposure of nephrotoxic medications is common but not accounted for in kidney injury scores. Current severity of illness measures do not include fluid overload, a well-described mortality risk factor. We hypothesized that a multidimensional measure of renal status would better characterize renal severity of illness while maintaining or improving on correlation measures with adverse outcomes, when compared with traditional acute kidney injury staging. DESIGN: A novel, real-time, multidimensional, renal status measure, combining acute kidney injury, fluid overload greater than or equal to 15%, and nephrotoxin exposure, was developed (Fluid Overload Kidney Injury Score) and prospectively applied to all patient encounters. Peak Fluid Overload Kidney Injury Score values prior to discharge or death were used to measure correlation with outcomes. SETTING: Quarternary PICU of a freestanding children's hospital. PATIENTS: All patients admitted over 18 months. INTERVENTION: None. RESULTS: Peak Fluid Overload Kidney Injury Score ranged between 0 and 14 in 2,830 PICU patients (median age, 5.5 yr; interquartile range, 1.3-12.9; 55% male), 66% of patients had Fluid Overload Kidney Injury Score greater than or equal to 1. Fluid Overload Kidney Injury Score was independently associated with PICU mortality and PICU and hospital length of stay when controlled for age, Pediatric Risk of Mortality-3, ventilator, pressor, and renal replacement therapy use (p = 0.047). Mortality increased from 1.5% in Fluid Overload Kidney Injury Score 0 to 40% in Fluid Overload Kidney Injury Score 8+. When urine output points were excluded, Fluid Overload Kidney Injury Score was more strongly correlated with mortality than fluid overload or acute kidney injury definitions alone. CONCLUSION: A multidimensional score of renal disease burden was significantly associated with adverse PICU outcomes. Further studies will evaluate Fluid Overload Kidney Injury Score as a warning and decision support tool to impact patient-centered outcomes.

Accelerated Regeneration of ATP Level after Irradiation in Human Skin Fibroblasts by Coenzyme Q10.

Human skin is exposed to a number of harmful agents of which the ultraviolet (UV) component of solar radiation is most important. UV-induced damages include direct DNA lesions as well as oxidative damage in DNA, proteins and lipids caused by reactive oxygen species (ROS). Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, coenzyme Q10 (CoQ10 ) is used as a potent therapeutic in a number of diseases. We analyzed the mitochondrial respiration profile, the mitochondrial membrane potential and cellular ATP level in skin fibroblasts after irradiation. We observed an accelerated regeneration of cellular ATP level, a decrease in mitochondrial dysfunction as well as a preservation of the mitochondrial membrane potential after irradiation in human skin fibroblasts by treatment with CoQ10 . We conclude that the faster regeneration of the ATP level was achieved by a preservation of mitochondrial function by the addition of CoQ10 and that the protective effect of CoQ10 is primarily mediated via its antioxidative function. We suggest also that it might be further dependent on a stimulation of DNA repair enzymes by CoQ10 .

Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

Acute Kidney Injury in Pediatric Heart Failure.

Acute kidney injury (AKI) is very common in pediatric medical and surgical cardiac patients. Not only is it an independent risk factor for increased morbidity and mortality in the short run, but repeated episodes of AKI lead to chronic kidney disease (CKD) especially in the most vulnerable hosts with multiple risk factors, such as heart transplant recipients. The cardiorenal syndrome, a term coined to emphasize the bidirectional nature of simultaneous or sequential cardiac-renal dysfunction both in acute and chronic settings, has been recently described in adults but scarcely reported in children. Despite the common occurrence and clinical and financial impact, AKI in pediatric heart failure outside of cardiac surgery populations remains poorly studied and there are no large-scale pediatric specific preventive or therapeutic studies to date. This article will review pediatric aspects of the cardiorenal syndrome in terms of pathophysiology, clinical impact and treatment options.

Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum.

The cereal pathogen Fusarium graminearum threatens food and feed production worldwide. It reduces the yield and poisons the remaining kernels with mycotoxins, notably deoxynivalenol (DON). We analyzed the importance of gamma-aminobutanoic acid (GABA) metabolism for the life cycle of this fungal pathogen. GABA metabolism in F. graminearum is partially regulated by the global nitrogen regulator AreA. Genetic disruption of the GABA shunt by deletion of two GABA transaminases renders the pathogen unable to utilize the plant stress metabolites GABA and putrescine. The mutants showed increased sensitivity against oxidative stress, GABA accumulation in the mycelium, downregulation of two key enzymes of the TCA cycle, disturbed potential gradient in the mitochondrial membrane and lower mitochondrial oxygen consumption. In contrast, addition of GABA to the wild type resulted in its rapid turnover and increased mitochondrial steady state oxygen consumption. GABA concentrations are highly upregulated in infected wheat tissues. We conclude that GABA is metabolized by the pathogen during infection increasing its energy production, whereas the mutants accumulate GABA intracellularly resulting in decreased energy production. Consequently, the GABA mutants are strongly reduced in virulence but, because of their DON production, are able to cross the rachis node.

Poly(ADP-ribose)-mediated interplay of XPA and PARP1 leads to reciprocal regulation of protein function.

Poly(ADP-ribose) (PAR) is a complex and reversible post-translational modification that controls protein function and localization through covalent modification of, or noncovalent binding to target proteins. Previously, we and others characterized the noncovalent, high-affinity binding of the key nucleotide excision repair (NER) protein XPA to PAR. In the present study, we address the functional relevance of this interaction. First, we confirm that pharmacological inhibition of cellular poly(ADP-ribosyl)ation (PARylation) impairs NER efficacy. Second, we demonstrate that the XPA-PAR interaction is mediated by specific basic amino acids within a highly conserved PAR-binding motif, which overlaps the DNA damage-binding protein 2 (DDB2) and transcription factor II H (TFIIH) interaction domains of XPA. Third, biochemical studies reveal a mutual regulation of PARP1 and XPA functions showing that, on the one hand, the XPA-PAR interaction lowers the DNA binding affinity of XPA, whereas, on the other hand, XPA itself strongly stimulates PARP1 enzymatic activity. Fourth, microirradiation experiments in U2OS cells demonstrate that PARP inhibition alters the recruitment properties of XPA-green fluorescent protein to sites of laser-induced DNA damage. In conclusion, our results reveal that XPA and PARP1 regulate each other in a reciprocal and PAR-dependent manner, potentially acting as a fine-tuning mechanism for the spatio-temporal regulation of the two factors during NER.

Shortwave UV-induced damage as part of the solar damage spectrum is not a major contributor to mitochondrial dysfunction.

Because of the absence of a nucleotide excision repair in mitochondria, ultraviolet (UV)-induced bulky mitochondrial DNA (mtDNA) lesions persist for several days before they would eventually be removed by mitophagy. Long persistence of this damage might disturb mitochondrial functions, thereby contributing to skin ageing. In this study, we examined the influence of shortwave UV-induced damage on mitochondrial parameters in normal human skin fibroblasts. We irradiated cells with either sun-simulating light (SSL) or with ultraviolet C to generate bulky DNA lesions. At equivalent antiproliferative doses, both irradiation regimes induced gene expression of mitochondrial transcription factor A (TFAM) and matrix metallopeptidase 1 (MMP-1). Only irradiation with SSL, however, caused significant changes in mtDNA copy number and a decrease in mitochondrial respiration. Our results indicate that shortwave UV-induced damage as part of the solar spectrum is not a major contributor to mitochondrial dysfunction.

Mitochondrial DNA copy number - but not a mitochondrial tandem CC to TT transition - is increased in sun-exposed skin.

Mitochondrial DNA (mtDNA) mutations are causatively associated with photo-ageing and are used as biomarkers of UV exposure. The most prominent mitochondrial mutation is the common deletion (CD), which is induced in many tissues by oxidative stress. More photo-specific mutations might be CC to TT tandem transitions which arise from UV-induced cyclobutane pyrimidine dimers. As nucleotide excision repair is absent in mitochondria, this DNA damage can presumably not be repaired resulting in high mitochondrial mutation levels. Here, we analysed levels of the CD, a mitochondrial and a chromosomal tandem transition in epidermis and dermis from exposed and less UV-exposed skin. We also analysed mtDNA copy number, for which changes as a result of oxidative stress have been described in different experimental settings. Whereas mitochondrial tandem transition levels were surprisingly low with no discernible correlation with UV exposure, mtDNA copy number and CD were significantly increased in UV-exposed samples.

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts.

BACKGROUND: The Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing). RESULTS: In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. CONCLUSIONS: The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.