The Impact of Chronic Disease on the Corrected QT (QTc) Value in Women in a British Columbia First Nations Population.

BACKGROUND: Indigenous women have higher rates of chronic disease than Indigenous men and non-Indigenous women. Long QT syndrome (LQTS) can be inherited or acquired; the latter may occur with chronic disease. A prolonged corrected QT value (QTc) is an independent risk factor for ventricular arrhythmias and sudden death, but few studies have quantified the impact of chronic disease on the QTc. We assessed the association between chronic disease and QTc prolongation in a population of First Nations women previously ascertained to study a high rate of inherited LQTS due to a unique genetic (founder) variant in their community. METHODS: This substudy focusing on women expands on the original research where patients with clinical features of LQTS and their relatives were assessed for genetic variants discovered to affect the QTc. Medical records were retrospectively reviewed and chronic diseases documented. Using multivariate linear regression, adjusting for the effect of genetic variants, age, and QTc-prolonging medications, we evaluated the association between chronic disease and the QTc. RESULTS: In total, 275 women were included. After adjustments, a prolonged QTc was associated with coronary artery disease (26.5 ms, 95% confidence interval [CI] 9.0-44.1 ms; P = 0.003), conduction system disease (26.8 ms, 95% CI 2.2-51.4 ms; P = 0.033), rheumatoid arthritis (28.9 ms, 95% CI 12.7-45.1 ms; P = 0.001), and type 2 diabetes mellitus (17.9 ms, 95% CI 3.6-32.3 ms; P = 0.015). CONCLUSIONS: This quantification of the association between chronic disease and QTc prolongation in an Indigenous cohort provides insight into the nongenetic determinants of QTc prolongation. Corroboration in other populations will provide evidence for generalisability of these results.

Effects of Sensorimotor Rhythm Modulation on the Human Flexor Carpi Radialis H-Reflex.

People can learn over training sessions to increase or decrease sensorimotor rhythms (SMRs) in the electroencephalogram (EEG). Activity-dependent brain plasticity is thought to guide spinal plasticity during motor skill learning; thus, SMR training may affect spinal reflexes and thereby influence motor control. To test this hypothesis, we investigated the effects of learned mu (8-13 Hz) SMR modulation on the flexor carpi radialis (FCR) H-reflex in 6 subjects with no known neurological conditions and 2 subjects with chronic incomplete spinal cord injury (SCI). All subjects had learned and practiced over more than 10 < 30-min training sessions to increase (SMR-up trials) and decrease (SMR-down trials) mu-rhythm amplitude over the hand/arm area of left sensorimotor cortex with ≥80% accuracy. Right FCR H-reflexes were elicited at random times during SMR-up and SMR-down trials, and in between trials. SMR modulation affected H-reflex size. In all the neurologically normal subjects, the H-reflex was significantly larger [116% ± 6 (mean ± SE)] during SMR-up trials than between trials, and significantly smaller (92% ± 1) during SMR-down trials than between trials (p < 0.05 for both, paired t-test). One subject with SCI showed similar H-reflex size dependence (high for SMR-up trials, low for SMR-down trials): the other subject with SCI showed no dependence. These results support the hypothesis that SMR modulation has predictable effects on spinal reflex excitability in people who are neurologically normal; they also suggest that it might be used to enhance therapies that seek to improve functional recovery in some individuals with SCI or other CNS disorders.

UVA-induced carbon-centred radicals in lightly pigmented cells detected using ESR spectroscopy.

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.

Mitochondrial protein-linked DNA breaks perturb mitochondrial gene transcription and trigger free radical-induced DNA damage.

Breakage of one strand of DNA is the most common form of DNA damage. Most damaged DNA termini require end-processing in preparation for ligation. The importance of this step is highlighted by the association of defects in the 3'-end processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) and neurodegeneration and by the cytotoxic induction of protein-linked DNA breaks (PDBs) and oxidized nucleic acid intermediates during chemotherapy and radiotherapy. Although much is known about the repair of PDBs in the nucleus, little is known about this process in the mitochondria. We reveal that TDP1 resolves mitochondrial PDBs (mtPDBs), thereby promoting mitochondrial gene transcription. Overexpression of a toxic form of mitochondrial topoisomerase I (TOP1mt*), which generates excessive mtPDBs, results in a TDP1-dependent compensatory up-regulation of mitochondrial gene transcription. In the absence of TDP1, the imbalance in transcription of mitochondrial- and nuclear-encoded electron transport chain (ETC) subunits results in misassembly of ETC complex III. Bioenergetics profiling further reveals that TDP1 promotes oxidative phosphorylation under both basal and high energy demands. It is known that mitochondrial dysfunction results in free radical leakage and nuclear DNA damage; however, the detection of intermediates of radical damage to DNA is yet to be shown. Consequently, we report an increased accumulation of carbon-centered radicals in cells lacking TDP1, using electron spin resonance spectroscopy. Overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) reduces carbon-centered adducts and protects TDP1-deficient cells from oxidative stress. Conversely, overexpression of the amyotrophic lateral sclerosis-associated mutant SOD1G93A leads to marked sensitivity. Whereas Tdp1 knockout mice develop normally, overexpression of SOD1G93A suggests early embryonic lethality. Together, our data show that TDP1 resolves mtPDBs, thereby regulating mitochondrial gene transcription and oxygen consumption by oxidative phosphorylation, thus conferring cellular protection against reactive oxygen species-induced damage.

Reproducibility and biological variability of HDL's vascular functional assays.

BACKGROUND: Recent failures of clinical trials promoting HDL-elevating therapies have prompted research groups to focus on its functional activity in disease. Endothelial effects of HDL can be measured with in vitro cell assays. The reproducibility and biological relevance of these assays have not been explored both in healthy individuals and those at increased cardiovascular (CV) risk. METHODS: HDL dependent nitric oxide (NO) bioavailability, superoxide (SO) production and serum paraoxonase-1 (PON-1) activity were measured in 35 healthy adults (34.37 24-49) and 8 patients (43.56 37-49) suffering from a chronic inflammatory condition (periodontitis-PD). Assay reproducibility was assessed by independent technicians on consecutive days to determine inter and intra analyser variability for each assay. The 35 healthy individuals were further divided into young (n = 16) and middle aged (n = 19) groups and compared with regards to HDL functions. Within-subject biological variation of HDL function was determined in a sub-group of 25 healthy volunteers at intervals of one day and 1 month, and in 8 patients at intervals of one day and 1 week. Power curves were also generated to estimate the number of patients that would be required for HDL functional assays in a cross-over and parallel study design. RESULTS: NO bioavailability was the most reproducible assay in healthy adults (coefficient of variation = 1.72%, 1.92 - intra and inter respectively) and PD patients (CV = 4.4% and 5.5%). All measures demonstrated no statistical difference between young and healthy middle aged population. No single assay demonstrated significant variations over time, indicating that within patient variations are negligible. Our power curves for NO bioavailability and PON-1 activity suggest that low number of patients will be required to detect significant differences in HDL function in a cross over and parallel study design. CONCLUSION: This study suggests that in vitro HDL functional assays are reliable and can be used to assess HDL functionality in healthy and diseased populations. NO bioavailability was the most reproducible assay, but PON-1 activity remains the most practical for application in clinical trials due to its capacity for scale.

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method.

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.

Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells.

Skin cancer incidence is dramatically increasing worldwide, with exposure to ultraviolet radiation (UVR) a predominant factor. The UVA component initiates oxidative stress in human skin, although its exact role in the initiation of skin cancer, particularly malignant melanoma, remains unclear and is controversial because there is evidence for a melanin-dependent mechanism in UVA-linked melanoma studies. Nonpigmented (CHL-1, A375), moderately pigmented (FM55, SKmel23), and highly pigmented (FM94, hyperpigmented FM55) human melanoma cell lines have been used to investigate UVA-induced production of reactive oxygen species using FACS analysis, at both the cellular (dihydrorhodamine-123) and the mitochondrial (MitoSOX) level, where most cellular stress is generated. For the first time, downstream mtDNA damage (utilizing a quantitative long-PCR assay) has been investigated. Using UVA, UVB, and H(2)O(2) as cellular stressors, we have explored the dual roles of melanin as a photoprotector and photosensitizer. The presence of melanin has no influence over cellular oxidative stress generation, whereas, in contrast, melanin protects against mitochondrial superoxide generation and mtDNA damage (one-way ANOVA with post hoc Tukey's analysis, P<0.001). We show that if melanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (P<0.001), providing evidence for the dual roles of melanin.

Intensity-dependent direct solar radiation- and UVA-induced radical damage to human skin and DNA, lipids and proteins.

Skin can be exposed to high-intensity UV-radiation in hot countries and during sunbed use; however, the free-radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free-radical generation in ex vivo human skin/substitutes +/- the spin-trap 5,5 dimethyl-1-pyrroline N-oxide (DMPO) exposed to solar-irradiation equivalent to Mediterranean sunlight. Skin-substitutes, model DNA-photosensitizer systems, lipids and proteins were also irradiated with low-intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail-keratin, tendon-collagen, phospholipid and DNA+melanin or riboflavin. In addition to lipid-derived (tentatively tert-alkoxyl/acyl-) and protein radicals detected with DMPO at lower intensities, isotropic carbon-, additional oxygen- and hydrogen-adducts were detected in solar-irradiated skin/substitutes at higher intensities. Carbon-adducts were detected in UVA-irradiated human skin cells, DNA+melanin or riboflavin and soybean-phospholipid. Anisotropic protein-adducts, comparable to adducts in solar-irradiated tendon-collagen, were absent in UVA-irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen-adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon- and oxygen-radicals) and part extracellular (protein- and hydrogen/H(+)+e(-) ), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid-oxidation is direct, DNA and protein-oxidation require photosensitisation.

Comparable photoreactivity of hair melanosomes, eu- and pheomelanins at low concentrations: low melanin a risk factor for UVA damage and melanoma?

Melanin is known to be photoreactive and photoprotective, but its function in skin in vivo is still debated. Data is lacking of the effects of UVA irradiation on human skin melanosomes of different pigmentation, which have not been extensively degraded by isolation procedures. We have shown previously that melanosomes isolated from human oriental and black cat hair, and synthetic eumelanins, are photoreactive producing superoxide at low concentrations when exposed to UVA irradiation comparable to UK levels of sunlight. Here we investigated the UVA-irradiation of melanosomes, isolated from different colored human hair samples, using electron spin resonance spectroscopy and spin trapping. Comparable irradiation of synthetic pheomelanins synthesized from L-dopa and L-cysteine was also studied. An alkali method (5 min NaOH at 90 degrees C) could be used to isolate oriental hair melanosomes but was not suitable for auburn and blonde hair. Dithiothreitol and proteinase K resulted in melanin release from possible over-digestion of melanosomes; however, dithiothreitol and papain resulted in no melanin release and good melanosome yields with separation from residual keratin for brown, auburn and blonde hair. Melanosomes isolated by the latter method and synthetic pheomelanins were similar in UVA-photoreactivity at low concentrations, independent of hair color, and broadly comparable to synthetic melanins. Melanosome concentration at constant fluence may be more significant with respect to photodamage and UVA photocarcinogenesis (melanoma) via superoxide radical production than pigment type.

Protein, lipid, and DNA radicals to measure skin UVA damage and modulation by melanin.

Afro-Caribbeans have a lower incidence of skin cancer than Caucasians, but the effectiveness of melanin as a photoprotective pigment is debated. We investigated the UVA and solar irradiation of ex vivo human skin and DMPO using electron spin resonance spectroscopy, to determine whether pigmented skin is protected by melanin against free radical damage. Initial ascorbate radicals in Caucasian skin were superseded by lipid and/or protein radical adducts with isotropic (a(H)=1.8 mT) and anisotropic spectra comparable to spectra in irradiated pig fat (a(H)=1.9 mT) and BSA. DNA carbon-centered radical adducts (a(H)=2.3 mT) and a broad singlet were detected in genomic DNA/melanin but were not distinguishable in irradiated Caucasian skin. Protein and lipid radicals (n=6 in Caucasian skin) were minimal in Afro-Caribbean skin (n=4) and intermediate skin pigmentations were variable (n=3). In irradiated Afro-Caribbean skin a shoulder to the melanin radical (also in UVA-irradiated pigmented melanoma cells and genomic DNA/melanin and intrinsic to pheomelanin) was detected. In this sample group, protein (but not lipid) radical adducts decreased directly with pigmentation. ESR/spin trapping methodology has potential for screening skin susceptibility to aging and cancer-related radical damage and for measuring protection afforded by melanin, sunscreens, and antiaging creams.

Synthetic melanin is a model for soluble natural eumelanin in UVA-photosensitised superoxide production.

Studies to UV-irradiate natural eumelanins in vitro have used insoluble pigment obtained by acid hydrolysis, which lacks melanoprotein. Eumelanin synthesised in the presence of a protein is not insoluble, and the insoluble form of melanin from acid hydrolysis may not have the same physicochemical properties as the natural pigment synthesised in vivo in the melanosome. Here we investigated radical production by three natural eumelanins exposed to solar levels of UVA; sepia melanin from Sepia officinalis, and eumelanins isolated from Oriental human and domestic cat hair. UVA irradiation of sepia melanin in solution at pH 4.5 in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave hydroperoxyl and hydroxyl radical-adducts, maximal at 0.6-2.5 mg/ml melanin concentrations. Hydroperoxyl radical production was relatively low in acetate buffer, but detected in aqueous suspensions of sepia melanin. Hair eumelanins were photoreactive with hydroperoxyl radical-adduct production at low concentrations (0.1-0.4 mg/ml melanin). Synthetic pigment after synthesis undergoes photo-oxidation (producing superoxide) at low concentrations (0.3 mg/ml) and its oxidation increases the photoreactivity at higher melanin concentrations. These findings may be physiologically relevant to the properties and function of eumelanin in vivo when it is at low concentration (found in a small proportion of Caucasian melanocytes), and suggest that synthetic melanin has the potential for the basis of a model for natural eumelanin.

Relevance of sunscreen application method, visible light and sunlight intensity to free-radical protection: A study of ex vivo human skin.

With the continued rise in skin cancers worldwide there is a need for effective skin protection against sunlight damage. It was shown previously that sunscreens, which claimed UVA protection (SPF 20+), provided limited protection against UV-induced ascorbate radicals in human skin. Here the results of an electron spin resonance (ESR) investigation to irradiate ex vivo human skin with solar-simulated light are reported. The ascorbate radical signal in the majority of skin samples was directly proportional to the irradiance over relevant sunlight intensities (0.9-2.9 mW cm(-2)). Radical production (substratum-corneum) by UV (wavelengths < 400 nm) and visible components (> 400 nm) was approximately 67% and 33% respectively. Ascorbate radicals were in steady state concentration at low irradiance (approximately 1 mW cm(-2) equivalent to UK sunlight), but at higher irradiance (approximately 3 mW cm(-2)) decreased with time, suggesting ascorbate depletion. Radical protection by a four star-rated sunscreen (with UVA protection) was optimal when applied as a thin film (40-60% at 2 mg cm(-2)) but less so when rubbed into the skin (37% at 4 mg cm(-2) and no significant protection at 2 mg cm(-2)), possibly due to cream filling crevices, which reduced film thickness. This study validates ESR determinations of the ascorbate radical for quantitative protection measurements. Visible light contribution to radical production, and loss of protection when sunscreen is rubbed into skin, has implications for sunscreen design and use for the prevention of free-radical damage.

An experimental and theoretical model for solar UVA-irradiation of soluble eumelanin: towards modelling UVA-photoreactions in the melanosome?

A model is developed for the UVA-irradiation of soluble eumelanin exposed to levels of irradiation comparable to sunlight. Radical production was determined in soluble dl- and l-dopa melanins exposed to solar levels of UVA, using electron spin resonance spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Steady-state concentrations of DMPO-O(2)H(.-), which increased up to 0.3 mg/ml melanin, and then declined above 0.3 mg/ml, were detected at pH 4.5. The kinetic model incorporated the photosensitizing and radical-scavenging reactions of eumelanin, and assumed semiquinone radical reduction of oxygen to be fast compared to disproportionation. The model is consistent with experimental data for melanin concentrations <0.1 mg/ml; but >0.1 mg/ml melanin is consistent only with data at raised oxygen tension. The rate-constant for reaction of the melanin semiquinone-radical and oxygen is estimated to be 10(3) mol(-1)dm(3)s(-1). In this model, where DMPO competes with melanin for HO(2)(.-), at ambient oxygen levels, eumelanin exposed to solar levels of UVA photosensitizes superoxide at concentrations <0.3 mg/ml melanin, and is increasingly stable towards oxidation when >0.3 mg/ml concentration. Eumelanin could have a negligible screening effect <0.1 mg/ml and very strong screening >1 mg/ml. This model would be biologically relevant if soluble forms of eumelanin were shown to exist in vivo, and is potentially useful for studies of the photochemistry and photophysics of eumelanin and phaeomelanin and to explore the effects of metal-ions, proteins and lipids in a model system.

Differences in production of melanin radicals by 694 nm ruby laser and UVA radiation.

BACKGROUND AND OBJECTIVES: The 694 nm ruby laser is used clinically for hair removal and the mechanism is predominantly photo thermal via melanin targeting. We investigated 694 nm laser-irradiation of human hair, and laser-irradiation of synthetic dopa melanin to establish whether photolysis and oxygen radical production is also contributory, and which may have side effects. STUDY DESIGN/MATERIALS AND METHODS: Ultraviolet-A (UVA) irradiation of melanin was used as a positive control for radical production. Laser- and UVA-irradiated hair samples, and synthetic dopa melanin in media of different viscosity, were analyzed using electron spin resonance spectroscopy, and compared. The spin trap 5,5-dimethyl- 1-pyrroline N-oxide (DMPO) was used to probe laser-irradiated dopa melanin for superoxide radical production. RESULTS: Comparable to UVA, laser-irradiation of hair increased the signal-intensity of the intrinsic melanin radical. UVA-induced radicals decay rapidly; however, laser-induced radicals decayed slowly and did not fully revert to original levels after 24 hours. Laser-induced radicals were increasingly stable with viscosity of the medium. Superoxide radicals were detected using DMPO in UVA- but not laser-irradiated synthetic dopa-melanin at pH 4.5. CONCLUSIONS: Laser-irradiation of melanin does not result in oxygen radical formation; however, a paramagnetic species, long-lived in rigid media, is detected which is worth further investigation.

Sunscreens inadequately protect against ultraviolet-A-induced free radicals in skin: implications for skin aging and melanoma?

Sunscreens are employed to mitigate the adverse effects of sunlight on skin but are primarily designed to prevent ultraviolet-B-associated burning and damage. The increasingly recognized role of ultraviolet A in aging, and possibly melanoma, highlights the need to include ultraviolet A screens; however, validation remains difficult. We have used a novel method to establish the efficacy of sunscreens, by measuring ultraviolet-A-induced free-radical production (thought to contribute towards ultraviolet-A-related aging and malignant change). Electron spin resonance spectroscopy was used to detect free radicals directly in human Caucasian skin during irradiation with levels of ultraviolet comparable to solar intensities. Using this system the protection afforded by three high factor sunscreens (sun protection factor 20+) that claim ultraviolet A protection was examined. Each sunscreen behaved similarly: at recommended application levels (> or = 2 mg per cm2) the ultraviolet-induced free radicals were reduced by only about 55%, and by about 45% at 0.5-1.5 mg per cm (0.5 mg per cm2 reported for common usage). A "free-radical protection factor" calculated on the basis of these results was only 2 at the recommended application level, which contrasts strongly with the erythema-based sun protection factors (mainly indicative of ultraviolet B protection) quoted by the manufacturers (20+). The disparity between these protection factors suggests that prolonged sunbathing (encouraged by use of these creams) would disproportionately increase exposure to ultraviolet A and consequently the risk of ultraviolet-A-related skin damage.