Incidence and Relative Survival of Patients with Merkel Cell Carcinoma in North Rhine-Westphalia, Germany, 2008-2021.

BACKGROUND: To date, only a few population-representative studies have been carried out on the rare Merkel cell carcinoma (MCC). We provide incidence and survival estimates of MCC, including the conditional relative survival. METHODS: We analyzed data from the cancer registry of North Rhine-Westphalia, Germany, 2008-2021, covering a population of 18 million. We included all newly diagnosed MCCs and calculated age-standardized (old European Standard population) incidence rates and unconditional and conditional relative survival. RESULTS: Our analysis included 2164 MCC patients. The age-standardized incidence of MCC was 5.2 (men) and 3.8 (women) per million person-years. The 5-year relative survival was 58.8% (men) and 70.7% (women). Survival was lower among men than women in all age-sex groups and was highest for MCC of the upper extremity in both men (68.2%) and women (79.3%). The sex difference in survival is particularly due to the better survival of women with MCC of the head and neck. In terms of survival, the first two years are particularly critical. CONCLUSIONS: Our data validate the worse survival among men and highlights a more favorable prognosis for MCCs located on the limbs. The first two years after diagnosis of MCC are the years with the highest excess mortality.

Time trends in survival and causes of death in multiple myeloma: a population-based study from Germany.

BACKGROUND: Steady evolution of therapies has improved prognosis of patients with multiple myeloma (MM) over the past two decades. Yet, knowledge about survival trends and causes of death in MM might play a crucial role in long-term management of this patient collective. Here, we investigate time trends in myeloma-specific survival at the population level over two decades and analyse causes of death in times of prolonged survival. METHODS: Age-standardised and age group-specific relative survival (RS) of MM patients aged < 80 years at diagnosis was estimated for consecutive time periods from 2000-2019 using data from the Cancer Registry of North Rhine-Westphalia in Germany. Conditional RS was estimated for patients who already survived one to five years post diagnosis. Causes of death in MM patients were analysed and compared to the general population using standardised mortality ratios (SMR). RESULTS: Three thousand three hundred thirty-six MM cases were included in the time trend analysis. Over two decades, age-standardised 5-year RS increased from 37 to 62%. Age-specific survival improved from 41% in period 2000-2004 to 69% in period 2015-2019 in the age group 15-69 years, and from 23 to 47% in the age group 70-79 years. Conditional 5-year RS of patients who survived five years after diagnosis slightly improved as compared to unconditional 5-year RS at diagnosis. MM patients are two times more likely to die from non-myeloma malignancies (SMR = 1.97, 95% CI 1.81-2.15) and from cardiovascular diseases (SMR = 2.01, 95% CI 1.86-2.18) than the general population. CONCLUSIONS: Prognosis of patients with MM has markedly improved since the year 2000 due to therapeutic advances. Nevertheless, late mortality remains a major concern. As survival improves, second primary malignancies and cardiovascular events deserve increased attention.

The Epidemiology of Colorectal Cancer in Younger and Older Patients.

BACKGROUND: Recent studies have shown that the incidence of colorectal cancer among younger persons is rising. We investigated incidence trends and survival in the German federal state of North Rhine-Westphalia. METHODS: Cancer registry data from the period 2008-2019 were classified into two age groups (15-54 and 55-99). In each age group, the standardized incidence, average annual percent change (AAPC), and relative 5-year survival (RS) were calculated and stratified according to the site, histology, size, and grade of the colorectal tumor. RESULTS: 167 919 cases were included, with adenocarcinoma accounting for 86.4%. In 2019, the age-standardized incidence per 100 000 person-years was 13.8 and 10.3 among men and women (respectively) in the younger age group, compared with 197.9 and 126.3 in the older age group. Over the study period from 2008 to 2019, the incidence declined among older men and women (AAPC -2.6% and -2.9%) but remained nearly constant among younger men and women (-0.5% and -0.4%). The incidence of neuroendocrine, T1, and G2 tumors rose in both age groups (AAPC range: 2.3%-12.2%; 2.2%-8.3%, 6.3%-8.8%). Younger patients have a better RS, with the largest difference between age groups being found for neuroendocrine tumors (88% and 83% in younger men and women, 65% and 61% in older men and women). CONCLUSION: The incidence of colorectal tumors has remained constant in persons under age 55 and declined in persons aged 55 and older. Nonetheless, the incidence of neuroendocrine tumors and of small and well-differentiated tumors has risen in both age groups. The trends among younger persons and the rise in neuroendocrine tumors merit further study.

Nickel Release, ROS Generation and Toxicity of Ni and NiO Micro- and Nanoparticles.

Occupational exposure to airborne nickel is associated with an elevated risk for respiratory tract diseases including lung cancer. Therefore, the increased production of Ni-containing nanoparticles necessitates a thorough assessment of their physical, chemical, as well as toxicological properties. The aim of this study was to investigate and compare the characteristics of nickel metal (Ni) and nickel oxide (NiO) particles with a focus on Ni release, reactive oxygen species (ROS) generation, cellular uptake, cytotoxicity and genotoxicity. Four Ni-containing particles of both nano-size (Ni-n and NiO-n) and micron-size (Ni-m1 and Ni-m2) were tested. The released amount of Ni in solution was notably higher in artificial lysosomal fluid (e.g. 80-100 wt% for metallic Ni) than in cell medium after 24h (ca. 1-3 wt% for all particles). Each of the particles was taken up by the cells within 4 h and they remained in the cells to a high extent after 24 h post-incubation. Thus, the high dissolution in ALF appeared not to reflect the particle dissolution in the cells. Ni-m1 showed the most pronounced effect on cell viability after 48 h (alamar blue assay) whereas all particles showed increased cytotoxicity in the highest doses (20-40 µg cm2) when assessed by colony forming efficiency (CFE). Interestingly an increased CFE, suggesting higher proliferation, was observed for all particles in low doses (0.1 or 1 µg cm-2). Ni-m1 and NiO-n were the most potent in causing acellular ROS and DNA damage. However, no intracellular ROS was detected for any of the particles. Taken together, micron-sized Ni (Ni-m1) was more reactive and toxic compared to the nano-sized Ni. Furthermore, this study underlines that the low dose effect in terms of increased proliferation observed for all particles should be further investigated in future studies.

Optimization of an air-liquid interface exposure system for assessing toxicity of airborne nanoparticles.

The use of refined toxicological methods is currently needed for characterizing the risks of airborne nanoparticles (NPs) to human health. To mimic pulmonary exposure, we have developed an air-liquid interface (ALI) exposure system for direct deposition of airborne NPs on to lung cell cultures. Compared to traditional submerged systems, this allows more realistic exposure conditions for characterizing toxicological effects induced by airborne NPs. The purpose of this study was to investigate how the deposition of silver NPs (AgNPs) is affected by different conditions of the ALI system. Additionally, the viability and metabolic activity of A549 cells was studied following AgNP exposure. Particle deposition increased markedly with increasing aerosol flow rate and electrostatic field strength. The highest amount of deposited particles (2.2 µg cm(-2) ) at cell-free conditions following 2 h exposure was observed for the highest flow rate (390 ml min(-1) ) and the strongest electrostatic field (±2 kV). This was estimated corresponding to deposition efficiency of 94%. Cell viability was not affected after 2 h exposure to clean air in the ALI system. Cells exposed to AgNPs (0.45 and 0.74 µg cm(-2) ) showed significantly (P < 0.05) reduced metabolic activities (64 and 46%, respectively). Our study shows that the ALI exposure system can be used for generating conditions that were more realistic for in vitro exposures, which enables improved mechanistic and toxicological studies of NPs in contact with human lung cells.Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.

Copper-based nanoparticles induce high toxicity in leukemic HL60 cells.

From the increasing societal use of nanoparticles (NPs) follows the necessity to understand their potential toxic effects. This requires an in-depth understanding of the relationship between their physicochemical properties and their toxicological behavior. The aim of the present work was to study the toxicity of Cu and CuO NPs toward the leukemic cell line HL60. The toxicity was explored in terms of mitochondrial damage, DNA damage, oxidative DNA damage, cell death and reactive oxygen species (ROS) formation. Particle characteristics and copper release were specifically investigated in order to gain an improved understanding of prevailing toxic mechanisms. The Cu NPs revealed higher toxicity compared with both CuO NPs and dissolved copper (CuCl2), as well as a more rapid copper release compared with CuO NPs. Mitochondrial damage was induced by Cu NPs already after 2 h exposure. Cu NPs induced oxidation at high levels in an acellular ROS assay, and a small increase of intracellular ROS was observed. The increase of DNA damage was limited. CuO NPs did not induce any mitochondrial damage up to 6 h of exposure. No acellular ROS was induced by the CuO NPs, and the levels of intracellular ROS and DNA damage were limited after 2 h exposure. Necrosis was the main type of cell death observed after 18 h exposure to CuO NP and dissolved copper.

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).

DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Intracellular uptake and toxicity of Ag and CuO nanoparticles: a comparison between nanoparticles and their corresponding metal ions.

UNLABELLED: An increased understanding of nanoparticle toxicity and its impact on human health is essential to enable a safe use of nanoparticles in our society. The aim of this study is to investigate the role of a Trojan horse type mechanism for the toxicity of Ag-nano and CuO-nano particles and their corresponding metal ionic species (using CuCl2 and AgNO3 ), i.e., the importance of the solid particle to mediate cellular uptake and subsequent release of toxic species inside the cell. The human lung cell lines A549 and BEAS-2B are used and cell death/membrane integrity and DNA damage are investigated by means of trypan blue staining and the comet assay, respectively. Chemical analysis of the cellular dose of copper and silver is performed using atomic absorption spectroscopy. Furthermore, transmission electron microscopy, laser scanning confocal microscopy, and confocal Raman microscopy are employed to study cellular uptake and particle-cell interactions. The results confirm a high uptake of CuO-nano and Ag-nano compared to no, or low, uptake of the soluble salts. CuO-nano induces both cell death and DNA damage whereas CuCl2 induces no toxicity. The opposite is observed for silver, where Ag-nano does not cause any toxicity, whereas AgNO3 induces a high level of cell death. IN CONCLUSION: CuO-nano toxicity is predominantly mediated by intracellular uptake and subsequent release of copper ions, whereas no toxicity is observed for Ag-nano due to low release of silver ions within short time periods.

The effects of hemodialysis treatment on the level of DNA strand breaks and oxidative DNA lesions measured by the comet assay.

Hemodialysis patients have a higher risk for oxidative stress-related complications, such as cardiovascular disease and cancer. The increased level of oxidative stress is due to several factors, e.g., the hemodialysis treatment itself and the uremic state. In the present study, the effects of dialysis treatment on the level of DNA breaks and oxidative DNA lesions in mononuclear cells were measured with the comet assay. Factors possibly affecting DNA damage (reported as % DNA in tail) such as the duration of dialysis, time since last dialysis session, years of dialysis treatment, nutritional status (measured as protein catabolic rate), age, and diabetes were also investigated. The levels of DNA breaks (13.6 ± 4.7 before dialysis) and oxidative DNA lesions (7.9 ± 4.8 before dialysis) were significantly higher in dialysis patients (n = 31) compared to the levels of DNA breaks (5.8 ± 1.1) and oxidative DNA lesions (3.4 ± 1.7) in 10 healthy controls (P < 0.001). A decrease of DNA breaks was observed after dialysis (P = 0.038), and the level of oxidative DNA lesions was higher when the time between two treatment sessions were 68 hours compared to 44 hours (P < 0.001). Older subjects had a higher level of DNA breaks (P = 0.003), a good nutritional status predicted a lower level of DNA breaks (P < 0.001), and the duration of the dialysis session was inversely correlated with oxidative DNA lesions (P = 0.014). Diabetes or years of dialysis treatment did not affect DNA damage. The observations in the present study suggest that accumulation of uremic toxins induce DNA damage. The hemodialysis treatment seems to change the DNA damage.

The effect of sea buckthorn supplement on oral health, inflammation, and DNA damage in hemodialysis patients: a double-blinded, randomized crossover study.

OBJECTIVE: Chronic kidney disease is associated with inflammation, oxidative stress, malnutrition, poor oral health, and mouth dryness. The objective of this study was to evaluate effects of sea buckthorn oil (SBO) extract, which is rich in vitamins, phytochemicals, and polyunsaturated fatty acids, on oxidative stress, saliva production, and inflammation in hemodialysis patients. DESIGN SETTING AND SUBJECTS: This was a randomized, double-blinded, and placebo-controlled crossover study (2 × 8 weeks, 4-week washout). The study subjects were hemodialysis patients (n = 45) recruited from the Department of Renal Medicine at Karolinska University Hospital in Stockholm. INTERVENTION AND MAIN OUTCOME MEASURES: The patients received 4 capsules per day, each containing 500 mg of SBO or placebo, for 8 weeks. They were then crossed over to the other treatment after a 4-week washout period. Salivary gland biopsies, saliva, and blood samples were collected before and after each treatment period. Main outcomes were DNA breaks and oxidative DNA lesions in minor accessory salivary glands, salivary flow rates, and inflammation markers in blood (high-sensitivity C-reactive protein, antitrypsin, orosomucoid in plasma, leukocytes in blood). Blood markers including creatinine, urea in plasma, and hemoglobin in blood were investigated. RESULTS: The results showed no significant changes in DNA breaks, oxidative DNA lesions, salivary flow rates, or inflammation after SBO supplementation. However, plasma levels of phosphate and sodium increased and plasma levels of iron decreased. CONCLUSION: In conclusion, SBO supplementation as performed in this study did not protect against oxidative stress, nor improve oral health or inflammation status in hemodialysis patients.

Cellular dose of partly soluble Cu particle aerosols at the air-liquid interface using an in vitro lung cell exposure system.

BACKGROUND: There is currently a need to develop and test in vitro systems for predicting the toxicity of nanoparticles. One challenge is to determine the actual cellular dose of nanoparticles after exposure. METHODS: In this study, human epithelial lung cells (A549) were exposed to airborne Cu particles at the air-liquid interface (ALI). The cellular dose was determined for two different particle sizes at different deposition conditions, including constant and pulsed Cu aerosol flow. RESULTS: Airborne polydisperse particles with a geometric mean diameter (GMD) of 180 nm [geometric standard deviation (GSD) 1.5, concentration 10(5) particles/mL] deposited at the ALI yielded a cellular dose of 0.4-2.6 µg/cm(2) at pulsed flow and 1.6-7.6 µg/cm(2) at constant flow. Smaller polydisperse particles in the nanoregime (GMD 80 nm, GSD 1.5, concentration 10(7) particles/mL) resulted in a lower cellular dose of 0.01-0.05 µg/cm(2) at pulsed flow, whereas no deposition was observed at constant flow. Exposure experiments with and without cells showed that the Cu particles were partly dissolved upon deposition on cells and in contact with medium. CONCLUSIONS: Different cellular doses were obtained for the different Cu particle sizes (generated with different methods). Furthermore, the cellular doses were affected by the flow conditions in the cell exposure system and the solubility of Cu. The cellular doses of Cu presented here are the amount of Cu that remained on the cells after completion of an experiment. As Cu particles were partly dissolved, Cu (a nonnegligible contribution) was, in addition, present and analyzed in the nourishing medium present beneath the cells. This study presents cellular doses induced by Cu particles and demonstrates difficulties with deposition of nanoparticles at the ALI and of partially soluble particles.

Inter-laboratory variation in DNA damage using a standard comet assay protocol.

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals.

Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, ß-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine.

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. ß-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

DNA damage in salivary gland tissue in patients with chronic kidney disease, measured by the comet assay.

OBJECTIVE: The aim of this observational study was to investigate the relationship between DNA damage in minor accessory salivary glands, hyposalivation, and inflammation in patients with chronic kidney disease (CKD). STUDY DESIGN: DNA strand breaks and oxidative DNA lesions in salivary glands, inflammatory markers, and uremic state were measured in 79 patients with CKD and matched controls. RESULTS: CKD patients not yet on dialysis had significantly more, and dialysis patients significantly less, DNA strand breaks in salivary tissue compared with controls. All measured inflammatory markers were higher in patients with CKD compared with controls. Salivary secretion rates were significantly lower in dialysis patients compared with controls. A high level of salivary secretion rate at rest significantly predicted a high level of DNA strand breaks in patients with CKD. CONCLUSIONS: Dialysis patients had fewer DNA strand breaks in minor accessory salivary glands than controls, suggesting that peripheral tissue is differently affected by CKD than leukocytes.

Effect of sonication and serum proteins on copper release from copper nanoparticles and the toxicity towards lung epithelial cells.

Abstract Different methodological settings can influence particle characteristics and toxicity in nanotoxicology. The aim of this study was to investigate how serum proteins and sonication of Cu nanoparticle suspensions influence the properties of the nanoparticles and toxicological responses on human lung epithelial cells. This was investigated by using methods for particle characterization (photon correlation spectroscopy and TEM) and Cu release (atomic absorption spectroscopy) in combination with assays for analyzing cell toxicity (MTT-, trypan blue- and Comet assay). The results showed that sonication of Cu nanoparticles caused decreased cell viability and increased Cu release compared to non-sonicated particles. Furthermore, serum in the cell medium resulted in less particle agglomeration and increased Cu release compared with medium without serum, but no clear difference in toxicity was detected. Few cells showed intracellular Cu nanoparticles due to fast release/dissolution processes of Cu. In conclusion; sonication can affect the toxicity of nanoparticles.

Supplementation with a combination of antioxidants does not affect glycaemic control, oxidative stress or inflammation in type 2 diabetes subjects.

The present clinical trial examined the influence of a supplement, containing a combination of antioxidants extracted from fruit, berries and vegetables, on levels of plasma antioxidants (tocopherols, carotenoids and ascorbate), glycaemic control (blood glucose, HbA1c, insulin), oxidative stress biomarkers (F(2)-isoprostane, malondialdehyd, nitrotyrosine, 8-oxo-7, 8-dihydro-2'-deoxyguanosine, formamidopyrimidine glycosylase sites, frequency of micronucleated erythrocytes) and inflammatory markers (interleukin-6, C-reactive protein, prostaglandin F(2α)-metabolite) in type 2 diabetes. Forty subjects were randomly assigned to control, single or double dose group and completed the study. In summary, 12 weeks of antioxidant supplementation did neither affect glycaemic control nor the levels of biomarkers of oxidative stress or inflammation, despite substantially increased plasma concentrations of antioxidants. The absence of an effect may be explained by the selected study subjects with relatively well-controlled diabetes, a high intake of fruit and vegetable and levels of plasma antioxidants, biomarkers of oxidative stress and inflammatory markers comparable to those found in healthy subjects.