Toxicity of nickel in the marine calanoid copepod Acartia tonsa: Nickel chloride versus nanoparticles.

Nickel compounds are widely used in industries and have been massively introduced in the environment in different chemical forms. Here we report the effect of two different chemical forms of nickel, NiCl2 and nickel nanoparticles (NiNPs), on the reproduction of the marine calanoid copepod Acartia tonsa. The behavior of nickel nanoparticles was analyzed with different techniques and with two protocols. In the "sonicated experiment" (SON) NiNP solution was sonicated while in the "non-sonicated experiment" (NON-SON) the solution was vigorously shaken by hand. Final nominal concentrations of 5, 10 and 50mgL(-1) and 1, 5 and 10mgL(-1) NiNPs were used for the acute and semichronic tests, respectively. Nanoparticle size did not change over time except for the highest concentration of 50mgL(-1) NiNPs, in which the diameter increased up to 843nm after 48h. The concentration of Ni dissolved in the water increased with NP concentration and was similar for SON and NON-SON solutions. Our results indicate that sonication does not modify toxicity for the copepod A. tonsa. Mean EC50 values were similar for NON-SON (20.2mgL(-1)) and SON experiments (22.14mgL(-1)) in the acute test. Similarly, no differences occurred between the two different protocols in the semichronic test, with an EC50 of 7.45mgL(-1) and 6.97mgL(-1) for NON-SON and SON experiments, respectively. Acute and semichronic tests, conducted exposing A. tonsa embryos to NiCl2 concentrations from 0.025 to 0.63mgL(-1), showed EC50 of 0.164 and 0.039mgL(-1), respectively. Overall, A. tonsa is more sensitive to NiCl2 than NiNPs with EC50 being one order of magnitude higher for NiNPs. Finally, we exposed adult copepods for 4 days to NiCl2 and NiNPs (chronic exposure) to study the effect on fecundity in terms of daily egg production and naupliar viability. Egg production is not affected by either form of nickel, whereas egg viability is significantly reduced by 0.025mgL(-1) NiCl2 and by 8.5mgL(-1) NiNPs. At NiNP concentration below the acute EC50 (17mgL(-1)) only 9% of embryos hatched after 4 days. Interestingly, the percentage of naupliar mortality (>82%) observed in the semichronic test at the nominal concentration of 10mgL(-1) NiNPs corresponding to almost 0.10mgL(-1) of dissolved Ni, was similar to that recorded at the same Ni salt concentration. Electron microscopical analyses revealed that A. tonsa adults ingest NiNPs and excrete them through fecal pellets. To the best of our knowledge, this is the first study investigating the toxicity of two different forms of Ni on the reproductive physiology of the copepod A. tonsa and showing the ability of the calanoid copepod to ingest nanoparticles from seawater.

The healthcare system and the provision of oral healthcare in European Union member states. Part 2: Spain.

Spain is the second largest EU Member State with an area of 504,645 km(2) and is the fifth most populated one with a total of 46.5 million inhabitants. The number of dentists working in Spain has grown rapidly in the last 20 years. In December 2014, there were 33,346 practising dentists with a ratio of one dentist for every 1394 inhabitants. Oral health of children has improved; with a fall in the national mean DMFT index (decayed, missing and filled permanent teeth) among 12-year-olds, from 4.20 in 1984 to 1.12 in 2010. The percentage of the population that has visited a dentist within the last three months has risen from 13.5% (1987) to 16.9% (2011-2012). Forty-three percent of the Spanish population visited a dentist in the last year in 2009. The Spanish National Health System (SNS) provides comprehensive cover for general health, but very little oral healthcare for adults. Only emergency care and oral surgery (dental extractions) for adults are provided in publicly funded clinics. The vast majority of oral health care is provided in the private sector and over 90% of dental professionals work in the private sector. Nevertherless, children aged 7-15 years are covered (with some restrictions) by publicly funded oral healthcare with different care models, depending on the local health authority, and some of them are funded by a capitation system which was introduced 25 years ago.

Comparing outcomes and costs between contingent and combined first-trimester screening strategies for Down's syndrome.

OBJECTIVE: To compare a contingent strategy with a combined strategy for prenatal detection of Down's syndrome (DS) in terms of cost, outcomes and safety. STUDY DESIGN: The contingent strategy was based on a simulation, removing measurement of the free beta subunit of human chorionic gonadotropin (free ßhCG) and calculating the DS risk retrospectively in 32,371 pregnant women who had been screened with the combined strategy in the first trimester. In the contingent strategy, a risk between 1:31 and 1:1000 in the first trimester indicated further testing in the second trimester (alpha-fetoprotein, inhibin A, unconjugated oestriol and free ßhCG). The cut-off risk values for the contingent and combined strategies in the first trimester were 1:30 and 1:250, respectively, and the cut-off risk value for integrated screening in the second trimester was 1:250. Costs were compared in terms of avoided DS births, and the ratio of loss of healthy fetuses following invasive procedures per avoided DS birth was calculated. RESULTS: The combined strategy had sensitivity of 40/44 (90.9%) and a false-positive rate of 2.8%. Corresponding values for the contingent strategy were 39/44 (88.6%) and 1.3%, respectively. Only 11% of pregnant women required tests in the second trimester, and the approximate cost reduction for each avoided DS birth was 5000€. The ratio of lost healthy fetuses following invasive procedures per avoided DS birth improved by up to 0.65. CONCLUSION: The contingent strategy has similar effectiveness to the combined strategy, but has lower costs and fewer invasive procedures.

Moderate consumption of red wine, but not gin, decreases erythrocyte superoxide dismutase activity: a randomised cross-over trial.

BACKGROUND AND AIMS: Several studies have shown that moderate alcohol consumption reduces the risk of coronary heart disease, a disease related to oxidative stress. However, the effects of different alcoholic beverages on antioxidant status are not fully known. Our aim was therefore to compare the effects of a moderate intake of an alcoholic beverage with high polyphenol content (red wine) and another without polyphenol content (gin) on plasma antioxidant vitamins, lipid profile and oxidability of low-density lipoprotein (LDL) particles. METHODS AND RESULTS: Forty healthy men (mean age, 38 years) were included in a randomised cross-over trial. After a 15-day washout period, subjects received 30 g/ethanol/d as either wine or gin for 28 days. Diet and exercise were monitored. Before and after each intervention, we measured serum vitamins, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase activities, lipid profile, oxidized LDL and LDL resistance to ex-vivo oxidative stress. Compared to gin intervention, wine intake reduced plasma SOD activity [-8.1 U/gHb (95% confidence interval, CI, -138 to -25; P=0.009)] and MDA levels [-11.9 nmol/L (CI, -21.4 to-2.5; P=0.020)]. Lag phase time of LDL oxidation analysis also increased 11.0 min (CI, 1.2-20.8; P=0.032) after wine, compared to gin, whereas no differences were observed between the two interventions in oxidation rate of LDL particles. Peroxide concentration in LDL particles also decreased after wine [-0.18 nmol/mL (CI, -0.3 to-0.08;P=0.020)], as did plasma oxidized LDL concentrations [-11.0 U/L (CI,-17.3 to -6.1; P=0.009)]. CONCLUSION: Compared to gin, red wine intake has greater antioxidant effects, probably due to its high polyphenolic content.

Combining fetal nuchal fold thickness with second-trimester biochemistry to screen for trisomy 21.

OBJECTIVE: To assess second-trimester screening for trisomy 21 by combining ultrasound nuchal fold (NF) measurement with maternal serum biochemistry. METHODS: NF, maternal serum alpha-fetoprotein (AFP) and free beta-human chorionic gonadotropin (beta-hCG) were determined concurrently at 14-19 weeks' gestation in a study population comprising 1813 women with singleton pregnancies, including 1257 unselected women undergoing serum screening for trisomy 21 (1999-2002), and 556 high-risk pregnancies prior to amniocentesis (2003-2005), 402 of whom had positive serum screening tests. The results were expressed in multiples of the gestation-specific normal median (MoMs). RESULTS: There were 1799 unaffected singleton pregnancies, and their NF values approximately fitted a log Gaussian distribution over a wide range. There was a weak but statistically significant correlation between log NF and log AFP (r = - 0.069, P < 0.005) and the correlation coefficient between log NF and log free beta-hCG was even smaller and not statistically significant (r = 0.038, P = 0.11). Among the seven trisomy 21 pregnancies, the median NF level was 1.53 MoM (geometric mean 1.75 MoM), a highly statistically significant increase compared with unaffected pregnancies (P < 0.0001, one-tail Wilcoxon Rank Sum Test). In pregnancies referred because of positive serum screening tests (391 unaffected, seven cases of trisomy 21, one of monosomy X and three other chromosomal anomalies) the use of NF to modify the serum screening risk would have reduced the invasive procedures in unaffected pregnancies by 46% without affecting the detection rate of trisomy 21 or other anomalies. Statistical modeling predicted that adding NF to AFP and free beta-hCG would increase detection more than would adding unconjugated estriol as well as inhibin-A, an analyte that is difficult to measure with precision. CONCLUSIONS: The addition of NF measurement to second-trimester biochemical markers improves screening performance, and could overcome drawbacks in the implementation of inhibin-A assay in clinical practice.

Effect of apolipoprotein E polymorphism on renal transplantation.

INTRODUCTION: Dyslipidemia is an important cardiovascular risk factor and is implicated in the pathogenesis of chronic graft failure in renal transplant recipients. Apolipoprotein E (apoE), a hepatic glycoprotein involved in lipid metabolism, has been associated with hypercholesterolemia and premature coronary disease. AIM: This study assessed the impact of apoE polymorphism on the evolution of renal transplant recipients. METHODS: A total of 517 patients (age, 47 +/- 14 years; 62% men), who had undergone renal transplantation at least 12 months before enrollment, were assessed (mean follow-up, 5.4 +/- 2.2 years). ApoE polymorphisms (E2, E3, and E4 alleles) were analyzed using polymerase chain reaction (PCR) using genomic DNA. Donor-recipient clinical variables were assessed using univariate methods and Cox multivariate regression model. RESULTS: Genotype frequency was as follows: E2/E2 <1%, E2/E3 10%, E3/E3 71%, E2/E4 2%, E3/E4 16%, and E4/E4 1%, with no differences between sexes. In the univariate study, E2/E4, E3/E4, and E4/E4 genotypes were related with poorer patient survival (P = .0045). In the multivariate study, the E4 allele was associated with a higher independent risk of graft loss (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.44-7.21; P < .0001) and death of the patient (OR, 16.03; 95% CI, 3.28-75.18; P < .0001), but only in patients older than 60 years of age. In patients with the E4 allele, 45% of deaths were due to cardiovascular causes. CONCLUSIONS: The genetic polymorphism of apoE (E4 allele) has an independent negative impact on patient and graft survival in the long term, particularly in older patients.

Comparison of a high-carbohydrate and a high-monounsaturated fat, olive oil-rich diet on the susceptibility of LDL to oxidative modification in subjects with Type 2 diabetes mellitus.

AIMS: To compare the effects of a high-carbohydrate (CHO) diet and a high-monounsaturated fatty acid (MUFA) diet on LDL oxidative resistance in free-living individuals with Type 2 diabetes mellitus. METHODS: Twenty-two men and women out-patients with Type 2 diabetes, with mean age 61 years and in fair metabolic control (HbA1c<8.0%), were enrolled at a university hospital lipid clinic in a randomized, crossover feeding trial comparing two isocaloric diets for 6 weeks each: CHO (fat, 28% energy) and MUFA (fat, 40% energy) based on virgin olive oil. Outcome measurements were changes in LDL susceptibility to oxidation, body weight, glycaemic control, and lipoprotein profiles. RESULTS: Planned and observed diets were well matched. Participants preferred the MUFA diet over the CHO diet. The lag time of conjugated diene formation during Cu2+-induced LDL oxidation was similar after the CHO and MUFA diets (36.4 +/- 12.2 min and 36.0 +/- 13.7 min, respectively). Body weight, glycaemic control, total triglycerides, and total, LDL- and HDL-cholesterol levels also were similar after the two diets. Compared with the CHO diet, the MUFA diet lowered VLDL-cholesterol by 35% (P=0.023) and VLDL triglyceride by 16% (P=0.016). CONCLUSIONS: Natural food-based high-CHO and high-MUFA diets have similar effects on LDL oxidative resistance and metabolic control in subjects with Type 2 diabetes. A MUFA diet is a good alternative to high-CHO diets for nutrition therapy of diabetes because it also has a beneficial effect on the lipid profile and superior patient acceptance.

Effect of apolipoprotein polymorphisms in renal transplant recipients.

Cardiovascular disease (CVD) is the main cause of mortality among long-term renal transplant recipients (RTR). On the other hand, allograft chronic nephropathy is the primary cause of graft loss among long-term RTR. Hyperlipidemia is a predisposing factor for both conditions. Polymorphisms of the apolipoproteins modulate lipid metabolism. The aim of the study was to evaluate the effect of apo A-I, apo A-IV and apo C-III genotypes on the long-term results of renal transplantation. Clinical assessment (renal allograft and patient survival) and genotyping for apo A-I (+83C/T), apo C-III (Sst I), and apo A-IV (Thr347Ser and Gln360His) polymorphisms were evaluated in 516 kidney transplant patients and correlated with the clinical evolution over 12 months. The distribution of the apo A-I (+83C/T) polymorphisms was: CC 91.9%, CT 7.9%, and TT 0.2%. The apo C-III genotype showed: S1S1 84.4%, S1S2 15.2%, and S2S2 0.4%. The apo A-IV (Pvu II) polymorphism was: GG 82%, GT 18%, and 0% TT. Finally, the frequency of apo A-IV (Hinf I) polymorphism was: AA 69%, AT 27%, and TT 4%. The frequency of polymorphisms was similar between men and women. In conclusion, there was no significant influence of apolipoprotein polymorphisms on renal and patient survival.

Effects of atorvastatin on glucose homeostasis, postprandial triglyceride response and C-reactive protein in subjects with impaired fasting glucose.

AIMS: To investigate the effects of atorvastatin on glucose homeostasis, the basal and postprandial lipid profiles and the CRP levels (C reactive protein) in subjects with impaired fasting glucose (IFG). METHODS: Thirty-three subjects (22 men and 11 women) were included in our study. All displayed an IFG (fasting plasma glucose between 6.1 and 7.0 mmol/l) on at least two occasions during the last 6 months prior the study. They were randomly assigned to receive either 40 mg atorvastatin/day (n=16) or placebo (n=17) over 16 weeks, in a double-blind design. Before and after the end of the study all participants underwent on three consecutive days: a 75-g oral glucose tolerance test, a frequent sampling intravenous glucose tolerance test with Minimal Model analysis and a meal tolerance test (glucose, insulin and triglycerides). CRP was measured before and after the treatment period. RESULTS: CRP decreased significantly in the atorvastatin-treated group compared with the placebo group (percent change respect initial values; -42.3 %[-21.5 to - 63.1] and -9.6%[15.0 to -34.0], respectively, p<0.01). Atorvastatin treatment did not produce any change in oral glucose tolerance categories or induce any change in glucose and insulin response in OGTT. The statin produced a trend towards a significant improvement in insulin sensitivity as expressed by a change in Si from baseline to the end of treatment. Atorvastatin reduced the postprandial response of triglycerides to the meal test compared with placebo (19-26 % across the meal test, p<0.05) correlating with the amelioration observed in Si (-0.34, p<0.05; percentage changes). CONCLUSION: Our results suggest that the use of statins in subjects with IFG seems to include other potentially beneficial actions in addition to their cholesterol-lowering effects.

Effects of estradiol, cyproterone acetate, tibolone and raloxifene on uterus and aorta atherosclerosis in oophorectomized cholesterol-fed rabbits.

BACKGROUND: Different hormonal replacement regimens are used for treating climacteric complaints; however, not all of them have the same clinical profile. Cardiovascular disease (CVD) is a major health problem and tibolone, raloxifene, estradiol (alone or with cyproterone acetate) have been added to cholesterol-fed rabbits to study atherosclerosis. METHODS: A total of 48 cholesterol-fed New Zealand white rabbits were studied for 4 months. Forty rabbits underwent bilateral ovariectomy and the other eight were sham operated (group S). The ovariectomized rabbits were allocated to five groups of eight animals each receiving tibolone (Group T, 6 mg/day), raloxifene (R, 35 mg/day), estradiol valerate (E, 3 mg/day), estradiol valerate plus cyproterone acetate (EC, 3+0.5 mg/day, respectively), and no treatment for the control group (C). The sham group received no treatment too. RESULTS: After 4 months the percentage of the extent of atherosclerosis in the aorta was 30.4% in C group, 24.5% in S group, 10.2% in T group, 30.3% in R group, 17.9% in E group and 28.1% in EC group (P<0.05 T vs. C, R, EC). The aortic cholesterol content compared with aortic weight was 8.55 microg/mg in C group, 11.97 microg/mg in S group, 1.86 microg/mg in T group, 3.82 microg/mg in R group, 2.86 microg/mg in E group and 5.24 microg/mg in EC group (P<0.05 T vs. EC, C, S; R vs. C, S; E vs. C, S). Uterine weights in grams were: 1.89 (C group), 2.24 (S), 7.38 (T), 1.94 (R), 9.92 (E), and 5.94 (EC); P<0.05 (C, S, R, vs. T, E, EC; T vs. E; EC vs. T, E). CONCLUSION: Our study showed a decrease in the extent of aortic atherosclerosis in oophorectomized cholesterol-fed rabbits treated with tibolone or estradiol, and a decrease in aortic cholesterol content in rabbits treated with tibolone, raloxifene and estradiol. However, rabbits treated with tibolone showed an increased uterine weight, which is contrary to that observed in humans.

Polymorphism in intron 2 of islet amyloid polypeptide gene is associated with lower low-density lipoprotein cholesterol in nondiabetic subjects and in type 2 diabetic patients.

The aim of this study was to investigate the presence of mutations in the islet amyloid polypeptide (IAPP) gene in a Spanish population with type 2 diabetes and gestational diabetes mellitus (GDM). Using polymerase chain reaction single-stranded conformation polymorphism, we examined the coding region and the 5'-untranslated region (UTR) of the IAPP gene in 177 unrelated type 2 diabetic patients, 110 healthy control subjects, 38 women with GDM, and 38 gestational control subjects. Mutations were confirmed by DNA sequencing. A heterozygous C-to-A nucleotide substitution at +79 bp in intron 2 of the IAPP gene was detected. The frequencies of the +79-bp polymorphism (A allele) were 6.8% in type 2 diabetic patients, 7.7% in nondiabetic control subjects, 11.8% in women with GDM, and 9.2% in gestational control subjects. No AA genotypes were detected. Nondiabetic subjects and patients with type 2 diabetes bearing the CA genotype had lower low-density lipoprotein (LDL) cholesterol levels than subjects bearing wild genotype. Multivariate logistic regression analysis showed an independent association (p < 0.001; odds ratio: 0.33; 95% confidence interval: 0.17-0.63). We did not detect any sequence variant within exons 1 or 2. One diabetic patient was heterozygous for a silent mutation at codon 31 of exon 3 (Asn31 AAC --> AAT). Our findings indicate that the presence of the +79-bp polymorphism of the IAPP gene in nondiabetic subjects and in patients with type 2 diabetes is associated with lower levels of LDL cholesterol. Furthermore, abnormalities of the coding regions or the 5'-UTR of the IAPP gene are not associated with type 2 diabetes or GDM in the Spanish population.

Bone mineral density in young, hypothalamic oligoamenorrheic women treated with oral contraceptives.

OBJECTIVE: To evaluate whether decreasing doses of ethinyl estradiol affect bone loss related to hypothalamic amenorrhea. STUDY DESIGN: Sixty-four women with hypothalamic oligoamenorrhea were allocated to two therapy groups: group A (n = 24) received an OC containing 0.030 mg of ethinyl estradiol (EE) and 0.15 mg of desogestrel. Group B (n = 22) received an OC containing 0.020 mg of EE and 0.15 mg of desogestrel. Eighteen women who did not wish to use hormonal therapy constituted the control group (C). Calcium, phosphate and osteocalcin were measured basally and at 6 and 12 months of follow-up. Bone mineral density at the lumbar spine was determined before initiation of the study and at 12 months by dual energy X-ray absorptiometry. RESULTS: Serum calcium, phosphate and osteocalcin were significantly reduced by both active treatment regimens, whereas no differences were observed in the control group. Bone mineral density at 12 months showed an increase in both therapy groups (A, 2.4%; B, 2.5%), while group C showed a significant decrease (1.2%, P < .05). CONCLUSION: Both doses of EE were equally effective in preventing bone loss related to hypoestrogenism in hypothalamic oligoamenorrheic subjects.

Serum antibodies to oxidized low-density lipoprotein in pregnant women with preeclampsia and chronic hypertension: lack of correlation with lipid peroxides.

OBJECTIVES: To evaluate the circulating levels of antibodies to oxidized low-density lipoprotein (LDL) and their correlation with the lipid peroxide/vitamin E ratio in pregnant women with preeclampsia and chronic hypertension. METHODS: Antibodies to oxidized LDL were measured by enzyme-linked immunoassay, lipid peroxides (malondialdehyde), and vitamin E were measured by high-pressure liquid chromatography. Patients were 25 healthy pregnant women, 20 previously nonhypertensive women diagnosed with preeclampsia, and 20 women with uncomplicated chronic hypertension. RESULTS: Serum levels of antibodies to LDL in preeclamptic patients were similar to controls, whereas women with chronic hypertension showed a trend for increased mean levels. Lipid peroxides in serum were significantly increased and vitamin E levels were significantly decreased in preeclampsia with respect to nonhypertensive pregnancy, but no differences were observed for chronic hypertensive women. CONCLUSIONS: Our results suggest that preeclampsia is not accompanied by increased levels of antibodies to oxidized LDL. By contrast, and according to previous studies in nonpregnant patients, chronic hypertensive patients showed a trend for elevated levels.

Inhibin A serum levels in proteinuric and nonproteinuric pregnancy-induced hypertension: evidence for placental involvement in gestational hypertension?

OBJECTIVES: To evaluate the serum levels of inhibin A in pregnant women with different types of hypertension. METHODS: A case-control study, including 60 cases (20 women with preeclampsia, 20 with mild gestational hypertension, and 20 with chronic hypertension), and 60 gestational-age- and parity-matched controls. Inhibin A was measured in duplicate by enzyme-linked immunosorbent assay in serum samples frozen at -80 degrees C. RESULTS: As compared to controls, inhibin A levels were significantly elevated in women with preeclampsia ¿2.32 standard deviation (SD) 1.4¿ versus 0.50 (0.29) ng/mL, p < 0.001) and gestational hypertension [1.09 (0.73) versus 0.55 (0.29) ng/mL, p < 0.05], but not in the group of chronic hypertension [0.88 (0.69) versus 0.54 (0.39) ng/mL, p = 0.08]. Overlap in inhibin A values between cases and controls was observed in 20% (4/20) of women with preeclampsia and 55% (11/20) with gestational hypertension. CONCLUSIONS: Increased serum inhibin A may indicate that a proportion of mild nonproteinuric hypertension cases are associated with placental involvement.

Aging is associated with increased lipid peroxidation in human hearts, but not with mitochondrial respiratory chain enzyme defects.

BACKGROUND: Aging is associated with increased oxidative damage at multiple cellular and tissular levels. A decrease in mitochondrial function has repeatedly been advocated as a primary key event, especially on the basis of analysis of skeletal muscle mitochondria. However, some doubts on this issue have arisen when confounding variables (such as physical activity or smoking habit) have been taken into account in the analysis of mitochondrial respiratory chain (MRC) enzyme activities or when additional analytical parameters such as enzyme ratios have been considered. OBJECTIVE: To determine whether oxidative damage and enzyme activities of the MRC are influenced by the aging process in human hearts. PATIENTS AND METHODS: We studied cardiac muscle obtained from 59 organ donors (age: 56+/-12 years, 75% men). Oxidative membrane damage was evaluated through the assessment of lipid peroxidation. Absolute and relative enzyme activities (AEA and REA, respectively) of complex I, II, III and IV of the MRC were spectrophotometrically measured. Stoichiometric relationships among MRC complexes were also assessed through calculating MRC ratios. Linear regression analyses were employed to disclose any potential correlation between mitochondrial dysfunction and aging. RESULTS: We found a progressive, significant increase of heart membrane lipid peroxidation with aging (P<0.05). Conversely, neither AEA nor REA decreased with age (P=n.s. for all complexes). Similarly to observations in other tissues, we found that stoichiometry of the MRC enzymes is maintained within a narrow range in human hearts. When the effects of aging on MRC ratios were explored, we failed again in demonstrating any subtle disarray. CONCLUSION: MRC enzymes remain preserved in heart with aging, and thus they cannot be considered the main cause of the increased oxidative damage associated with aging.