Olive oil and health: summary of the II international conference on olive oil and health consensus report, Jaén and Córdoba (Spain) 2008.

Olive oil (OO) is the most representative food of the traditional Mediterranean Diet (MedDiet). Increasing evidence suggests that monounsaturated fatty acids (MUFA) as a nutrient, OO as a food, and the MedDiet as a food pattern are associated with a decreased risk of cardiovascular disease, obesity, metabolic syndrome, type 2 diabetes and hypertension. A MedDiet rich in OO and OO per se has been shown to improve cardiovascular risk factors, such as lipid profiles, blood pressure, postprandial hyperlipidemia, endothelial dysfunction, oxidative stress, and antithrombotic profiles. Some of these beneficial effects can be attributed to the OO minor components. Therefore, the definition of the MedDiet should include OO. Phenolic compounds in OO have shown antioxidant and anti-inflammatory properties, prevent lipoperoxidation, induce favorable changes of lipid profile, improve endothelial function, and disclose antithrombotic properties. Observational studies from Mediterranean cohorts have suggested that dietary MUFA may be protective against age-related cognitive decline and Alzheimer's disease. Recent studies consistently support the concept that the OO-rich MedDiet is compatible with healthier aging and increased longevity. In countries where the population adheres to the MedDiet, such as Spain, Greece and Italy, and OO is the principal source of fat, rates of cancer incidence are lower than in northern European countries. Experimental and human cellular studies have provided new evidence on the potential protective effect of OO on cancer. Furthermore, results of case-control and cohort studies suggest that MUFA intake including OO is associated with a reduction in cancer risk (mainly breast, colorectal and prostate cancers).

Prescription data improve the medication history in primary care.

BACKGROUND: Incomplete medication lists increase the risk of medication errors and adverse drug effects. In Denmark, dispensing data and pharmacy records are available directly online to treating physicians. We aimed (1) to describe if use of pharmacy records improved the medication history among patients consulting their general practitioner and (2) to characterise inconsistencies between the medication history reported by the patient and the general practitioner's recordings. METHODS: Patients attending a general practitioner clinic were interviewed about their current medication use. Subsequently, the patients were contacted by phone and asked to verify the medication list previously obtained. Half of the patients were randomly selected for further questioning guided by their dispensing data: during the telephone interview, these patients were asked to clarify whether drugs registered in their pharmacy records were still in use. Pharmacy records show all drugs acquired on prescription from any national pharmacy in the preceding 2 years. The medication list was corrected accordingly. In all patients, the medication lists obtained on the in-clinic and telephone interviews were compared to the general practitioner's registrations. RESULTS: The 150 patients included in the study had a median age of 56 years (range 18-93 years), and 90 (60%) were women. Patients reported use of 849 drugs (median 5, range 0-16) at the in-clinic interview. Another 41 drugs (median 0, range 0-4) were added during the telephone interview. In the subgroup of 75 patients interviewed guided by pharmacy records, additionally 53 drugs (10%) were added to the 474 drugs already mentioned. The 27 patients adding more drugs guided by pharmacy records were significantly older and used more drugs (both p<0.05) than the 48 patients not adding drugs. When the medication lists were compared with the general practitioner's lists, specifically use of over-the-counter products and prescription-only medications from Anatomical Therapeutic Chemical Classification System group J, A, D, N and R were not registered by the general practitioner. DISCUSSION: Dispensing data provide further improvement to a medication history based on thorough in-clinic and telephone interviews. Use of pharmacy records as a supplement when recording a medication history seems beneficial, especially among older patients treated with polypharmacy.

MTHFR polymorphisms and 5-FU-based adjuvant chemotherapy in colorectal cancer.

BACKGROUND: Methylenetetrahydrofolate reductase is a pivotal enzyme in folate metabolism and 5-fluorouracil (5-FU) cytotoxicity. Two common single-nucleotide polymorphisms (SNPs), MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), reduce enzyme activity. Initially, these SNPs were claimed to predict clinical efficacy, but further studies have yielded contradictory results. We tested whether these two polymorphisms are determinants of clinical outcome in a large patient group with a long follow-up time. PATIENTS AND METHODS: We included 331 patients who had been treated with adjuvant 5-FU/leucovorin chemotherapy after intended curative resection between 1997 and 2003. Clinical data, including relapse rates, overall survival, and tumor stage, were collected. DNA was extracted from formalin-fixed tumor tissue and analyzed for the MTHFR 677C>T and 1298A>C SNPs with real-time PCR. RESULTS: The MTHFR 677C>T and 1298A>C polymorphisms were not associated with survival or relapse-free survival (P > 0.2). The 677 CC genotype was associated to toxicity (odds ratio = 1.83, P = 0.01). CONCLUSIONS: The MTHFR 677C>T and 1298A>C polymorphisms probably do not predict efficacy of adjuvant 5-FU treatment in colorectal cancer after complete resection; however, the 677C>T polymorphism may be associated with lower toxicity in 5-FU treatment. Implementation of SNP analysis for these polymorphisms for individualized treatment is premature.

Antibiotic resistance in Pseudomonas aeruginosa strains with increased mutation frequency due to inactivation of the DNA oxidative repair system.

The chronic Pseudomonas aeruginosa infection of the lungs of cystic fibrosis (CF) patients is characterized by the biofilm mode of growth and chronic inflammation dominated by polymorphonuclear leukocytes (PMNs). A high percentage of P. aeruginosa strains show high frequencies of mutations (hypermutators [HP]). P. aeruginosa is exposed to oxygen radicals, both those generated by its own metabolism and especially those released by a large number of PMNs in response to the chronic CF lung infection. Our work therefore focused on the role of the DNA oxidative repair system in the development of HP and antibiotic resistance. We have constructed and characterized mutT, mutY, and mutM mutants in P. aeruginosa strain PAO1. The mutT and mutY mutants showed 28- and 7.5-fold increases in mutation frequencies, respectively, over that for PAO1. These mutators had more oxidative DNA damage (higher levels of 7,8-dihydro-8-oxodeoxyguanosine) than PAO1 after exposure to PMNs, and they developed resistance to antibiotics more frequently. The mechanisms of resistance were increased beta-lactamase production and overexpression of the MexCD-OprJ efflux-pump. Mutations in either the mutT or the mutY gene were found in resistant HP clinical isolates from patients with CF, and complementation with wild-type genes reverted the phenotype. In conclusion, oxidative stress might be involved in the development of resistance to antibiotics. We therefore suggest the possible use of antioxidants for CF patients to prevent the development of antibiotic resistance.

Pharmacokinetics and allometric scaling of levormeloxifene, a selective oestrogen receptor modulator.

The pharmacokinetics of a new selective oestrogen receptor modulator levormeloxifene was investigated in mice, rats, cynomolgus monkeys and humans by compartmental pharmacokinetics. Levormeloxifene was administered as an oral solution in all studies. Allometric scaling was used to predict human pharmacokinetic parameters and the performance of the approach was evaluated. Mean values of clearance confounded by F(CL/F) were 0.073, 0.29, 3.18 and 2.4 l/h in mice, rats, monkeys and humans, respectively. Values of distribution volume at steady state confounded by F(V(ss)/F) were 0.073 and 7.5 l in mice and rats. In monkeys, values of the central volume F(V(c)/F) and volume at steady state F(V(ss)/F) were 28.9 and 57.9 l, respectively. In humans, values of V(c)/F and V(ss)/F were 106 and 587 l, respectively. Predicted CL/F and V(ss)/F showed a linear relationship when plotted vs BW on a log-log scale; for CL/F, r was 0.95-0.98 and for V(ss)/F, r was 0.99. Using allometric scaling the predicted human V(ss)/F deviated 3-fold from the experimentally determined values. Observed values of CL/F deviated 21-25 fold from the predicted, the latter depending on the scaling method. Confidence intervals for the predicted parameters showed major lack of precision for all the allometric scaling methods.

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo.

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near future, it has become essential that studies reported in this journal reflect an adequate level of scientific scrutiny. Therefore a set of essential characteristics of studies has been defined. These basic requirements are default properties rather than non-negotiables: deviations are possible and useful, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9) Statistics; and (10) Quality Assurance.

Endothelium-dependent vasorelaxation in inhibited by in vivo depletion of vascular thiol levels: role of endothelial nitric oxide synthase.

Thiols like glutathione may serve as reducing cofactors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 micrograms/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular superoxide anion (O2.-) production in conscious chronically catheterized rats. Thiol depletion induced by buthionine sulfoximine (BSO, 1 g i.p. within 24 h) decreased the hypotensive effect of Ach by 30% (MAP reduction before BSO 27 +/- 3 mmHg, 19 +/- 3 mmHg after BSO, (mean +/- SEM), p < .05, n = 8). The impaired effect of Ach was associated with a significant reduction in eNOS activity (control: 7.7 +/- 0.8, BSO: 3.9 +/- 0.4 pmol/min/mg protein (p < .05), n = 6). In contrast, neither NADH/NADPH driven membrane-associated oxidases nor lucigenin reductase activity were significantly (p < .05) affected by BSO (BSO: 4415 +/- 123, control: 4105 +/- 455 counts/mg; n = 6) in rat aorta. It is concluded that in vivo thiol depletion results in endothelial dysfunction and a reduced receptor-mediated vascular relaxation. This effect is caused by reduced endothelial NO formation.

N-acetylcysteine attenuates oxidative burst by neutrophils in response to ergometer rowing with no effect on pulmonary gas exchange.

This study evaluated whether the reduction of the neutrophil oxidative burst by N-acetylcysteine improves pulmonary gas exchange during a six minute maximal ergometer row. Healthy trained oarsmen were double-blinded randomized to either N-acetylcysteine (6 g daily for three days) or placebo groups. As determined by the relative changes of the zymosan-stimulated luminol-enhanced chemiluminescence response, N-acetylcysteine suppressed the exercise-induced enhanced neutrophil oxidative burst response to rowing (-7 +/- 6% vs. 17 +/- 8%; P < 0.05). This was the case although the concentration of neutrophils remained similarly elevated above the pre-exercise level in both trials (to 5.4+/-0.5 vs. 5.9+/-0.6 x 10(9) cells x l(-1), respectively, P>0.05). In the placebo and N-acetylcysteine groups, pulmonary ventilation increased and the arterial CO2 partial pressure decreased to the same extent during exercise. Also, at the end of exercise the arterial O2 partial pressure (77 1 vs. 78+/-1 mmHg), haemoglobin O2 saturation (92 +/- 1% vs. 93 +/- 1%) and O2 uptake (5.0 +/- 0.2 vs. 4.9 +/- 0.21 x min(-1)) were not significantly affected by N-acetylcysteine. Equally, two hours after exercise, the pulmonary diffusion capacity was reduced by 7 +/- 2% below the pre-exercise with no significant influence of N-acetylcysteine. We conclude that the neutrophil oxidative burst to exercise does not influence pulmonary gas exchange during and after maximal rowing.

Effects of a Brussels sprouts extract on oxidative DNA damage and metabolising enzymes in rat liver.

The apparent anticarcinogenic effect of cruciferous vegetables found in numerous epidemiological and experimental studies has been associated with their influence on phase I and phase II metabolising enzymes as well as on the antioxidant status. In the present study we investigated the effect of administration of a Brussels sprouts extract on the expression at the mRNA level and/or catalytic activity in rat liver of three phase I enzymes [cytochrome P450-1A2 (CYP1A2),-2B1/2 (CYP2B1/2) and-2E1 (CYP2E1)] and two phase II enzyme [NADPH:quinone reductase (QR) and glutathione S-transferase pi 7 (GSTpi)], all previously suggested to be induced by vegetables. We also examined the activity and/or expression of several important antioxidant enzymes: glutathione peroxidase (GPx), catalase and gamma-glutamyl-cysteine synthetase (GCS) and the activity of the repair enzyme 8-oxoguanine DNA glycosylase (OGG1). QR, GPx and catalase activity was also assessed in the kidneys. In order to examine a possible effect of the Brussels sprouts related to oxidative stress, we measured oxidative DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and lipid peroxidation in terms of malondialdehyde (MDA) formation in the liver. Oral administration of an aqueous Brussels sprouts extract for 4 days was found to induce the expression of GST 1.3-fold (P < 0.05) and the activity of QR 2.6-fold in rat liver (P < 0.05). No significant differences were seen in the expression of the phase I enzymes. No differences in antioxidant enzyme activity/expression or OGG1 activity were observed. In a second experiment, administration of the Brussels sprouts extract for 3 or 7 days was found to increase the level of 8-oxodG in rat liver from 0.75 to 0.97 per 10(5) dG and from 0.81 to 0.97 per 10(5) dG, respectively (P < 0.05). No effects on MDA levels were found. The present results support the data obtained in several studies that consumption of cruciferous vegetables is capable of inducing various phase II enzyme systems. However, the observed increase in oxidative DNA damage raises the question of whether greatly increased ingestion of cruciferous vegetables is beneficial.

Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry.

A method for the determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and urine by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry is described. For the urine samples there is no sample preparation except for addition of buffer and internal standards followed by redissolvation of precipitate containing 8-oxo-2'-deoxyguanosine and a centrifugation step before the samples are injected onto the HPLC column. The detection limit for 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine is approximately 0.3 nM corresponding to 7.5 fmol injected. Long runs, that is, > 50 samples, can be analyzed with only minimal loss of sensitivity. The concentrations excreted into urine samples from humans are between 1 and 100 nM for 8-oxo-2'-deoxyguanosine and below 0.3 nM for 8-oxo-2'-deoxyadenosine. In calf thymus DNA levels down to about 1 oxidized guanosine and adenosine per 10(6) unmodified bases can be detected. High levels of 8-oxo-2'-deoxyguanosine were found, 30 per 10(6) 2'-deoxyguanosine, levels of 8-oxo-2'-deoxyadenosine are at or below the detection limit. These findings indicate that High Performance Liquid Chromatography-Tandem Mass Spectrometry is a highly sensitive and specific method for analysis of oxidative DNA modifications in tissue as well as for analysis of excretion of oxidized nucleotides into urine that ensures a minimum artifact formation.

Autoprotection in acetaminophen intoxication in rats: the role of liver regeneration.

Autoprotection by acetaminophen, i.e. increased resistance to toxic effects caused by pretreatment, is a well-known phenomenon. The purpose of the present work was to identify mechanisms for increased acetaminophen tolerance induced by pretreatment of rats. One group of female Wistar rats (pretreated rats) received acetaminophen orally in increasing doses (1 to 4.3 g/kg) twice a week for 3 weeks, one group (naïve rats) received the vehicle. At time zero pretreated rats received a toxic dose of 7.5 g/kg (100% lethal in naïve rats), and naïve rats received a toxic dose of 4.3 g/kg. Blood and liver tissue were collected before and 12, 24, 36, and 48 hr after the toxic dose and were analysed for hepatic glutathione and cysteine contents, hepatic glutathione-S-transferase and blood alanine aminotransferase activity, as well as acetaminophen concentration in plasma. Steady-state mRNA levels of proteins involved in acetaminophen detoxification, cell division and acute phase response were measured, liver tissue was examined for proliferating cell nuclear antigen and degree of hepatocyte necrosis. Six naïve rats not receiving acetaminophen served as controls. The mortality was the same in pre-treated and naïve rats (33 percent). Thus, pretreatment increased the tolerance twice. Before the toxic dose pretreated rats compared to control rats had higher activity of glutathione-S-transferase (liver) and alanine aminotransferase (serum), higher hepatic mRNA level of glutathione-S-transferase and gamma-glutamylcysteine synthetase heavy and light chain subunits, and lower hepatic concentration of glutathione, cysteine and mRNA of CYP1A2 than control rats. After the toxic dose, the mRNA levels of glutathione-S-transferase, gamma-glutamylcysteine synthetase heavy and light chain subunits, and CYP1A2 in naïve rats rose, approaching those of pretreated rats. Proliferating cell nuclear antigen labelling was high in pretreated rats, while only slightly increased in a few of the naïve rats. Necrotic hepatocytes were found at all time intervals in pretreated rats, and in naïve rats they appeared after 12 hr, peaking after 36 hr. Pretreatment increased the tolerance to acetaminophen toxicity twice, as estimated by mortality. The data indicate that pretreatment may reduce the relative production of toxic metabolites, but it primarily enhances the protection against these metabolites by regenerating hepatocytes.

Supplementation with vitamin E but not with vitamin C lowers lipid peroxidation in vivo in mildly hypercholesterolemic men.

Although the use of vitamin E supplements has been associated with a reduction in coronary events, assumed to be due to lowered lipid peroxidation, there are no previous long-term clinical trials into the effects of vitamin C or E supplementation on lipid peroxidation in vivo. Here, we have studied the long-term effects of vitamins C and E on plasma F2-isoprostanes, a widely used marker of lipid peroxidation in vivo. As a study cohort, a subset of the "Antioxidant Supplementation in Atherosclerosis Prevention" (ASAP) study was used. ASAP is a double-masked placebo-controlled randomized clinical trial to study the long-term effect of vitamin C (500 mg of slow release ascorbate daily), vitamin E (200 mg of D-alpha-tocopheryl acetate daily), both vitamins (CellaVie), or placebo on lipid peroxidation, atherosclerotic progression, blood pressure and myocardial infarction (n = 520 at baseline). Lipid peroxidation measurements were carried out in 100 consecutive men at entry and repeated at 12 months. The plasma F2-isoprostane concentration was lowered by 17.3% (95% CI 3.9-30.8%) in the vitamin E group (p = 0.006 for the change, as compared with the placebo group). On the contrary, vitamin C had no significant effect on plasma F2-isoprostanes as compared with the placebo group. There was also no interaction in the effect between these vitamins. In conclusion, long-term oral supplementation of clinically healthy, but hypercholesterolemic men, who have normal vitamin C and E levels with a reasonable dose of vitamin E lowers lipid peroxidation in vivo, but a relatively high dose of vitamin C does not. This observation may provide a mechanism for the observed ability of vitamin E supplements to prevent atherosclerosis.

Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: a randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis.

OBJECTIVES: To study the efficacy of vitamin E and C supplementation on the progression of carotid atherosclerosis, hypothesizing an enhanced preventive effect in men and in smokers and synergism between vitamins. DESIGN AND SUBJECTS: Double-masked two-by-two factorial trial, randomization in four strata (by gender and smoking status) to receive twice daily either 91 mg (136 IU) of d-alpha-tocopherol, 250 mg of slow-release vitamin C, a combination of these or placebo for three years. A randomized sample of 520 smoking and nonsmoking men and postmenopausal women aged 45-69 years with serum cholesterol >/= 5.0 mmol L-1 were studied. SETTING: The population of the city of Kuopio in Eastern Finland. INTERVENTION: Twice daily either a special formulation of 91 mg of d-alpha-tocopherol, 250 mg of slow-release vitamin C, a combination of these (CellaVie(R)) or placebo for three years. MEASUREMENTS: Atherosclerotic progression, defined as the linear regression slope of ultrasonographically assessed common carotid artery mean intima-media thickness (IMT), was calculated over semi-annual assessments. RESULTS: The average increase of the mean IMT was 0.020 mm year-1 amongst men randomized to placebo and 0.018 mm year-1 in vitamin E, 0.017 mm year-1 in vitamin C and 0.011 mm year-1 in the vitamin combination group (P = 0.008 for E + C vs. placebo). The respective means in women were 0.016, 0.015, 0.017 and 0.016 mm year-1. The proportion of men with progression was reduced by 74% (95% CI 36-89%, P = 0.003) by supplementation with the formulation containing both vitamins, as compared with placebo. CONCLUSIONS: Our study shows that a combined supplementation with reasonable doses of both vitamin E and slow-release vitamin C can retard the progression of common carotid atherosclerosis in men. This may imply benefits with regard to other atherosclerosis-based events.

Long-term effects of vitamin E, vitamin C, and combined supplementation on urinary 7-hydro-8-oxo-2'-deoxyguanosine, serum cholesterol oxidation products, and oxidation resistance of lipids in nondepleted men.

We studied the long-term effects of vitamins E and C and their combination on lipid peroxidation in vivo and in vitro. The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) trial is a double-masked placebo-controlled randomized clinical trial to study the effects of vitamin C (500 mg of slow release ascorbate per day), vitamin E (182 mg of RRR-alpha-tocopherol acetate per day), and the combination of both antioxidants. Lipid peroxidation measurements were carried out for 48 male participants at entry and at 12 and 36 months. Compared with placebo, vitamin E and the vitamin combination increased plasma lipid-standardized alpha-tocopherol during the first 12 months by 68.2% and 65.2% (P:<0. 001 for both), respectively, and reduced serum 7beta-hydroxycholesterol by 50.4% (P:=0.013) and 44.0% (P:=0.041), respectively. The net change of lipid standardized alpha-tocopherol was 63.8% after 36 months of vitamin E supplementation and 43.3% for the combination. Vitamin C supplementation elevated plasma total ascorbate level by 30.1% (P:=0.043) in 12 months and by 91.1% (P:=0. 001) in 36 months. Neither vitamin E, vitamin C, nor the combination influenced the urinary excretion rate of 7-hydro-8-oxo-2'-deoxyguanosine or the antioxidative capacity of plasma. Vitamin E and the combination of vitamins E and C enhanced the oxidation resistance of isolated lipoproteins and total serum lipids. Our data indicate that long-term supplementation of nondepleted men with a reasonable dose of vitamin E alone or in combination with slow release vitamin C reduces lipid peroxidation in vitro and in vivo, whereas a relatively high dose of vitamin C alone does not.

Inhibition of oxidative DNA damage in vitro by extracts of brussels sprouts.

Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light. The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC50 values were 6-20 microg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C. Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.

Iron-induced oxidative DNA damage in rat sperm cells in vivo and in vitro.

We investigated whether acute iron intoxication causes oxidative DNA damage, measured in terms of 7-hydro-8-oxo-2'-deoxyguanosine, 8-oxodG, in nuclear DNA in testes and epididymal sperm cells in vivo and in vitro in rats. In addition, we investigated levels of the modified nucleoside in liver and kidney and measured its urinary excretion. Sperm cells were isolated from the epididymides and the testes cells were isolated after homogenisation. In vitro, the sperm and testes cells were incubated with increasing concentrations of FeCl2 ranging from 0 to 600 microM. The median (range) levels of 8-oxodG/10(5) dG in the epididymal sperm cells increased from 0.48 (0.42-0.90) to 15.1 (11.4-17.6) (p < 0.05), whereas the level rose from 0.63 (0.22-0.81) to 8.8 (4.5-11.6) (p < 0.05) at 0 and 600 microM, respectively, in the testicular cells. In vivo groups of 7-8 rats received 0, 200 or 400 mg iron/kg as dextran i.p. After 24 h, epididymal sperm cells, testes, kidneys and liver were collected for analysis. Kidney and sperm DNA showed a significant increase in 8-oxodG in the iron-treated animals. The median (range) values of the 8-oxodG/10(5) dG in the epididymal sperm cells rose from 0.66 (0.38-1.09) to 1.12 (0.84-5.88) (p < 0.05) at 0 and 400 mg iron/kg, respectively, whereas the values in the testes and liver showed no significant change. In the kidneys the 8-oxodG/10(5) dG median (range) values were 0.98 (0.73-1.24), 1.21 (1.13-1.69) and 1.34 (1.12-1.66) after 0, 200 and 400 mg iron/kg, respectively (p < 0.05). The 8-oxodG-excretion rate was measured in 24h urine before and after iron treatment. The rate of urinary 8-oxodG excretion increased from 129 (104-179) pmol/24 h before treatment to 147 (110-239) pmol/24 h after treatment in the group receiving 400 mg iron/kg (p < 0.05). The results indicate that acute iron intoxication may increase oxidative damage to sperm and kidney DNA.

Enhanced benzene-induced DNA damage in PMA-stimulated cells in vitro and in LPS-treated animals.

The present study investigated the interaction between inflammatory reactions and benzene in vitro and in vivo with respect to oxidative DNA damage. In the in vitro models the oxidative burst of cells was induced by the pretreatment with phorbol myristate acetate (PMA) and in the in vivo models of inflammation mice were pretreated with lipopolysaccharide (LPS). The oxidative DNA damage was indicated by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and strand breaks as assessed by alkaline single cell gel electrophoresis (SCGE, Comet assay). The results showed that combination of PMA and benzene enhanced the level of 8-oxodG in DNA from mouse bone marrow cells by 197%, from human lymphocytes by 188% and from human neutrophils by 205% (p < .05). Pretreatment of mice with LPS and benzene resulted in an enhanced Comet score formation in bone marrow cells by 98% and in lymphocytes by 39% in Comet score (p < .05) and in an enhanced 8-oxodG level in bone marrow cells by 290%. The effects of the combined treatment with PMA/LPS and benzene exceeded the sum of the effects induced by PMA/LPS or benzene alone. The production of nitrate/nitrite showed a two fold increase in the supernatant from incubation of benzene and PMA-pretreated neutrophils. The increase in the 8-oxodG level in the human neutrophil incubation system demonstrated a correlation with nitrate/nitrite production, indicating a possible relationship with the generation of reactive nitrogen species.

Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers.

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2'-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated on the workday. Repeated measures ANOVA and multifactorial ANCOVA with CYP1A2 activity, age and UDS as covariates were used for statistical evaluation. On the workday, the 8-oxodG excretion was 190+/-108 and 146+/-89 pmol/kg 24 h in the bus drivers from central and the suburban/rural areas Copenhagen, respectively (p<0.05). The 8-oxodG excretion was not significantly different between the workday and the day off. CYP1A2 activity was not affected by driving area but was correlated with the 8-oxodG excretion on the workday (r=0.53; p<0.05). UDS was not significantly affected by driving area or correlated with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggests that exposure to ambient air pollution causes oxidative damage to DNA. This effect may be modified by the activity of CYP1A2 or a coregulated enzyme.

Dexamethasone ameliorates oxidative DNA damage induced by benzene and LPS in mouse bone marrow.

Mice were grouped to receive vehicle, dexamethasone (DEX), lipopolysaccharide (LPS), benzene (BZ, 200 mg/kg) and combinations: LPS + DEX, BZ + DEX, LPS + BZ, LPS + DEX + BZ. The DNA damage in bone marrow cells from BZ group was enhanced 2.8-fold measured by nuclear 8-hydroxy-2 '-deoxyguanosine (8-oxodG) and 1.4-fold measured by Comet score (index of DNA breaks) (p < 0.05). In the BZ + DEX group, 8-oxodG level and the Comet score were lowered to 65% and 76% respectively of that in the BZ group (p < 0.05). The BZ + LPS caused a 3.9-fold increase in 8-oxodG and a 1.6-fold increase in the Comet score (p < 0.05). The LPS + DEX + BZ lowered 8-oxodG level and the Comet score to 50% and 78% of the values in the LPS + BZ group, respectively (p < 0.05). Nitrate/nitrite levels in serum were higher after BZ + LPS treatment than after all other treatments. Both 8-oxodG level and the Comet scores were correlated to the serum nitrate/nitrite level across all the treatments (r = 0.55, p < 0.01 and r = 0.69, p < 0.01, respectively). In bone marrow cells the 8-oxodG correlated with the Comet scores (r = 0.80, p < 0.01). We conclude that DEX administration can reduce the DNA damage from BZ treatment and from the combination of BZ and LPS. The correlation of DNA damage with nitrate/nitrite indicates the possible involvement of reactive nitrogen species (RNS) in the interaction between BZ and the inflammatory reaction stimulated by LPS. The 8-oxodG determination is more sensitive than strand break analysis by the Comet assay in bone marrow in vivo in mice for measuring the BZ-induced DNA damage.