Changes in Dry State Hemoglobin over Time Do Not Increase the Potential for Oxidative DNA Damage in Dried Blood

BACKGROUND: Hemoglobin (Hb) is the iron-containing oxygen transport protein present in the red blood cells of vertebrates. Ancient DNA and forensic scientists are particularly interested in Hb reactions in the dry state because both regularly encounter aged, dried bloodstains. The DNA in such stains may be oxidatively damaged and, in theory, may be deteriorated by the presence of Hb. To understand the nature of the oxidative systems potentially available to degrade DNA in the presence of dried Hb, we need to determine what molecular species Hb forms over time. These species will determine what type of iron (i.e. Fe(2+)/Fe(3+)/Fe(4+)) is available to participate in further chemical reactions. The availability of "free" iron will affect the ability of the system to undergo Fenton-type reactions which generate the highly reactive hydroxyl radical (OH*). The OH* can directly damage DNA. METHODOLOGY/PRINCIPAL FINDINGS: Oxygenated Hb (oxyHb) converts over time to oxidized Hb (metHb), but this happens more quickly in the dry state than in the hydrated state, as shown by monitoring stabilized oxyHb. In addition, dry state oxyHb converts into at least one other unknown species other than metHb. Although "free" iron was detectable as both Fe(2+) and Fe(3+) in dry and hydrated oxyHb and metHb, the amount of ions detected did not increase over time. There was no evidence that Hb becomes more prone to generating OH* as it ages in either the hydrated or dry states. CONCLUSIONS: The Hb molecule in the dried state undergoes oxidative changes and releases reactive Fe(II) cations. These changes, however, do not appear to increase the ability of Hb to act as a more aggressive Fenton reagent over time. Nevertheless, the presence of Hb in the vicinity of DNA in dried bloodstains creates the opportunity for OH*-induced oxidative damage to the deoxyribose sugar and the DNA nucleobases

Oxygen-derived species: their relation to human disease and environmental stress.

Free radicals and other reactive oxygen species (ROS) are constantly formed in the human body, often for useful metabolic purposes. Antioxidant defenses protect against them, but these defenses are not completely adequate, and systems that repair damage by ROS are also necessary. Mild oxidative stress often induces antioxidant defense enzymes, but severe stress can cause oxidative damage to lipids, proteins, and DNA within cells, leading to such events as DNA strand breakage and disruption of calcium ion metabolism. Oxidative stress can result from exposure to toxic agents, and by the process of tissue injury itself. Ozone, oxides of nitrogen, and cigarette smoke can cause oxidative damage; but the molecular targets that they damage may not be the same

Analysis of plasma indices of redox homeostasis in dairy cows reared in polluted areas of Piedmont (northern Italy)

Steel manufacturing is responsible for the emission of pollutants, including dioxins and transition metals, inducing reactive oxygen species generation and DNA damage. Dioxin pollution represents the major cause of milk and dairy product contamination, in Italy, and is associated with oxidative stress-related processes, that may impair health and performance of cows.We evaluated the effect of exposure to different concentrations of pollutants derived from steel manufacturing on blood redox homeostasis of bovine cows. We analyzed two groups of dairy cows (A, B), reared in two different polluted areas, and a control group of cows bred in an industry free area. The extent of exposure to contaminants was defined by measuring dioxin level in bulk milk samples collected from animals of each farm. This level was lower in milk of group A than in group B. Plasma concentrations of retinol, alpha-tocopherol and ascorbate, the total antioxidant capacity, and the activities of superoxide dismutase and glutathione peroxidase were higher in control group than in exposed groups. In particular, retinol and tocopherol levelswere higher in the groupwith lower milk dioxin level. Plasma titers of protein-bound carbonyls (PC), nitro-tyrosine, and hydroperoxideswere lower in control group than in A or B. Hydroperoxides and PC plasmaconcentrationswere increased in the groupwith higher milk concentration of dioxin. Our results demonstrate that, irrespective of the nature of chemicals inducing oxidativemodifications, the extent of damage to plasmaprotein and lipid, is correlatedwith the concentration of dioxin in milk. So, the characterization of blood redox status might be a useful tool for identifying animals exposed to environmental pollutants. Plasma concentrations of retinol, alpha-tocopherol, PC and hydroperoxides could therefore represent good indices of the extent of animal exposure, as they significantly change in groups with different milk concentrations of dioxi

Effect of Turmeric and Carrot Seed Extracts on Serum Liver Biomarkers and Hepatic Lipid Peroxidation, Antioxidant Enzymes and Total Antioxidant Status in Rats

Introduction: Pathogenic role of free radicals are well known in various metabolic diseases. They originate from internal and external sources of body. Essential roles of antioxidant defense system for cellular redox regulation and free radical scavenging activity were described in this study. Many in vitro investigations have shown that turmeric (TE) and carrot seed extract (CSE) exhibits to possess antioxidant activities. In this study, we evaluated the antioxidant potentials of ethanolic TE and CSE based on in vivo experiment in the rats. Methods: Animals were assigned to six groups: the 1st and 2nd groups were control groups and 2nd group received 0.2 ml dimethyl sulphoxide as vehicle treated group; other four experimental groups received different doses of TE (100, 200 mg/kg b.w.) and CSE (200, 400 mg/kg b.w.) by gavages, respectively for a period of one month. The indicators of oxidative stress, lipids peroxidation, markers of hepatocyte injury and biliary function markers were measured. Results: The levels of superoxide dismutase, catalase, and glutathione peroxidase were significantly stimulated in the hepatic tissue of treatment groups. The malondialdehyde contents of liver tissue were significantly reduced in the groups fed with TE and CSE. Serum levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, in treated groups were found to be significantly decreased, whereas albumin and total protein increased as compared to the control groups (P<0.05). Conclusion: this study showed that the regular intake of TE and CSE through the diet can improve antioxidant status and inhibit peroxidation activity in the liver tissue so that using these extracts may protect tissue oxidative stress

Gene-Environment Interactions: The Case of Asbestosis

It is becoming evident that both environmental/lifestyle and genetic factors may influence the development of many diseases. This chapter highlights the importance of considering gene-environment interactions, which is shown on the example of our studies into asbestosis, one of the most frequent asbestos-related diseases. Asbestos fibres induce generation of reactive oxygen and nitric species (ROS and RNS), and it is generally accepted that ROS and RNS are involved in the pathogenesis of asbestos-related diseases. Human tissues contain specific enzymes that metabolise ROS and RNS, such as superoxide dismutases (SODs), catalase (CAT), glutathione-S-transferases (GSTs) and inducible nitric oxide synthase (iNOS). As these enzymes are encoded by polymorphic genes, genetic variability in an individual’s capacity to detoxify these reactive species may modify the risk for disease. Our previous studies into asbestosis showed that the associations between the risk of asbestosis and MnSOD Ala-9Val polymorphism and between asbestosis and iNOS genotypes were modified by CAT −262C>T polymorphism. A strong interaction was also found between smoking (lifestyle factor) and GSTM1-null polymorphism, between smoking and iNOS (CCTTT)n polymorphism and between cumulative asbestos exposure (environmental factor) and iNOS (CCTTT)n polymorphism. The findings of our studies and other studies indicate that in addition to environmental and/or occupational exposure to different hazards and lifestyle factors, genetic factors as well as the interactions between different genotypes, between genotypes and lifestyle factors and between genotypes and environmental/occupational exposure to hazards may also have an important role on the development of diseases and should be further investigated

Metal Chelating Activity Of Rice Bran And Rice Husk

Free radical-induced oxidative stress is the root cause for many human diseases. Naturally occurring antioxidant supplements from plants are vital to counter the oxidative damage in cells. The main objective of the present study was to explore and compare the antioxidant activity of rice bran and rice husk extracted from rice milling waste. N-hexane extracts of rice bran and rice husk were used to study their in vitro antioxidant activities using metal chelating activity (iron (II)-phenanthroline complex). Vitamin E was used as standard material. The ability of the sample to chelate metal ion (ferrous ion) was calculated relative to the control and expressed as % inhibition. % inhibition of two samples were analyzed with student test (P=0.05). The results have shown that at the same concentration (10 ppm), rice husk extract, rice bran extract and vitamin E have the different activity, i.e.: 0.51%, 2.27% and 5.55% in inhibition of chelat formation, respectively. In conclusion, metal chelating activity of rice husk extract is smaller than rice bran extract. Activity of rice bran extract is almost a half from vitamin E, so thi

Characterization and Partial Purification of a Factor Supporting NADPH-Dependent Lipid Peroxidation.

The NADPH-dependent lipid peroxidation in rat liver microsomal membrane was studied by using an effective and sensitive "reconstituted lipid peroxidation assay", which closely resembled the microsomal system. The NADPH-dependent lipid peroxidation was monitored by Thiobarbituric Acid Reactive Substances (TBARS) formation in the reconstituted assay system, initially using a 1.2%-0.4% (w/v) differentially cholate solubilized fraction (CCMs), which had a 3-fold increase in relative specific activity compared to microsomes. The lipid peroxidation activity in the reconstituted assay was demonstrated to be dependent on the presence of NADPH, NADPH-Cytochrome P-450 reductase enzyme, CCMs and extracted microsomal membrane phospholipids. The optimal concentrations of these reagents in the reconstituted assay, were determined. It was shown that the factor supporting NADPH-lipid peroxidation activity was heat labile, lending support to the possible protein nature of this factor. By using Gas Chromatography, it was demonstrated that arachidonate and docosahexanoate fatty acids were the preferred substrates in the assay. Both phosphate and cholate detergent inhibited lipid peroxidation activity. The lipid peroxidation active fraction was extremely labile. Solubilization with alternative detergents such as 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), or addition of protease inhibitors such as phenylmethylsulfonylfluoride, did not improve its labile nature. However, addition of bovine serum albumin did improve stability but then further purification steps were required. Nevertheless, some degree of purification of the lipid peroxidation activity was achieved. An 11-, 10-, 5- and 4- fold purification was achieved (compared to microsomes) using 75%-45% (w/v) ammonium sulphate precipitation method, diethylaminoethyl-, hydroxyapatite- and laurate-sepharose- chromatographies, respectively. All the post chromatography eluate fractions with measurable lipid peroxidation activity, had an increased level of “background” TBARS. This resulted in an overall decrease in their lipid peroxidation activity. Although an electrophoretically homogeneous preparation was not produced, partial progress has been made, providing a basis for future purification and characterisation of this enzyme system