Smoking and low antioxidant levels increase oxidative damage to sperm DNA.

Our previous studies have shown that men with low ascorbate intake have markedly increased oxo8dG in the DNA of their sperm. Because cigarette smoke is high in oxidants and depletes plasma and tissue antioxidants, oxidative DNA damage in sperm and tocopherol and ascorbate levels in seminal plasma were determined in smokers and non-smokers. The level in sperm DNA of oxo8dG, an oxidative lesion of guanine, was 50% higher in smokers compared to nonsmokers (p = 0.005). The concentration of alpha-tocopherol in seminal plasma was decreased in smokers by 32% (p = 0.03). Smoking and low antioxidant levels increase oxidative damage to sperm DNA. We discuss the possibility that paternal smoking causes mutations in sperm that lead to cancer, birth defects, and genetic diseases in offspring.

Oxidative susceptibility of low density lipoprotein subfractions is related to their ubiquinol-10 and alpha-tocopherol content.

The conjugated polyene fatty acid parinaric acid (PnA) undergoes a stoichiometric loss in fluorescence upon oxidation and can be used to directly monitor peroxidative stress within lipid environments. We evaluated the course of potentially atherogenic oxidative changes in low density lipoproteins (LDL) by monitoring the oxidation of PnA following its incorporation into buoyant (p = 1.026-1.032 g/ml) and dense (p = 1.040-1.054 g/ml) LDL subfractions. Copper-induced oxidation of LDL-associated PnA exhibited an initial lag phase followed by an increased rate of loss until depletion. Increased PnA oxidation occurred immediately after the antioxidants ubiquinol-10 and alpha-tocopherol were consumed but before there were marked elevations in conjugated dienes. Despite differences in sensitivity to early oxidation events, PnA oxidation and conjugated diene lag times were correlated (r = 0.582; P = 0.03), and both indicated a greater susceptibility of dense than buoyant LDL in accordance with previous reports. The greater susceptibility of PnA in dense LDL was attributed to reduced levels of ubiquinol-10 and alpha-tocopherol, which were approximately 50% lower than in buoyant LDL (mol of antioxidant/mol of LDL) and together accounted for 80% of the variation in PnA oxidation lag times. These results suggest that PnA is a useful probe of LDL oxidative susceptibility and may be superior to conjugated dienes for monitoring the initial stages of LDL lipid peroxidation. Differences in oxidative susceptibility among LDL density subfractions are detected by the PnA assay and are due in large part to differences in their antioxidant content.

Measurement of antioxidants in human blood plasma.

The concentration of antioxidants in human blood plasma is important in investigating and understanding the relationship between diet, oxidant stress, and human disease. The HPLC-EC technique combines selectivity with high sensitivity for measuring both water- and lipid-soluble antioxidants. The excellent sensitivity of the methods described here allows one to measure a panel of antioxidants in a small volume of plasma.

The oxidation of blood plasma and low density lipoprotein components by chemically generated singlet oxygen.

Human blood plasma and freshly isolated LDL were exposed to singlet oxygen (1O2) by thermal decomposition of synthetic endoperoxides. Exposure of blood plasma to 20 mM water-soluble 1O2 generator resulted in the depletion of ascorbate (100%), urate (75%), ubiquinol-10 (65%), protein thiols (50%), and bilirubin (25%), whereas under these conditions the levels of alpha-tocopherol, beta-carotene, and lycopene remained unchanged. The following rates of depletion were obtained by kinetic analysis (moles depleted per 100 mol of 1O2 consumed): protein thiols (5), urate (5), ascorbate (4), bilirubin (1), and ubiquinol-10 (0.008). In contrast, the rates of depletion using the lipid-soluble 1O2 generator were faster for bilirubin (13-fold), protein thiols (9-fold), ubiquinol-10 (8-fold), and ascorbate (5-fold), and slower for urate (2-fold). The formation of lipid hydroperoxides, including mostly cholesteryl linoleate hydroperoxide, was observed in 1O2-treated plasma (0.007-0.009 mol/100 mol 1O2) and LDL solutions (0.086 mol/100 mol 1O2). Based on competition kinetics, we estimate that 98% of 1O2 generated in the aqueous phase of plasma is quenched by components in this phase, mostly by plasma protein (63%; 6% by protein thiols), urate (9%; 5% by chemical quenching), and bilirubin (5%; 1% by chemical quenching). Ascorbate and ubiquinol-10 do not contribute to 1O2 quenching in plasma, and their oxidation is probably mediated secondary species. The remaining 1O2 generated in plasma (2%) diffuses into lipoprotein leading to the formation of lipid hydroperoxides with an efficiency of about 100-fold greater than that compared to aqueous generated 1O2. The principal 1O2 quenchers in LDL include apoB (42%), lycopene and beta-carotene (40%), and alpha-tocopherol (17%). The importance of carotenoids in the quenching of 1O2 in lipoprotein suggest that the beneficial effects of these compounds in health may in part be due to the elimination of this species in biology and medicine.

Toxic hydroperoxides in intravenous lipid emulsions used in preterm infants.

The unsaturated fatty acids that make up a large component of the lipid emulsion Intralipid are highly susceptible to peroxidation, and the products of this reaction could explain the toxicity that has been associated with the administration of some emulsions. Lipid peroxidation produces hydroperoxides, which can alter arachidonic acid metabolism or react to form organic free radicals, which then stimulate a cascade of damage to endogenous lipids. The lipid hydroperoxides and their breakdown products are also mutagens and carcinogens. To determine the degree of lipid peroxidation in Intralipid, we measured the lipid hydroperoxide content of three lots of 20% Intralipid using high-performance liquid chromatography with chemiluminescence detection. The average concentration was 290 +/- 29 mumol/L (SEM) lipid hydroperoxides (n = 15), a large portion of which was made up of trilinoleate derivatives. Measurements made on Intralipid samples collected from the end of the intravenous tubing after a 20-hour infusion cycle were not significantly different from measurements made on newly opened bottles. The lipid hydroperoxide content of some lipid emulsions may represent a clinically significant risk to premature infants, particularly those with preexisting lung disease.

Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation.

Exposure of human plasma to gas-phase (but not to whole) cigarette smoke (CS) produces oxidative damage to lipids [Frei, Forte, Ames & Cross (1991) Biochem. J. 277, 133-138], which is prevented by ascorbic acid. The ability of CS to induce protein damage was measured by the carbonyl assay and by loss of enzyme activity and protein -SH groups. Both whole and gas-phase CS caused formation of carbonyls in human plasma, which was partially inhibited by GSH but not by ascorbic acid or metal-ion-chelating agents. Isolated albumin exposed to CS showed much faster carbonyl formation (per unit protein) than did whole plasma; damage to isolated albumin was partially prevented by chelating agents. Isolated creatine kinase (CK) lost activity upon exposure to CS much faster than did CK in plasma. Direct addition to plasma of mixtures of some or all of the aldehydes reported to be present in CS caused protein carbonyl formation and inactivation of CK, but neither occurred to the extent produced by CS exposure.

Oxidative damage to plasma constituents by ozone.

The reaction of ozone (O3) with human blood plasma was studied to help understand possible events that could occur in the respiratory tract. Uric acid (quantitatively the most important scavenger) and ascorbic acid were oxidized quickly, protein-SH groups were lost more slowly, and there was no loss of bilirubin or alpha-tocopherol. There was little formation of lipid hydroperoxides and no detectable formation of 4-hydroxynoneal, hexanal or nonanal, or changes in lipoprotein electrophoretic mobility. Uric acid in human upper airway secretions may play a significant role in removing inhaled O3. Oxidative damage to lipids must not be assumed to be the key mechanism of respiratory tract O3 toxicity.

Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.

Multiple selenocysteine content of selenoprotein P in rats.

Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport.

Selenium-containing proteins of rat kidney and liver microsomes.

Selenium (Se)-containing proteins in microsomal fractions of rat kidney and liver were investigated after isotopic labeling of rats with [75Se]selenite. More than 85% of the 75Se in the solubilized microsomal extracts precipitated with protein after trichloroacetic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), used to separate the labeled protein subunits in the solubilized microsomal extracts, revealed several 75Se-containing proteins in addition to glutathione peroxidase. 75Se-labeled subunits with molecular weights of 55, 30, 26, 22, 19, and 17 kDa were present in microsomal fractions of kidney and liver. The 75Se-labeled tryptic peptide of the 55 kDa subunit had the same Rf value on a 17% SDS-PAGE gel as the peptide from plasma selenoprotein P. A time-course study of the labeling of individual protein subunits in kidney and liver microsomes from Se-supplemented and Se-deficient rats showed that most of the 75Se was associated with the 55 kDa subunit 3 hr after injection. The amount of 75Se associated with this protein subunit decreased by 12 hr, with a concurrent increase in the labeling of lower molecular-weight subunits. The results support the hypothesis that there is a mechanism for transfer of Se from the 55 kDa subunit to other Se-containing proteins.

Rat plasma selenoprotein P properties and purification.

A selenoprotein in rat plasma, selenoprotein P, was fractionated and characterized. Plasma collected from rats 3 h post injection of 75SeO3(2-) contained one 75Se-labeled protein, selenoprotein P. Selenoprotein P was fractionated using salt precipitation, Affi-Gel Blue, and DEAE chromatography. The 75Se-containing subunit of selenoprotein P was purified to 90% homogeneity using SDS-polyacrylamide gel electrophoresis followed by electroelution. This isolation resulted in an 850-fold purification of the 75Se-containing subunit of selenoprotein P with a 15% yield of 75Se radioactivity. The molecular weight of selenoprotein P in plasma was 98,000. The 75Se-containing subunit of selenoprotein P had a molecular mass of 57 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing under nondenaturing conditions resulted in a band of 75Se radioactivity at pH 5.4. A comparison of Coomassie Blue- and silver-staining properties of selenoprotein P in SDS-polyacrylamide gels was made. Reverse-phase HPLC and Sephadex G-50 chromatography of tryptic peptides of the 57 kDa subunit of selenoprotein P yielded several peaks of 75Se radioactivity. These results indicate that 75Se is present in several locations within the 57 kDa subunit of selenoprotein P.

Halogenated hydrocarbon and hydroperoxide-induced peroxidation in rat tissue slices.

Tissue slices were used to compare relative peroxidation capacity of bromotrichloromethane (BrCCl3) and t-butyl hydroperoxide (BHP) by measurement of both peroxidation products and biochemical indices of damage. In liver and testes slices, BHP increased thiobarbituric acid reactive-substances (TBARS) and total aldehydes, measured as cyclohexanedione-reactive substances (CHDRS), to a greater extent than did an equimolar amount of BrCCl3. GSH was decreased more by BHP than by BrCCl3. Neither compound released lactate dehydrogenase or glutamic-pyruvic transaminase from liver slices. Treatment of rats with cyanamide, an aldehyde dehydrogenase inhibitor, increased the total CHDRS in liver slices and medium after incubation with BHP or BrCCl3. HPLC of the CHDRS showed hexanal and propanal increased to the greatest extent. The hydroperoxide, BHP, which does not require metabolism to an active species, was a better initiator of peroxidation than the halogenated hydrocarbon, BrCCl3, which must be metabolized to a radical species before it can initiate peroxidation.