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 eaition of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxoIdG), 1 of "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 A-A levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo'dG were correlated with low seminal plasma AA. The endogenous level of oxodG in this group was 13 fmol per jzg of DNA or "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 oxoedG 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.The damage produced by endogenously generated oxygen radicals has been proposed to be a major contributing factor in aging and the many degenerative processes associated with it including cancer, heart disease, and cognitive dysfunction (1-6). Under physiological conditions, endogenous oxidants are produced at a high rate, resulting in extensive oxidative damage to proteins, lipids, and DNA (5-8). Oxidative damage to DNA, based on the urinary excretion ofDNA adducts, occurs at an estimated rate of 105 hits per cell per day in the rat (8) and 104 hits per cell per day in the human (9).Endogenous oxidative damage to germline DNA is likely to lead to heritable mutations and increased incidence of birth defects, genetic diseases, and cancer in offspring. Ionizing radiation, an oxidative mutagen, damages gamete DNA, resulting in mutations that are transmitted to the progeny in experimental animals (10). Most, but not all, DNA damage is expected to be repaired, and damaged residues that remain may be converted to mutations during the DNA replication that accompanies cell division in spermatogenesis, oogenesis, and embryogenesis. Studies of genetic abnormalities and cancers believed to arise from germline mutations show a higher frequency of paternal than maternal origin (11), such as the increased frequency of paternal origin of germline mutations in the retinoblastoma gene (12). This is consistent with the markedly higher number of cell divisions that occur during spermatogenesis (relative to oogenesis), resulting in an increase in the risk for mutation in sperm. Once spermatogen...

No abstract

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

No abstract

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.