The use of alkaline elution procedures to measure DNA damage in spermiogenic stages of mice exposed to methyl methanesulfonate

The ability of methyl methanesulfonate (MMS) to induce DNA breakage in spermiogenic stages of the mouse was studied using an alkaline elution technique. At daily intervals over a 3-week period following i.p. injection of 50 mg MMS/kg, mature spermatozoa were recovered from treated (3H-labeled) and control (14C-labeled) animals, lysed together on polycarbonate filters, and eluted with a high pH (12.2) buffer. Elution of germ-cell DNA from MMS-treated animals was found to increase in stages in which genetic damage from MMS is greatest. In general, the pattern of DNA elution from treated, spermiogenic stages paralleled the pattern of sensitivity to dominant lethals, specific-locus mutations and heritable translocations found by other investigators. It also paralleled the pattern of sperm-head methylation and protamine methylation measured in an earlier study (Sega and Owens, 1983). At 9 days post treatment (sperm sampled were in mid-to late-spermatid stages at the time of MMS exposure) the elution of sperm DNA did not change significantly over a pH range of 11.6-12.8, suggesting that, at the time of assay, DNA breaks were already present in the sperm. Because of the parallelism found between increased sperm DNA elution and increased genetic damage after mutagen treatment, alkaline elution may prove useful in monitoring potential genetic damage in human sperm.     

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.     

Detection of DNA damage in spermiogenic stages of mice treated with enriched uranyl fluoride by alkaline elution

Summary DNA breakage in spermiogenic stages of mice treated with enriched uranyl fluoride (UO₂F₂) was studied using an alkaline elution technique. Mature spermatozoa were sampled from the animal's vas and eluted with a buffer (PH 12.2) at 3-day intervals over a 33-day period after i.p. injection of 2 mg U0₂F₂/kg and always at 36 days after thymidine labeling in the testes. Elution of sperm DNA from treated animals varied with spermiogenic stages. At 12 days after exposure the amount of the elution of sperm DNA was found highest and increased with the increasing U0₂F₂ dose up to 6 mg/kg.     

Conformation of the fowl protamine, galline, and its binding properties to DNA

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.     

Dynamics of protamine 1 binding to single DNA molecules

Protamine molecules bind to and condense DNA in the sperm of most vertebrates, packaging the sperm genome in an inactive state until it can be reactivated following fertilization. By using methods that enable the analysis of protamine binding to individual DNA molecules, we have monitored the kinetics of DNA condensation and decondensation by protamine 1 (P1) and synthetic peptides corresponding to specific segments of the bull P1 DNA binding domain. Our results show that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex prior to the formation of inter-protamine disulfide cross-links. The high affinity of P1 for DNA is achieved by the coordinated binding of three anchoring domains, which together in bull P1 contain 19 Arg residues. The single DNA molecule experiments show that sequences containing two or more anchoring domains have an off-rate that is at least 3 orders of magnitude slower than those containing a single domain. The use of Arg, rather than Lys residues, and the inclusion of Tyr or Phe residues in the hinge regions between anchoring domains provide additional stability to the complex.     

The binding of protamines to DNA; Role of protamine phosphorylation

The thermodynamics of protamine-DNA interaction was investigated with clupeine Z from herring labeled at its amino terminus with fluorescein. The ionic strength dependence, the influence of protamine phosphorylation, of the native DNA conformation, using native and heat-denatured DNA, and of the protamine primary structure, using two oligoarginine peptides of similar length as the clupeine, was thoroughly studied. The unusually high cooperativity of interaction found is strictly correlated to the native DNA conformation and the protamine primary structure. Cooperativity is explained by cross-linking of DNA segments resulting in an increase of the negative charge density. The importance of protamine phosphorylation lies in the fact that thermodynamically governed interaction with DNA and favorable cross-linking of DNA are shifted to physiologically reasonable ionic strengths.Abbreviations FITC fluorescein isothiocyanate - FTC-clupeine clupeine labeled at its amino terminus with fluorescein via a thiocarbamate bond     

Characterization of the fluorescence of the protamine thynnine and studies of binding to double-stranded DNA

The protamine thynnine is an arginine-rich protein approximately 30 amino acids long with a tyrosine in the middle of its sequence. Its fluorescence decay kinetics can be described by a biexponential function with lifetimes of 0.52 and 2.1 ns, with almost equal preexponential factors. The fluorescence quencher CsCl does not affect the short lifetime but shifts the equilibrium between the long and short lifetime toward the short one and reduces the long lifetime. In nature, thynnine is found complexed with chromosomal DNA. In vitro complexes of thynnine with double-stranded (ds) DNA are stable at physiologic ionic strength but dissociate at high NaCl concentration. This dissociation can be monitored by steady-state fluorescence. From the salt concentration dependence of the dissociation of the complex of thynnine with ds-DNA 145 bp long, it can be concluded that only 4 of 21 possible full electrostatic bonds are involved in thynnine-DNA binding. In addition, the binding constant at 1M NaCl is of the order of 10⁶, indicating a strong nonelectrostatic component in arginine-DNA binding.     

Measurement of DNA breakage in spermiogenic germ-cell stages of mice exposed to ethylene oxide, using an alkaline elution procedure

DNA breakage in spermiogenic stages of the mouse was studied after exposure to ethylene oxide (EtO), using an alkaline elution technique. At daily intervals over a 23-day period following i.p. injection of 100 mg EtO/kg, mature spermatozoa were recovered from treated ([3H]dThd-labeled) and control ([14C]dThd-labeled) animals, lysed together on polycarbonate filters, and the DNA was eluted with a high pH (12.2) buffer. Elution of germ-cell DNA from EtO-exposed animals increased (more DNA strand breaks) in stages sensitive to the genetic effects of EtO (late spermatids to early spermatozoa). The stage-related pattern of EtO-induced DNA breakage paralleled the pattern of sperm alkylation and protamine alkylation found to be produced by EtO in an earlier study (Sega and Owens, 1987). At 9 days posttreatment (sperm sampled were in late-spermatid stages at the time of EtO exposure) the amount of sperm DNA eluted did not change significantly over a pH range of 11.6-12.8, indicating that, at the time of assay, DNA breaks were already present in the sperm.     

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.     

The binding mode of a mammalian (boar) protamine to DNA

The binding modes of mammalian and fish protamines to DNA were studied by reconstitution experiments from dansylated protamines and DNA, using fluorescence spectroscopy, thermal denaturation and sedimentation. Both boar and fish protamines showed strong positive cooperativity in binding to DNA. Binding parameters of the protamines were determined in 0.1 M NaCl, 50 mM Tricine-HCl, pH 7.4, at 37 degrees C: in the boar protamine, the cooperative binding constant (Kc) = 3.4 X 10(6) M-1 and the cooperative factor (q) = 667, in the fish protamine, Kc = 1.8 X 10(7) M-1 and q = 304. The boar protamines bound to DNA with two functional domains, but the fish protamines bound directly to DNA as a single linear molecule.     

Bleomycin-induced alterations in DNA replication: relationship to DNA damage

Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).     

Cellular DNA damage by the antitumor protein macromomycin and its relationship to cell growth inhibition

Macromomycin, an antitumor protein, after purification inhibited HeLa S₃ cell growth at low nanomolar concentrations. DNA synthesis was inhibited at drug levels that left RNA and protein synthesis unaffected. Incubation of macromomycin with HeLa S₃ cells resulted in a rapid fragmentation of cellular DNA that was detectable at low nanomolar drug levels. Heat denaturation of macromomycin showed that both its ability to inhibit cell growth and to fragment cellular DNA were lost at similar rates.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

DNA damage in mouse lymphocytes exposed to curcumin and copper

Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate thein vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 μM curcumin and various concentrations of copper (10 μM, 100 μM and 200 μM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 μM curcumin in the presence of 100–200 μM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 μM H₂O₂ in mouse lymphocytes. Moreover, 50 μM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 μM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.     

Genomic DNA damage in mouse transgenesis

Creating transgenic mammals is currently a very inefficient process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In the present study, we demonstrate that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by microinjection of pronuclei, intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF)-mediated transgenesis, and correlated these findings with the abilities of embryos to develop in vitro and yield transgenic morulas/blastocysts. Chromosomal analysis was performed after microinjection of the pronuclei in zygotes, as well as in parthenogenetic and androgenetic embryos. In all the pronuclei injection groups, significant oocyte arrest and increased incidence of chromosome breaks were observed after both transgenic DNA injection and sham injection. This indicates that the DNA damage is a transgene-independent effect. In ICSI-mediated transgenesis, there was no significant oocyte arrest. The observed chromosomal damage was lower than that after pronuclei microinjection in zygotes and was dependent upon the presence of exogenous DNA. The occurrence of DNA breaks, as measured by comet assay performed on the sperm prior to ICSI, showed that DNA damage was present in the sperm before fertilization. Embryonic development in vitro and transgene expression at the morula/blastocyst stage were higher in ICSI-mediated transgenesis than after microinjection of pronuclei into zygotes. Sperm-mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development, but did not yield any transgenic embryos. The present study demonstrates that DNA damage occurs after both the microinjection of pronuclei and ICSI-mediated transgenesis, albeit through different mechanisms.     

DNA damage and repair in brain: relationship to aging.

The usefulness of conducting DNA damage and repair studies in a postmitotic tissue like brain is emphasized. We review studies that use brain as a tissue to test the validity of the DNA damage and repair hypothesis of aging. As far as the accumulation of age dependent DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting and equally divided. Possible reasons for this discrepancy are discussed. It is suggested that assessment of the repair capacity of neurons with respect to a specific type of damage in a specific gene might yield more definitive answers regarding the role of DNA repair potential in the aging process and as a longevity assurance system.     

DNA packaging in mouse spermatids : Synthesis of protamine variants and four transition proteins

A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.