[Technical study on inactivating/removing virus in collagen sponge].

OBJECTIVE: To verify the technics of inactivating/removing virus in collagen sponge derived from bovine Achilles tendon. METHODS: Possible pathogen species were determined according to the raw material of bovine Achilles tendon used in production, then vesicular stomatitis virus (VSV), theiler's mouse encephalomyelitis virus (TEMV), pseudorabies virus (PRV), and simian vacuolating virus 40 (SV40) were selected as indicator virus. Virus suspension was prepared in accordance with Technical Standard for Disinfection. 60Co radiation 25 kGy of collagen sponge was determined as inactivating/removing virus process according to the analysis of the manufacture process, the virus inactivation/removal effect was verified by the measurement of median tissue culture infective dose (TCID50) and showed by virus reduction factor (sample average values of numerical difference before and after processing). RESULTS: Reduction factors of VSV, TEMV, PRV, and SV40 after 60Co radiation 25 kGy were 5.646, 4.792, 5.042, and 5.292 logTCID50/0.1 mL (logs), respectively. Reduction factor of each indicator virus was greater than 4 logs, showing that 60Co irradiation 25 kGy can effectively inactivate and remove viruses. CONCLUSION: 60Co radiation 25 kGy of collagen sponge derived from bovine Achilles tendon can be used as the technics of inactivating/removing virus during the preparation process of collagen sponge to guarantee the safety of the product.

DNA strand breakage by bivalent metal ions and ionizing radiation.

PURPOSE: To investigate mechanisms of DNA breakage via the interaction of bivalent metal ion, thiol reducing agent and ionizing radiation, in *OH scavenging abilities comparable to those in cells. MATERIALS AND METHODS: We measured the effects of 10 min exposure to 200 microM Fe2+ vs. Fe3+ on the induction of single (SSB) and double (DSB) strand breaks in unirradiated and oxically irradiated SV40 DNA, in aqueous solution containing 75 or 750 mM glycerol and/or 5 mM glutathione (GSH). RESULTS: Fe2+ or GSH alone produced little DNA damage. However, their combination produced a dramatic increase in the production of both SSB and DSB. Experiments with ferric ion suggest that it produces DNA damage only after partial reduction to ferrous by GSH. Induction efficiencies for SSB in the presence of Fe2+/GSH showed additivity of the effects of radiation alone with those from Fe2+/GSH. However, the corresponding induction efficiencies for DSB demonstrated a 2.5-fold enhancement. CONCLUSIONS: Our results are consistent with a model in which reduced bivalent metal ions plus thiols, in the presence of O2, produce DSB in DNA primarily via local clusters of hydroxyl radicals arising from site specific Fenton reactions. The synergism observed between DSB production by Fe/GSH and by ionizing radiation, also believed to occur via local clusters of hydroxyl radicals, is consistent with this model. Our results suggest that both normally present intracellular iron and ionizing radiation may be important sources of oxidative stress in cells.

Virus inactivation and protein recovery in a novel ultraviolet-C reactor.

BACKGROUND AND OBJECTIVES: Ultraviolet-C (UVC) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. A reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled UVC treatment. The objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor. MATERIALS AND METHODS: Virus- and mock-spiked Alpha1-proteinase inhibitor (Alpha1-PI) solutions were treated with UVC. The virus samples were assayed for residual infectivity and amplified by the polymerase chain reaction (PCR). The mock-spiked samples were assayed for protein integrity. RESULTS: Greater than 4 log10 of all test viruses were inactivated, regardless of the type of nucleic acid or presence of an envelope. Unlike previous studies, viruses with the smallest genomes were found to be those most sensitive to UVC irradiation, and detection of PCR amplicons > or = 2.0 kb was correlated to viral infectivity. Doses that achieved significant virus inactivation yielded recovery of > 90% protein activity, even in the absence of quenchers. CONCLUSIONS: The results demonstrate the effectiveness of UVC treatment, in the novel reactor, to inactivate viruses without causing significant protein damage, and confirm the utility of large PCR amplicons as markers for infectious virus.

Wild-type p53 inhibits replication-associated homologous recombination.

In mammalian cells homologous recombination is stimulated, when the replication fork stalls at DNA breaks or unrepaired lesions. The tumor suppressor p53 downregulates homologous recombination independently of its transcriptional transactivation function and has been linked to enzymes of DNA recombination and replication. To study recombination with respect to replication, we utilized a SV40 virus based assay, to follow the synchronous events after primate cell infection. gamma-ray treatment at different times after viral entry unveiled an increase of interchromosomal exchange frequencies, when the damage was introduced during DNA synthesis. Elevated recombination frequencies were fully suppressed by p53. With respect to the downregulation of spontaneous recombination, we noticed a requirement for active p53 molecules, when replication started. After a transient treatment with replication inhibitors, we observed inhibition of the drug induced recombination by p53, particularly for the elongation inhibitor aphidicolin. Consequently, we propose that p53 is a surveillance factor of homologous recombination at replication forks, when they stall as a consequence of endogenous or of exogenously introduced damage.

Genomic DNA regions whose complementary strands are prone to UV light-induced crosslinking.

We prepared an extensive set of DNA restriction fragments, irradiated them with UV light, and detected crosslinked complementary strands by electrophoresis in denaturing agarose gels. These experimental data were quantified by densitometry to determine tetranucleotide contributions to crosslinking. The tetranucleotide contributions were used to predict genomic maps of the crosslinking probability that permitted us to identify two strongly crosslinking genomic regions having 295 and 389 base pairs in length. The two sequences shared the (ATTTTATA).(TATAAAAT) octamer, which is a candidate for the hotspot of UV light-induced crosslinking between the complementary strands of DNA.

Roles of replication protein A and DNA-dependent protein kinase in the regulation of DNA replication following DNA damage.

Exposure of mammalian cells to DNA damage-inducing agents (DDIA) inhibits ongoing DNA replication. The molecular mechanism of this inhibition remains to be elucidated. We employed a simian virus 40 (SV40) based in vitro DNA replication assay to study biochemical aspects of this inhibition. We report here that the reduced DNA replication activity in extracts of DDIA-treated cells is partly caused by a reduction in the amount of replication protein A (RPA). We also report that the dominant inhibitory effect is caused by the DNA-dependent protein kinase (DNA-PK) which inactivates SV40 T antigen (TAg) by phosphorylation. The results demonstrate that RPA and DNA-PK are involved in the regulation of viral DNA replication after DNA damage and suggest that analogous processes regulate cellular DNA replication with the DNA-PK targeting the functional homologues of TAg.

Inhibition of initiation of simian virus 40 DNA replication during acute response of cells irradiated by ultraviolet light.

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.

Yield of strand breaks as a function of scavenger concentration and LET for SV40 irradiated with 4He ions.

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

Induction of DNA breaks in SV40 by heavy ions.

Simian virus (SV40) DNA was used to study the induction of DNA strandbreaks by heavy ions varying in LET. DNA was exposed to X-rays and to accelerated particles either in dilute solution or in the presence of different radical scavengers. Relative proportions of the intact supercoiled DNA, nicked form arising from single strand breaks (SSB) and linear molecules produced by double strandbreaks (DSB) were quantified on the base of their electrophoretic mobility in agarose gels. Cross sections for the induction of SSBs and DSBs were calculated from the slope of dose effect curves. Mercaptoethanol was found to protect more efficiently against DNA strand breakage than Tris. When the biological efficiency, i.e. the number of strand breaks per unit dose and molecule weight was evaluated as a function of LET, curves for SSB induction always showed a continuous decrease. For DSB induction, an increase in the yield of DSBs with a maximum around 500 keV/micrometer was observed in the presence of radical scavenger. This peak of biological efficiency gradually disappeared when the radiosensitivity of the system was increased, and was no longer apparent in the dilute buffer system, where DNA showed a high susceptibility to strand breakage. When the relative biological efficiency was plotted versus LET, the curve for DSB induction observed in a low radical scavenging environment paralleled the curve obtained for SSB induction.

Relative induction of cyclobutane dimers and cytosine photohydrates in DNA irradiated in vitro and in vivo with ultraviolet-C and ultraviolet-B light.

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus.

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vectors. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA than virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.

Singlet molecular oxygen induced mutagenicity in a mammalian SV40-based shuttle vector.

We have determined the deleterious effects of singlet oxygen (1O2), generated by thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene)dipropionate (NDPO2), on plasmid DNA. By following the electrophoretic mobility of DNA on agarose gels, we detected single and double strand breaks induced by treatment with NDPO2. The vector employed was a mammalian shuttle vector and the mutagenic consequences of these damages were investigated, using as mutation target the supF suppressor tRNA gene. A high increase of the mutation frequency, over the background, was observed in plasmids transfected in bacteria or after passage through mammalian cells. Trapping agents and quencher effects and other controls confirm the involvement of 1O2 in DNA damage and mutagenicity. These findings indicate that 1O2 can induce DNA lesions which are repaired by an error-prone process in prokaryotic and eukaryotic cells.

Inhibition of in vitro SV40 DNA replication by ultraviolet light.

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble human cell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m2. The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication.

UV-enhanced reactivation of UV-damaged SV40 is due to the restoration of viral early gene function.

Mammalian cells respond to UV-radiation by inducing an increased ability to support the survival of UV-damaged virus. We have tested whether the induction of enhanced viral reactivation (ER) reflects heightened UV-resistance of specific viral functions. For this, we examined the extent of ER for SV40 containing UV-damage in three functionally distinct regions of the SV40 genome: (i) the viral regulatory region, (ii) the early genes region and (iii) the late genes region. ER corresponding to a dose reduction factor of 43% was observed for damage in the early genes region. No ER was observed for damage in the regulatory or late genes regions. We conclude that ER in SV40 reverses the lethal disruption of an essential function peculiar to the viral early genes region. This function is almost certainly transcription.

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells.

UV light induces DNA lesions which are mutagenic in mammalian cells. We used simian virus 40 tsB201 (unable to produce viral capsid at the restrictive temperature of 41 degrees C because of a point mutation in the VP1 gene) to analyze the mutagenic potency of the two major UV-induced lesions, pyrimidine dimers (Py-Py) and pyrimidine (6-4) pyrimidones [Py(6-4)Py], which are formed on the same nucleotide sites. The mutagenesis criterion was the reversion toward a wild-type growth phenotype. After UV irradiation (mainly at 254 nm), part of the DNA was treated with the photoreactivating enzyme of Escherichia coli, which monomerizes Py-Py but does not modify the Py(6-4)Py photoproduct. Higher survival and lower mutation frequency rates for the photoreactivated DNA indicated that the two lesions were lethal and mutagenic. The VP1 gene of some mutants was entirely sequenced. The mutation spectra showed that the two lesions did not induce the same mutation hot spots, although some sites were common to both. The induced mutation hot spots were not only correlated with lesion hot spots but seemed partially directed by local DNA structures.

The influence of radiation quality on the formation of DNA breaks.

We have aimed to present a comprehensive review of our understanding to date of the formation of DNA strand breaks induced by high LET radiation. We have discussed data obtained from DNA in solution as well as from the formation and "repair" of strand breaks in cell DNA. There is good agreement, qualitatively, between these two systems. Results were evaluated for two parameters: (1) effectivity per particle, the cross section (sigma) in micrometers 2/particle; and (2) the strand break induction frequency as number of breaks per Gy per unit DNA (bp or dalton). A series of biological effects curves (one for each Z-number) is obtained in effectivity versus LET plots. The relationships between induction frequencies of single-strand breaks, or double-strand breaks, or the residual "irrepairable" breaks and LET-values have been evaluated and discussed for a wide spectrum of heavy ions, both for DNA in solution and for DNA in the cell. For radiation induced total breaks in cell DNA, the RBE is less than one, while the RBE for the induction of DSBs can be greater than one in the 100-200 keV/micrometers range. The level of irrepairable strand breaks is highest in this same LET range and may reach 25 percent of the initial break yield. The data presented cover results obtained for helium to uranium particles, covering a particle incident energy range of about 2 to 900 MeV/u with a corresponding LET range of near 16 to 16000 keV/micrometers.