Aminothiols protect endothelial cell proliferation against inhibition by lipopolysaccharide.

Lipopolysaccharide (LPS) is a primary agent of sepsis that damages the vascular endothelium. Endothelial cell proliferation is key to the repair of damaged endothelium, and drugs that counteract the antiproliferative impact of LPS on endothelial cells should be beneficial. Because LPS exerts much of its cytotoxicity by generating reactive oxygen and nitrogen intermediates, it would be helpful to know whether therapeutic antioxidant thiols maintain cell proliferation in injured endothelium. In this study, it was found that LPS inhibited bovine aortic endothelial cell proliferation by inducing apoptosis and by decreasing DNA synthesis. Because of its benefit to irradiated endothelial cells, we then treated the cells with a radio- and chemoprotective aminothiol, WR-1065 ([N-2-mecaptoethyl]-1-3-diaminopropane, the active form of Amifostine/Ethyol). WR-1065 attenuated the inhibition of DNA synthesis caused by LPS exposure. The disulfide of WR-1065, WR-33278, was tested and shown to both promote DNA synthesis and inhibit apoptosis. The effectiveness of the disulfide suggests that the reduction of cytotoxicity does not necessarily result from the scavenging of free radicals. These findings demonstrate a novel role for aminothiols in promoting DNA synthesis and lowering apoptosis in endothelium injured with LPS.

Subdenaturing (pH 11.1) filter elution: more sensitive quantification of DNA double-strand breaks.

The apparent biological significance of DNA double-strand breaks (DSBs) has stimulated considerable effort toward quantification of this lesion. The neutral (or nondenaturing) filter elution assay at pH 7.2 or 9.6 has long been a standard method for the measurement of double-strand breakage and rejoining in eukaryotic cells, with a threshold dose for detection of DSBs of 5-10 Gy. Agarose gel electrophoresis, either pulsed- or constant-field, can detect DSBs induced by as little as 1 Gy of ionizing radiation, but electrophoresis assays may have inherent problems in measurement of break rejoining, and may be more susceptible than elution to factors other than break frequency, such as cell cycle stage or bromodeoxyuridine substitution. We report here that filter elution performed at pH 11.1 can detect DSBs produced by only 1 Gy of ionizing radiation, but is insensitive to the single-strand breaks that are formed when cells are exposed to hydrogen peroxide. Double-strand breaks produced in permeabilized cells by the restriction endonuclease HaeIII were used to demonstrate that the increase in the pH of the eluting solution from 9.6 to 11.1, although increasing assay sensitivity by a factor of five, converts few additional alkali-labile sites to DSBs. Thus validated, the pH 11.1 filter elution assay was applied to a low-dose measurement of induction and rejoining of DSBs in 9L cells.

WR-1065 and radioprotection of vascular endothelial cells. I. Cell proliferation, DNA synthesis and damage.

Normal tissue toxicity limits radiation therapy and could depend on the extent of damage to the vascular endothelium Aminothiols such as WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] provide radioprotection for normal tissues, but little is known about how the aminothiols specifically affect the endothelium. Bovine aortic endothelial cells in culture were exposed to WR-1065 for 2 h before irradiation (137Cs gamma rays, 1 Gy/min). Alone, WR-1065 demonstrated an antiproliferative effect that was related to dose (0.5-4 mM) and was evident by lowered counts of adherent cells 48 h after exposure. WR-1065 was clearly radioprotective when assessed by colony formation and incorporation of [3H]thymidine. However, when the number of adherent cells was evaluated, radioprotection appeared to be slight and evident only in logarithmically growing cells. WR-1065 at 2 mM suppressed single-strand DNA breaks after 3 Gy by 22% and double-strand breaks after 9 Gy by 47%. Also in the irradiated cells, WR-1065 more than doubled the rate of progression of cells from G1 to S phase. WR-1065 pretreatment elevated cellular glutathione (GSH) content more than twofold. Although pretreatment with buthionine sulfoximine inhibited the elevation of GSH, the radioprotective impact of WR-1065 on total DNA strand breaks and colony formation was unaffected. These results suggest that WR-1065 may enable tissue recovery from irradiation by promoting the replication of endothelial cells, possibly by mechanisms independent of GSH.

The relaxation of supercoiled DNA molecules as a biophysical dosimeter for ionizing radiations: a feasibility study.

In this paper we explore the feasibility of using DNA molecules as a biophysical radiation dosimeter. Supercoiled phi X174 bacteriophage DNA molecules were irradiated with different gamma radiation doses. The strand breakage produced by ionizing radiation within supercoiled double-stranded DNA molecules (RFI) yields relaxed circular DNA molecules (RFII) and linear DNA molecules (RFIII) as a result of single-strand breaks and double-strand breaks, respectively. The irradiated samples were subjected to electrophoresis on agarose gels to separate the three forms. A proprietary fluorescent dye was used to detect DNA bands within the gel, which was photographed under UV transillumination. The negative was scanned with a computerized imaging densitometric system for DNA band quantitation. The relative fractions of the three molecular forms are dose dependent, and can be modeled mathematically with five parameters. The values of the parameters were determined by optimizing the fit of the model to the data, using a nonlinear regression procedure of a commercial statistical analysis package. Once the parameters of DNA breakage have been determined, absorbed dose can be measured by this technique, which we have termed supercoil relaxation dosimetry. The average accuracy of dose determination for our system over the range of 1-40 Gy was about 5%. Supercoil relaxation dosimetry may be well suited to certain difficult dosimetric problems.

A 5-4 pyrimidine-pyrimidone photoproduct produced from mixtures of thymine and 4-thiouridine irradiated with 334 nm light.

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.

Evidence from nondenaturing filter elution that induction of double-strand breaks in the DNA of Chinese hamster V79 cells by gamma radiation is proportional to the square of dose.

We have used nondenaturing filter elution performed at both pH 7.2 and pH 9.6 to measure the induction of double-strand breaks (DSBs) in the DNA of Chinese hamster V79 cells by 60Co gamma-radiation doses between 10 and 120 Gy. The absolute DSB yields as measured by this assay were determined by using our recent calibration of the assay based upon disintegrations of 125I incorporated into the DNA. An analysis of the dose-response relationship for the induction of DSBs by 60Co gamma rays showed that the number of DSBs induced per dalton of DNA was proportional to the square of the applied dose throughout the dose range used. The contribution made by the dose to the first power was small at pH 9.6 and negligible at pH 7.2. These results suggest that DSB induction in cells by gamma rays may be entirely a two-hit event.

Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA.

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.

Measurement of double-strand breaks in Chinese hamster cell DNA by neutral filter elution: calibration by 125I decay.

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.

The role of hydroxyl radical quenching in the protection by acetate and ethylenediaminetetraacetate of supercoiled plasmid DNA from ionizing radiation-induced strand breakage.

A supercoiled plasmid of 7300 base pairs was isolated and exposed in various aqueous environments to 60Co gamma-radiation. Conversion of the supercoiled form to the relaxed circular and linear forms was monitored by agarose gel electrophoresis and quantified by fluorescence scanning of the gel. Acetate, which has been reported to affect the conformation of DNA in solution, decreased the radiosensitivity of the supercoil in a concentration-dependent manner. Acetate, formate, and azide anions, as well as mannitol, all protected the supercoil from relaxation in approximate proportion to the rate at which their solutions quench the hydroxyl radical. At concentrations greater than 300 mmol dm-3, however, the efficiency of acetate radioprotection is reduced. Disodium ethylenediaminetetraacetate protected the supercoil more efficiently than would be expected from the published value of its rate constant for quenching the hydroxyl radical.