Tests of the double-strand break, lethal-potentially lethal and repair-misrepair models for mammalian cell survival using data for survival as a function of delayed-plating interval for log-phase Chinese hamster V79 cells.

Our data (Reddy et al., Radiat. Res. 141, 252-258, 1995) on the kinetics of the repair of potentially lethal damage in log-phase Chinese hamster V79 cells are used to test some predictions which arise from the different assumptions of the repair-misrepair (RMR) (C. A. Tobias, Radiat. Res. 104, S77-S95, 1985), lethal-potentially lethal (LPL) (S. B. Curtis, Radiat. Res. 106, 252-270, 1986) and double-strand break (DSB) (J. Y. Ostashevsky, Radiat. Res. 118, 437-466, 1989) models. The LPL model defines the time available for repair of PLD (t(rep)) as the time taken to reach maximal survival in a delayed-plating recovery experiment. Those data show that after this time has elapsed, contrary to the expectation of the LPL model, survival can be increased by changing the medium used for delayed plating from fresh growth medium to conditioned medium. According to the RMR model, all potentially lethal lesions should also be committed by that time and be unavailable for repair in the new medium. Only the DSB model correctly predicted that PLD (= DSBs) would still be available for repair after that time. Second, data for split-dose recovery are used to predict the first-order kinetics time constant for DSB repair (tau(DSBR)) using the DSB model (24 +/- 1.5 min). This value is nearly identical to the value of 27 +/- 1 min determined from the data obtained by Cheong et al. using pulsed-field gel electrophoresis (PFGE) (Mutat. Res. 274, 111-122, 1992). The value based on PFGE is used to calculate the value of t(rep) predicted by the DSB model (2.6 +/- 0.1 h), which agrees with the value determined experimentally as the time when changing the delayed-plating medium from growth medium to conditioned medium no longer gives the full recovery seen with delayed plating in conditioned medium (2.5 h). However, some recovery was seen for a change in the medium (growth medium to conditioned medium) up to 5-6 h postirradiation. Reanalysis of the original data on DSB repair shows that they are consistent with two first-order repair rates (18 +/- 7 min and about 52 min). These results are consistent with two pools of DSBs (or cells), each with their own t(rep). The early t(rep), associated with tau(fast), is predicted to be 1.7 +/- 0.7 h, and the late t(rep), associated with tau(slow), is predicted to be about 5 h. Both values are in excellent agreement with the times at which changing from growth medium to conditioned medium no longer gives the full recovery seen in conditioned medium only (the early t(rep)), and the time when changing from growth medium to conditioned medium produces no further increase in survival (the late t(rep)), respectively. It is noted that attempts to correlate radiosensitivity with the rates of DSB repair, rather than using an explicit model such as the DSB model, are unlikely to be productive since survival depends on both tau(DSBR) and t(rep) (as defined in the DSB model) and the latter may be the more important determinant of radiosensitivity (as it appears to be for ataxia telangiectasia cells compared to normal fibroblasts and for irs compared to V79 cells).

Nutrient dilution before and after X irradiation increases the radioresistance of log- and plateau-phase Chinese hamster V79 cells.

Previous observations have shown that cells cultured in standard growth medium (100%) demonstrated similarly enhanced survival when incubated postirradiation either in non-growth-promoting conditioned medium or in growth-promoting 40% growth medium (Reddy and Lange, Radiat, Res. 119, 338-347, 1989). From these results, it was suggested that nutrient dilution altered radiosensitivity by a mechanism independent of progression of cells through the cell cycle. In this study, we have examined the effects on radiosensitivity of incubation in 40% growth medium prior to irradiation on both log- and plateau-phase Chinese hamster V79 cells and the effects on the distribution of cells in the cell cycle of incubation in 40% or 100% growth medium before and after irradiation. Radioresistance increased by a factor of 1.5-1.6 compared to 100% growth medium for both log-phase and plateau-phase cells cultured in 40% growth medium prior to X irradiation and incubated in either 40% growth medium or conditioned medium after X irradiation. The cell cycle distributions of log-phase cells in 100% and 40% growth medium before irradiation were identical. The change in cell cycle distribution induced by 10 Gy did not differ among log-phase cells incubated for 3 h postirradiation in 100% growth medium, 40% growth medium or conditioned medium. These results, in addition to supporting our previous conclusions, demonstrate that culturing prior to irradiation in 40% growth medium alone increases cell survival and that incubation in 40% growth medium before and after irradiation maximizes the survival of V79 cells.

Chinese hamster V79 cells harbor potentially lethal damage which is neither fixed nor repaired for long times after attaining maximal survival under growth conditions.

The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium and attained the same survival as that of cells in conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in growth medium for different times followed by incubation in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth medium but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation.

Analysis of spine motion variability using a computerized goniometer compared to physical examination. A prospective clinical study.

A prospective clinical study was carried out to determine whether the CA-6000 Spine Motion Analyzer was more reliable in measuring spine motion compared to previously described methods of physical examination. One-hundred-twenty age-controlled patients with a negative back history were tested with the CA-6000 to establish normal values. Interexaminer and intraexaminer correlation coefficients and coefficients of variation were determined for 30 patients by three different examiners, each testing the patients three times each. Each test consisted of examining flexion, extension, lateral flexion, and rotation. For flexion, the CA-6000 was compared to the double inclinometer and Schober methods. For extension and lateral flexion, the CA-6000 was compared to the double inclinometer and Moll methods. For rotation, the CA-6000 was compared to the double inclinometer method. The interexaminer and intraexaminer variability using the CA-6000 was significantly less (P < or = 0.025) than other methods for all motions except flexion. In flexion, the differences were not significantly different. The data from this experiment indicate that the CA-6000 has significantly less variability in measuring spine motion clinically than the other methods tested.

Gender differences in age-related decline in DNA double-strand break damage and repair in lymphocytes.

DNA repair capacity has been suggested to be a genetic mechanism which influences the rate of ageing and length of life. We recently reported an age-related decline in DNA double-strand break (DSB) repair in unstimulated human lymphocytes (Mayer, Lange, Bradley, and Nichols 1989, 1990). In that work peripheral lymphocytes isolated from whole blood donated by 20 normal, healthy subjects aged 23-78 years, were X-irradiated (30 Gy) on ice and incubated at 37 degrees C for repair times of 15, 30, and 120 min, and neutral filter elution was used to assay DSB induction and completeness of DSB rejoining. Further analysis reveals that the decrease in DSB rejoining appears to be more pronounced in older women than in older men (and may begin after age 65 years). Moreover the reported age-related decline in DSB induction occurs more rapidly (by a factor of ca. 2) in women than in men. We had previously demonstrated that, independently of in vivo age, cells with lower radiosensitivity (i.e. lesser DSB induction) appear to be less repair-proficient (Mayer et al. 1990). The present analysis reveals a gender-specific pattern in this correlation between extent of DSBs induced and percentage of DSBs rejoined: at comparable levels of DSB induction, cells from men rejoin a higher percentage of DSBs than do cells from women. This preliminary study suggests the following hypothesis: age-related DSB effects (i.e. induction and rejoining) are due to changes in chromatin structure and males are less sensitive than females to the influence which age-related alterations in chromatin exert on DSB effects.

Mechanism of disc rupture. A preliminary report.

Lumbar intervertebral disc herniation is thought to be related to senescent changes in the nucleus pulposus except in rare instances of trauma. This investigation provides the first in vitro model of disc prolapse that reliably ruptures discs under physiologically reasonable stress. Fourteen vertebral motion segments with intact posterior elements were loaded repetitively at 1.5 Hz in a combination of flexion (7 degrees), rotation (less than 3 degrees), and compression (1,334 N) for an average of 6.9 hours (range, 3.0-13.0 hours) in a materials testing machine. Loading was terminated when reaction force leveled off for more than 1 hour. Ten discs failed through annular protrusions, and four failed by nuclear extrusion through annular tears, supporting the hypothesis that intervertebral disc prolapse is peripheral in origin. The annulus fibrosus is the site of primary pathologic change.

Investigation of the neutral filter elution technique I. Effects of pore density and pore diameter on elution rate at pH 9.6.

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.

The saturated repair kinetics of Chinese hamster V79 cells suggests a damage accumulation--interaction model of cell killing.

The aim of this study is to determine whether the repair process in log-phase Chinese hamster V79 cells exposed to X rays is unsaturated, saturable, or saturated. The kinetics of recovery from damage induced by 2 to 14 Gy of 250 kVp X rays was studied by treating cells with 0.5 M hypertonic saline for 20 min at different postirradiation repair intervals. From the kinetic data, the repair half-time (t1/2), the repair time (time needed to attain maximal survival), and the recovery ratio were calculated. The results show that the t1/2 (1.42 min/Gy) and the repair time (6.04 min/Gy) increase linearly with dose, the logarithm of the recovery ratio increases linear-quadratically with dose, and the D0 increases linearly with repair interval at a rate of 2.4 cGy/min. From these results we suggest a model: the repair of damage (undefined lesions) necessary for cell survival is effected by a repair process (t 1/2 of 1.42 min/Gy) which is saturated at doses as low as 2.4 cGy; repair saturation leads to a dose-dependent accumulation of repairable lesions; and interaction among accumulated repairable lesions results in the induction of irreparable (lethal) lesions. We call this the accumulation-interaction model of cell killing by low-LET radiation.

Coagulopathies associated with major spinal surgery.

Coagulation disorders occurring during major spinal surgery may be more common than previously thought. Patients who develop clinical disseminated intravascular coagulation (DIC) during spinal surgery represent one extreme of this process. The etiology of the coagulopathy seems to be unrelated to age, gender, or etiology of the spinal deformity, e.g., idiopathic, congenital, neuromuscular, or degenerative. Acute bleeding diathesis may occur during or shortly after surgery and may be difficult to control. The consumptive coagulopathy is not a self-limiting process. Preoperative, intraoperative, and postoperative monitoring of coagulation studies in patients with prolonged spinal procedures may help to identify those patients at risk for fulminant DIC and allow for prompt therapeutic intervention. In the presence of active hemorrhage or in those patients who cannot tolerate large volume transfusion, cryoprecipitate is the replacement product of choice for fibrinogen replacement.

Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes.

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.

The effect of mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) on DNA damage, repair and aging of human diploid fibroblasts.

We used mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) to examine aging at the cellular level in relation to DNA damage and repair. With the filter elution technique we monitored spontaneous single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA during in vitro aging at 34 degrees C, 37 degrees C and 39 degrees C of normal human diploid fibroblasts (HDF). DNA repair was assessed after ionizing and non-ionizing (ultraviolet) radiation of HDF at different population doubling levels (PDLs): the former was assayed by filter elution and the latter by unscheduled DNA synthesis. Survival was assessed by trypan blue dye exclusion and colony formation. Cells at 37 degrees C achieve a higher cumulative PDL (67 +/- 6) than cells at 39 degrees C (60 +/- 5) or at 34 degrees C (55 +/- 6). The level of spontaneous SSBs and DSBs, and radiosensitivity of DNA to either 6 Gy or 100 Gy gamma rays, do not change with in vitro age at any of the three temperatures. Repair of SSBs (induced by 6 Gy) and DSBs (induced by 100 Gy) does not change with in vitro age: rejoining is 86-104% complete by 60 min repair and generally does not differ across temperatures. Response to non-ionizing radiation (254 nm, 75, 150, 300 ergs/mm2) does not change with in vitro age at 37 degrees C or 39 degrees C, whereas excision repair increases with age at 34 degrees C even though cell survival does not. The results do not support the rate of living theory of aging (Pearl, R., The Rate of Living, University of London Press, London, 1928) as applied to temperature effects on HDF aging in vitro (as measured by proliferative lifespan) and on their response to radiation-induced DNA damage.

Hypersensitivity of cells from a new chromosomal-breakage syndrome to DNA-damaging agents.

Cells derived from a patient with severe chromosomal breakage, immunodeficiency, and growth retardation were found to resemble those from individuals with ataxia telangiectasia (A-T) in terms of their sensitivity to cell killing and the induction of cytogenetic abnormalities by X-rays. Their response to other DNA-damaging agents, including 254-nm UV light, mitomycin C, MNNG, and bleomycin was also A-T-like. In contrast to classical A-T, however, X-irradiated cells exhibited a G1 block after release from density inhibition of growth that was not significantly different from that of normal controls.

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes.

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro.

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.

Developmental time and adult longevity in two strains of Drosophila melanogaster in a constant low-stress environment.

This study examines the life spans of wild type flies (Sevelen strain, n = 2991) and post-replication DNA repair-deficient flies (mei-41D5, n = 1607) as members of age-synchronized birth cohorts and as members of full sibships. Flies were individually housed from the time they were laid as eggs until they died as imagoes; environmental conditions were constant throughout the life span of these virgins. In this minimal stress experiment, developmental time and imaginal longevity were negligibly correlated (r = -0.09 to -0.25), even though developmental time was canalized at different levels in both strains. Previous research involving environmental perturbations during the life span of Drosophila (e.g. temperature, diet, population density) has demonstrated significant strong correlations, mostly positive, between developmental time and adult longevity. We have demonstrated that the duration of development and aging are not coupled in the absence of such stresses. We therefore suggest that related changes in the duration of the pre-imaginal and the adult stages of the lifecycle of Drosophila may have evolved through adaptation to environmental stresses.

Chylothorax: an unusual complication of anterior thoracic interbody spinal fusion.

Chylothorax as a complication of anterior thoracic spine fusion is described in the case of a male with congenital kyphosis. Treatment with protracted chest tube drainage was required, along with dietary modification in which fat ingestion was restricted. This complication is reported to occur in 0.2% of intrathoracic operations and may give rise to a 50% mortality unless properly recognized and managed.

Post-poliomyelitis paralytic scoliosis. A review of curve patterns and results of surgical treatments in 118 consecutive patients.

Curve patterns and surgical treatment in 118 consecutive patients with post-poliomyelitic paralytic scoliosis are reviewed. The typical curve pattern was double structural curves, usually including the lumbar spine. Modifying the incision and altering the sequence of instrumentation facilitated both the exposure and instrumentation of the fourth and fifth lumbar bodies. Anterior fusion and Dwyer's instrumentation yield most of the correction of pelvic obliquity and significant correction of both the upper and lower curves. Two weeks of halofemoral traction did not achieve significant curve correction. It is suggested that staged anterior and posterior procedures result in structural correction of pelvic obliquity and paralytic scoliosis in severe deformities equal to the degree of correction obtained using posterior fusion and instrumentation alone in less severe cases.