A study of chromosomal aberrations and chromosomal fragility after recombinant growth hormone treatment.

Increased chromosomal rearrangements and chromosomal fragility have been previously observed in lymphocytes of children treated with human GH, implying that treatment could predispose to malignancy. Twenty-four children with classic GH deficiency, neurosecretory GH dysfunction, and Turner syndrome were treated with recombinant human GH (0.3 mg x kg(-1) x wk(-1)). Metaphase cells were assessed for spontaneous chromosomal and chromatid aberrations at baseline and 6 mo into treatment. There were no significant differences in aberrations between baseline and the 6-mo samples. However, the mean frequency of chromatid-type aberrations on a per cell basis was significantly higher than at baseline, 0.0088 versus 0.0064 aberrations per cell (p < 0.024). Two patients contributed inordinately to this increase. A third sample from these two patients was almost identical to their baseline samples. Cells were also irradiated in vitro (3 Gy) to assess chromosomal fragility. After irradiation, no patient showed a significant difference for any aberration type, although there was a significantly lower frequency of ring chromosomes on a per cell basis in the 6-mo samples (p < 0.001). We find no evidence that GH therapy influences spontaneous chromosomal aberrations or chromosomal fragility.

Desferrioxamine enhances the effects of gamma radiation on clonogenic survival and the formation of chromosomal aberrations in endothelial cells.

Vascular injury and endothelial damage contribute to the efficacy and complications of radiotherapy. Iron chelation protects against iron-catalyzed oxidative injury, but it also inhibits DNA synthesis in proliferating cells and can cause apoptosis. We examined the prevailing effects of iron chelation on the survival of gamma-irradiated human umbilical vein endothelial cells by treating monolayers, primarily in the G1/G0 phase of the cell cycle, with the iron chelator desferrioxamine for 24 h prior to gamma irradiation. Desferrioxamine treatment alone diminished plating efficiency by inducing apoptosis and delaying proliferation; this effect disappeared by 48 h. Desferrioxamine treatment reduced clonogenic survival after exposure to 2.5 Gy gamma radiation, but neither iron loading with hemin nor treatment with another iron chelator, 2,2-dipyridyl, which is a potent inhibitor of ribonucleotide reductase, had an effect on survival after irradiation. Clonogenic survival and chromosomal aberrations were measured in parallel in endothelial cells treated with desferrioxamine after increasing doses of gamma radiation. In a linear-quadratic model of survival, desferrioxamine treatment did not change the occurrence of directly lethal lesions, but it significantly increased sublethal injury. Desferrioxamine was not clastogenic alone, but it increased the frequency of formation of chromosomal rings and of excess acentric fragments after gamma irradiation.

Chromosomal adaptive response in human lymphocytes.

It has been almost a decade since the initial report of Olivieri et al. (Science 223, 594-597, 1984) on the phenomenon they termed "adaptive response of human lymphocytes to ionizing radiation." Although a number of reports have appeared since then, our understanding of this response is still incomplete. In this paper, the author presents an analysis of the area using published data in the literature as well as unpublished data from the author's laboratory. Most of the data come from measurements of the effects of low-dose radiation on chromatid-type aberrations induced in late S/early G2 phase cells. Exposure of lymphocytes to low doses of ionizing radiation can affect a certain fraction of aberrations induced by a subsequent high dose. Chemicals have been substituted for ionizing radiation as either inducers or challenging agents; however, their use has not provided specific information about inducing signals or target lesions. The working hypothesis in studies on adaptive response is that a repair activity is induced that acts on lesions in DNA. Although there is promising evidence that new and/or altered synthesis of proteins is required to observe reductions in aberrations, the gap between hypothesis and evidence is still wide. Co-ordinate analysis of different end points in individual cells should help to close this gap. While an adaptive response can be induced under a range of conditions, there is no good explanation for the inter/intradonor variability observed. The contributors to this variation need to be identified.

Evidence that the adaptive response of human lymphocytes to ionizing radiation acts on lethal damage in nonaberrant cells.

In a previous study we found that a cytogenetic adaptive response could lead to increases in survival if there was a sufficient increase in nonaberrant cells (Shadley and Dai, 1992). Since the high challenge doses used produced mainly multiply aberrant cells, we suggested using challenge doses that gave mainly singly aberrant cells in order to improve detection of a survival adaptive response. To test this, human lymphocytes from 6 donors were exposed in the first G1 phase to 5 cGy of X-rays, followed by 100 cGy 6 h later. Nearly all of the aberrant cells bore only one chromosome aberration with this challenge dose, and in agreement with our proposal, survival adaptive responses were seen in 4 of 6 donors. A near 1:1 relationship between the % nonaberrant cells and % survival was found with 100 cGy, suggesting that the lymphocyte populations scored in the survival and aberration assays were representative of each other. However, the increase in nonaberrant cells was not sufficient to account for the increase in survival. Thus, a large fraction of the increase in survival was due to a decrease in lethal damage in cytologically nonaberrant cells. Such damage could range from sub-microscopic lesions, to larger alterations not visible in Giemsa-stained cells. In conjunction with adaptive response studies of others, these results intimate that the adaptive response affects damage at different levels of chromosomal hierarchy (i.e. from the chromosome to DNA). The process(es) responsible for the effects observed in this study may act on lethal, rather than mutagenic lesions.

Cytogenetic and survival adaptive responses in G1 phase human lymphocytes.

Human lymphocytes from six donors were treated with 5 cGy of X-rays followed by 200 or 400 cGy in the first G1 phase after PHA stimulation, and assayed for cytogenetic aberrations and cell survival. Four donors showed cytogenetic adaptive responses with 400 cGy, and one with both 200 cGy and 400 cGy. Both exchanges and deletions were reduced, indicating that the cytogenetic adaptive response acts by restitution of chromosome breaks. Good correlations were found between nonaberrant cells and survival, although the former were often higher than the later, especially with the 400 cGy dose. In four of six donors, 5 cGy alone had significant effects on cell survival; however, this was independent of the 5 cGy effect on high-dose-induced responses. Two donors had survival adaptive responses with 5 + 200 cGy, while none were found with the 5 + 400 cGy treatment. The comparisons between the cytogenetic and survival responses suggests that a survival adaptive response will only be seen with a sufficient increase in nonaberrant cells. To date, adaptive responses to ionizing radiation have been reported to occur in G0, G1 and late S/early G2 human lymphocytes.

Differential locus sensitivity to mutation induction by ionizing radiations of different LETs in Chinese hamster ovary K1 cells.

Mutation induction after exposures to 250 kVp X-rays, alpha-particles from the radon daughter 212Bi, and fission-spectrum neutrons from the JANUS reactor was studied in Chinese hamster ovary (CHO) K1 cells and in CHO-10T5, a K1 derivative containing the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt). Mutation induction was analyzed at three genetic loci: the gpt locus, the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, and the thymidine kinase (tk) locus. After X-irradiation, mutants were induced at the tk loci at approximately 8-9 times the rate of mutant induction at the hprt locus, and the rate of mutant induction at the gpt locus was 8-10 times greater than that at the hprt locus. Neutron and alpha-radiation were more effective mutagenic agents. Mutant frequencies were approximately 4- to 6-fold higher than for X-rays at the hprt and gpt loci and greater than 12-fold greater than X-rays at the tk locus. The greater sensitivity of the tk locus to mutation induction by ionizing radiation (especially neutron and alpha-particle radiation) compared to the hprt locus is likely to be due to the recovery of an additional class of mutants, possibly ones containing larger-sized mutational events. Approximately half of the X-ray-induced tk-1- mutants were small-colony mutants, and 75% of the alpha- and neutron-induced tk-1- mutants were small-colony mutants. The increase in the proportion of small-colony mutants seen with increasing radiation linear energy transfer (LET) suggests that the radiation quality influenced the type of mutation recovered at this locus. There is probably a different reason for the hypersensitivity of the gpt locus because the frequency of gpt mutants, compared to the hprt locus, was independent of radiation quality. Therefore, the LET dependence of mutant induction is gene specific and not necessarily related to the size of deletion recoverable.

The effects of radon daughter alpha-particle irradiation in K1 and xrs-5 CHO cell lines.

We investigated the radiobiological effects of the radon daughter bismuth-212 (212Bi) in Chinese hamster ovary (CHO) K1 cells and in xrs-5 cells, which are X-ray sensitive and deficient in the ability to rejoin DNA double-strand breaks. The cells were exposed to 250 kVp X-rays or to 212Bi chelated to diethylene triamine pentaacetic acid (DTPA); chelation of 212Bi to DTPA prevented its attachment to or entry into the cells. Cytotoxic, clastogenic, and mutagenic responses of the cells were measured and RBEs (D10, 2 chromatid aberrations/cell and 10 induced 6-thioguanine-resistant mutants) were calculated to be 3.8, 3.5, and 3.9, respectively for K1, and 1.4, 0.8, and 5.1, respectively, for xrs-5. With the exception of the RBE of less than 1 for alpha-induced aberrations in xrs-5, the results are consistent with the following conclusions: (1) alpha-particles are in general more effective cytotoxic, clastogenic and mutagenic agents than X-rays; (2) the primary lethal and clastogenic lesion induced by both X-rays and alpha-particles is probably a DNA double-strand break; (3) DNA double-strand breaks induced by alpha-radiation are less well repaired than those induced by X-rays, although a portion of alpha-induced damage is repairable; and (4) deficiencies in rejoining DNA double-strand breaks affect the clastogenic and cytotoxic effects of X-rays and alpha-radiation, not their mutagenic effects. The RBE of 0.8 for aberration induction in xrs-5 cells could reflect a deficiency in the ability of these cells to convert alpha-induced damage to chromosome aberrations. Alternatively, the RBE of less than 1 might reflect an unusual sensitivity of xrs-5 cells to alpha-induced G2 delays.

Comparison of radon-daughter-induced effects in repair-proficient and repair-deficient CHO cell lines.

The radiobiological effects of the radon daughter 212Bi were investigated in the Chinese hamster ovary cell line AA8 and its radiosensitive derivative EM9. EM9 cells rejoin radiation-induced DNA strand breaks more slowly than do AA8 cells. Three endpoints were examined: cell killing, G2-induced chromosome aberration frequency, and mutation induction at the hypoxanthine (guanine) phosphoribosyltransferase (HGPRT) locus. Cells were exposed to the alpha-emitter 212Bi chelated to diethylenetriaminepentaacetic acid (212Bi-DTPA). As expected, 212Bi-DTPA was more effective than X-rays in producing cytotoxicity, chromosome aberrations, and gene mutations. The relative biological effectiveness (RBE) for all three endpoints ranged from about 2 for chromosome aberrations to 4.4 for mutation induction. EM9 was more sensitive than AA8 cells to the cytotoxic and clastogenic effects of both X-rays and 212Bi-DTPA, suggesting that the repair deficiency in EM9 cells affects response to low- and high-linear energy transfer (LET) radiation for these endpoints. There was no significant difference between these two cell lines in their mutagenic response to X-rays and AA8 was slightly more sensitive to the mutagenic effects of alpha radiation. These results suggest that alterations in DNA repair ability may affect response of cells to both low- and high-LET radiation-induced cytotoxicity and clastogenicity, but they appear to have little effect on gene mutation induction.

Induction of the adaptive response by X-rays is dependent on radiation intensity.

Human lymphocytes pretreated with low (0.01 Gy) but not high (0.5 Gy) doses of X-rays become somewhat refractory to the induction of chromatid deletions by subsequent exposure to high (1.5 Gy) doses of X-rays (i.e. the yield of chromatid deletions is less than the sum of the yields induced by the pre-exposure and the subsequent challenge doses). This adaptive response can also be induced by pretreating the cells with very low, or even high, concentrations of tritiated thymidine. Because high concentrations of tritiated thymidine result in high doses of radiation that are delivered at very low dose-rates (i.e. less than 0.01 Gy/min), the lack of adaptation following high pre-treatment doses of X-rays could be attributed to their higher dose-rates. To test the effect of X-ray intensity on the induction of the adaptive response, lymphocytes were irradiated with 0.5 Gy of X-rays at 0.005-0.5 Gy/min at 28-30 h of culture, and then irradiated with 1.5 Gy at 48 h. Chromatid deletions were measured 6 h later. The results show that 0.5 Gy of X-rays given at low dose-rates (0.005 or 0.01 Gy/min), but not at high dose-rates (0.1, 0.2, or 0.5 Gy/min), are capable of inducing the adaptive response. Furthermore, experiments in which a male subject's cells exposed to 0.5 Gy given at 0.005 Gy/min were cocultivated with a female subject's cells irradiated with 0.5 Gy at 0.5 Gy/min showed that cells exposed to radiation at low and high intensity progress to metaphase equally and, therefore, that the lack of an adaptive response at high dose-rates cannot be attributed to selection of radioresistant cells. Although the induction of the adaptive response at higher X-ray doses occurs at low radiation intensity, there seems to be a minimum dose required for this effect; e.g., 0.01-Gy pretreatments induced the adaptive response when given at 0.2 Gy/min, but not at 0.005 Gy/min. Thus, the adaptive response is dependent both on the total dose of the pretreatment and on the rate at which the dose is given.

Characterization of the adaptive response to ionizing radiation induced by low doses of X rays to human lymphocytes.

In previous studies we have shown that low doses of radiation from incorporated tritiated thymidine can make human lymphocytes less susceptible to the genetic damage manifested as chromatid breakage induced by a subsequent high dose of X rays. We have also shown that this adaptive response to ionizing radiation can be induced by very low doses of X rays (0.01 Gy; i.e., 1 rad) delivered during S phase of the cell cycle. To see if a low dose of X rays could induce this response in cells at other phases of the cell cycle, human lymphocytes were irradiated with 0.01 or 0.05 Gy before stimulation by phytohemagglutinin (G0) or with 0.01 Gy at various times after stimulation (G1), followed by 1.5 Gy (150 rad) at G2 phase. Although G0 lymphocytes failed to exhibit an adaptive response, G1 cells irradiated as early as 4 h after stimulation did show the response. Experiments were also carried out to determine how long the adaptive response induced by 0.01 Gy could persist. A 0.01-Gy dose was delivered to lymphocytes in the first S phase, followed by 1.5 Gy in the same or subsequent cell cycles. Lymphocytes receiving a 1.5-Gy dose at 40, 48, or 66 h after stimulation exhibited an adaptive response, whereas those receiving a 1.5-Gy dose at 90 or 114 h did not. Duplicate cultures containing bromodeoxyuridine showed that at 40 h all the lymphocytes were in their first cell cycle after stimulation, at 48 h half of the lymphocytes were in their first cell cycle and half in their second, and at 66 h 80% of the lymphocytes were in their third cell cycle. Thus the adaptive response persists for at least three cell cycles after it is induced by 0.01 Gy of X rays. In other experiments, the time necessary for maximal expression of the adaptive response was determined by delivering 0.01 Gy at hourly intervals 1-6 h before the 1.5-Gy dose. While a 4-h interval was enough for expression of the adaptive response, shorter intervals were not.

Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation.

Cultured human lymphocytes exposed to very low doses of X-rays become less susceptible to subsequent higher doses of X-rays. Cells exposed to doses as low as 0.5 rad (cGy) or 1 rad of X-rays at 32-34 h of culture become adapted so that less cytogenetic damage in the form of chromosome breakage is induced by 150 rad administered at 48 h. This response, which does not occur after high initial doses of X-rays, can be eliminated by 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase.

Potentiation of sister chromatid exchange by 3-aminobenzamide is not modulated by topoisomerases or proteases.

Poly(ADP-ribose) is synthesized in response to DNA strand breaks and covalently modifies numerous intracellular proteins. We have proposed that this modification regulates, i.e., inhibits, the activity of these enzymes, e.g., topoisomerases and proteases, which could otherwise cause additional DNA damage or alterations in chromatin structure. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide (3AB) in cells exposed to DNA-damaging agents would, according to this proposal, eliminate the regulatory role of ADP-ribosylation. When Chinese hamster ovary cells are cultured with methyl methanesulfonate (MMS) and 3AB, a synergistic increase in sister chromatid exchange frequency is observed. We investigated the regulatory role of poly(ADP-ribose) polymerase to see if topoisomerases or proteases are involved in this synergistic increase. Cells were exposed to MMS or the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), 3AB, and either the topoisomerase inhibitor novobiocin or the protease inhibitor antipain. Neither novobiocin nor antipain affected the synergistic response of MMS and 3AB or the additive response of m-AMSA and 3AB. These results suggest that topoisomerases or proteases do not account for the effect of 3AB on sister chromatid exchange frequency after DNA damage.