Genotoxic effects of radiotherapy and chemotherapy on circulating lymphocytes in patients with Hodgkin's disease

Chemical mutagenesis of Caenorhabditis elegans has relied primarily on EMS to produce missense mutations. The drawback of EMS mutagenesis is that the molecular lesions are primarily G/C --> A/T transitions. ENU has been shown to produce a different spectrum of mutations, but its greater toxicity to C. elegans makes it a difficult mutagen to use. We describe here methods for minimizing ENU toxicity in C. elegans. Methods include preparing ENU stocks in absolute ethanol and storing stock solutions for not more than 2 weeks at -20 degrees C. To maintain reasonable brood sizes of mutagenized animals, mutagenic solutions should not exceed 1.0mM ENU. We provide data which suggest ENU is degraded or altered to more toxic products in aqueous solution, but less so in solvents such as absolute ethanol.     

Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. II. Alteration of DNA repair and chromosome radiosensitivity

Lymphocytes from 43 breast cancer patients were tested for their DNA-repair ability and in vitro chromosomal radiosensitivity. Lymphocytes were collected before and after treatment with radiotherapy or chemotherapy or both, and then irradiated in vitro. The aim was to detect alterations of these 2 indicators of radiosensitivity, in relation to cancer status or medical treatment. Patients before treatment were significantly deficient in DNA-repair ability but had a normal chromosomal radiosensitivity as compared to healthy donors. When assessed after treatment, DNA-repair ability and the frequency of in vitro-induced chromosome anomalies were modified according to the type of treatment. A reduced DNA-repair ability was observed for patients after radiotherapy but not after chemotherapy. In vitro-induced dicentrics and acentrics were not modified to the same extent according to the treatment. A decreased number of acentrics (the most frequently observed alteration) was preferentially associated with a more reduced DNA-repair ability. Interindividual differences of response to in vitro irradiation tested by both assays were observed between patients who had undergone similar treatments. The possibility that these assays could be used for predicting individual susceptibility to radiation or chemotherapy drug exposure is discussed.     

Chromosome aberrations induced in human lymphocytes by in vitro and in vivo X-rays

A dose-effect curve is presented obtained by analysis of dicentric chromosomes and centric ring chromosomes in lymphocyte metaphase spreads of three healthy volunteers after in vitro 100 kV X-ray-irradiation of peripheral blood samples. This calibration curve follows a linear quadratic equation, y=c+alpha D+beta D(2), with the coefficients: y=(0.0005+/-0.0001)+(0.0355+/-0.0066)D+(0.0701+/-0.0072)D(2). The model is based on 13.231 first-division metaphases analyzed after in vitro exposure to doses ranging from 0.1 to 2.0 Gy at a dose rate of 0.4 Gy min(-1). Significant overdispersion of the observed chromosomal aberrations was evident for dose points 1.0 and 2.0 Gy, respectively. The calibration curve was applied to derive equivalent whole body doses of three subjects after suspected extensive exposure to diagnostic X-rays.     

Chromosome aberrations induced in human lymphocytes by neutron irradiation.

In vitro dose--response curves of unstable chromosome aberrations in human lymphocytes have been obtained for neutron spectra of mean energies 0-7, 0-9, 7-6 and 14-7 MeV. The aberration yields have been fitted to the quadratic function Y = alphaD + betaD2, which is consistent with the single-track and two-track model of aberration formation. However with high-LET radiation, the linear component of yield, corresponding to damage caused by single tracks, predominants, and this term becomes more dominant with increasing LET, so that for fission spectrum neutrons the relationship is linear, Y = alphaD. At low doses, such as those recieved by radiation workers, limiting r.b.e. values between 13 and 47 are obtained relative to 60Co gamma-radiation. At higher doses, as used in radiotherapy, the values are much lower; ranging from 2-7 to 8 at 200 rad of equivalent gamma-radiation. Both sets of r.b.e. values correlate well with track-averaged LET but not with dose-averaged LET. When the numbers of cells without aberrations are plotted against radiation dose, curves are obtained which are similar in shape to those for conventional cell-survival experiments with comparable neutron spectra. The Do values obtained in the present study are close to those from other cell system.     

A comparison of chromosomal aberrations induced by in vivo radiotherapy in human sperm and lymphocytes

Chromosomal aberrations in human sperm and lymphocytes were compared before and after in vivo radiation treatment of 13 cancer patients. The times of analyses after radiotherapy (RT) were 1, 3, 12, 24, 36, 48 and 60 months. The median total radiation dose was 30 Gy and the testicular dose varied from 0.4 to 5.0 Gy. Human sperm chromosome complements were analysed after fusion with golden hamster eggs. There were no abnormalities in sperm or lymphocytes before RT. Following RT there was an increase in the frequency of numerical and structural chromosomal abnormalities in both lymphocytes and sperm. For structural abnormalities there were more rejoined lesions (dicentrics, rings) in lymphocytes and more unrejoined lesions (chromosome breaks, fragments) in sperm. After RT there was a dramatic increase in the frequency of chromosomal abnormalities in lymphocytes: at 1 mo. the frequency was 42%, at 3 mo. 25%, at 12 mo. 14%, at 24 mo. 11%, at 36 mo. 9%, at 48 mo. 7% and at 6 mo. 4%. Since the majority of men were azoospermic after RT, there is little data on sperm chromosome complements before the analyses performed at 24 mo. post-RT. At 24 mo. the frequency of abnormalities was 13%, followed by 21% at 36 mo., 12% at 48 mo. and 22% at 60 mo. Thus it appears that the frequency of lymphocyte chromosomal abnormalities had an initial marked increase after RT followed by a gradual decrease with time whereas the frequency of sperm chromosomal abnormalities was elevated when sperm production recovered and remained elevated from 24 to 60 mo. post-RT. This difference in the effect of time makes it very difficult to compare abnormality rates in lymphocytes and sperm and to use analysis of induced damage in somatic cells as surrogates for germ cells since the ratio between sperm and lymphocytes varied from 1:1 (at 24 mo. post-RT) to 5:1 (at 60 mo. post-RT).     

Chromosome aberrations induced in human lymphocytes by radiation from 252Cf.

In vitro dose--response curves of unstable chromosome aberrations in human lymphocytes have been obtained for 252Cf neutron radiation. The aberration yields fitted best to the linear function Y=aD, which is consistent with the single-track model of aberration formation for high LET radiation. The curves have been compared with others previously produced in this laboratory for several energies of neutrons and for 60Co gamma radiation. The r.b.e. for 252Cf with respect to 60Co is 27 at very low doses, decreasing to 6 at an aberration yield equivalent to 400 rad of 18 rad/hour gamma radiation. A profile of chromosome-aberration induction with depth in a perspex phantom was obtained by placing blood samples at several distances over the range 0.65-2.0 cm from the californium source. This profile was compared with depth-damage calculations for a radium needle. The r.b.e. of 252Cf radiation relative to 226Ra gamma radiation increased with the distance from the source, implying that californium is more effective at greater distances in destroying the ability of cells to divide, which may be an advantage in the treatment of large tumours.     

Genotoxic effects induced by fotemustine and vinorelbine in human lymphocytes

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 microg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 microg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

Chromosome aberrations produced in human lymphocytes by in vivo and in vitro irradiation

The production of chromosome aberrations in vivo has been studied in lymphocytes from a patient undergoing a wholebody treatment with gamma-radiation up to a cumulative dose of 1.4 Gy. These results were compared with the observations performed on whole blood samples irradiated in vitro with doses from 0.05 up to 2 Gy of gamma-rays. The frequency of chromosome aberrations, particularly the dicentrics, was found to be similar in vivo and in vitro. The yield of dicentrics could be best related to the dose by using a linear-quadratic model in both cases, the ratio of the coefficients a/b being of 0.56 and 0.69 Gy, respectively in vivo and in vitro. These observations confirm that in vitro dose response curves may be used to evaluate accurately an in vivo absorbed dose.     

Unstable chromosome aberrations in peripheral blood lymphocytes from patients with cervical uterine cancer following radiotherapy.

Scoring of unstable chromosomes aberrations (dicentrics, rings and fragments) in circulating lymphocytes is the most extensively studied biologic system for estimating individual exposure to ionizing radiation. In this work, blood samples from 5 patients, with cervical uterine cancer, were analyzed by conventional cytogenetic in order to correlate the frequency of chromosome aberrations in lymphocytes with the dose absorbed by the patient, as a result of radiotherapy with 60Co gamma. The samples were collected in three phases of the treatment: before irradiation, 24 hr after receiving 0.08 Gy and 1.8 Gy, respectively. On the basis of the frequencies of unstable aberrations observed, a good agreement was obtained between doses estimated by calibration curve and the doses previously planned to radiotherapy. This report discusses the methodology employed as an important tool for dose assessment as a result of partial-body exposure to ionizing radiation.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation

The induction of chromosome aberrations in eggs of mice fertilized with X-irradiated sperm was performed by using an in vitro fertilization technique. Capacitated mature sperm was irradiated with various doses of X-rays and cytological analysis of the first cleavage metaphase of in vitro fertilized eggs was made. The frequencies of chromosome aberrations increased exponentially with dose and the dose-response relationship for overall breaks fitted well to a quadratic equation. The chromosome aberrations were mainly chromosome-type (82.1%), and the majority of aberrations were fragments.     

Chromosome aberrations in the blood lymphocytes of astronauts after space flight.

Cytogenetic analysis of the lymphocytes of astronauts provides a direct measurement of space radiation damage in vivo, which takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. Chromosome exchanges were measured in the blood lymphocytes of eight crew members after their respective space missions, using fluorescence in situ hybridization (FISH) with chromosome painting probes. Significant increases in aberrations were observed after the long-duration missions. The in vivo dose was derived from the frequencies of translocations and total exchanges using calibration curves determined before flight, and the RBE was estimated by comparison with individually measured physical absorbed doses. The values for average RBE were compared to the average quality factor (Q) from direct measurements of the lineal energy spectra using a tissue-equivalent proportional counter (TEPC) and radiation transport codes. The ratio of aberrations identified as complex was slightly higher after flight, which is thought to be an indication of exposure to high-LET radiation. To determine whether the frequency of complex aberrations measured in metaphase spreads after exposure to high-LET radiation was influenced by a cell cycle delay, chromosome damage was analyzed in prematurely condensed chromosome samples collected from two crew members before and after a short-duration mission. The frequency of complex exchanges after flight was higher in prematurely condensed chromosomes than in metaphase cells for one crew member.     

Caffeine effects on the chromosomal aberrations induced in vivo by sarcolysine and methotrexate

Wistar adult rats bearing Guérin T8 ascite tumours were intravenously inoculated with 3 mg/kg b.w. sarcolysine and respectively 5 mg/kg b.w. methotrexate on the 7th day after ascite cells grafting. Four hours after cytostatic administration, 5 mg/kg b.w. caffeine was intravenously given. Cytogenetic observations concerning the frequency and the type of induced chromosomal aberrations were performed 24 hrs after cytostatic administration, both in the animals treated with only sarcolysine, methotrexate and caffeine and in those double-treated with cytostatics and caffeine. Chromosome examinations were also performed in untreated controls. Both in the sarcolysine- and methotrexate- treated tumors, the induced chromosome lesions were enhanced by caffeine administration, but this effect was very obvious in the methotrexate experiments and rather weak in the sarcolysine treatments (see table). This different effect of caffeine might be due to the different mechanisms by which sarcolysine and methotrexate are interfering in DNA replicating processes.     

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.     

Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations

Synchronous cultures of V₇₉ Chinese hamster cells were irradiated in G₁ with 300 rad of X-rays. Cells were collected for 2-h intervals after synchronization to include the first three post-irradiation divisions and were scored for chromosome aberrations. After the first post-irradiation division, asymmetrical exchanges were distributed according to the Poisson formula and both the asymmetrical exchange frequency and the acentric fragment frequency exhibited significant variations with collection time. Formulae derived from a previous mathematical analysis were used in conjunction with the aberration frequencies observed at the first, second, and third post-irradiation divisions to predict transmission and survival parameters for specific chromosomal aberrations.The probability, 2T, that an acentric fragment will be transmitted to a daughter cell at anaphase was found to be 0.57. The probability, W, that a two-break aberration (asymmetrical exchange) will be transmitted and observed at the next division was 0.56. Finally, the probability, P, that a cell will survive to a subsequent mitosis after losing a single acentric fragment was about 1.0 for one post-irradiation generation but somewhat less for two generations.     

Chromosome aberrations induced in vitro by low doses of radiation: nondisjunction in lymphocytes of young adults.

Epidemiological studies suggest radiation exposure as a cause of meiotic non-disjunction in humans, but experimental evidence with cytological proof has been lacking. Our results indicate that mitotic nondisjunction of lymphocyte chromosomes can also be induced by exposure to a low dose of radiation. Abnormal segregation can be induced not only when the cells are irradiated but also when nonirradiated cells are incubated with irradiated cell-free plasma or serum. The X and no. 21 chromosomes appear particularly susceptible to nondisjunction.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.