Analysis by FISH of the spectrum of chromosome aberrations induced by X-rays in G0 human lymphocytes and their fate through mitotic divisions in culture.

The induction, distribution, and persistence of chromosome aberrations in human lymphocytes exposed to X-rays in G0 were analyzed in 48-, 70-, and 94-hr cultures by conventional metaphase analysis and painting of chromosomes 1, 2, and 4 by FISH. All cells that had been scored by FISH were relocated to determine by differential staining of chromatids whether they had passed through 1, 2, or > or =3 divisions. FISH revealed a dose-dependent induction of stable and unstable aberrations, while chromatid labeling showed mitotic lag caused by irradiation in G0. Relative to their DNA contents, there was a small but significant overrepresentation of chromosome 4 and underrepresentation of chromosome 2 among the aberrations involving chromosomes 1, 2, and 4. FISH slightly underestimated the genomic frequency of unstable aberrations measured by conventional metaphase analysis. There was a slight excess of translocations relative to dicentrics, but the data are compatible with the 1:1 ratio expected from cytogenetic theory. Many of the translocations were apparently incomplete (i.e., nonreciprocal). Incomplete translocations were more frequent at higher X-ray dose and in first division, suggesting that they may be associated with complex damage and are more apt to be lost in mitosis than complete translocations. Among the incomplete translocations, t(Ab) outnumbered t(Ba) -- a difference ascribable to the FISH technique. Aberration frequencies declined as the cells divided in culture. The overall decline in the frequency of aberrant cells (approximately 29% per cell generation) reflects a rapid decline in dicentrics and fragments (approximately 60% per cell generation) and the relative stability of translocations. The frequency of translocation-bearing cells underwent a modest decline in culture (approximately 13% per cell generation).

Do recorded doses overestimate true doses received by Chernobyl cleanup workers? Results of cytogenetic analyses of Estonian workers by fluorescence in situ hybridization.

Studies of workers who were sent to Chernobyl after the 1986 reactor accident are being conducted to provide a better understanding of the effects of chronic low-dose radiation exposures. A crucial component to these investigations is an accurate assessment of the radiation doses received during the cleanup activities. To provide information on biological measurements of dose, fluorescence in situ hybridization (FISH) with whole-chromosome painting probes has been applied to quantify stable chromosome aberrations (translocations and insertions) among a defined cohort of 4,833 cleanup workers from Estonia. Cytogenetic analysis of 48-h lymphocyte cultures from 118 Estonian cleanup workers (10.3 cGy mean recorded dose; 25 cGy maximum), 29 Estonian population controls and 21 American controls was conducted by three laboratories. More than 258,000 painted metaphases were evaluated. Overall, we observed lower translocation frequencies than has been reported in previous studies using FISH among Chernobyl cleanup workers. In our data, a clear association with increased levels of translocations was seen with increasing age at blood drawing. There was no correlation, however, between aberration frequency and recorded measurements of physical dose or any category of potential high-dose and high-dose-rate exposure such as being sent to Chernobyl in 1986, working on the roof near the damaged nuclear reactor, working in special zones or having multiple tours. In fact, the translocation frequency was lower among the exposed workers than the controls, though not significantly so. To estimate the level of effect that would have been expected in a population of men having an average dose of approximately 10 cGy, blood from six donors was exposed to low-LET radiation, and more than 32,000 metaphases were scored to estimate dose-response coefficients for radiation-induced translocations in chromosome pairs 1, 2 and 4. Based on these results, we estimate that had this group of 118 men received an average whole-body dose of 10-11 cGy, as chronic or acute exposures, an increase in the mean frequency of chromosome translocations of more than 40-65% would have been observed in their lymphocytes compared to findings in nonirradiated controls. In spite of evaluating more than a quarter of a million metaphases, we were unable to detect any increase in the mean, median or range in chromosome aberrations in lymphocyte cultures from a group of Estonian men who took part in the cleanup of the Chernobyl nuclear power site and those who did not. We conclude that it is likely that recorded doses for these cleanup workers overestimate their average bone marrow doses, perhaps substantially. These results are consistent with several negative studies of cancer incidence in Chernobyl cleanup workers and, if borne out, suggest that future studies may not be sufficiently powerful to detect increases in leukemia or cancer, much less distinguish differences between the effects of chronic compared to brief radiation exposures.

Thyroid nodularity and cancer among Chernobyl cleanup workers from Estonia.

Thyroid examinations, including palpation, ultrasound and, selectively, fine-needle aspiration biopsy, were conducted on nearly 2,000 Chernobyl cleanup workers from Estonia to evaluate the occurrence of thyroid cancer and nodular thyroid disease among men with protracted exposure to ionizing radiation. The examinations were conducted in four cities in Estonia during March-April 1995, 9 years after the reactor accident. The study population was selected from a predefined cohort of 4,833 cleanup workers from Estonia under surveillance for cancer incidence. These men had been sent to Chernobyl between 1986 and 1991 to entomb the damaged reactor, remove radioactive debris and perform related cleanup activities. A total of 2,997 men were invited for thyroid screening and 1,984 (66%) were examined. Estimates of radiation dose from external sources were obtained from military or other institutional records, and details about service dates and types of work performed while at Chernobyl were obtained from a self-administered questionnaire. Blood samples were collected for assay of chromosomal translocations in circulating lymphocytes and loss of expression of the glycophorin A (GPA) gene in erythrocytes. The primary outcome measure was the presence or absence of thyroid nodules as determined by the ultrasound examination. Of the screened workers, 1,247 (63%) were sent to Chernobyl in 1986, including 603 (30%) sent in April or May, soon after the accident. Workers served at Chernobyl for an average of 3 months. The average age was 32 years at the time of arrival at Chernobyl and 40 years at the time of thyroid examination. The mean documented radiation dose from external sources was 10.8 cGy. Biological indicators of exposure showed low correlations with documented dose, but did not indicate that the mean dose for the population was higher than the average documented dose. Ultrasound examinations revealed thyroid nodules in 201 individuals (10.2%). The prevalence of nodules increased with age at examination, but no significant associations were observed with recorded dose, date of first duty at Chernobyl, duration of service at Chernobyl, building the sarcophagus or working on the roof of neighboring buildings or close to the damaged reactor. Nodularity showed a nonsignificant (p(1) = 0.10) positive association with the proportion of lymphocytes with chromosome translocations, but associations with the frequency of variant erythrocytes in the GPA assay were weak and unstable (p(1) > or = 0.46). The majority of fine-needle biopsies taken on 77 study participants indicated benign nodular disease. However, two cases of papillary carcinoma and three benign follicular neoplasms were identified and referred for treatment. Both men with thyroid cancer had been sent to Chernobyl in May of 1986, when the potential for exposure to radioactive iodines was greatest. Chernobyl cleanup workers from Estonia did not experience a markedly increased risk of nodular thyroid disease associated with exposure to external radiation. Possible reasons for the apparent absence of effect include low radiation doses, the protracted nature of the exposure, errors in dose measurement, low sensitivity of the adult thyroid gland or the insufficient passage of time for a radiation effect to be expressed.

Methods for improving the yield and quality of metaphase preparations for FISH probing of human lymphocyte chromosomes.

Procedures are described for the in vitro culture of human lymphocytes, which have been concentrated by density gradient centrifugation, and for a modified slide-making technique for the fixed cells. The method yields improved percentages of mitotic cells which are largely synchronized at harvest. Controlled placement of fixed cells on slides produces well-spread metaphase preparations with little background material to interfere with fluorescence in situ hybridization (FISH) probe procedures. The FISH reagents and microscope scanning time required are minimized by concentrating cells in a defined area of the slide.

Delayed gastric emptying as a factor in delayed postprandial glycaemic response in pregnancy.

OBJECTIVE: To determine whether an alteration in gastric emptying contributes to an altered postprandial glycaemic response in normal pregnancy. DESIGN: A longitudinal study in normal pregnancy and postpartum. SETTING: Teaching hospital in Sheffield. SUBJECTS: Primigravid women with uncomplicated pregnancies. INTERVENTIONS: Simultaneous meal tolerance and paracetamol absorption tests. MAIN OUTCOME MEASURES: 1. Gastric emptying: maximum concentration (Cmax) and time to maximum concentration (Tmax) of paracetamol; 2. glycaemic response: Cmax, Tmax, and area under the curve of plasma glucose; 3. insulinaemic response: Cmax, Tmax, area under the curve of plasma insulin. RESULTS: An increased, but not delayed, insulin response, and an increased initial glucose response to a test meal in the third trimester were not accompanied by any simultaneous delay in gastric emptying. CONCLUSION: Gastric emptying does not seem to be a factor in the glycaemic response of pregnancy.

Cytogenetic damage in marrow cells of mice after injections of yttrium-90-labeled monoclonal antibody.

Chromosome aberrations, micronuclei and sister-chromatid exchanges were quantified in marrow cells of athymic nude and B6C3F1 mice at various times up to 14 days after injection of 90Y-labeled monoclonal antibody CO17-1A. Aberrations, predominantly of the chromatid type, were sharply elevated at 24 h post-injection then declined in a curvilinear fashion over the 14 days. Micronucleus numbers among polychromatic erythrocytes peaked 3-4 days after treatment, then declined exponentially but remained at higher than expected levels. Sister-chromatid exchanges were roughly double the control rate with no apparent relation to post-treatment time.

Negative dose-response relationship for radiation-induced micronuclei in polychromatic erythrocytes of mice.

Micronuclei were assessed among the bone marrow PCEs of mice 28 hr after exposures to 1 to 8 Gy of X-rays and at 6-hr intervals from 12-60 hr after exposures to 2 or 6 Gy. At 28 hr, the frequency of micronuclei declined as the exposure level increased from 1 to 8 Gy. The peak proportion of micronucleated PCEs appeared much later following 6 Gy than after 2 Gy exposures, implicating cell cycle delay as the cause of the negative dose-response relationship.

Induction of chromosomal damage in mammalian cells in vitro and in vivo by sulfapyridine or 5-aminosalicylic acid.

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.

In vivo cytogenetic activity of diphenylhydantoin in mice.

Diphenylhydantoin was tested in vivo in mice using a variety of cytogenetic endpoints to evaluate its genotoxicity. Injected doses of 125, 250 and 500 mg/kg failed to increase the number of chromosome aberrations in marrow cells at 17 h post-treatment, and 37.5, 75 and 150 mg/kg doses were likewise ineffective at 36 h. SCEs were significantly increased by doses of 125 mg/kg (but not 250 mg) after 23 h and modestly, in relation to dose, at 42 h. No increase in the number of micronuclei among marrow PCEs was seen following single i.v. injections ranging from 0.1 to 20 mg/kg. Three daily i.p. injections of doses up to 70 mg/kg also failed to increase the number of micronuclei in either marrow or peripheral blood PCEs. Some cytotoxic effect was evident following relatively high doses.

Chromosomal effects of theophylline measured in mouse marrow cells in vivo.

Doses of 62.5, 125 and 250 mg/kg of theophylline were administered to male B6C3F1 mice by intraperitoneal injection. Chromosome aberrations were scored in first-division metaphases of marrow cells 18 and 36 h post-treatment and sister-chromatid exchanges were quantified in second-division metaphases at 24 h. A modest but statistically significant increase in the number of SCEs occurred, but chromosome aberrations were not significantly different from controls following treatment with any level of the drug at either time period.

Mouse dominant lethal and bone marrow micronucleus studies on methyl vinyl sulfone and divinyl sulfone.

Methyl vinyl sulfone and divinyl sulfone were tested for the induction of dominant lethal mutations and micronucleated bone-marrow erythrocytes in male mice. These chemicals were chosen for study because of their similarities in structure and chemical reactivity to acrylamide which is known to induce both effects. Following administration of the test compounds by intraperitoneal injection at the maximum tolerated doses, no evidence of induced dominant lethal mutations or micronucleated bone-marrow cells was observed for either chemical. It is concluded that structures and Michael reactivities similar to acrylamide are not sufficient to impart similar in vivo genetic toxicity to MVS and DVS.

Modified system outcome score and outcome index--method of monitoring patient care in a special care area.

A scoring system intended both to assess mortality risk and permit surveillance, evaluation, and comparison of medical care was developed in our surgical ICU. Five simple clinical indices of organ system failure were selected and weighted according to their statistically validated relationship to mortality, resulting in a daily System Outcome Score (SOS). Cluster analysis was used to divide the creation data set of 2,777 patients into suitable groupings of scores to predict mortality; the clustering was confirmed for reproducibility with a validation set of an additional 2,860 patients. Based on this validation of the scoring system, two computer-controlled patient care surveillance techniques were developed. The first involved the definition of three unfavorable SOS patterns evolving during the course of a patient's admission. Detection of one or more of these patterns, described by the acronym SDL, permits review of the care administered to the specific patient generating the pattern. A global assessment of care is achieved with the Outcome Index (OI), which relates overall mortality risk in the ICU to the actual mortality rate over a given time period. Effectiveness of care can then be compared between different time periods within the one unit or between different units with similar patient mix. The overall system offers the potential for a surveillance-based quality assurance system with widespread applicability.

Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells.

4 chemicals, with various modes of clastogenic action were used to evaluate induced chromosomal aberrations in mouse bone marrow at different times after intraperitoneal injection. Aberration frequencies induced by mitomycin C, cyclophosphamide and dimethylbenz[a]anthracene increased with increasing time between treatment and sampling until those time points (approximately 18 h) when significant proportions of second-division metaphases were among the cells being scored; this increase was not obvious following treatment with 4-nitroquinoline 1-oxide. When BrdUrd tablets were implanted prior to treatment and scoring was restricted to first-division metaphases, aberration rates continued to increase for as long as 24 h post-treatment. The presence of BrdUrd did not affect significantly the rate of aberration induction by the chemicals. Our data indicate that the sensitivity of the in vivo mouse marrow assay for clastogenic chemicals can be greatly increased by utilizing BrdUrd to insure the scoring of only first-division metaphases at post-treatment times of approx. 18 h.

A replicated five cluster MMPI typology of morbidly obese female candidates for gastric bypass.

This study clusters empirically into subgroups the preoperative MMPI profiles of morbidly obese females seeking a gastric bypass, assesses the replicability of clusters across different subject populations and tests the hypotheses that cluster inclusion bears a significant relationship to weight loss and post-operative follow-up behavior. One hundred seventy women participated: 85 were patients at The University of Texas Health Science Center at San Antonio, Texas (UTHSCSA), and 85 were from the Cherryhill Medical Surgical Center (CMSC), Albion, Michigan. A hierarchical cluster analytic technique was applied using the cosine measure and average linkage within groups method. Purification of the two samples resulted in seven groups with 59 cases in the UTHSCSA sample and six groups with 62 cases in the CMSC sample. Five groups containing 94 subjects (55 percent) replicated across the two samples (UTHSCSA = 44; CMSC = 50). Discriminant analysis yielded four statistically significant functions. The chi 2 test was employed to discern significant differences based on group inclusion. Demographic differences between the two geographic samples are discussed. One year follow-up studies found that personality type bears a significant relationship to indexed weight loss, follow-up appointment frequency, and follow-up duration for the San Antonio sample.

The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo.

Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time (-1, +1 or +8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and CP/MMC are probably due to DMBA's slower absorption/distribution kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.

Caprolactam and benzoin: tests for induction of chromosome aberrations and SCEs in mouse bone marrow.

CAP and ZOIN were tested in vivo for their ability to induce sister-chromatid exchanges (SCE) and chromosome aberrations in mouse bone marrow cells. Single intraperitoneal injections of ZOIN to a maximum of 3000 mg/kg body weight failed to increase the number of SCEs in metaphases recovered 24 h post-treatment, and doses of 1500 mg/kg did not induce measurable increases in chromosome-aberration levels among first-division metaphases at 18 h. Similarly, neither endpoint showed a significant increase following near-lethal doses of 700 mg/kg of CAP. Two lower doses of each chemical were also ineffective. Under the in vivo conditions of our test system, both chemicals were cytogenetically inactive.

The outcome index. A method of quality assurance in the special care area.

A method of quality assurance for a surgical intensive care unit is described. A system outcome score is devised, incorporating only easily obtained objective components that reflect the likelihood of death. Through the use of a derived outcome index, the actual mortality rate is compared with the predicted mortality rate as a method of monitoring the quality of care provided. Subroutines exist to identify errors in data entry, to detect malicious interference in patient care, to add nonscoring components for the purposes of clinical studies, and to facilitate retrieval of a concise summary of the major events during the stay of every patient admitted to the intensive care unit.

Assays of three carcinogen/non-carcinogen chemical pairs for in vivo induction of chromosome aberrations, sister chromatid exchanges and micronuclei.

Three pairs of structurally similar carcinogenic/non-carcinogenic chemicals were tested for in vivo genotoxic activity in B6C3F1 mice. The carcinogenic/non-carcinogenic pairs, respectively, were o-toluidine hydrochloride/o-anthranilic acid, 4-chloro-o-phenylenediamine/4-nitro-o-phenylenediamine, and 3-(chloromethyl)pyridine hydrochloride/2-(chloromethyl)pyridine hydrochloride. Bone marrow cells from mice given intraperitoneal injections of up to the maximum tolerated dose were evaluated for chromosomal aberration, sister chromatid exchange, and micronucleus induction, o-anthranilic acid and o-toluidine hydrochloride did not increase the frequency of chromosomal aberrations or micronuclei. o-Toluidine hydrochloride increased the frequency of sister chromatid exchanges in two successive trials, while o-anthranilic acid had a positive effect on sister chromatid exchanges in two of three trials. Both 2-(chloromethyl) and 3-(chloromethyl)pyridine hydrochloride were negative for all three endpoints. Assays for chromosomal aberrations and micronuclei each distinguished between 4-chloro-o-phenylenediamine and its non-carcinogenic companion, 4-nitro-o-phenylenediamine. In the aberration test, 4-chloro-o-phenylenediamine produced a few cells with very large numbers of aberrations rather than an even distribution of damage among cells.