mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (56Fe ions).

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. 56Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu 56Fe ions (0, 0.1, 0.5 and 1.0 Gy) or (137)Cs gamma rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to 56Fe ions or 137Cs gamma rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of 56Fe ions or 3 Gy of 137Cs gamma rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to 56Fe ions or 137Cs gamma rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, 56Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than 137Cs gamma rays in inducing chromosomal damage.

Frequencies of hprt mutant lymphocytes in marijuana-smoking mothers and their newborns.

Reports of increases in the prevalence of marijuana smoking, especially among young people, have led to concerns about possible genotoxic effects from marijuana use due to exposure to the mutagenic and carcinogenic agents present in marijuana smoke. Prior studies of the adverse health consequences of marijuana smoking, using disease outcomes, have sometimes been confounded by the fact that most marijuana smokers also smoke tobacco. In the present study, the potential mutagenic effects of marijuana smoking were investigated with a somatic cell mutation assay that detects mutations occurring in vivo in the hprt gene. Subjects were volunteers recruited from a prenatal clinic that performs urine drug screens on all consenting patients. Blood samples were collected from 17 subjects whose drug screens indicated marijuana use, but who did not smoke tobacco or use cocaine or opiates, and 17 non-smokers with negative drug screens. Absence of tobacco use was confirmed by plasma cotinine tests. Cord blood samples were collected from newborns of 5 of the marijuana smokers and 5 non-smokers. Lymphocytes were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The frequency of variant (mutant) lymphocytes (Vf) in the 17 non-smokers (+/- standard error) was 1.93 (+/- 0.17) per million evaluatable cells. The Vf of 17 marijuana smokers was more than three-fold higher, 6.48 (+/- 0.48) x 10(-6), a significant difference, p < 0.001. Cord blood lymphocytes from 5 newborns of non-smokers had a Vf of 0.85 (+/- 0.23) x 10(-6), compared to 2.55 (+/- 0.60) x 10(-6) for 5 newborns of marijuana smokers, significantly higher, p < 0.05. Because of the known association between increases in somatic mutations and the development of malignancies, this study indicates that marijuana smokers may have an elevated risk of cancer. For pregnant marijuana smokers, there is also concern for the possibility of genotoxic effects on the fetus, resulting in heightened risk of birth defects or childhood cancer.

In vitro and in vivo genotoxic activity of miral, an organophosphorus insecticide used in Colombia.

Miral 500 CS (CAS# 42509-80-8), an organophosphorus insecticide, has been widely used in Columbia to fumigate coffee plantations. Therefore, there is extensive human exposure to this pesticide. Miral's mutagenic and genotoxic activities, however, are not known. In this study, such activities of the pesticide were evaluated using the Salmonella TA98/S9 test and the chromosome aberration assay in bone marrow cells of Swiss albino CD1 male mice. All doses tested with Salmonella in the presence of S9 mix (3.2, 16, 80, 400 and 2000 micrograms/plate) induced a mutagenic response that was three times the spontaneous mutation frequency. The mutagenic response without S9 was twice the spontaneous frequency. Based on a 4-day treatment (i.p.) of mice with Miral, the median lethal dose (LD50) and the maximum tolerated dose (MTD) were 912.5 mg/kg and 730 mg/kg, respectively. A significant dose-dependent cell cycle delay (r2 = 0.85, p < 0.01) was observed in bone marrow cells when mice were treated for 24 h with 73, 146, 219, 292, 365, 438, 511, 584, 657 and 730 mg/kg. Significant increase in mitotic indices (p < 0.02) and chromosome aberrations (p < 0.05) were induced in bone marrow cells, when mice were treated for 18 h with the highest dose 511 mg/kg. Our results indicate that Miral is a mutagenic compound in Salmonella and is capable of inducing chromosome aberrations at high doses in mice. Additional genotoxicity studies in farmers exposed to Miral should be conducted to determine the potential human health risk resulting from chronic low-dose exposures to this pesticide.

Frequencies of hprt mutant lymphocytes in smokers, non-smokers, and former smokers.

Previous work with the autoradiographic mutant lymphocyte assay has provided information about the time-course of development of hprt mutations and the persistence of detectable mutant cells in human subjects following therapeutic exposures to genotoxic agents. These early studies also revealed elevations in frequencies of mutant cells in pretreatment blood samples from patients who were current tobacco smokers, but no information was available on former smokers. In the present study, blood samples were obtained from 21 healthy former tobacco smokers who had quit smoking at least 1 year before sampling, 42 subjects who had never smoked, and 23 tobacco smokers. Plasma from all samples was tested for cotinine, a metabolite of nicotine. Current smokers were categorized as heavy smokers (> or = 10 cigarettes per day, cotinine > or = 90 ng/ml plasma) and light smokers (< 10/day, cotinine < 90 ng/ml). Lymphocytes from the blood samples were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The 21 former tobacco smokers had a mean variant (mutant) frequency (Vf +/- standard error) of 1.97 (+/-0.13) per million evaluatable cells. The Vf of 42 subjects who had never smoked was 1.74 (+/-0.13) x 10(-6), not significantly different from the former smokers. The smokers had Vfs of 8.09 (+/-0.78) x 10(-6) for 18 heavy smokers and 5.22 (+/-1.02) x 10(-6) for five light smokers. The two categories of smokers had frequencies of mutant cells significantly different from each other, and each was significantly higher than non-smokers and former smokers (P < 0.05). Vfs were significantly correlated with both cotinine concentrations and the number of cigarettes smoked per day, P < 0.001. This study demonstrates the sensitivity of the autoradiographic hprt assay for detecting mutagenic effects related to chronic low-level exposures to genotoxins, and indicates that this assay is more likely to detect the effects of recent rather than past exposures.

Biological monitoring for mutagenic effects of occupational exposure to butadiene.

The use of biological markers in the evaluation of human exposure to hazardous agents has increased rapidly in recent years. Because 1,3-butadiene is a mutagenic carcinogen, existing occupational levels of exposure may be appropriately evaluated using somatic cell mutation as a biomarker. Previously, we have described a biomarker study of workers in a butadiene monomer plant (Ward et al., 1994). We now report results from a second study of the same group of workers, conducted after plant modernization, and present preliminary results from a study of exposures in a styrene butadiene rubber (SBR) plant. Air levels of butadiene were determined using either charcoal tubes with air pumps or passive badge dosimeters. The quantity of a butadiene metabolite in the urine was used as a biomarker of exposure and the mutagenic effects of exposure were measured using the autoradiographic hprt mutant lymphocyte assay. In all three studies, the frequencies of hprt mutants were significantly elevated in workers from the areas of highest exposure when compared to workers from lower exposure areas or non-exposed subjects. The concentration of the urinary metabolite was significantly increased in high-exposed workers in the first study of monomer plant workers but not in the second. In the first monomer plant study, historical air concentrations of butadiene were higher in the production units than in the central control unit. While concurrent determined air concentrations were not elevated in the second monomer plant study, they were elevated in high exposure areas in the SBR plant study. Mutant frequencies in the lower-exposure and the non-exposed groups were consistent with historical values for non-smoking individuals who were not exposed to known mutagens. The use of biomarkers, including the hprt mutant lymphocyte assay, may be of great value in determining an appropriate occupational exposure limit for butadiene.

Gestational pyelonephritis--associated Escherichia coli isolates represent a nonrandom, closely related population.

OBJECTIVE: A select group of Escherichia coli strains known as uropathogenic cause pyelonephritis in nonpregnant individuals. We investigated whether Escherichia coli from gestational pyelonephritis represent a random population or possess common uropathogenic characteristics. STUDY DESIGN: Repetitive element sequence-based polymerase chain reaction, plasmid profiles, hemolysin, and O serotypes were assayed from Escherichia coli isolates of 57 pregnant patients with acute pyelonephritis at different gestational ages. RESULTS: The majority of the first trimester isolates fell primarily into repetitive element sequence-based patterns 1 and 3 and O6, O15, and O75 serotypes. Second-trimester isolates had multiple patterns with high-frequency repetitive element sequence-based polymerase chain reaction 1 and 5 and an unknown (OX) serotype. Pattern 3, predominantly O75 serotype, was found primarily among third-trimester isolates. CONCLUSION: It is likely that Escherichia coli associated with acute pyelonephritis during different trimesters of pregnancy represents nonrandom closely related isolates, and some of these strains may be characteristic in pregnant patients only.

Biomarker monitoring of a population residing near uranium mining activities.

We investigated whether residents residing near uranium mining operations (target population), who are potentially exposed to toxicants from mining waste, have increased genotoxic effects compared with people residing elsewhere (reference population). Population surveys were conducted, and 24 target and 24 reference residents were selected. The selected subjects and controls were matched on age and gender and they were nonsmokers. Blood samples were collected for laboratory studies. The standard cytogenetic assay was used to determine chromosome aberration frequencies, and the challenge assay was used to investigate DNA repair responses. We found that individuals who resided near uranium mining operations had a higher mean frequency of cells with chromosome aberrations and higher deletion frequency but lower dicentric frequency than the reference group, although the difference was not statistically significant. After cells were challenged by exposure to gamma-rays, the target population had a significantly higher frequency of cells with chromosome aberrations and deletion frequency than the reference group. The latter observation is indicative of abnormal DNA repair response in the target population.

Chromosome aberrations and response to gamma-ray challenge in lymphocytes of workers exposed to 1,3-butadiene.

An integrated population monitoring study was initiated to investigate whether occupational exposure to current low levels of butadiene is mutagenic to workers. Ten exposed workers (mean production area concentration of 3.5 ppm) and 10 matched plant controls (mean exposure to 0.03 ppm) were selected and blood samples were collected for our study. The standard cytogenetic assay was used to determine chromosome aberration frequencies. In addition, a challenge assay was used to determine response to gamma-rays as an indication of DNA repair deficiencies. In the latter assay, cells were exposed to gamma-rays at the G1 phase of the cell cycle in vitro and the frequencies of chromosome aberrations in the first post-irradiation metaphase cells were quantitated. Based on results of the cytogenetic assay, the exposed group had a higher frequency of cells with chromosome aberrations and higher chromatid breaks per 100 cells compared with the control. However, the difference was not significant (p > 0.1). With the challenge assay, the exposed group had a higher frequency of aberrant cells (p < 0.04), chromatid breaks (p < 0.05), deletions (p < 0.07), and dicentrics (p < 0.02) than the controls. In addition, the dicentric frequencies from workers were significantly correlated with the presence of a butadiene metabolite [1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane] in urine with a correlation of coefficient of 0.6 (p < 0.01). Two outliers were identified and our interpretation of their responses will be discussed. This study indicates that the workers had exposure-induced mutagenic effects. Together with the observation of gene mutation in a subset of the present population, this study indicates that the current occupational exposure to butadiene may not be safe to workers.

hprt mutant lymphocyte frequencies in workers at a 1,3-butadiene production plant.

1,3-Butadiene is a major industrial chemical that has been shown to be a carcinogen at multiple sites in mice and rats at concentrations as low as 6.25 ppm. Occupational exposures have been reduced in response to these findings, but it may not be possible to determine by using traditional epidemiological methods, whether current exposure levels are adequate for protection of worker health. However, it is possible to evaluate the biological significance of exposure to genotoxic chemicals at the time of exposure by measuring levels of genetic damage in exposed populations. We have conducted a pilot study to evaluate the effects of butadiene exposure on the frequencies of lymphocytes containing mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in workers in a butadiene production plant. At the same time, urine specimens from the same individuals were collected and evaluated for the presence of butadiene-specific metabolites. Eight workers from areas of the plant where the highest exposures to butadiene occur were compared to five workers from plant areas where butadiene exposures were low. In addition, six subjects with no occupational exposure to butadiene were also studied as outside controls. All of the subjects were nonsmokers. An air sampling survey conducted for 6 months, and ending about 3 months before the study, indicated that average butadiene levels in the air of the high-exposure areas were about 3.5 +/- 7.5 ppm. They were 0.03 +/- 0.03 ppm in the low-exposure areas. Peripheral blood lymphocytes from the subjects were assayed using an autoradiographic test for hprt mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Insudative lesions--their pathogenesis and association with glomerular obsolescence in diabetes: a dynamic hypothesis based on single views of advancing human diabetic nephropathy.

Kidneys from 74 consecutive, primarily non-insulin-dependent diabetics at autopsy and 59 age-, sex, and ethnic group-matched controls were examined qualitatively and semiquantitatively to determine the prevalence and severity of insudative lesions (ILs) and obsolescent glomeruli with (OGcFC) and without (OGsFC) insudative (fibrin cap) lesions. A subset of 25 cases with advanced diabetic changes was examined using serial sections, immunohistochemical stains, and electron microscopy to determine the pathogenesis of ILs and OGcFCs. Insudative lesions consisted of intramural accumulations (hereafter called deposits) of presumably imbibed plasma proteins and lipids within renal arterioles, glomerular capillaries, Bowman's capsule, and proximal convoluted tubules. Insudative lesions in Bowman's capsule are called capsular drop lesions (CDs), in glomerular capillaries they are called fibrin cap lesions (FC), and in afferent and efferent arterioles they are called hyalinized afferent (HA) and hyalinized efferent (HE) arterioles, respectively. All ILs were much more numerous and/or larger in diabetics than in controls. Contrary to previous opinion, CDs and HE arterioles were not specific for diabetes, being present in small numbers in nine (15%) controls. Controls with CD/HE arterioles had far more HA arterioles and focal mesangiolyses (FMs) than those without. Insudative lesions consisted of the well known homogenous eosinophilic deposits (homogenous eosinophilic ILs) and the less familiar foamy, reticulated, and vacuolar deposits (heterogenous lucent ILs). Homogenous eosinophilic ILs were predominant in afferent arterioles and more so in efferent arterioles, and were segregated into globules of varying density with the denser deposits located peripherally. Two types of CDs, which differed sharply in location and composition, were found. The first was mostly homogenous eosinophilic, usually without capsular adhesions and located near the vascular pole close to preglomerular arterioles. The second was mostly heterogenous lucent, located away from the vascular pole, and consistently connected by adhesions to the capillary tuft usually near FMs and/or Kimmelstiel-Wilson (KW) nodules. The latter ILs sometimes extended in continuity along the internal surface of the basement membrane from Bowman's capsule into the proximal convoluted tubule. It was hypothesized that ILs traveled centrifugally through the walls of preglomerular arterioles to form the first type of CD and longitudinally within the walls of afferent arterioles and glomerular capillaries and through adhesions to form the second. Contrary to previous opinion, FCs were consistently intramural. When numerous, FCs were associated with a form of glomerular obsolescence called OGcFC.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevated frequencies of hprt mutant lymphocytes in cigarette-smoking mothers and their newborns.

Maternal cigarette smoking during pregnancy has been associated with increased perinatal mortality and low birth weight. Several epidemiological studies have demonstrated an association between smoking during pregnancy and an elevated risk of hematopoietic cancer in the child, but other studies have failed to confirm this association. We have used an assay for somatic cell mutation to evaluate the in utero effects of exposure to maternal cigarette smoking. Cord blood samples were obtained from 10 newborns whose mothers smoked cigarettes during pregnancy and 10 newborns of non-smoking mothers. Blood samples were also obtained from 5 of the smoking and 5 of the non-smoking mothers. Smoking status was confirmed in all samples by testing the blood plasma for cotinine. The frequency of lymphocytes containing mutations at the hypoxanthine phosphoribosyltransferase (hprt) locus was determined with an autoradiographic assay using cells that had been cryopreserved. The mothers who were smokers had a mean frequency (+/- SE) of 3.08 (+/- 0.55) variant (mutant) cells per 10(6) evaluatable lymphocytes. The frequency (Vf) in non-smokers was 1.07 (+/- 0.17) x 10(-6). The Vf of newborns of smokers was 2.17 (+/- 0.24) x 10(-6), and newborns of non-smokers had a Vf of 0.77 (+/- 0.13) x 10(-6). In both mothers and newborns the difference in Vf between smokers and non-smokers was statistically significant (p < 0.05). Maternal and newborn Vfs were significantly correlated (r = 0.88; p < 0.004), and there was a positive association (r = 0.86; p < 0.001) between the reported number of cigarettes smoked per day and the Vfs. This study provides further evidence that maternal smoking may be hazardous to the future health of children exposed in utero to mutagenic agents in cigarette smoke.

Circadian dependence of interferon antitumor activity in mice.

BACKGROUND: Chronobiological studies with anticancer drugs have shown that their effectiveness and/or toxicity is significantly influenced by the time of their administration in the circadian cycle. Previous studies also have shown that the myelotoxicity of interferons is similarly influenced. PURPOSE: This study was undertaken to evaluate the antitumor activity of interferons as a function of their administration to animals at defined points in the circadian cycle with equal light and dark periods. METHODS: A murine tumor model was employed. Following adaptation to alternating cycles of 12 hours of light and 12 hours of dark for a period of 2-3 weeks, C57BL/6 mice were inoculated with B16 melanoma cells intraperitoneally at different hours after light onset. Exactly 24 hours after inoculation, each group received intraperitoneal injections of either recombinant human interferon alpha (rHuIFN-alpha A/D), recombinant murine IFN-gamma (rMuIFN-gamma), or interferon-carrier solution as control (once a day for 5 days) and were monitored for the length of their survival. RESULTS: The antitumor activity (calculated as percent increased life span) of both rHuIFN-alpha A/D and rMuIFN-gamma varied with the points at which they were administered in the circadian cycle. However, the points showing minimum and maximum activity for rHuIFN-alpha A/D (12-16 and 0-4 hours after light onset, respectively) did not correspond with the points for the rMuIFN-gamma (0-8 and 16 hours after light onset, respectively). To generate maximum antitumor activity, approximately fivefold higher amounts of rHuIFN-alpha A/D were required at 12 than at 4 hours after light onset (dose range, 3333-90,000 IU/d) (P < .0001). Similarly, for rMuIFN-gamma at least 8.5-fold greater amounts were required at 8 than at 16 hours after light onset (dose range, 667-6000 IU/d) (P < .01). CONCLUSIONS: In the murine tumor model, administration of rHuIFN-alpha A/D at 4 hours after light onset and rMuIFN-gamma at 16 hours after light onset may produce maximum antitumor activity.

Focal mesangiolysis and the pathogenesis of the Kimmelstiel-Wilson nodule.

Kidneys from 74 consecutively autopsied primarily non-insulin-dependent diabetes cases and 59 age-, sex-, and ethnic group-matched controls were examined qualitatively and semiquantitatively to determine whether focal mesangiolyses (FMs), Kimmelstiel-Wilson (KW) nodules, and glomerular capillary microaneurysms (GCMs) were related lesions, to determine their extent and pathogenic sequence, and to look for associations with structural and functional factors. Light microscopic examination of serial sections, immunohistochemical stains, image analysis, and electron microscopy were used. Focal mesangiolyses, KW nodules, and GCMs occurred in 31 of the 74 diabetes cases (27 had FMs, 29 had KW nodules, and nine had GCMs) and were positively correlated with each other semiquantitatively (r = .71, .70, and .68, respectively). Numerous FMs were found, involving 62% and 78% of the glomeruli in the two most severely affected cases. Most FMs were located at the periphery of KW nodules, but de novo FMs were documented in six cases. Glomerular capillary microaneurysms were deemed occasional complications of FMs because they were much less common, and 25 of the 27 GCMs identified were contiguous with FMs. Focal mesangiolyses and GCMs were deemed transient lesions, being absent in end-stage kidneys. Both FMs and KW nodules consisted of a spectrum of lesions. For the sake of clarity they were arbitrarily divided into two types: edematous and proliferative FMs and simple and complicated KW nodules. Their characteristics suggested the following pathogenic sequence: edematous FM-->proliferative FM-->focal nodular mesangial expansion-->simple KW nodule-->recurrent FM-->complicated KW nodule. Complicated nodules were associated with marked alterations in the lobular capillary. The number of mesangial cells was increased in FMs and they were thought to be responsible for increased matrix production. Focal mesangiolyses and KW nodules were positively associated with diabetes, proteinuria, and hyalinization of afferent and efferent arterioles, but were weakly or not associated with hypertension, arcuate and interlobular artery stenosis, hydroenphrosis, acute pyelonephritis, renal arterial atheromatous emboli, glomerular platelet-fibrin thromboemboli, and congestive heart failure.

The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice.

Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.

Chromosome rearrangements in fumigant appliers: possible relationship to non-Hodgkin's lymphoma risk.

Appliers of pesticides (n = 18) who are exposed to the fumigant phosphine or who have a mixed exposure to other pesticides and phosphine demonstrate a significant increase in chromosome rearrangements in G-banded chromosomes from peripheral blood compared to control subjects (n = 26). Appliers who had discontinued using phosphine for at least 8 months prior to specimen collection (n = 5) do not demonstrate significant increases in chromosome rearrangements compared to controls. Breakpoint analysis of 6,138 metaphases from all subjects demonstrates 196 breaks per 3605 metaphases in exposed subjects and 102 breaks per 2,533 metaphases in control subjects. Bands with significantly more breaks than expected based on band length in all study subjects were 1q32, 3p14, 7p15, and 14q11. Three of these four bands had significantly more breaks than expected in the exposed group, and all four bands had a significant excess of breaks in the control group. There are four bands with a significant excess of breaks in the exposed group and no breaks in the control group; each of these occurs in a known protooncogene region. These are 1p13 (NRAS), 2p23 (NMYC), 14q32 (ELK2), and 21q12 (ETS-2). Most breaks at bands 1p13, 14q32, and 21q22 are associated with chromosome rearrangements and occurred in appliers who have a mixed exposure to phosphine and other pesticides. Cytogenetic abnormalities, i.e., rearrangements and/or deletions involving bands 1p13, 2p23, and 14q32, are associated with non-Hodgkin's lymphoma. We speculate that these findings could relate to the risk of evolution of a neoplastic clone in these workers. Epidemiological studies of similarly exposed workers indicate an excess of non-Hodgkin's lymphoma.

Highly purified human alpha-thrombin promotes morphological transformation of BALB/c 3T3 cells.

Purified human alpha-thrombin stimulates phosphoinositide turnover as a necessary step in its stimulation of fibroblast cell proliferation. Since phosphoinositide turnover releases diacylglycerol, which activates protein kinase C, we postulated that long-term exposure to thrombin might promote cellular transformation in a manner similar to long-term exposure to phorbol esters, which also activate protein kinase C. The present studies show that chronic exposure of BALB/c 3T3 cells (subclone A31-1-13) to thrombin (5 micrograms/ml) led to a 4- to 20-fold increase in the frequency of morphological transformation over controls as determined by induced foci in monolayer cultures. The foci appeared to represent true transformants as cells from randomly selected foci grew in soft agar and had saturation densities 2- to 3-fold higher than control cells. Acute thrombin treatment for 24 h resulted in small but statistically significant (P less than 0.05) increases in morphological transformation with or without promotion by phorbol myristate acetate, indicating that thrombin can act as a weak initiator or complete carcinogen in this test system. Initiation of cells with low levels of 3-methylcholanthrene followed by promotion with thrombin caused a greater enhancement of morphological transformation (P less than 0.005). Thus, it appears that most of the stimulation of in vitro cell transformation by thrombin may be due to its promotional activity. These results raise the possibility that thrombin released locally following tissue injury or chronic irritation may play a role in cellular transformation and tumor development, especially in tissues sensitized by exposure to initiating carcinogens.

Comparison of hprt variant frequencies and chromosome aberration frequencies in lymphocytes from radiotherapy and chemotherapy patients: a prospective study.

The autoradiographic 6-thioguanine-resistant mutant lymphocyte assay and a chromosome aberration assay were used to determine the time-course of appearance and persistence of elevated frequencies of hprt variants and dicentric chromosomes in patients receiving x-irradiation therapy. Twelve cancer patients, treated with 180-200 cGy/day, 5 days/wk, for 3-7 wk, were studied before treatment, at various weekly intervals during treatment, and after treatment. The hprt mutation assays were done with frozen/thawed lymphocytes isolated from aliquots of the same blood samples used for the chromosome aberration assays. The hprt variant frequencies (Vfs) of only 4 of the 7 patients assayed at 2 wk of treatment were elevated over pre-treatment Vfs, but during the 3rd and 4th weeks of treatment there were significant (P less than 0.01) 5- to 15-fold increases in all Vfs. By 6-32 wk after treatment Vfs had fallen to levels only slightly higher than the mean pre-treatment Vf. The frequencies of cells with dicentric chromosomes were significantly increased (P less than 0.01) after 1 wk of radiotherapy, continued to increase during therapy, and remained elevated after treatment. Five multiple sclerosis patients were also studied before and at 2 and 4 wk intervals after treatment with monthly i.v. doses of 750 mg/m2 of cyclophosphamide (CP). There were no significant elevations in chromosome aberrations at these post-treatment sample times. Previous assays for hprt mutants, done with aloquots of the same blood samples (Ammenheuser et al.: Mutat Res 204:509-520, 1988), had shown 8- to 20-fold increases in Vfs 2 wk after the 1st CP treatment. Our results demonstrate the complementary nature of these two human monitoring assays and emphasize the importance of careful selection of optimal sampling times.

Male-mediated behavioral abnormalities.

The assessment of behavioral development in the progeny of males exposed to known mutagenic chemicals is a potentially sensitive endpoint for detecting transmissible abnormalities. A genetic component is demonstrated as these behavioral abnormalities can also be passed on to the F2 generation. In this review, experimental studies exploring transmission of behavioral deficits from paternal exposure to drugs, chemicals and radiation are addressed. Additionally included is a brief synopsis of recent work performed in our laboratory investigating such abnormalities in offspring from male rats exposed to ionizing radiations. The implications of these behavioral endpoints to humans is also discussed.