Search

The goal of this study was to determine if prolonged exposure to perceptual-motor mismatch increased adaptability and retention of balance in older adults. Sixteen adults, aged 66 to 81 years, were randomized to one of two groups: either the control group (n = 8) or the experimental group (n = 8). Both groups first completed six trials of walking an obstacle course. Participants then trained twice a week for 4 weeks. In the training, the control group walked on a treadmill for 20 minutes while viewing a static visual scene and the experimental group walked on a treadmill for 20 minutes while viewing a rotating visual scene that provided a perceptual-motor mismatch. Following training, both groups were post-tested on the obstacle course. The experimental group moved faster through the obstacle course with fewer penalties. This training effect was retained for 4 weeks. Exposure to perceptual-motor mismatch induced an adaptive training effect that improved balance and locomotor control in older adults. [ABSTRACT FROM AUTHOR]

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 μg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection., Competing Interests: I.M.-P. is an employee of Cumberland Pharmaceuticals, Inc., manufacturer of the test compound (telavancin); however, the project was funded entirely by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Human Health Services, under contract number HHSN272201700040I/75N93019F00271 (PMCID Task Order A29).

The use of antibiotics is a vital means of treating infections caused by the bacteria Bacillus (B.) anthracis . Importantly, with the potential future use of multidrug-resistant strains of B. anthracis as bioweapons, new antibiotics are needed as alternative therapeutics. In this blinded study, we assessed the protective efficacy of teixobactin, a recently discovered antibiotic, against inhalation anthrax infection in the adult rabbit model. New Zealand White rabbits were infected with a lethal dose of B. anthracis Ames spores via the inhalation route, and blood samples were collected at various times to assess antigenemia, bacteremia, tissue bacterial load, and antibody production. Treatments were administered upon detection of B. anthracis protective antigen in the animals' sera. For comparison, a fully protective dose of levofloxacin was used as a positive control. Rabbits treated with teixobactin showed 100% survival following infection, and the bacteremia was completely resolved by 24-48 h post-treatment. In addition, the bacterial/spore loads in tissues of the animals treated with teixobactin were either zero or dramatically less relative to that of the negative control animals. Moreover, microscopic evaluation of the tissues revealed decreased pathology following treatment with teixobactin. Overall, these results show that teixobactin was protective against inhalation anthrax infection in the rabbit model, and they indicate the potential of teixobactin as a therapeutic for the disease.

Little is known about plasma proteins that can be used as biomarkers for early and late responses to radiation. The purpose of this study was to determine a link between depletion of plasma gelsolin (pGSN) and cell-death as well as inflammatory responses in the lung (one of the tissues known to be radiosensitive) of the same exposed CBA/CaJ mice after exposure to heavy silicon ( 28 Si) ions. To prevent the development of multiple organ dysfunctions, pGSN (an important component of the extracellular actin-scavenging system) is responsible for the removal of actin that is released into the circulation during inflammation and from dying cells. We evaluated the levels of pGSN in plasma collected from groups of mice (5 mice in each) at 1 week (wk) and 1 month (1 mo) after exposure whole body to different doses of 28 Si ions, i.e. 0, 0.1, 0.25, or 0.5 Gy (2 fractionated exposures, 15 days apart that totaled each selected dose). In the same mouse, the measurements of pGSN levels were coupled with the quantitation of injuries in the lung, determined by (a) the levels of cleaved poly (ADP-ribose) polymerase (cleaved-PARP), a marker of apoptotic cell-death, (b) the levels of activated nuclear factor-kappa B (NF-κB) and selected cytokines, i.e. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6, from tissue-lysates of the lung. Further, the ratio of neutrophils and lymphocytes (N/L) was determined in the same mouse. Our data indicated: (i) the magnitude of pGSN depletion was dependent to radiation dose at both harvest times, (ii) a persistent depletion of pGSN up to 1 mo post-exposure to 0.25 or 0.5 Gy of 28 Si ions, (iii) an inverse-correlation between pGSN depletion and increased levels of cleaved-PARP, including activated NF-κB/pro-inflammatory cytokines in the lung, and (iv) at both harvest times, statistically significant increases in the N/L ratio in groups of mice exposed to 0.5 Gy only. Our findings suggested that depletion in pGSN levels reflects not only the responses to 28 Si-ion exposure at both harvest times but also early and late-occurring damage., (Copyright © 2018. Published by Elsevier Ltd.)

Although the lung is one of the target organs at risk for cancer induction from exposure to heavy ions found in space, information is insufficient on cellular/molecular responses linked to increased cancer risk. Knowledge of such events may aid in the development of new preventive measures. Furthermore, although it is known that germinal cells are sensitive to X- or γ-rays, there is little information on the effects of heavy ions on germinal cells. Our goal was to investigate in vivo effects of 1 GeV/n (48)Ti ions (one of the important heavy ions found in the space environment) on somatic (lung) and germinal (testis) tissues collected at various times after a whole body irradiation of CBA/CaJ mice (0, 0.1, 0.25, or 0.5 Gy, delivered at 1 cGy/min). We hypothesized that (48)Ti-ion-exposure induced damage in both tissues. Lung tissue was collected from each mouse from each treatment group at 1 week, 1 month, and 6 months postirradiation. For the testis, we collected samples at 6 months postirradiation. Hence, only late-occurring effects of (48)Ti ions in the testis were studied. There were five mice per treatment group at each harvest time. We investigated inflammatory responses after exposure to (48)Ti ions by measuring the levels of activated nuclear factor kappa B and selected pro-inflammatory cytokines in both tissues of the same mouse. These measurements were coupled with the quantitation of the levels of global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Our data clearly showed the induction of chronic inflammation in both tissues of exposed mice. A dose-dependent reduction in global 5hmC was found in the lung at all time-points and in testes collected at 6 months postirradiation. In contrast, significant increases in global 5mC were found only in lung and testes collected at 6 months postirradiation from mice exposed to 0.5 Gy of 1 GeV/n (48)Ti ions. Overall, our data showed that (48)Ti ions may create health risks in both lung and testicular tissues.

Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to (28)Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1 cGy/min) to various doses of 300 MeV/n (28)Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5 Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p<0.01), indicating the induction of genomic instability after exposure of mice to 300 MeV/n (28)Si ions. Slight increases in the levels of 5 mC were observed in all treatment groups, as compared to the sham-control level. In contrast, there was a significant reduction in levels of 5 hmC (ANOVA, p<0.01). Since these endpoints were evaluated in the same mouse, our data suggested for the first time a link between a reduction in 5 hmC and genomic instability in HSPC-derived myeloid colonies of CBA/CaJ mice exposed to 300 MeV/n (28)Si ions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Little is known about in vivo cytogenetic effects of protons delivered at the dose and dose rates encountered in space. We determined the effects of 100MeV protons, one of the most abundant type of protons produced during solar particle events (SPE), on the induction of chromosome aberrations (CAs) in bone marrow (BM) cells collected at early (3 and 24h) and late (6 months) time-points from groups of BALB/cJ mice (a known radiosensitive strain) exposed whole-body to 0 (sham-controls), 0.5, or 1.0Gy of 100MeV protons, delivered at 0.5 or 1.0cGy/min. These doses and dose-rates are comparable to those produced during SPE events. Additionally, groups of mice were exposed to 0 or 1Gy of (137)Cs γ rays (delivered at 1cGy/min) as a reference radiation. The kinetics of formation/reduction of gamma-histone 2-AX (γH2AX) were determined in BM cells collected at 1.5, 3, and 24h post-irradiation to assess the early-response. There were five mice per treatment-group per harvest-time. Our data indicated that the kinetics of γH2AX formation/reduction differed, depending on the dose and dose rate of protons. Highly significant numbers of abnormal cells and chromatid breaks (p<0.01), related to those in sham-control groups, were detected in BM cells collected at each time-point, regardless of dose or dose-rate. The finding of significant increases in the frequencies of delayed non-clonal and clonal CAs in BM cells collected at a late time-point from exposed mice suggested that 0.5 or 1Gy of 100MeV protons is capable of inducing genomic instability in BM cells. However, the extent of effects induced by these two low dose rates was comparable. Further, the results showed that the in vivo cytogenetic effects induced by 1Gy of 100MeV protons or (137)Cs γ rays (delivered at 1cGy/min) were similar., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It has been well established that the bone marrow (BM) is a radiosensitive tissue, but the radiosensitivity of the heart is poorly understood. In this study, we investigated the comparative effects of ²⁸Silicon (²⁸Si) ions (one type of heavy ion found in space) on tissue from the heart and the BM of exposed mice. We gave adult male CBA/CaJ mice a whole-body exposure to a total dose of 0, 0.1, 0.25, or 0.5 Gy of 300 MeV/nucleon (n) ²⁸Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). The heart and BM were collected from 5 mice per treatment group at various times up to 6 months post-irradiation. In each mouse, we obtained tissue lysates from the heart and from the total population of BM cells for measuring the levels of cleaved poly (ADP-ribose) polymerase (cleaved PARP, a marker of apoptotic cell death) and the levels of activated nuclear factor-kappa B (NF-κB) and selected NF-κB-regulated cytokines known to be involved in inflammatory responses. Our data showed that, up to 6 months post-irradiation, the levels of apoptotic cell death and inflammatory responses in tissues from the heart and BM collected from exposed mice were statistically higher than those in sham controls. Hence, these findings are suggestive of chronic apoptotic cell death and inflammation in both tissues after exposure to ²⁸Si ions. In summary, our data are indicative of a possible association between exposure to ²⁸Si ions during space flight and long-term health risk.

The goal of this study was to determine if exposure to sensorimotor adaptation training improved head stabilization in older adults. Sixteen participants, age 66-81 yr, were assigned at random to the control group (n = 8) or the experimental group (n = 8). Both groups first completed 6 trials of walking a foam pathway consisting of a moveable platform that induced a lateral perturbation during walking. Head-in-space and trunk-in-space angular velocities were collected. Participants from both groups then trained twice per week for 4 wk. Both groups walked on a treadmill for 20 min. The control group viewed a static scene. The experimental group viewed a rotating visual scene that provided a perceptual-motor mismatch. After training, both groups were retested on the perturbation pathway test. The experimental group used a movement strategy that preserved head stabilization compared with the controls (p < .05). This training effect was not retained after 4 wk.

In spite of extensive research, assessment of potential health risks associated with exposure to low-dose (≤ 0.1 Gy) radiation is still challenging. We evaluated the in vivo induction of genomic instability, expressed as late-occurring chromosome aberrations, in bone-marrow cells of two strains of mouse with different genetic background, i.e. the radiosensitive BALB/cJ and the radioresistant C57BL/6J strains following a whole-body exposure to varying doses of (137)Cs gamma rays (0, 0.05, 0.1, and 1.0 Gy). A total of five mice per dose per strain were sacrificed at various times post-irradiation up to 6 months for sample collections. Three-color fluorescence in situ hybridization for mouse chromosomes 1, 2, and 3 was used for the analysis of stable-aberrations in metaphase-cells. All other visible gross structural-abnormalities involving non-painted-chromosomes were also evaluated on the same metaphase-cells used for scoring the stable-aberrations of painted-chromosomes. Our new data demonstrated in bone-marrow cells from both strains that low doses of low LET-radiation (as low as 0.05 Gy) are incapable of inducing genomic instability but are capable of reducing specific aberration-types below the spontaneous rate with time post-irradiation. However, the results showed the induction of genomic instability by 1.0 Gy of (137)Cs gamma rays in the radiosensitive strain only.

Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals' heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins' direct impact on the heart.

Inhalation injury frequently occurs in burn patients and contributes to the morbidity and mortality of these injuries. Arterial carboxyhemoglobin has been proposed as an indicator of the severity of inhalation injury; however, the interrelation between arterial carboxyhemoglobin and histological alterations has not yet been investigated. Chronically instrumented sheep were subjected to a third degree burn of 40% of the total body surface area and inhalation of 48 breaths of cotton smoke. Carboxyhemoglobin was measured immediately after injury and correlated to clinical parameters of pulmonary function as well as histopathology scores from lung tissue harvested 24 hours after the injury. The injury was associated with a significant decline in pulmonary oxygenation and increases in pulmonary shunting, lung lymph flow, wet/dry weight ratio, congestion score, edema score, inflammation score, and airway obstruction scores. Carboxyhemoglobin was negatively correlated to pulmonary oxygenation and positively correlated to pulmonary shunting, lung lymph flow, and lung wet/dry weight ratio. No significant correlations could be detected between carboxyhemoglobin and histopathology scores and airway obstruction scores. Arterial carboxyhemoglobin in sheep with combined burn and inhalation injury are correlated with the degree of pulmonary failure and edema formation, but not with certain histological alterations including airway obstruction scores.

Background: Recent evidence suggests that nitric oxide produced via the neuronal nitric oxide synthase is involved mainly in the early response to sepsis, whereas nitric oxide derived from the inducible nitric oxide synthase is responsible during the later phase. We hypothesized that early neuronal and delayed inducible nitric oxide synthase blockade attenuates multiple organ dysfunctions during sepsis., Methods: Sheep were randomly allocated to sham-injured, nontreated animals (n = 6); injured (48 breaths of cotton smoke and instillation of Pseudomonas aeruginosa into the lungs), nontreated animals (n = 7); and injured animals treated with a neuronal nitric oxide synthase inhibitor from 1 to 12 h and an inducible nitric oxide synthase inhibitor from 12 to 24 h postinjury (n = 6)., Results: The injury induced arterial hypotension, vascular leakage, myocardial depression, and signs of renal and hepatic dysfunctions. The treatment significantly attenuated, but did not fully prevent, the decreases in mean arterial pressure and left ventricular stroke work index. Although the elevation of creatinine levels was partially prevented, the decreases in urine output and creatinine clearance were not affected. The injury-related increases in bilirubin levels, international normalized ratio, and lipid peroxidation in liver tissue were significantly attenuated. Although plasma nitrite/nitrate levels were significantly increased versus baseline from 12-24 h in controls, plasma nitrite/nitrate levels were not increased in treated animals., Conclusions: The combination treatment shows potential benefit on sepsis-related arterial hypotension and surrogate parameters of organ dysfunctions in sheep. It may be crucial to identify the time course of expression and activation of different nitric oxide synthase isoforms in future investigations.

Background and Purpose: The present study investigated whether the pathophysiological changes induced by burn and smoke inhalation are modulated by parenteral administration of Na(2)S, a H(2)S donor., Experimental Approach: The study used a total of 16 chronically instrumented, adult female sheep. Na(2)S was administered 1 h post injury, as a bolus injection at a dose of 0.5 mg.kg(-1) and subsequently, as a continuous infusion at a rate of 0.2 mg.kg(-1).h(-1) for 24 h. Cardiopulmonary variables (mean arterial and pulmonary arterial blood pressure, cardiac output, ventricular stroke work index, vascular resistance) and arterial and mixed venous blood gases were measured. Lung wet-to-dry ratio and myeloperoxidase content and protein oxidation and nitration were also measured. In addition, lung inducible nitric oxide synthase expression and cytochrome c were measured in lung homogenates via Western blotting and enzyme-linked immunosorbent assay (elisa) respectively., Key Results: The H(2)S donor decreased mortality during the 96 h experimental period, improved pulmonary gas exchange and lowered further increase in inspiratory pressure and fluid accumulation associated with burn- and smoke-induced acute lung injury. Further, the H(2)S donor treatment reduced the presence of protein oxidation and 3-nitrotyrosine formation following burn and smoke inhalation injury., Conclusions and Implications: Parenteral administration of the H(2)S donor ameliorated the pulmonary pathophysiological changes associated with burn- and smoke-induced acute lung injury. Based on the effect of H(2)S observed in this clinically relevant model of disease, we propose that treatment with H(2)S or its donors may represent a potential therapeutic strategy in managing patients with acute lung injury.

Bacillus anthracis, the causative agent of anthrax, is a category A priority pathogen that causes extensive damage in humans. For this reason, B. anthracis has been the focus of numerous studies using various animal models. In this study, we explored physiologic parameters in Dutch belted rabbits with inhalation anthrax to characterize the disease progression in this model. To this end, we infected Dutch belted rabbits with 100 LD(50) B. anthracis Ames spores by nasal instillation and continuously recorded various physiologic parameters by using telemetry. In addition, samples were collected at selected times for serum chemistry, hematology, and blood gas analysis. The animals exhibited hemodynamic and respiratory changes that coincided with those reported in human cases of inhalational anthrax infection, including hypotension, altered heart rate, and respiratory distress. Likewise, hematology, serum chemistry, and blood gas analysis revealed trends comparable to human anthrax-related pathophysiology. The Dutch belted rabbit model of inhalational anthrax exhibited most of the physiologic, hematologic, and biochemical sequelae noted in human cases. Therefore, this rabbit model fulfills several of the criteria of a useful animal model for studying disease pathogenesis and evaluating therapeutics during inhalational anthrax.

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.

In this study, we examined the tissue specificity of inflammatory and oxidative responses and mitochondrial dysfunction in mice infected by Trypanosoma cruzi. In acute mice, parasite burden and associated inflammatory infiltrate was detected in all tissues (skeletal muscle>heart>stomach>colon). The extent of oxidative damage and mitochondrial decay was in the order of heart>stomach>skeletal muscle>colon. In chronic mice, a low level of parasite burden and inflammation continued in all tissues; however, oxidant overload and mitochondrial inefficiency mainly persisted in the heart tissue (also detectable in stomach). Further, we noted an unvaryingly high degree of oxidative stress, compromised antioxidant status, and decreased mitochondrial respiratory complex activities in peripheral blood of infected mice. A pair-wise log analysis showed a strong positive correlation in the heart-versus-blood (but not other tissues) levels of oxidative stress markers (malonyldialdehyde, glutathione disulfide), antioxidants (superoxide dismutase, MnSOD, catalase), and mitochondrial inhibition of respiratory complexes (CI/CIII) in infected mice. T. cruzi-induced acute inflammatory and oxidative responses are widespread in different muscle tissues. Antioxidant/oxidant status and mitochondrial function are consistently attenuated in the heart, and reflected in the peripheral-blood of T. cruzi-infected mice. Our results provide an impetus to investigate the peripheral-blood oxidative responses in relation to clinical severity of heart disease in chagasic human patients.

The development of extra efferent vessels (EEV) is a little-known feature of diabetic glomerulopathy. The only previous large study [Min W, Yamanaka N. Three-dimensional analysis of increased vasculature around the glomerular vascular pole in diabetic nephropathy. Virchows Archiv A Pathol Anat 1993; 423:201-7] known to us found that up to 5 EEV per glomerulus (glom) each drained a separate lobule. Most EEV connected to the second- and third-order branches of the afferent arteriole (AA), and drained into peritubular capillaries. Although not so stated, the illustrations suggested that some EEV could be shunts, and thus detrimental to glom function, and possibly glom health. There was no correlation between the unquantitated presence of increased EEV at the vascular pole (VP) and the severity of the major diabetic glomerular (glom) lesions. The authors opined that efferent arteriole (EA) stenosis by insudative lesions (IL) stimulated the formation of EEV. To confirm and extend these findings, we have repeated the study in 18 diabetic cases with mild to severe, but not end-stage, diffuse and nodular lesions (DL and NL), 8 controls, and the 2 normal traumatic nephrectomy cases. Up to 18 EEV per glom were found in diabetic cases along with occasional EEV in controls. EEV contained muscle and were almost identical to the EA in structure. Nearly all EEV connected with efferent glom capillaries at the VP, where they exited the glom through apparently preexisting gaps in the Bowman's capsule and/or glomerular capillary basement membranes (BCBM/GCBM). The EA exited through a similar gap, so the exit of EEV was accomplished without altering the anatomical relationships between the exiting vessels and the components of the VP thought to be important in the control of glom outflow. The largest number of EEV occurred in long-standing T2DM cases with mild to moderate DL and NL. Complete photographic glom reconstructions revealed numerous anastomoses among efferent glom capillaries in normal and diabetic gloms with mild to moderate DL and NL. No disproportionately dilated EEV were seen. The findings just cited confirm that EEV are common and surprisingly numerous in diabetic gloms. They suggest that EEV formation served to preserve glom function, and that EEV could neither shunt nor restrict glom outflow locally. In our opinion, the formation of EEV represents a significant, possibly hemodynamically induced, remodeling of the glom that should be added to the list of changes that occur in diabetes. It is hypothesized that EEV develop because of increased glom inflow, and that the latter may be attributable to AA muscle damage that impairs its contractile ability.

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.

The potential use of flavonoids as a radioprotector is of increasing interest because of their high antioxidant activity and abundance in the diet. The aim of this study is to examine genotoxic and radioprotective effects of one of the most common flavonoids, apigenin, on radiation-induced chromosome aberrations in human lymphocytes. The cytokinesis-block micronucleus (CBMN) assay was used to evaluate such effects of apigenin. Blood samples were collected from two non-smoking healthy male volunteers who had no history of previous exposure to other clastogenic agents. Isolated lymphocytes were cultured. There were two tubes per concentration for all treatments. To evaluate the genotoxicity of apigenin, cells were first treated with different concentrations of apigenin (0, 2.5, 5, 10 and 25 microg/mL) at 24 h after culture initiation, followed by cytochalasin-B (Cyt-B) treatment (3 microg/mL) and cell harvest at 44 and 72 h, respectively. Secondly, to investigate the radioprotective effect, cell cultures were exposed to different concentrations of apigenin as described above for 30 min before being irradiated to 2 Gy of 137Cs gamma rays (at a dose rate of 0.75 Gy/min). In all instances, the frequency of MN was scored in binucleated (BN) cells. The nuclear proliferation index also was calculated. We did not detect an increase in the frequency of MN in non-irradiated human lymphocyte cultures treated with 2.5, 5.0 or 10 microg/mL apigenin; although, we did observe an increase in cultures treated with 25 microg/mL apigenin (the highest concentration of apigenin used in our study). We also observed a significant increase in the frequency of MN in irradiated cells overall; however, the frequency was decreased as the concentration of apigenin increased, suggesting a radioprotective effect. These findings provide a basis for additional studies to help clarify the potential use and benefit of apigenin as a radioprotector.

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.

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.

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.

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.

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.

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.

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.

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)

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)

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.

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.

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.

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.

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.

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.

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.

Down syndrome (DS) individuals are known to be predisposed to develop leukemia and their lymphocytes are highly sensitive to the induction of chromosome aberrations by X-rays. A study was conducted to identify the chromosome breakpoints and to evaluate whether site specificity for chromosome breakage and rearrangement may exist which may explain the predisposition phenomenon. DS lymphocytes at the G1 phase of the cell cycle were irradiated with 300, 450, and 600 rad of X-rays. Cells were harvested after 3 days in culture and 193 G-banded karyotypes were analyzed to identify the induced chromosome abnormalities. Out of 273 breakpoints identified, 122 were involved in the formation of stable chromosome rearrangements and 151 in the formation of unstable abnormalities. The Poisson analysis of these breakpoints demonstrated that 16 chromosome bands located in chromosomes 1, 3, 7, 12, 17, 19 and X were preferentially involved in breakage and rearrangement (P less than 0.05). These 16 bands are also found to be locations of "cancer breakpoints," oncogenes, or fragile sites. Many abnormal cells were observed to carry stable chromosome rearrangements only. Therefore, these cells are presumed to be compatible with survival and to be "initiated" in the transformation process. We propose that similar stable and site-specific chromosome rearrangements may exist in proliferating cells in DS individuals after exposure to clastogens and that this abnormality predisposes them to develop leukemia.

The present investigation was undertaken to standardize the early diagnosis of Gram-negative septicemia in burned children. Data were collected by means of a matrix which encompassed eight clinical variables routinely monitored by nursing personnel. These variables were evaluated according to their severity using a numerical scale of 0 to 3. A sepsis score was thus calculated for each of 243 burned patients, three times a day throughout their entire hospitalization. Eighty patients with suspiciously high scores (controls) were subjected to a battery of ten laboratory tests aimed at confirming the presence or absence of septicemia. During the 26 months of the study 16 patients (22 episodes) had clinical and laboratory evidence of Gram-negative septicemia. Multiple regression and discriminant analysis techniques were then used to develop statistical models for early diagnosis of septicemia. The two most practical and reliable of these are reported herein. Model I and II would have predicted the diagnosis of sepsis, 83% and 86% of the time, respectively, 1 day before the diagnosis was made using conventional methods. The false positive rates of Models I and II were 7% and 3%, respectively. On the basis of this information it seems possible and rewarding to utilize decision-making charts for monitoring and diagnosis of septicemia.

We conducted dominant lethal studies with chemical mutagens IMS and TEM to investigate the influence which different treated male stocks might have upon individual female response. In both studies, treated males from a CD-1 random bred stock and each of two F1 hybrid stocks (C57BL/6N X C3H; C57BL/6J X BALB/c) were mated to untreated females from the two F1 hybrid stocks. We found that variation in response due to the treatment effect was modulated by the specific male stock/female stock combination involved. The male-dependent variation observed prevented any meaningful evaluation of relative female stock sensitivity but it was obvious that factors other than differences in repair capacity of female germ cells contributed to the response differences observed.

Six cytogenetics laboratories joined in a collaborative study of rat chromosome aberrations to measure interlaboratory variation in results of standardized procedures and to devise methods to minimize interlaboratory differences. A preliminary workshop was held to resolve scoring differences, to develop a joint protocol and common glossary, and to reach agreement on uniform reporting methods. Osborne-Mendel rats from a common source were sent to each laboratory. Triethylenemelamine (TEM) was used at doses of 100, 200, 300 and 400 microgram/kg to induce clastogenic effects; results were compared to those of a control group of untreated animals. Femoral bone marrow cells were evaluated with the scorers unaware of the dosage. Final results showed highly significant dose effects with the test compound, and most laboratories showed a similar pattern of dose response. This study illustrates that rat cytogenetic analysis can be an effective test system for evaluation of a compound for mutagenic potential, particularly for the index reflecting the proportion of abnormal cells, but that results should be interpreted cautiously when arbitrary values are assigned for some of the categories being analyzed, as was done in this project for the category of severely damaged cells.

Cytogenetic analysis has been used for several years in attempts to detect the mutagenic effects of suspect agents. Yet there remain many questions related to statistical analysis. While several procedures have been and are being used, many unfortunately are not always appropriate for the types of data actually obtained. Two general analysis approaches are discussed together with ways to insure that the test assumptions are reasonably met. Because assay sensitivity is a major concern, the sample size issue is discussed with examples for specific situations.

Release of histamine from human basophils was induced by activation of complement using zymosan. The histamine-releasing factor resembled C5a on the basis of m.w. (15,000) as well as previous studies showing inactivation by anti-C5. Complement-induced release of histamine was compared with allergic release of histamine which is mediated through appropriate allergens and reaginic IgE. Previously we demonstrated that the former reaction occurred more quickly. Both reactions were inhibited by drugs which increase intracellular concentrations of cAMP3 (theophylline, prostaglandin E1, and histamine) or which mimic the action of cAMP (its dibutyrly derivative). Calcium was required for complement-mediated release of histamine and an increasing response was observed up to physiologic concentrations (2 mM). Magnesium (0 to 1 mM) did not affect the amount of histamine released. Also, glycolysis was probably required for optimal release by complement, since both 2-deoxyglucose and iodoacetamide were inhibitory. When basophils were partly enriched by depletion of neutrophils and eosinophils, the percentage of histamine released by complement was unchanged. Finally, it was shown that activated complement desensitized basophils from responding to a second challenge by the same stimulus. Cross-desensitization was not observed between complement and pollen allergens.

Carbon dioxide urethral pressure profiles were analyzed under the same conditions for 113 spina bifida and 87 non-spina bifida patients categorized by sex and age. Either duplicate or triplicate measurements were obtained from each patient for parameters of continence length, closure pressure and functional length. The degree of reproducibility of each parameter varied depending on age, sex and diagnosis. Except for 1 age-sex category closure pressure exhibited the smallest degree of relative variation for spina bifida patients. Results for non-spina bifida patients were mixed in that the measurement of no one parameter resulted in a uniformly smallest degree of relative variation. Reproducibility based on within patient variability generally was least for the continence length.

Cytogenetics data resulting from one laboratory in a multiple-laboratory study were analyzed to determine if 5 well-trained scorers produced significantly different results using metaphase scoring procedures. Although the scorers reached the same general conclusion, results show that scorer differences exists (p less than 0.01). Consequently, all participating scorers in a laboratory should be used equally in all treatment groups and the results should be analyzed accordingly to account for scorer variations. This is easily accomplished in controlled prospective experiments; however, it is often difficult in retrospective studies using data which exists. In such studies, every effort should be made to analyze and interpret the data so that scorer differences are taken into account. For severely damaged cells not only were there scorer differences but the difference were greater at higher doses. This phenomenon may be related to the operational definition of a severely damaged cell, since scorers who identify more damage than other scorers would logically tend to classify more cells as severely damaged both overall and at lower doses.

Parameters of the carbon dioxide urethral pressure profilometry were examined in detail previously for reproducibility within subjects. In the current study we have extended that analysis to determine the relationship of these parameters between patient subgroups. Mean values for non-spina bifida male subjects showed the same pattern of increase across age groups, regardless of the measured parameter. These increases were significant for continence and functional lengths but not for closure pressure. Male subjects with spina bifida showed mixed responses across age but age was not a statistically significant factor for any of the 3 parameters. Female subjects were quite uniform in the mean values across age for continence and functional lengths regardless of diagnosis. Mean values for female subjects were not uniform for closure pressure and tended to decrease with age in contrast to continence and functional lengths. The parameter continence length has a between subject standard deviation that is relatively stable across age, sex and pathology, and may be used for a basis of comparison. However, standard deviations for functional length is least variable in most subgroups of sex, age and diagnosis.