A focus group study of DES daughters: implications for health care providers.

A focus group study of women exposed to diethylstilbestrol (DES) in utero (DES daughters) was conducted to gain understanding about exposure to this drug from a patient perspective. Focus group participants reported that learning about their DES exposure was devastating; they experienced strains in their family relationships, emotional shock, a feeling that their health concerns were not appreciated by others and, to some degree, a sense of social isolation. Although many were aware of the need for special gynecological exams and high-risk prenatal care, they were frustrated by what they felt was a lack of reliable and clear information about the effects of DES exposure. Most expressed questions and anxiety about their health. Many found their communication with physicians about their DES exposure unsatisfying. They felt that physicians lacked information about the long-term health effects of DES exposure and as a result did not give them accurate information. Furthermore, they felt that physicians were dismissive of their concerns and often gave what they felt to be false reassurances. Consequently, the women developed an enduring distrust of the medical profession. The results of the study suggest implications for the delivery of health care to DES daughters.

A study of breast cancer detection practices and beliefs in black women attending public health clinics.

In this study, breast cancer knowledge, beliefs and practices in low income black women were examined. First, focus groups were held with a total of 33 participants. Information gathered from the focus groups was used to develop a telephone survey which was partially based on the Health Belief Model (HBM) and administered to 92 subjects. Utilization rates of mammography and breast self-examination (BSE) were quite high; 66.3% of survey participants reported having at least one mammogram and 72.5% performed BSE. Because low-cost mammograms were available to the survey participants, these results suggest that women in this target population will utilize accessible and affordable mammograms. Several knowledge deficiencies that need to be addressed were also identified. Most of the health beliefs were not significantly associated with mammography or BSE utilization. Because the HBM has never been extensively tested on this population, its appropriateness as a behavior model for low-income women is examined. Implications for future research and interventions are discussed.

Acute endotoxin-induced lymphocyte subset sequestration in sheep lungs.

Endotoxemia is associated with an early phase of pulmonary hypertension and a later increase in microvascular permeability. These physiologic changes are attended by peripheral blood and lung lymph leukopenia and a rapid accumulation of both granulocytes and lymphocytes in the peripheral lung. In the present study, the numbers of lymphocytes in blood, lung lymph, and lung tissue after infusion of endotoxin were determined by fluorescent labeling of lymphocyte populations with monoclonal antibodies to sheep T1, T4, T8, or leukocyte common antigen and with rabbit anti-sheep immunoglobulins (B cells). Peripheral blood, lung lymph, and lung tissue samples were collected at baseline and 15, 30, 60, 120, 180, and 240 minutes after the start of intravenous infusion of E. coli endotoxin (1.25 micrograms/kg, N = 6) or saline (N = 4) from open-chest anesthetized sheep. Pulmonary artery pressure and lung lymph flow were also monitored at these times. Endotoxin caused marked reductions in the number of T and B lymphocytes in blood and lung lymph. As compared with baseline, total blood leukocytes and granulocytes were significantly reduced below control levels from 30 minutes of endotoxin, and lymphocyte numbers were reduced from 60 minutes. T1, T4, T8 and B lymphocyte subsets contributed to the fall in blood lymphocytes. Endotoxin caused a significant fall in number of lung lymph leukocytes (T1, T4 and T8 cells) from 120 minutes; numbers of B lymphocytes were also reduced. Counts of the number of lymphocytes in the biopsy tissue revealed a significant rise in T lymphocytes sequestered in the lung. We conclude endotoxemia in sheep causes a reduction in lymphocytes in blood and lung lymph and an increase in these cell types in peripheral lung tissue. These findings suggest that lymphocyte subpopulations accumulate in the lungs during periods of pulmonary hypertension and increased permeability and may participate in endotoxin-induced lung injury.

Airway responsiveness in isolated perfused rat lungs: effect of thoracic irradiation.

We developed techniques for assessing airway reactivity in isolated perfused rat lungs by measuring the lung mechanics changes produced by injection of ACh into the pulmonary circulation. Lung resistance (RL) and dynamic compliance (Cdyn) changed in a dose-response fashion after ACh. We used the preparation to examine the effect of thoracic irradiation on airway responsiveness and pulmonary inflammation. Groups of rats were studied after sham irradiation or 24 h or 72 h after a single dose of 1500 rads. Thoracic irradiation did not alter baseline lung mechanics, but did increase the responsiveness of rat lungs to ACh 72 h after radiation. Radiation was not associated with an increase in neutrophils in lung lavage, airways or peripheral lung tissue. We conclude that thoracic irradiation alters airways reactivity without causing overt pulmonary inflammation, and that isolated perfused lungs can be useful for measurement of airway reactivity.

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells.

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals.

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.

Endotoxin-induced neutrophilic alveolitis and macrophage chemotaxin production in sheep.

The mechanism(s) responsible for endotoxin-induced pulmonary leukostasis has not yet been elucidated. We studied ten unanesthetized sheep by performing serial bronchoalveolar lavages (BAL) before and after infusing Escherichia coli endotoxin to determine whether or not neutrophils appeared in the airways and whether or not endotoxemia stimulated alveolar macrophages to produce neutrophil chemotactic activity. The absolute number and percentage of neutrophils recoverable by BAL increased significantly at 24 hours after infusion of endotoxin. Alveolar macrophages isolated from the BAL of sheep after endotoxemia produced a substance that is chemotactic for neutrophils, a response that is also maximal 24 hours after endotoxin infusion. We conclude that endotoxemia causes migration of neutrophils into lung air spaces and that this response may result from in vivo production of a chemotactic factor(s) by activated alveolar macrophages.

Acute effects of thoracic irradiation on lung function and structure in awake sheep.

To investigate the acute physiological and structural changes after lung irradiation, the effects of whole-lung irradiation were investigated in fourteen sheep. Ten sheep were prepared with vascular and chronic lung lymph catheters, then a week later were given 1,500 rad whole-lung radiation and monitored for 2 days. Four sheep were given the same dose of radiation and were killed 4 h later for structural studies. Lung lymph flow increased at 3 h after radiation (14.6 +/- 2.1 ml/h) to twice the base-line flow rate (7.5 +/- 1.3), with a high lymph-to-plasma protein concentration. Pulmonary arterial pressure increased twofold from base line (18 +/- 1.6 cmH2O) at 2 h after radiation (33 +/- 3.8). Cardiac output and systemic pressure in the aorta did not change after lung radiation. Arterial O2 tension decreased from 85 +/- 3 to 59 +/- 4 Torr at 1 day after radiation. Lymphocyte counts in both blood and lung lymph decreased to a nadir by 4 h and remained low. Thromboxane B2 concentration in lung lymph increased from base line (0.07 +/- 0.03 ng/ml) to peak at 3 h after radiation (8.2 +/- 3.7 ng/ml). The structural studies showed numerous damaged lymphocytes in the peripheral lung and bronchial associated lymphoid tissue. Quantitative analysis of the number of granulocytes in peripheral lung showed no significant change (base line 6.2 +/- 0.8 granulocytes/100 alveoli, 4 h = 10.3 +/- 2.3). The most striking change involved lung airways. The epithelial lining of the majority of airways from intrapulmonary bronchus to respiratory bronchiolus revealed damage with the appearance of intracellular and intercellular cell fragments and granules. This new large animal model of acute radiation lung injury can be used to monitor physiological, biochemical, and morphological changes after lung radiation. It is relevant to the investigation of diffuse oxidant lung injury as well as to radiobiology per se.

Effects of N-nitrosodimethylamine on cell-mediated immunity.

Dimethylnitrosamine (DMN) exposure altered the cell-mediated immune response of B6C3F1 adult female mice as assessed by several immunological assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice exhibited a depression in their lymphoproliferative response to the T-cell mitogens concanavalin A and phytohemagglutinin, and in their mixed lymphocyte response to mitomycin-treated DBA-2 spleen cells. The delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH), as measured by vascular permeability changes, was decreased by over 50% at the 5.0-mg/kg dose. When the DHR to KLH was measured by an influx of endogenously 125I-iododeoxyuridine (IUdR)-labelled monocytes, there was a 300% increase in the response of the 5.0-mg-DMN/kg group. Adoptive transfer studies using exogenously radiolabelled (51Cr) bone marrow cells from either vehicle- or DMN-treated (5 mg/kg) donors indicated a greater than 60% reduction in the DHR to KLH in DMN-treated mice (5.0 mg/kg level) regardless of the donor treatment. Animals exposed to DMN exhibited a decreased susceptibility to Listeria monocytogenes. The dichotomy in the results of the KLH DHR measured by monocyte influx and the increased resistance to the bacterial challenge were interpreted to reflect an effect on bone marrow. The numbers of granulocyte/monocyte stem cells were increased in a dose-related fashion in bone marrow from DMN-treated mice. The results indicate that DMN-treatment impairs cell-mediated immunity while increasing the number of bone marrow cells differentiating to form granulocytes or monocytes with an apparent enhancement in functional activity.

Effects of N-nitrosodimethylamine on tumor susceptibility.

Dimethylnitrosamine (DMN) exposure altered the activity of the macrophage and natural killer (NK) cell defense mechanisms against the B16F10 melanoma in B6C3F1 adult female mice as assessed by several immunologic assays. Following 14 daily exposures (i.p.) to 1.5, 3.0, or 5.0 mg DMN/kg, mice were injected (i.v.) with B16F10 melanoma. The incidence and number of lung nodules, determined 18 days after challenge, were decreased in the DMN-exposed animals. The initial observation indicated the mice exposed to 3 mg/kg DMN were afforded the greatest protection. However, when mice exposed to the highest dose of DMN were divided into subgroups of mice with or without ascites, there was a degree protection seen in the 5-mg/kg-treated mice without ascites that was comparable to that of the 3-mg/kg group. The development of ascites is an overt toxic effect reflecting damage to the liver and was frequently associated with exposure to 5 mg/kg DMN. Exposure to DMN produced only slight changes in the activity of splenic NK cells as determined by the cytotoxicity of 51Cr-labelled YAC-1 cells. The activity was significantly increased only in mice exposed to 3 mg/kg DMN and only at effector:target (E:T) ratios of 30:1 and 10:1. Interestingly, the activity of the NK cells was significantly decreased at all E:T ratios in mice exposed to 5 mg/kg that developed ascites. The number of peritoneal exudate cells was decreased, albeit nonsignificantly, in a dose-related fashion. The phagocytic activity, as measured by the uptake of fluorescent latex beads, was increased in a dose-related fashion with significance noted at the highest dose of DMN. The role of the macrophage in the increased resistance to the tumor challenge was assessed with bone marrow derived macrophages. The cytostatic activity versus B16F10 tumor cells, as measured by the uptake of 3H-thymidine, was markedly increased in the bone marrow derived macrophages from DMN (5mg/kg) mice when compared to vehicle controls. These results suggest that exposure to DMN alters bone marrow, particularly the differentiation of effector tumoricidal cells, which renders the host more resistant to metastatic tumor formation.

Naphthalene toxicity in CD-1 mice: general toxicology and immunotoxicology.

Random bred CD-1 mice were used to evaluate the acute oral toxicity and subchronic toxicity of naphthalene administered in corn oil. The acute oral LD50 of naphthalene was 533 and 710 mg/kg in male and female mice, respectively. Subchronic toxicity was evaluated with 14- and 90-day daily oral gavage studies. Doses utilized in the 14-day study were 27, 53, and 267 mg/kg, with the latter representing one-half of the male LD50. Both males and females demonstrated a 5-10% mortality and depressed body weight at the high dose only. Males had decreased thymus weights, and females had decreased spleen and increased lung weights at the high dose only. Other organ weights were unaffected at any dosage level. Serum enzyme and electrolyte levels were not altered in a dose-related manner. To assess the potential immunotoxicity of naphthalene the following screen was utilized: humoral immune response, response to mitogens, delayed hypersensitivity response, popliteal lymph node response, bone marrow stem cell number, and DNA synthesis. No evidence of immunotoxicity was demonstrated. The 90-day study employed daily oral doses of 5.3, 53, and 133 mg/kg. There was no treatment-related mortality in either sex, nor was body weight affected. Organ weights were not affected in males, and females showed reduced spleen weights only at the high dose. Serum enzyme and electrolyte levels, as well as the immunotoxicity screen, indicated that naphthalene doses up to one-fourth the LD50 for 90 days failed to elicit consistent statistically significant and biologically relevant compound-related effects. A screen of the effects of the 90-day naphthalene treatment on various aspects of the hepatic drug metabolizing system indicated no alterations, with the exception of a specific dose-related inhibition of aryl hydrocarbon hydroxylase activity in both male and female mice.

Effects of diethylstilbestrol and cyclophosphamide on the pathogenesis of experimental Cryptococcus neoformans infections.

Treatment of mice with a single dose of cyclophosphamide 24 h before challenge with Cryptococcus neoformans increased host survival, whereas treatment with 10 daily exposures of cyclophosphamide, starting 2 days before challenge, markedly reduced survival in mice challenged on the second day of drug treatment. Treatment with 14 daily exposures of diethylstilbestrol before challenge with C. neoformans did not markedly affect host survival. A correlation was sought between the distribution of radiolabeled C. neoformans and host survival. Radiolabeled C. neoformans administered intravenously was cleared rapidly from the blood of naive mice and accumulated in the lungs, liver and kidney within 1 h. The radiolabeled yeasts were subsequently cleared from the lungs. The distribution of radiolabeled C. neoformans among organs was generally the same in control mice and mice treated with diethylstilbestrol of various cyclophosphamide regimens after 3 or 24 h. The distribution of C. neoformans measured as colony forming units was generally in agreement with results from radioactivity measurements for animals sacrificed 3 or 24 h after challenge. One week after challenge, C. neoformans colonies were grown from the brain, liver and kidneys. C. neoformans was found in the brain within 1 h after i.v. challenge, suggesting that the central nervous system disease in mice challenged i.v. resulted from a primary infection of the brain.

Synthesis and biological evaluation of sparsomycin analogues.

Three series of sparsomycin analogues were prepared and examined for their ability to inhibit DNA or protein synthesis in bone marrow, P388 lymphocytic leukemia, and P815 mastocytoma cells. The compounds of series I and II, distinguished by the inclusion or exclusion of a hydroxymethyl functional group, were designed to elucidate the effect on activity of replacing the oxodithioacetal side chain of sparsomycin with 4-substituted benzyl groups. The series III analogues, which excluded the hydroxymethyl group and replaced the oxodithioacetal moiety of sparsomycin with a benzyl amide group, were designed to investigate the potential interaction of an amide oxygen in contrast to the sulfoxide oxygen of sparsomycin. Overall, the bromobenzyl-substituted analogues imparted the greatest inhibitory activity in the protein synthesis assay, while the methoxybenzyl-substituted analogues displayed the least. The methylbenzyl and the unsubstituted benzyl compounds were intermediate in inhibitory potential. The activity in the protein synthesis assay may correspond to the lipophilic and electronic characteristics of the substituents on the benzyl moiety of the analogues. All of the compounds were inactive in the DNA synthesis assay.

Subchronic toxicology of diethystilbestrol in the mouse.

This study evaluated the subchronic (14-day) toxicity of selected (0.2, 1.0, and 4.0 mg/kg) daily subcutaneous injections of diethylstilbestrol (DES) in female (C57B1/6 X C3H)F1 mice. Parameters observed included body and organ weights, gross organ morphology, histopathology, clinical chemistry, and hepatic microsomal enzyme activities. The liver, bone marrow, and thymus are major target organs for DES. Liver enlargement, with associated histopathological changes consistent with mild hepatitis, centrolobular necrosis, and sinusoidal changes were observed. Supporting the histological changes were alterations in serum enzyme levels and microsomal enzyme activity. Bone marrow changes included decreases in the number of cells as well as the number of colony forming units per gram stem cells. Toxicity to the thymus was evidenced by decreased thymic weights and lymphocyte depletion. The hepatic and thymic effects were observed at the lowest (0.2 mg/kg) dose. Although all parameters were not assessed for recovery, those that were evaluated returned to control levels by thirty days after treatment.