The DNA of annexin V-binding apoptotic cells is highly fragmented.

Jurkat leukemia cells induced to undergo apoptosis by treatment with an antibody against the Fas receptor have two annexin V (AV)-binding subpopulations: (a) single-positive cells that bind AV but not propidium iodide (PI); and (b) double-positive cells that bind AV and PI. The single-positive population is thought to represent an early stage of apoptosis. We have examined the relationship between AV binding and a classical characteristic of apoptosis, DNA fragmentation. Time course studies with Jurkat cells treated for 1, 2, or 4 h with anti-Fas indicated that the proportion of AV-binding cells was increased after 2 h. A significant increase in DNA fragmentation was observed only at 4 h as measured by the mean tail moment determined with the alkaline single cell gel electrophoresis (comet) assay. This correlation suggests a temporal relationship between the two parameters, but does not provide direct evidence of what happens in individual cells. We developed a method to measure fluorescent markers of cellular structure or function with a laser scanning cytometer and then perform the comet assay on the same cells. Cells in each AV-binding subpopulation were re-examined before and after electrophoresis. Most AV-/PI- cells had no DNA damage, although a few cells showed a pattern of damage characteristic for apoptosis. Double-positive cells all had damaged DNA; approximately half had the apoptotic pattern, and the rest had a pattern typical for necrosis. Nearly all of the single-positive cells had damaged DNA with the apoptotic pattern. Both AV-positive populations contained cells with little or no detectable DNA after electrophoresis, indicating that the DNA was highly fragmented. These results indicate that AV binding is an excellent marker for apoptotic cells, but that these cells already have fragmented DNA.

Chronic glandular bronchitis in young individuals residing in a metropolitan area.

A study of 161 Los Angeles County residents aged 12-28 years old who had died sudden violent deaths showed frequent and severe chronic glandular bronchitis (CGB), that is to say grade > or =5 (0-10) chronic inflammation involving at least one, half or more, and all submucosal glands in 53.4%, 21%, and 4.4% of the main stem bronchi, respectively. The mean plasma cell/gland/bronchus was high (> or =5) for 22 subjects (13.7%), while only 2 bronchi (1.2%) had a correspondingly high lymphocyte mean (P<0.001). Of the bronchi, 75.2% were affected by glandular atrophy (> or =5 in 8.1%), 10.6% had neutrophil infiltration of glands, and 3.1% had acute sialadenitis. Of the total of 1040 glands, CGB was found in 83.8% (> or =5 in 26.5%). Of 25 non-smokers identified, 14 (56%) had some degree of CGB in > or =50% of the glands, severe in 7 (26%). Severe CGB in many young individuals raises concern that a subpopulation of living cohorts may have an increased susceptibility to disease and a rising incidence of chronic lung disease. Demographic analysis is pending, but respiratory infection, smoking, adverse socioeconomic factors, and air pollution are all potential causative factors. Since pollution in Los Angeles frequently exceeds air quality standards, an ongoing multicity study is attempting to distinguish between the suspected effects of air pollution and confounding variables.

Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage.

Folate deficiency causes massive incorporation of uracil into human DNA (4 million per cell) and chromosome breaks. The likely mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into DNA by DNA polymerase. During repair of uracil in DNA, transient nicks are formed; two opposing nicks could lead to chromosome breaks. Both high DNA uracil levels and elevated micronucleus frequency (a measure of chromosome breaks) are reversed by folate administration. A significant proportion of the U.S. population has low folate levels, in the range associated with elevated uracil misincorporation and chromosome breaks. Such breaks could contribute to the increased risk of cancer and cognitive defects associated with folate deficiency in humans.

Human sperm morphometry and smoking, caffeine, and alcohol consumption.

The purpose of this study is to determine whether sperm nuclear size, shape, and chromatin texture parameters are associated with lifestyle exposures including smoking, caffeine, and alcohol consumption. Eighty-six healthy male volunteers (ages 18-35), recruited through newspaper advertisements, provided a semen, blood, and urine sample and completed a questionnaire concerning demographic and lifestyle exposures. Sperm nuclear size, shape, and chromatin texture parameters were measured using computerized image analysis. Results indicated no associations between the sperm nuclear morphometric parameters and age, smoking, or alcohol consumption. There was weak evidence for an association with caffeine intake. In conclusion, the lifestyle factors smoking, caffeine intake, and alcohol consumption do not appear to significantly affect sperm nuclear size, shape, or chromatin texture in this study population.

Use of fluorescence in situ hybridization (FISH) to assess effects of smoking, caffeine, and alcohol on aneuploidy load in sperm of healthy men.

Aneuploidy is a common cause of poor reproductive outcomes in humans and is associated with severe medical problems in liveborn offspring, yet little is known about its underlying cause. A substantial amount of aneuploidy is known to be contributed by the father through cytogenetically abnormal sperm. The purpose of this cross-sectional, observational study was to investigate the potential contribution of common lifestyle exposures (smoking, caffeine, and alcohol) to the aneuploidy load in sperm from 45 healthy male volunteers 19-35 years of age. Sperm FISH (fluorescence in situ hybridization) was used to determine aneuploidy and diploidy frequencies for chromosomes X, Y and 18 across varying exposure levels of smoking, caffeine, and alcohol. Caffeine was significantly associated with increased frequencies of sperm aneuploidy XX18 and XY18, diploidy XY18-18 and the duplication phenotype YY18-18 controlling for alcohol, smoking and donor age. Alcohol was significantly associated with increased frequencies of sperm aneuploidy XX18, diploidy XY18-18 and the duplication phenotype XX18-18 controlling for caffeine, smoking and donor age. There was a suggestive, but unstable, association between smoking and XX18. Even within our truncated age range, we were able to confirm an increased risk for XX18 aneuploidy with increasing donor age. Sperm FISH proved to be a useful biomarker to detect and compare numerical cytogenetic abnormalities in human sperm cells across differing levels of exposure to smoking, caffeine, and alcohol.

Iron intake and the risk of colorectal cancer.

Prospectively gathered data from the National Health and Nutrition Examination Survey I and the National Health Evaluation Follow-Up Study were analyzed to evaluate the risk of colorectal cancer due to consumption of iron. Morbidity and mortality data due to colorectal cancer were available on 14,407 persons first interviewed in 1971 and followed through 1986. A total of 194 possible colorectal cancers occurred in this group over the 15-year period. Subsite analysis showed that the risk of colon cancer due to iron intake was elevated throughout the colon for both men and women, with the highest adjusted risks for the interquartile range seen in the proximal colon for females (relative risk, 1.51; 95% confidence interval, 1.41-1.60). The risk of rectal cancer was not significantly elevated for men or women. Elevated serum iron was also associated with increased risk; however, this effect was strongest in the distal (rather than proximal) colon and was significant only among females (adjusted relative risk, 1.73; 95% confidence interval, 1.03-2.92). The mean transferrin saturation was higher among cases than controls (30.7 versus 28.7%), but total iron-binding capacity did not seem to predict the occurrence of colorectal cancer. Proportional hazards models confirmed that the effects of iron and serum iron were not confounded by age, gender, energy consumption, fat intake, or other known risk factors for colorectal cancer. These data suggest that iron may confer an increased risk for colorectal cancer, and that the localization of risk may be attributable to the mode of epithelial exposure. It seems that luminal exposure to iron increases risk proximally, whereas humoral exposure increases risk distally. These differences may be due to such factors as oxidation state, binding proteins and the presence of other cofactors such as bile acids, products of bacterial metabolism.

A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been determined by measurement of DNA adducts in human tissues. Competitive enzyme-linked immunosorbent assays (ELISAs) using antisera recognizing benzo[a]pyrenediol-epoxide-modified DNA (BPDE-I-DNA) and color of fluorescence endpoint detection have been used extensively for quantifying PAH-DNA adducts. The fluorescence ELISA (limit of detection 1 adduct/10(8) nucleotides) was previously reported to be more sensitive than the color ELISA (1/10(7)) for measuring PAH adducts (Santella et al. (1988) Carcinogenesis, 9, 1265-1269). However, the fluorescence assay has the disadvantages of greater variation among the replicates and higher background levels than the color assay. Using a newly developed antiserum against BPDE-I-DNA, we have modified the color of ELISA so that it has the same sensitivity as the fluorescence ELISA and requires only 33% of the sample quantity needed for the fluorescence ELISA. The modifications included preincubation of the antiserum with the samples, using microtiter plates with half-size, flat bottom wells, and optimizing the assay conditions. The improved color ELISA was used to analyze DNA samples from human autopsy tissues, including heart, lung, liver, kidney, spleen, pancreas and stomach from smokers and nonsmokers. With the exception of spleen and stomach, all tissues from smokers showed higher PAH-DNA adducts (ranging from 0.3 to 19.0 adducts/10(7) nucleotides) than the tissues from the nonsmokers (0.3 to 3.7 adducts/10(7) nucleotides) in two separate experiments. Among the tissues from smokers, heart showed the highest level of DNA adducts. This study demonstrates that a stable color ELISA with high sensitivity can be useful in assessing human exposure to PAH.

Host determinants of cellular and biochemical constituents of bronchoalveolar lavage fluids. Implications for design of epidemiologic studies.

The effects of demographic characteristics, exercise, environmental exposures, and other host factors on cellular and biochemical constituents of human bronchoalveolar lavage (BAL) fluids were investigated by studying more than 300 specimens obtained from normal volunteers and assayed in a single center. The BAL data demonstrated associations with race, smoking, exercise, skin-test reactions, and blood constituents, and weak or no associations with age, sex, pulmonary function tests (PFT), or ambient ozone exposure. The effect of exercise was relatively strong and more clearly characterized than in previous studies. Smoking effects were similar to those observed in other studies; our ability to study age and ambient ozone effects was greatly limited because of the homogeneity of the population under study. Blood constituents of the subjects also showed an association with level of exercise. Analysis of intraindividual and interindividual variability in BAL constituents results suggested that matching, although desirable, is not essential for the maintenance of adequate statistical power in BAL studies, so observational studies of the effects from air pollution on BAL fluids in humans could be effectively conducted using cross-sectional designs.

Aspirin use and lung, colon, and breast cancer incidence in a prospective study.

A large body of experimental data and several recent epidemiologic studies indicate that aspirin use may decrease cancer risk. The experimental studies found effects at many anatomic sites, whereas the epidemiologic studies saw the greatest effect on mortality from digestive cancers. To provide further human data, we examined the association between aspirin use and cancer risk using data from the National Health and Nutrition Examination Survey I (NHANES I) and the NHANES I Epidemiologic Follow-up Studies (NHEFS). Characterization of aspirin use was based on questions in the baseline interview asking whether subjects used aspirin during the previous 30 days. Data were available from 12,668 subjects age 25-74, at time of initial examination for NHANES I, who were followed for an average of 12.4 years. Among these subjects, 1,257 were diagnosed with cancer more than 2 years after their NHANES I examination. Incidence of several cancers was lower among persons who reported aspirin use: the incidence rate ratios (and 95% confidence intervals) for all sites combined were 0.83 (0.74-0.93), lung cancer 0.68 (0.49-0.94), breast cancer in women 0.70 (0.50-0.96), and colorectal cancer in younger men 0.35 (0.17-0.73). These findings were not readily explained by potentially confounding factors. The data suggest an association between aspirin consumption and decreased cancer incidence at several cancer sites.

Comparison of DNA adduct levels in human placenta from polychlorinated biphenyl exposed women and smokers in which CYP 1A1 levels are similarly elevated.

Previous studies demonstrated that cigarette smoking is associated with high elevations in levels of both cytochrome P450 1A1 (CYP1A1) and DNA adducts in human placenta. To date, the identity of the smoking related DNA adducts is not known. The DNA adducts identified in placenta of smokers could result from chemicals present in cigarette smoke, substances formed by CYP 1A1 metabolic activation of endogenous compounds, noncigarette related exposures or a combination of these processes. Exposure to contaminated rice oil containing large doses of polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) also resulted in massive elevation of CYP 1A1 in human placenta but formation of DNA adducts directly from this exposure has not previously been reported. The purpose for comparing the two populations was to test the hypothesis that if CYP 1A1 induction results in the metabolic activation of endogenous compounds, then DNA adducts should also be present in PCB/PCDF exposed tissues exhibiting high CYP 1A1 activity and some of the adducts detected in the placental DNA from smokers may be identified as those derived from the metabolic activation of endogenous compounds. To test this hypothesis, we measured DNA adducts using 32P-postlabeling to analyze placental DNA from women exposed to PCB/PCDF and from cigarette smokers where levels of CYP 1A1 were similarly elevated. There was no evidence of DNA adducts among specimens obtained from PCB/PCDF exposed individuals. These data suggest that CYP 1A1 induction alone (in the absence of cigarette smoking) does not induce the formation of DNA adducts detectable by this approach, and that smoking related adducts are not a consequence of CYP 1A1 induction mediated activation of endogenous compounds or xenobiotics other than cigarette smoke.

32P-postlabeling analysis of DNA adducts in human sperm cells from smokers and nonsmokers.

To determine the feasibility of using human sperm cells for DNA 32P-postlabeling analyses, and to evaluate the baseline level and the possible presence of smoking-related DNA adducts in these cells, sperm DNA was isolated from specimens obtained from 12 heavy smokers, 12 light smokers, and 12 nonsmokers. Background levels of radioactivity were minimized by using magnet transfer of 32P-labeled mononucleotides to new polyethyleneimine cellulose plates. Compared with placental tissues, few adducts were observed. Diffuse radioactivity observed in some of the autoradiograms was minimally above background but the level of radioactivity expressed as putative adducts/nucleotide was not related to smoking status. It was not clear, in some cases, whether this radioactivity was associated with chemically bound adducts or was from nonspecifically bound chemicals, radiolabeled enzymes, or other proteins. One major discrete DNA adduct of unknown chemical structure was detected in three of the 36 samples analyzed (one nonsmoker and two smokers). Based on the level of radioactivity associated with various dilutions of a benzo(a)pyrene-derived adduct, our limit of sensitivity was at least 1.2 adducts/10(9) nucleotides. Our study emphasizes the need to more clearly define the significance of background radioactivity associated with DNA adduct maps where the measured adduct levels approximate detection limits defined by visual observance of adduct spots. This point is particularly relevant given that the 32P-postlabeling procedures rely, in part, on visual verification of the presence of DNA adducts.

Quantification and molecular characterization of hprt mutants of human T-lymphocytes.

Somatic mutations have been implicated as critical early events in carcinogenesis. Point mutations, deletions, and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. In human population monitoring, quantitative analysis of mutation events that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures and to those tissues (like blood) that are accessible for analysis. In an effort to determine the frequency and types of mutations that can be detected at the hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have used the T-cell cloning assay and have developed a strategy to propagate mutants and screen for point mutations and breakage events. Early in the clonal expansion of mutants, 1-2 x 10(4) cells are prepared as a crude cell lysate, and a sample is analyzed using the multiplex polymerase chain reaction (PCR). Those mutants that yield altered DNA fragments are then expanded for Southern blot hybridization, PCR, flanking probe isolation, and DNA sequencing. To date we have found presumed point mutations, intragenic deletions, and deletions that extend outside of the hprt gene. By analyzing mutations in selectable, nonessential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. An association of these specific genetic events with human diseases and the evaluation of the ability of environmental chemicals to induce these specific types of mutations will lead to a rational basis for evaluating risks from various chemical exposures.

Cotinine concentrations in semen, urine, and blood of smokers and nonsmokers.

Cotinine levels in the semen, urine, and blood of 88 male smokers and nonsmokers, aged 18 to 35, were analyzed via radioimmunoassay. Detectable cotinine levels were found in all three body fluids, and cotinine levels in all three fluids were highly correlated. Cotinine levels in semen and blood were of similar magnitude; cotinine levels in urine were an order of magnitude or more higher. In all three fluids, cotinine levels increased with an increase in cigarette smoke exposure.

Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems.

DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture.

Effect of residual splenic function and folate levels on the frequency of micronucleated red blood cells in splenectomized humans.

Frequencies of micronucleated erythrocytes in the peripheral blood of splenectomized individuals can be used as an index of genetic damage to erythrocyte precursor cells in the bone marrow. This is in contrast to non-splenectomized humans, whose micronucleated erythrocytes are removed by the spleen. Many subjects whose spleen has been removed surgically have residual spleen tissue and consequent residual spleen function (RSF), which can be measured by the percentage of 'pitted' peripheral red blood cells. In this study evidence of RSF was associated with decreased frequencies of micronucleated erythrocytes. Analysis of data limited to subjects with minimal spleen function suggested an inverse association between the incidence of micronucleated erythrocytes and serum folate levels that was not apparent in the absence of stringent control for RSF.

Induction, accumulation, and persistence of sister chromatid exchanges in women with breast cancer receiving cyclophosphamide, adriamycin, and 5-fluorouracil chemotherapy.

The induction, accumulation, and persistence of sister chromatid exchanges (SCEs) and high SCE frequency cells (HFCs) was measured in peripheral blood lymphocytes of women with breast cancer before chemotherapy and on multiple occasions during and after therapy. Chemotherapy consisted of i.v. infusion of cyclophosphamide, Adriamycin, and 5-fluorouracil, administered on day 1 of each of approximately six 21-day cycles. This treatment resulted in a highly significant induction of SCEs (1.8-fold, P less than 0.0001) and HFCs (5-fold, P less than 0.0001) measured in samples obtained 1 week after the first therapy. Accumulation of lesions leading to SCEs was measured by comparing samples surrounding the first and last rounds of therapy and was significant for both SCEs and HFCs in most comparisons. Persistence of lesions leading to SCEs was evaluated at multiple times until 9 months after completion of therapy, and both SCEs and HFCs remained significantly elevated throughout this time. Differences between donors were observed throughout the study, although they were not always consistent with time. Our results also indicate that the SCE frequency declines rapidly within a few weeks after treatment but that residual damage remains up to 9 months after the end of chemotherapy.

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes.

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.