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.

Geographic information systems: their use in environmental epidemiologic research.

Advances in geographic information system (GIS) technology, developed by geographers, provide new opportunities for environmental epidemiologists to study associations between environmental exposures and the spatial distribution of disease. A GIS is a powerful computer mapping and analysis technology capable of integrating large quantities of geographic (spatial) data as well as linking geographic with nongeographic data (e.g., demographic information, environmental exposure levels). In this paper we provide an overview of some of the capabilities and limitations of GIS technology; we illustrate, through practical examples, the use of several functions of a GIS including automated address matching, distance functions, buffer analysis, spatial query, and polygon overlay; we discuss methods and limitations of address geocoding, often central to the use of a GIS in environmental epidemiologic research; and we suggest ways to facilitate its use in future studies. Collaborative efforts between epidemiologists, biostatisticians, environmental scientists, GIS specialists, and medical geographers are needed to realize the full potential of GIS technology in environmental health research and may lead to innovative solutions to complex questions.

Environmental chemical exposures and risk of herpes zoster.

This study investigated whether residence in Aberdeen, North Carolina, the location of the Aberdeen pesticides dumps site (a national priority list Superfund site containing organochlorine pesticides, volatile organic compounds, and metals), is associated with immune suppression as indicated by a higher incidence of herpes zoster and recent occurrences of other common infectious diseases. Study participants included 1,642 residents, 18-64 years of age, who responded to a telephone survey concerning potential occupational and recreational exposures to pesticides and other chemicals, lifetime history of herpes zoster (shingles), and the recent occurrence of other common infectious diseases. Stratified and logistic regression analyses were used to compare the cumulative incidence of herpes zoster among Aberdeen residents and residents of nearby communities. There was little evidence of an overall increased risk of herpes zoster among Aberdeen residents during the period 1951-1994 [relative risk (RR), 1.3; 95% confidence interval (CI), 0.8-2.1]. However, an elevated risk of herpes zoster was noted consistently among Aberdeen residents of younger ages as compared to residents of the nearby communities. The RR was 2.0 (CI, 1.0-4.0) among those 18-40 years of age and was not affected by controlling for potential confounders. The RR of herpes zoster was also consistently elevated in all age groups for the period before 1985. No differences were noted between residents of Aberdeen and those of the nearby communities with respect to the recent occurrence of other common infectious diseases. These results support the plausibility of an association between exposure to the Aberdeen pesticides dumps site and immune suppression and the potential use of herpes zoster as a marker of immune suppression in studies of environmental chemical exposures.

Effects on the immune system associated with living near a pesticide dump site.

In this paper, we report results of the second phase of a larger study designed to evaluate the effects on the immune system of living near a Superfund site containing organochlorine pesticides, volatile organic compounds, and metals. Phase II was conducted to determine whether living near the site, consisting of six locations in Aberdeen, North Carolina, is associated with higher plasma organochlorine levels, immune suppression, or DNA damage. Each of 302 residents of Aberdeen and neighboring communities provided a blood specimen, underwent a skin test, and answered a questionnaire. Blood specimens were analyzed for organochlorine pesticides, immune markers, and micronuclei. Of 20 organochlorines tested, only DDE was detected in the blood of participants (except for one individual). Age-adjusted mean plasma DDE levels were 4.05 ppb for Aberdeen residents and 2.95 ppb (p = 0.01) for residents of neighboring communities. Residents of 40-59 years of age who lived within a mile of any site, but particularly the Farm Chemicals site, had higher plasma DDE levels than residents who lived farther away. Residents who lived near the Farm Chemicals site before versus after 1985 also had higher plasma DDE levels. Overall, there were few differences in immune markers between residents of Aberdeen and the neighboring communities. However, residents who lived closer to the dump sites had statistically significantly lower mitogen-induced lymphoproliferative activity than residents who lived farther away (p < 0.05). Residential location was not consistently associated with frequency of micronuclei or skin test responses. Although some statistically significant differences in immune markers were noted in association with residential location, the magnitude of effects are of uncertain clinical importance.

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.

Cigarette smoking and semen quality.

OBJECTIVE: To determine whether cotinine levels provide stronger evidence for an association between smoking and semen quality than the number of cigarettes smoked per day or years smoked controlling for potential confounders and effect modifiers. DESIGN: Cross-sectional study. SETTING: Male volunteers at the Reproductive Endocrinology-Fertility Laboratory. PARTICIPANTS: Eighty-eight men (ages 18 to 35 years) provided a semen, urine, and blood specimen and completed a self-administered questionnaire concerning smoking and demographic information as well as caffeine and alcohol consumption. Urine, blood, and semen cotinine levels were analyzed via RIA. MAIN OUTCOME MEASURE: Standard clinical semen analysis. RESULTS: Number of cigarettes smoked per day, years smoked, and log-transformed cotinine levels were associated negatively with semen quality (density, total count, and motility). The association was evident among men age > or = 22 years. For example, the correlation coefficient for the overall association between logged urine cotinine and logged sperm density was -0.23; those stratified by age were 0. 13 (age < 22 years) and - 0. 39 (age > or = 22 years). Potential confounders included in regression models did not diminish the associations. CONCLUSIONS: Smoking is associated with lowered semen quality.

Cigarette smoking and sperm density: a meta-analysis.

OBJECTIVE: To quantify, through meta-analysis techniques, the association between cigarette smoking and sperm density. METHODS: The logarithm of the ratio of mean sperm density for smokers to that for nonsmokers for the studies included in this meta-analysis was regressed against a constant, an indicator of study population source (infertility clinic patients or normal men), minimum number of cigarettes smoked per day among smokers (< 10, > or = 10), exclusion of azoospermic men (yes/no), number of semen specimens analyzed (one versus two), and blinding of laboratory personnel to the smoking status of the study participants (yes/no). Regression analyses were performed both unweighted and weighted inversely by study size. A qualitative and quantitative assessment of the relationship between the numbers of cigarettes smoked per day and sperm density was performed. RESULTS: Results of the meta-analysis indicate that smokers' sperm density is on average 13% to 17% (95% confidence interval = 8.0, 21.5) lower than that of nonsmokers. No other factors besides cigarette smoking were found to be independent predictors of sperm density. No clear dose-response relationships between the numbers of cigarettes smoked per day and sperm density emerged. Research conducted by the authors supports the findings of the meta-analysis. CONCLUSION: Cigarette smoking is associated with lowered sperm density. The inconsistency in the literature with regard to this conclusion appears to be the result of small sample sizes in most studies.

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.

Smoking and male reproduction: a review.

Because of the large number of men worldwide who smoke and the fact that cigarette smoke contains known mutagens and carcinogens, there has been concern that smoking may have adverse effects on male reproduction. A review of the epidemiological literature indicates that cigarette smoking is associated with modest reductions in semen quality including sperm concentration, motility and morphology. The associations between male smoking and sperm concentration and motility are stronger among studies of 'healthy' men (e.g. volunteers and sperm donors) than among men from infertility clinic populations. Smoking has also been associated with alterations in hormone levels in males, for example increases in the levels of oestrone and oestradiol. Despite modest reductions in semen quality and altered hormone levels among smokers compared to non-smokers, studies have not shown a reduction in male fertility in association with paternal smoking. There is some evidence to suggest that paternal smoking is associated with congenital anomalies and childhood cancer (with ORs in positive studies generally < 2.0). Smoking has not been shown to be mutagenic to human spermatozoa, although studies have been small and have had methodological shortcomings. It is concluded that, although smokers as a group may not experience reduced fertility, men with marginal semen quality who wish to have children may benefit from quitting smoking, since several small studies indicate the potential for improved semen quality after quitting smoking. More research on the benefits of quitting smoking among men with marginal semen quality is needed. Methodological improvements in studies aimed at clarifying the association between paternal smoking and reproductive and developmental outcomes in offspring should include obtaining accurate paternal smoking dose information, evaluating smoking exposure information in relation to various time windows (e.g. prior to conception, during gestation), and controlling for potential confounders and modifying factors such as age and maternal smoking habits. More sensitive and specific laboratory assays and increased sample sizes are required to establish whether smoking induces mutations in human spermatozoa.

Biological markers: their use in quantitative assessments.

Biological markers can be conceptualized in terms of categories of markers that form a continuum representing a sequence of events from exposure to disease. These categories include markers of internal dose, biologically effective dose, early response, and disease. Outside of this sequence are susceptibility factors that can act at any point along the way to modify the effects of external exposures on disease outcomes. Examples of the use of these different types of markers in epidemiologic research are provided. There are many factors that one must consider when selecting a biological marker for use in an epidemiologic study. These factors include: the objectives of the study, the availability and specificity of potential markers, the feasibility of measuring the markers in various biological media, the invasiveness of the techniques necessary to measure the markers, the amount of biological specimen needed for analysis, the time to appearance of the markers in the biological media, the persistence of the markers in biological media, the variability of marker levels within and between individuals, the stability of markers in storage, as well as the cost, sensitivity, specificity, and reliability of the assays used to measure the markers. Each of these characteristics is discussed. The usefulness of biological markers in an epidemiologic study depends on the objectives of the study and whether the properties of the markers fulfill the objectives of the study in a feasible and cost-effective manner.

Biologic markers of exposure: current status and future research needs.

Biologic markers of exposure have been used successfully in many instances and are potentially useful in future epidemiologic investigations. For exposure markers to realize their full potential, much work still must be done on a marker-by-marker basis to identify properties of markers and to validate their use in human populations. Although properties of some markers, such as cotinine and lead, are well characterized, more research is needed on properties of many molecular and cytologic markers of exposure before they can be employed effectively in epidemiologic research investigations.

Gender differences in response to the multitest CMI skin test in the general population.

BACKGROUND: Previous studies of gender differences in response to the Multitest CMI skin test have produced conflicting results. OBJECTIVE: To determine whether gender is associated with response to the Multitest CMI skin test. METHODS: Two-hundred ninety-seven adults, aged 18 to 64 years, recruited originally for a study of the immune effects associated with living near a hazardous waste site containing primarily organochlorine pesticides, underwent a skin test using the Multitest CMI skin test. Six of seven antigens were tested: tetanus toxoid, diphtheria toxoid, Candida, Tricophyton, Streptococcus, and Proteus. The tuberculin antigen was excluded. Lymphocyte function was also evaluated in vitro using standardized methods of mitogen stimulation with phytohemaglutinin (PHA), concanavalin A (CON-A), and pokeweed mitogen. RESULTS: The frequency of positive responses to the skin tests was significantly (P < .001) higher among males (80.4%) than among females (55.7%). Males were more likely than females to respond to all six antigens tested (P < .05). The mean diameter of positive skin test measurements for males statistically significantly (P < .05) exceeded female responses for tetanus and diphtheria. Although not statistically significant, male response size exceeded that of females for all other antigens except Trycophyton. Controlling for age, race, smoking, income, and plasma DDE levels did not change these results. Skin test positivity was not associated with mitogen stimulation assay results overall or within gender groups. CONCLUSION: Significant gender differences in response to the Multitest CMI skin test could limit its use as a marker of anergy in general population studies.

Types and duration of symptoms prior to diagnosis of invasive or borderline ovarian tumor.

OBJECTIVE: The objective was to describe and compare types and duration of symptoms among women with invasive versus borderline ovarian tumors. METHODS: Cases were women, ages 20-69 years, diagnosed with invasive (616) and borderline (151) epithelial ovarian tumors from 1994 to 1998. Symptoms were obtained using a standardized in-person interview. Differences in types and duration of symptoms, time to diagnosis after consulting a physician, and primary reason for diagnosis by invasive/borderline status and histologic type were determined using bivariate and regression analyses controlling for age. RESULTS: Borderline and invasive cases reported similar types of symptoms. However, borderline cases were twice as likely to report not having had symptoms as invasive cases (16 vs 8%, P = 0.005). Prediagnostic symptom duration was longer among borderline versus invasive cases (median: 6 vs 4 months, P < 0.001). The median time from first consultation with a physician to diagnosis (1 month) did not differ by invasive/borderline status. Borderline cases were twice as likely to be diagnosed through routine examination as invasive cases (28 vs 16%, P = 0.001). Invasive cases were more likely to be diagnosed because of symptoms (62 vs 48%, P = 0.002). CONCLUSIONS: Because most (90%) women with ovarian tumors have symptoms and median symptom duration is 4 months, greater awareness of symptoms by women and physicians is needed for the earlier detection of ovarian tumors. The lesser likelihood of being detected by routine examination and the shorter symptom duration for invasive versus borderline cases underscores the need for effective screening and preventive strategies.

Age at diagnosis and multiple primary cancers of the breast and ovary.

This nested case-control study assessed the relationship between a woman's age at the time of her initial primary breast or ovarian cancer diagnosis and the risk of a second primary cancer at the other of these two sites. Multiple primary breast and ovarian cancer cases whose initial breast or ovarian diagnosis occurred in 1970-1989 and a random sample of single primary breast or ovarian cancer controls diagnosed in the same years were identified through tumor registries at Duke University Medical Center and the University of North Carolina. Women diagnosed with an initial primary breast cancer at age < or = 50 years were 4.3 times (95% CI: 1.8-10.6) more likely to have developed a subsequent ovarian cancer compared to those diagnosed after age 50. A relationship between an early age at diagnosis (< or = 50) of ovarian cancer and subsequent diagnosis of breast cancer was not found (odds ratio (OR) = 0.6; 95% CI: 0.2-2.0). Adjustment for stage at diagnosis, treatment, year of diagnosis and length of follow-up using Cox Proportional Hazards modeling techniques supported these relationships, yielding a hazard ratio (HR) for the development of a second primary cancer at the alternate site of 4.6 (95% CI: 1.8-11.5) for women with an initial breast cancer diagnosis and 0.6 (95% CI: 0.2-2.2) for women with an initial ovarian cancer diagnosis. Multiple primary breast and ovarian cancer patients diagnosed with an initial breast cancer at or prior to age 50 may represent a distinct subgroup of women with a germline mutation that confers susceptibility to both breast and ovarian cancers.

Atypical metaplasia and incidence of bronchogenic carcinoma.

The prognostic implication of atypical squamous metaplasia of the respiratory tract has been uncertain, especially for mild atypia. The relation between degree of severity of atypical metaplasia as detected by sputum cytology and incidence of bronchogenic carcinoma was assessed among 14,414 men aged 45 years or older who smoked one or more packs of cigarettes per day. Trial participants underwent sputum cytologic evaluations every 4 months for an average of 7.4 years as part of the Cooperative Early Lung Cancer Detection Program of the National Cancer Institute and were followed for the development of lung cancer between 1971 and 1983 at three institutions: The Johns Hopkins University, the Memorial Sloan-Kettering Cancer Center, and the Mayo Clinic. Analysis with logistic regression controlling for age, race, occupational exposures to lung carcinogens, average number of cytology records per year, and smoking habits revealed that the estimate of the relative rate (RR) of developing bronchogenic carcinoma was greater among men who had mild atypia as compared with men who had negative cytology readings, but there were marked differences among institutions (RR = 1.1, 95% confidence interval (Cl) 0.8-1.5 at The Johns Hopkins University; RR = 1.6, 95% Cl 1.1-2.5 at the Memorial Sloan-Kettering Cancer Center; and RR = 2.5, 95% Cl 1.6-4.0 at the Mayo Clinic). Results suggest that mild atypia as detected by cytologic evaluation of sputum is an indicator of a modest elevation in risk of bronchogenic carcinoma.

Plasma 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) levels and immune response.

For determination of whether plasma 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) pesticide levels (< or =1-32 ppb) are associated with immune suppression or DNA damage in lymphocytes, 302 individuals residing in Moore County, North Carolina, in 1994-1996 provided a blood specimen, underwent a skin test, and answered a questionnaire concerning factors affecting plasma organochlorine pesticide levels and the immune system. The blood specimens were analyzed for levels of plasma DDE (a metabolite of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane), numbers and types of blood cells, immunoglobulin levels, mitogen-induced lymphoproliferative activity, and lymphocyte micronuclei. When DDE levels were categorized as 1 or less, more than 1 to 2, more than 2 to 4.3, more than 4.3 to 7.6, and more than 7.6 ppb, individuals with higher plasma DDE levels had lowered mitogen-induced lymphoproliferative activity (concanavalin A, range: 74,218 dropping to 55,880 counts per minute, p = 0.03) and modestly increased total lymphocytes (range: 2.0-2.3 x 10(3)/microl, p = 0.05) and immunoglobulin A levels (range: 210-252 mg/dl, p = 0.04). There were no consistent differences in response to the skin tests by plasma DDE levels. Plasma DDE levels were not associated with a higher frequency of micronuclei. The authors conclude that relatively low levels of plasma DDE are associated with statistically significant changes in immune markers, although the magnitude of the effects are of uncertain clinical importance.

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.