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.

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.

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 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.

Using a particular model of apoptosis, we here demonstrate the ability of the comet assay to differentiate between different cell populations. In our study, the natural killer Kurloff cells, used as effector cells, recognize and bind to the tumoral L2C target cells. Formation of such conjugates leads to the death of the target cells by apoptosis, as previously described by different conventional techniques. With the alkaline comet assay, a conjugate could directly be visualized as an association of an undamaged cell joined to a highly damaged cell. The modified comet assay used in this study comprises specific labelling of Kurloff cells with immunomagnetic beads, which are visible as grey-dull spheres against the bright-red staining of nuclear origin on the comet preparation. The use of such labelled effector cells suggest the potential of the comet assay to visually identify different cell populations in an unique test.

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.

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.

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.

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.

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.

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.

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

Studies characterized insulin and EGF receptors in human placental tissue from smokers and nonsmokers. Specific binding of 125I-labeled insulin and EGF to placental membranes was not different for nonsmokers compared with smokers. EGF and insulin receptor kinases were further studied using a wheat germ agglutinin-purified preparation of solubilized placental membrane proteins. In extracts from the nonsmoker group, EGF stimulated the active phosphorylation of Mr 170,000 and 140,000 protein bands, which was half-maximal (EC50) at 5 x 10(-8) M. In extracts from the smokers group, however, phosphorylation of these two protein bands was barely detectable over a range of 0 to 10(-6) M EGF. Thus, EGF-stimulated phosphorylation of the 170,000 and 140,000 bands was markedly decreased in placental membranes from smokers. In contrast, insulin stimulated the phosphorylation of a 95,000 protein that was immunoprecipitated with anti-insulin receptor antiserum in membrane preparations from both nonsmokers and smokers. Dose-response curves for autophosphorylation indicate that EC50 values were 2.6 and 7.0 nM insulin for nonsmokers and smokers, respectively. Laser densitometry scan of the 95,000 band on autoradiograms further showed that maximal 32P incorporation was 30% greater in smokers compared with nonsmokers. Analysis of the insulin-dependent phosphorylation of an exogenous substrate, poly(Glu,Tyr) (4:1), showed a similar pattern of values for nonsmokers versus smokers. These results indicate that insulin receptor autophosphorylation and tyrosine kinase activity were normal or increased, whereas EGF-stimulated kinase activity was markedly decreased in placental membrane proteins from smokers. Western blot analysis using an antiserum to the EGF receptor showed the presence of immunoreactive bands of 126,000 and 150,000-170,000 in receptor preparations from nonsmokers, whereas only the 126,000 protein was detected in preparations from smokers. Thus, the smoking-related deficiency in EGF receptor autophosphorylation appeared to be due to the absence of a 150,000-170,000 receptor protein. In conclusion, maternal cigarette smoking is associated with selective alterations in two major receptor-mediated pathways thought to be involved in cell growth and differentiation in human placenta.

Specimens of human placental DNA were tested for chemical addition products (adducts) by recently developed 32P-postlabeling and immunologic assays, and results were compared with data concerning maternal exposures and birth weight. A total of 7 different adducts were detected in the 53 specimens of human placental tissue examined by the 32P-postlabeling assay. Three of these adducts were found almost exclusively in smokers. Among smokers there were positive dose-response relationships between levels of the smoking-related adducts and biochemical estimates of doses of maternal exposure to cigarette smoke during pregnancy. Levels of 1 adduct found only in smokers appeared to relate directly to amounts of caffeine consumption by the mother. In addition to these relationships with maternal exposures, levels of smoking-related adducts were inversely associated with the birth weight of offspring. Results from this study suggest that even at their current formative stage of development, assays for DNA adducts may help identify determinants of DNA damage to human tissues and improve our ability to demonstrate dose-response relationships for the effects of environmental exposures to potentially carcinogenic agents.

Recent studies have demonstrated that in the absence of spleen function, frequencies of micronuclei (Howell-Jolly bodies) in peripheral blood rbcs can be used to measure in vivo cytogenetic damage. Among 20 subjects studied greater than or equal to 6 months after splenectomy, 1 had a frequency of micronucleated rbcs more than an order of magnitude higher than rates for the others. Initial data suggested that this subject was mildly folate-depleted, and a therapeutic trial with folate rapidly reduced the frequency of micronucleated rbcs to normal values. These observations suggest a need to evaluate further the contribution of mild levels of folate depletion to spontaneous chromosomal damage. The approach used here provides a sensitive index of clastogenic damage and offers unique opportunities for investigating the determinants of cytogenetic damage in humans.

It has been conjectured that single-gene mutations in the human sperm or egg may increase the risk of cancer in subsequent offspring. We propose an epidemiological test of this hypothesis, using an observation from paediatric genetics. Children with autosomal dominant disease are occasionally born into previously unaffected families. This signals the probable mutation of a parent's germ cell. Risk of producing these offspring is found to be related to father's age but not to mother's age. This suggests that sex differences in germ cell production lead to a greater accumulation of germ cell mutations in men than women. If germ cell mutations increase with father's age, and if germ cell mutations are associated with subsequent increase in cancer risk, then some association should exist between age of the father (but not the mother) and risk of cancer. We discuss the few available data, and suggest ways that this hypothesis might easily be tested.

Recent studies have shown that diethylstilbestrol (DES) induces sister chromatid exchanges (SCEs) in lymphocytes from pregnant and premenopausal women but has only a slight effect on lymphocytes from post-menopausal women or men. In this study blood specimens from premenopausal women were used to define the role of different metabolic pathways on DES-induced formation of SCEs and to determine whether conditions resulting in induction of SCEs also induced detectable levels of DNA adducts. Exposure of lymphocytes in vitro to 0-40 microM DES induced a concentration-dependent increase in SCEs. Addition of indomethacin to the cultures partially abolished DES-induced SCEs, suggesting involvement of prostaglandin synthetases in the formation of specific DES metabolites that cause SCEs. alpha-Naphthoflavone, an inhibitor of cytochrome P-450 monooxygenases, had no effect on DES-induced SCEs. Cells exposed to DES at doses sufficient to cause large increases in SCE induction did not have adducts detectable by a 32P-postlabeling assay capable of revealing adducts at a level of 1 adduct/10(9) normal nucleotides.