Melatonin enhances junctional transfer in normal C3H/10T1/2 cells.

Gap junctional intercellular communication is known to be involved in controlling cell proliferation and differentiation, and seems to play a crucial role in suppression of tumor promotion. The pineal gland and its hormone, melatonin, are believed to intervene in the control of neoplastic processes. Several possible mechanisms have been suggested to be potentially responsible for melatonin's oncostatic action; however, the actual mechanisms involved in melatonin's effects at the cellular level remain unidentified. In the present study low-density cultures of C3H/10T1/2 mouse embryo fibroblasts were incubated until relatively quiescent monolayers were established (17-18 days). Gap junctional intercellular communication in control samples and in cells treated with 10(-12) to 10(-8) M melatonin was determined by the scrape-loading assay using the fluorescent dye Lucifer yellow. The results showed that concentrations of melatonin considered physiological (10(-11) and 10(-10) M) induced a significant increase in the transfer of the dye to adjacent cells through gap junctions; both higher and lower concentrations were ineffective. These results suggest that melatonin could exert its putative oncostatic action, in part, by modulating the levels of gap junctional intercellular communication.

Extrathoracic and intrathoracic removal of O3 in tidal-breathing humans.

We measured the efficiency of O3 removal from inspired air by the extrathoracic and intrathoracic airways in 18 healthy, nonsmoking, young male volunteers. Removal efficiencies were measured as a function of O3 concentration (0.1, 0.2, and 0.4 ppm), mode of breathing (nose only, mouth only, and oronasal), and respiration frequency (12 and 24 breaths/min). Subjects were placed in a controlled environmental chamber into which O3 was introduced. A small polyethylene tube was then inserted into the nose of each subject, with the tip positioned in the posterior pharynx. Samples of air were collected from the posterior pharynx through the tube and into a rapidly responding O3 analyzer yielding inspiratory and expiratory O3 concentrations in the posterior pharynx. The O3 removal efficiency of the extrathoracic airways was computed with the use of the inspiratory concentration and the chamber concentration, and intrathoracic removal efficiency was computed with the use of the inspiratory and expiratory concentrations. The mean extrathoracic removal efficiency for all measurements was 39.6 +/- 0.7% (SE), and the mean intrathoracic removal efficiency was 91.0 +/- 0.5%. Significantly less O3 was removed both extrathoracically and intrathoracically when subjects breathed at 24 breaths/min compared with 12 breaths/min (P less than 0.001). O3 concentration had no effect on extrathoracic removal efficiency, but there was a significantly greater intrathoracic removal efficiency at 0.4 ppm than at 0.1 ppm (P less than 0.05). Mode of breathing significantly affected extrathoracic removal efficiency, with less O3 removed during nasal breathing than during either mouth breathing or oronasal breathing (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Dispersion of aerosol boluses in the human lung: dependence on lung volume, bolus volume, and gender.

The dispersion of aerosol boluses in the human lungs has been studied in health and disease, usually as a means of investigating convective mixing. However, there are limited data on the roles of critical factors, such as the volume of inhaled boluses, lung inflation, and gender on dispersion. To examine these factors, we measured the difference in volume variance between exhaled and inhaled boluses (sigma 2V) of a 0.5-micron aerosol in 11 healthy male and 12 healthy female subjects as a function of tidal volume (VT = 1,000 and 1,500 ml in females and 1,000 and 2,000 ml in males), bolus penetration volume (Vi at 250-ml increments over each VT), and bolus volume (target VBol = 75, 150, and 300 ml). Analysis of variance showed marginally significant gender effects (P = 0.073) on sigma 2V, with sigma 2V greater in males than in females. There was also a significant effect of VBol on sigma 2V (P < 0.001). A Vi-dependent mean volume shift between inhaled and exhaled boluses (delta V) was observed at all Vi except 500 ml. The observation of gender and VBol effects and the existence of a nonzero delta V suggest that convective mixing mechanisms other than longitudinal dispersion alone occur in the healthy lung. The lack of VT dependence suggests a minimal role of lung inflation above functional residual capacity on dispersion. The dependence of sigma 2V on Vi2 up to 1,750 ml and minimal VBol effects demonstrates that convective mixing processes continue far into the gas exchange regions of the lung and support a significant role for axial streaming.

Role of the parasympathetic nervous system in acute lung response to ozone.

We conducted an ozone (O3) exposure study using atropine, a muscarinic receptor blocker, to determine the role of the parasympathetic nervous system in the acute response to O3. Eight normal subjects with predetermined O3 responsiveness were randomly assigned an order for four experimental exposures. For each exposure a subject inhaled either buffered saline or atropine aerosol followed by exposure either to clean air or 0.4 ppm O3. Measurements of lung mechanics, ventilatory response to exercise, and symptoms were obtained before and after exposure. O3 exposure alone resulted in significant changes in specific airway resistance, forced vital capacity (FVC), forced expiratory flow rates, tidal volume (VT), and respiratory rate (f). Atropine pretreatment prevented the significant increase in airway resistance with O3 exposure and partially blocked the decrease in forced expiratory flow rates but did not prevent a significant fall in FVC, changes in f and VT, or the frequency of reported respiratory symptoms after O3. These results suggest that the increase in pulmonary resistance during O3 exposure is mediated by a parasympathetic mechanism and that changes in other measured variables are mediated, at least partially, by mechanisms not dependent on muscarinic cholinergic receptors of the parasympathetic nervous system.

Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats.

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254R, on 45Ca2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90-120 (adult) male and female Long-Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca2+-uptake in selected brain regions increased with age; PND 7

Adrenocorticotropin (ACTH) and corticosterone secretion by perifused pituitary and adrenal glands from rodents exposed to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD).

Although in utero maternal stress has been shown to have lasting effects on rodent offspring, fetal effects of chemically-induced alterations of the maternal hypothalamic-pituitary-adrenal axis (HPA) have not been well studied. This study examined the effects of in vivo 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on pituitary-adrenal function in the male rat, pregnant female rat and pregnant female mouse. The secretion of adrenocorticotropin (ACTH) and corticosterone (CORT) in pituitary and adrenal glands, respectively, was assessed in ex vivo perifusion cultures. Male and pregnant female (gestation day 8) Sprague-Dawley rats were gavaged once with 10 microgram/kg TCDD, pregnant female mice once with 24 microgram/kg TCDD, and euthanized 10 days later. Hemi-pituitary (rat) or whole anterior pituitaries (mice) and right adrenal glands from the same animal were quartered, perifused under baseline and stimulated conditions. In both males and pregnant females, TCDD did not affect corticotropin releasing hormone (CRH)-stimulated ACTH secretion. Neither total pituitary ACTH nor plasma ACTH was altered in either sex or species by TCDD treatment. ACTH-stimulated CORT secretion was not affected by TCDD in either sex or species, and adrenal tissue and plasma CORT levels were unchanged in males and pregnant females by TCDD. However, the plasma ACTH:CORT ratio was decreased about 46% in male rats treated with TCDD. Plasma CORT levels were 23-fold higher and plasma ACTH levels were 1.5-fold higher in pregnant females than in male rats. In male versus female rats, adrenal CORT and anterior pituitary ACTH tissue levels were about 7.5- and 1.75-fold higher and ACTH, respectively. Female mouse adrenal tissue CORT was about 4-fold greater than female rat. The reduced plasma ACTH:CORT ratio in the male rat suggests that TCDD disturbs HPA function. Exposure of male rat to a 5-fold higher dose in earlier studies clearly demonstrated effects of TCDD on male rat HPA. The present study identified substantial HPA performance differences between male and pregnant female rats. The failure to detect a response to TCDD in pregnant female rat and mouse could be a function of both TCDD dose and the high level of secretion of both ACTH and CORT in pregnant animals. For the rat or mouse, a single exposure to TCDD during pregnancy does not appear sufficient to induce maternally-mediated developmental, reproductive and behavioral toxicity via the HPA axis.

Methanol potentiation of carbon tetrachloride hepatotoxicity: the central role of cytochrome P450.

Evidence to explain the enhanced hepatotoxicity of carbon tetrachloride (CCl4) following methanol exposure by inhalation is presented. Hepatic microsomes prepared from male F344 rats exposed to methanol at concentrations up to 10,000 ppm showed increased p-nitrophenol hydroxylase activity but no increase in pentoxyresorufin-O-dealkylase or ethoxyresorufin-O-deethylase activities. Hepatic antioxidant levels, glutathione levels and glutathione-S-transferase activity in methanol-treated animals were not different from controls. In vitro metabolism of CCl4 was also increased in microsomes from methanol-treated animals. Pretreatment with allyl sulfone, a specific chemical inhibitor of cytochrome P450 2E1, abolished the difference in microsomal metabolism between exposed and control animals. This study shows that methanol exposure induces cytochrome P450 2E1, which appears to be the principal toxicokinetic mechanism responsible for the increased metabolism and thus the increased hepatotoxicity of CCl4.

Comparison of open and blind histopathologic evaluation of hepatic lesions.

This paper explores the controversy among scientists on whether microscopic evaluation of tissue slides should be done in an open or blind fashion. Definitions are given and discussed that provide a better focus to the problem. An experiment was conducted in which hepatocellular degeneration and necrosis in rats were assessed both openly and blindly. The results indicate that 'simple bias' is present when the slides are read openly. Valid comparisons among treatment groups are possible in the presence of simple bias, provided appropriate control groups have been incorporated into the experimental design.

Fasting for less than 24 h induces cytochrome P450 2E1 and 2B1/2 activities in rats.

Cytochrome P450 (CYP) 2E1 activity is induced after 24 h of fasting but no information is available for shorter fasting periods. We investigate the induction of CYP 2E1, 2B1/2 and 1A1 in young adult male F344 rats after 8, 16 and 24 h of fasting compared to control. Liver microsomes were analyzed for the following enzyme activities: p-nitrophenol hydroxylase (PNP) for CYP 2E1, pentoxyresorufin-O-dealkylase (PROD) for CYP 2B1/2 and ethoxyresorufin-O-deethylase (EROD) for CYP 1A1. After each fasting interval, the activities per mg microsomal protein for PNP and PROD increased but the activity of EROD remained unchanged. Western blots for CYP 2E1 and CYP 2B1 showed increases comparable to the PNP and PROD activities, respectively. On a whole organ basis, increases were found for PNP and PROD activities, while decreases were found for EROD activity and total microsomal protein. The results are consistent with an induction of CYP 2E1 and CYP 2B1/2 activities after as little as 8 h of fasting.

Cytogenetic effects of inhaled ozone in man.

Peripheral blood samples were collected from 30 normal male volunteers before and at intervals after inhaling 0.4 ppm ozone for 4 h. Data from 4 of the subjects were excluded from the analysis because of missing data points. The blood samples were cultured for 48 h, slides made and stained with a uniform Giemsa stain, and 100 metaphase spreads per subject per treatment scored for chromosome aberrations. Cells with suspected aberrations were photographed, destained, restained with a banding procedure and rephotographed to identify the specific chromosomes and regions involved. Pre-exposure, immediate post-exposure, 3 days post-exposure, 2 weeks post-exposure and 4 weeks post-exposure means for the percentage of cells with 46 chromosomes were 93.0, 93.6, 91.7, 94.5 and 94.2, respectively; in the same order, the mean number of cells with chromatid and/or chromosome breaks per order, the mean number of cells with chromatid and/or chromosome breaks per 100 cells was 0.96, 0.85, 1.00, 0.88 and 0.81 respectively, and for chromatid and/or chromosome gaps per 100 cells: 1.35, 0.96, 1.35, 0.81 and 0.77, respectively. The means for each of these parameters as well as the mean frequencies of complex aberrations are not statistically significantly different between blood sampling times. The distribution of aberrations by chromosome and light and dark bands is not significantly influenced by ozone exposure. These data indicate no apparent detectable human cytogenetic effect due to exposure to ozone under the conditions of this experiment.

Airborne exposures to PAH and PM2.5 particles for road paving workers applying conventional asphalt and crumb rubber modified asphalt.

Personal exposure monitoring was conducted for road paving workers in three states. A research objective was to characterize and compare occupational exposures to fine respirable particles (< 2.5 microns) and particle-bound polycyclic aromatic hydrocarbons (PAHs) for road paving workers applying conventional (petroleum derived) asphalt and asphalt containing crumb rubber from shredded tires. Workers not exposed to asphalt fume were also included for comparison (to support the biomarker component of this study). The rubber content of the crumb rubber modified (CRM) asphalt at the three study sites was 12, 15, and 20%. A comparison of some specific job categories from two sites indicates greater potential carcinogenic PAH exposures during CRM asphalt work, however, the site with the greatest overall exposures did not indicate any differences for specific jobs. A statistical analysis of means for fine particle, pyrene and total carcinogenic PAH personal exposure shows, with two exceptions, there were no differences in exposures for these three measurement variables. One site shows significantly elevated pyrene exposure for CRM asphalt workers and another site similarly shows greater carcinogenic PAH exposure for CRM asphalt workers. Conventional and CRM asphalt worker airborne exposures to the PAH carcinogen marker, BaP, were very low with concentrations comparable to ambient air in many cities. However, this study demonstrates that asphalt road paving workers are exposed to elevated airborne concentrations of a group of unknown compounds that likely consist of the carcinogenic PAHs benz(a)anthracene, chrysene and methylated derivatives of both. The research described in this article has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

The influence of vision on computerized neurobehavioral test scores: a proposal for improving test protocols.

Computerized tests of neurobehavioral function are frequently administered in neurotoxicological studies with little attention given to the optical properties of test stimuli or to the vision of subjects. Yet many test stimuli are small or briefly presented, and test endpoints often involve short reaction times. Stimulus detection and reaction time are known to be strongly dependent upon stimulus luminance, contrast, and size, as well as on the subject's visual abilities. The current study assessed the influence of visual contrast sensitivity on Neurobehavioral Evaluation System 2 (NES2) test results in three data sets. Analyses indicated that vision was associated with up to 24% of the variance (Hand Eye Coordination test) in NES2 scores, even when visual acuity was normal, and that vision often influenced the significance of group differences. It is suggested that researchers measure the luminance, contrast, and size of test stimuli, the distance from the subject's eyes to the monitor, and the subject's visual contrast sensitivity. The measurement and control of stimulus parameters and the inclusion of visual function scores in analysis models could reduce the variability among computerized test scores both within and between studies. Models that assess the influence of vision on computerized test results may help to identify the CNS domains and specialized functions adversely affected by neurotoxicant exposures.

Neurobehavioral Evaluation System (NES): comparative performance of 2nd-, 4th-, and 8th-grade Czech children.

The Neurobehavioral Evaluation System was designed for field studies of workers, but many NES tests can be performed satisfactorily by children as young as 7 or 8 years old and a few tests, such as simple reaction time, can be performed by preschool children. However, little comparative data from children of different ages or grade levels are available. Studies of school children in the Czech Republic indicate that 2nd-grade children could perform the following NES tests satisfactorily: Finger Tapping, Visual Digit Span. Continuous Performance, Symbol-Digit Substitution, Pattern Comparison, and simpler conditions of Switching Attention. Comparative scores of boys and girls from the 2nd, 4th, and 8th grades and power analyses to estimate appropriate sample size were presented. Performance varied systematically with grade level and gender. Larger samples were needed with younger children to achieve comparable levels of statistical power. Gender comparisons indicated that boys responded faster, but made more errors than girls.

Lead absorption and psychological function in Zagreb (Croatia) school children.

A cross-sectional study was performed on 275 pupils from the third and fourth grade of three elementary schools (three urban areas with different traffic conditions) in Zagreb. Lead exposure was environmental, mostly through leaded gasoline. The difference in traffic density around the schools was consistent with biological indicators of lead absorption. The aim of the study was to clarify the relationship between characteristic biological indicators of lead absorption including indicators of hematological status with some psychological functions. Lead absorption in pupils was relatively low (mean blood lead: 70.8 +/- 17.88 microgram/L). Pupils' socio-economic status was evaluated by parents' education. The results obtained indicate that gender and school were associated with both biological and psychological variables. After adjusting for age, parental education, and gender, lead appears to have no association with cognitive or psycho-motor measures. The nonstandardized regression coefficients for blood lead-as a measure of the size of lead effect on VIQ, NIQ, and IQ-were -0.016, -0.031, and -0.025, respectively, all nonsignificant.

Hepatocarcinogenicity in the male B6C3F1 mouse following a lifetime exposure to dichloroacetic acid in the drinking water: dose-response determination and modes of action.

Male B6C3F, mice were exposed to dichloroacetic acid (DCA) in the drinking water in order to establish a dose response for the induction of hepatocellular cancer and to examine several modes of action for the carcinogenic process. Groups of animals were exposed to control, 0.05, 0.5, 1, 2, or 3.5 g/L DCA in the drinking water for 90-100 wk. Mean daily doses (MDD) of 8, 84, 168, 315, and 429 mg/kg/d of DCA were calculated. The prevalence (percent of animals) with hepatocellular carcinoma (HC) was significantly increased in the 1-g/L (71%), 2-g/L (95%), and 3.5-g/L (100%) treatment groups when compared to the control (26%). HC multiplicity (tumors/animal) was significantly increased by all DCA treatments-0.05 g/L (0.58), 0.5 g/L (0.68), 1 g/L (1.29), 2 g/L (2.47), and 3.5 g/L (2.90)-compared to the control group (0.28). Based upon HC multiplicity, a no-observed-effect level (NOEL) for hepatocarcinogenicity could not be determined. Hepatic peroxisome proliferation was significantly increased only for 3.5 g/L DCA treatment at 26 wk. and did not correlate with the liver tumor response. The severity of hepatotoxicity increased with DCA concentration. Below 1 g/L, hepatotoxicity was mild and transient as demonstrated by the severity indices and serum lactate dehydrogenase activity. An analysis of generalized hepatocyte proliferation reflected the mild hepatotoxicity and demonstrated no significant treatment effects on the labeling index of hepatocytes outside proliferative lesions. Consequently, the induction of liver cancer by DCA does not appear to be conditional upon peroxisome induction or chemically sustained cell proliferation. Hepatotoxicity, especially at the higher doses, may exert an important influence on the carcinogenic process.

Effects of age, socioeconomic status, and menstrual cycle on pulmonary response to ozone.

The purpose of this study was to investigate the effects of age, socioeconomic status, and menstrual cycle phase on the pulmonary response to ozone exposure. Three hundred seventy-two healthy white and black young adults, between the ages of 18 and 35 y, were exposed only once to 0.0, 0.12, 0.18, 0.24, 0.30, or 0.40 ppm ozone for 2.3 h. Prior to and after exposure, pulmonary function tests were obtained. Prior to exposure, each subject completed a personal and family-history questionnaire. The responses to this questionnaire were used to investigate age, socioeconomic status, and menstrual cycle phase effects on pulmonary responsiveness to ozone. We concluded that the ages of subjects, within the age range studied, had an effect on responsiveness (i.e., decrements in forced expiratory volume in 1 s decreased as the subjects' ages decreased). Socioeconomic status, as reflected by education of fathers, also appeared to affect forced expiratory volume in 1-s responsiveness to ozone, with the middle socioeconomic group being the most responsive. The phase of menstrual cycle did not have an impact on individual responsiveness to ozone.

Exposure of humans to a volatile organic mixture. II. Sensory.

Time-course functions for symptoms of the sick building syndrome were derived from 66 healthy males who, during separate sessions, were exposed to clean air and to a volatile organic compound (VOC) mixture. The mixture contained 22 VOCs (25 mg/m3 total concentration) commonly found airborne in new or recently renovated buildings. Subjects rated the intensity of perceived irritation, odor, and other variables before, and twice during, 2.75-h exposure periods. Eye and throat irritation, headache, and drowsiness increased or showed no evidence of adaptation during exposure, whereas odor intensity decreased by 30%. These results indicate that irritation intensity and other symptoms are not related in any simple way to odor intensity, which suggests that the symptoms may not be a psychosomatic response to the detection of an aversive odor. Instead, subthreshold levels of VOCs may interact additively or hyperadditively and stimulate trigeminal nerve receptors. Also, air quality ratings improved by 18% during exposure, which suggests that both odor and irritation intensity may influence assessments of air quality.

Exposure of humans to a volatile organic mixture. I. Behavioral assessment.

Exposure to a low-level mixture of volatile organic compounds, typical of those found in new buildings, has been reported to impair neurobehavioral function in persons who have experienced sick building syndrome (SBS). Sixty-six healthy young males who had no history of chemical sensitivity were exposed for 2.75 h to a complex mixture of volatile organic compounds at 0 and 25 mg/m3. Even though subjects reported more fatigue and more mental confusion following exposure to volatile organic compounds than to clean air, performance on 13 neurobehavioral tests was not affected. Practice or learning effects were observed if administration of many behavioral tests were repeated. Further studies are needed to clarify the relationship of exposure to volatile organic chemicals, neurobehavioral performance, and subject characteristics, e.g., age, gender, and chemical sensitivity.

A survey of effects of gaseous and aerosol pollutants on pulmonary function of normal males.

A total of 231 normal male human subjects were exposed for 4 hr to air, ozone, nitrogen dioxide, or sulfur dioxide; to sulfuric acid, ammonium bisulfate, ammonium sulfate, or ammonium nitrate aerosols; or to mixtures of these gaseous and aerosol pollutants. Only one concentration of each pollutant was used. This study, therefore, represents a preliminary survey, intended to allow direct comparison of studies to plan future research. During exposure each subject had two 15-min exercise sessions on a treadmill at 4 mph and 10% grade. Environmental conditions were mildly stressful, i.e., temperature = 30 degrees C and relative humidity = 60%. A battery of 19 measurements of pulmonary function was performed just prior to exposure (air control); 2 hr into the exposure, following the first exercise session; 4 hr into the exposure, following the second exercise session; and 24 hr after exposure. Significant differences were noted in specific airway resistance (SRAW), forced vital capacity (FVC), and forced expiratory flow at 50% of FVC (FEF50) and in related measurements in those experimental groups exposed to ozone or to ozone plus aerosols. None of the aerosols alone, nitrogen dioxide or sulfur dioxide alone, or mixtures of nitrogen dioxide or sulfur dioxide with aerosols produced significant effects. A distribution analysis of subject responsivity to ozone gave a normal distribution among subjects not exposed to ozone, and a distribution shifted to the right and skewed to the right among those exposed to ozone alone or in mixture, with no evidence of bimodal distribution of ozone sensitivity.