Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

We have previously shown that type IV collagen (alpha1 (IV) and alpha2 (IV) collagen chains) (Col-IV) inhibits testosterone (T) production by Leydig cells (LC). The aim of this study was to analyze mechanism/s by which Col-IV exerts this effect. No significant differences in the specific binding of hCG to LH/hCG receptors in LC cultured on uncoated or Col-IV coated plates were observed. An inhibition of cAMP production in hCG-stimulated LC cultured on Col-IV was detected. The inhibition exerted by Col-IV on T production in response to hCG was also observed when cells were stimulated with 8Bromo-cAMP. In addition, conversion of steroid precursors to T in LC cultured on uncoated and Col-IV coated plates was similar. On the other hand, we detected an increase of ERK1/2 phosphorylation in hCG-stimulated LC cultured on Col-IV. Genistein added to LC cultures reduced the ability of Col-IV to increase ERK1/2 phosphorylation and reverted the inhibitory effect of Col-IV on T production. An inhibitor of MEK, PD98059 added to LC cultures also reverted the inhibitory effect of Col-IV on T production. A decrease of steroidogenic acute regulatory protein (StAR) expression in hCG-stimulated LC cultured on Col-IV coated plates that could be reverted by addition of PD98059 to the cultures was also demonstrated. All together these results suggest that Col-IV inhibits T production in LC by binding to integrins, activating ERK1/2, decreasing cAMP production and decreasing StAR expression.

Exposure to methyl mercury, a risk factor for neurodevelopmental toxicity, was assessed in U.S. children 1–5 years of age (n = 838) and women 16–49 years of age (n = 1,726) using hair mercury analysis during the 1999–2000 National Health and Nutrition Examination Survey (NHANES). The data are nationally representative and are based on analysis of cross-sectional data for the non-institutionalized, U.S. household population. The survey consisted of interviews conducted in participants’ homes and standardized health examinations conducted in mobile examination centers. Distributions of total hair mercury levels expressed as micrograms per gram hair Hg and the association of hair Hg levels with sociodemographic characteristics and fish consumption are reported. Geometric mean (standard error of the geometric mean) hair mercury was 0.12 μg/g (0.01 μg/g) in children, and 0.20 μg/g (0.02 μg/g) in women. Among frequent fish consumers, geometric mean hair mercury levels were 3-fold higher for women (0.38 vs. 0.11 μg/g) and 2-fold higher for children (0.16 vs. 0.08 μg/g) compared with nonconsumers. The NHANES 1999–2000 data provide population-based data on hair mercury concentrations for women and children in the United States. Hair mercury levels were associated with age and fish consumption frequency.

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin‐1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of α3, α5, and β1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG‐stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 μg/cm 2 ) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 μg/cm 2 ), compared to uncoated plates. Laminin‐1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro. Mol. Reprod. Dev. 61:493–503, 2002. © 2002 Wiley‐Liss, Inc.

During the Minnesota Children's Pesticide Exposure Study (MNCPES), comparisons were made between the insecticide/herbicide loadings obtained with two household dust/insecticide or herbicide samplers: the Edwards and Lioy (EL) press sampler (used for dust collection from carpets or other surfaces) and the Lioy, Wainman and Weisel (LWW) surface wipe sampler. The results were compared with hand rinse levels, and urine metabolite levels obtained from 102 children (ages 3–13). All measurements were made during a 1-week sampling period, and information was obtained on household pesticide use and each child's activities. Of the homes, <5% had recent spot uses of a pesticide but none had recent general applications. The analyses focused primarily on atrazine (a herbicide), and malathion, diazinon, and chlorpyrifos (insecticides). Metabolites were measured for atrazine, malathion and chlorpyrifos. The atrazine levels obtained using the EL indicate that this compound was transported into the home by an unquantified transport mechanism (e.g. tracking of soil). Two malathion hand rinse values exceeded >170 ng/cm 2 , suggesting that since indoor surface levels were low, these children had other sources of exposure. Atrazine, chlorpyrifos and malathion were detectable in >30% of the homes by the EL, LWW or hand rinse. Only chlorpyrifos had detectable levels in ≥50% of the samples for all types, i.e. compound or metabolite, which is consistent with it being a common household pesticide. The median (and maximum) chlorpyrifos levels for the EL surface, EL carpet, LWW surface (two rooms), hand rinse, and urine metabolites were: 0.07 (32.6) ng/cm 2 ; 0.07 (44.5) ng/cm 2 ; 0.34 (3.64) ng/cm 2 ; 0.42 (14.4) ng/cm 2 ; 0.03 (2.14) ng/hand and 6.9 (59.0) µg/g, respectively. A strong correlation was found for chlorpyrifos between the EL surface and carpet samples. Chlorpyrifos levels detected by LWW had a different distribution and concentration range than the EL, indicating that it collected more than the surface dislodgeable insecticide. EL was directly comparable to the hand rinse or urine levels, but only the LWW had a weak correlation with hand rinse levels, suggesting that the children had other sources of chlorpyrifos exposure. Thus, mechanistic exposure studies are needed to more accurately establish exposure–dose relationships in residential settings.

Melnvk, L. J.; Berry, M. R. Jr.; Sheldon, L.; Freeman, N.; Pellizzari, E. Author Information

Follicle-stimulating hormone (FSH) is involved in the regulation and maintenance of gametogenesis. It exists in multiple molecular forms with different oligosaccharide structures which in turn are influenced by the hormonal milieu. Previous studies from our laboratory demonstrated that antiandrogen administration to immature male rats altered the biological activity and the distribution profile of pituitary FSH isoforms. The aim of this study was to examine possible modifications in pituitary FSH polymorphism throughout sexual development (10-, 32- and 75-day-old rats). In addition, the effect of androgen deprivation by castration (32-day-old rats) and its replacement with a nonaromatizable androgen – dihydrotestosterone – on pituitary FSH polymorphism was determined. Concanavalin A affinity chromatography was used to isolate groups of FSH isoforms according to their carbohydrate inner structure. Radioimmunoassay and Sertoli cell bioassay were used to evaluate FSH immuno- and bioactivities. Androgen rise in serum was accompanied by a marked increase in pituitary bio- and immuno-FSH content in 32- and 75-day-old rats. However, FSH pituitary content did not vary despite the significant increment observed in serum FSH levels after castration and decrease to control levels after androgen replacement. The distribution profile of immuno- and bioactive FSH changed throughout sexual maturation. The proportion of pituitary FSH isoforms bearing complex oligosaccharide structures (triantennary, bisecting, complete and truncated biantennary) increased with age, with a concomitant decrease in the proportion of isoforms bearing incomplete carbohydrate chains. The distribution profile observed in castrated 32-day-old rats was similar to that determined in 10-day-old animals. Androgen replacement restored the distribution profile to normal. These results suggest that androgens regulate the incorporation of sugar residues to the carbohydrate chains of pituitary FSH favoring the biosynthesis of complex-type oligosaccharide structures.

The time/activity diary developed for use in the National Human Exposure Assessment Survey (NHEXAS) was completed by 249 participants in the Research Triangle Institute/Environmental and Occupational Health Sciences Institute (RTI/EOHSI) NHEXAS population-based pilot project conducted in the upper Midwest (EPA Region 5). The majority of participants successfully completed the diary during the 6-day study period. Participant responses showed internal consistency between related questions within the diary and between instruments used within the study. Comparison of response rates with the National Human Activity Pattern Survey, a nationwide population-based study, found consistent results when the same questions were used in both studies. Several questions identified age-specific activities. The value of the 6-day diary over 1-day surveys was apparent in discriminating between episodic and regularly conducted activities and in identifying subpopulations whose behavior may contribute to exposure to environmental pollutants.

Pellizzari, E.; Freeman, N.; Head, S.; Whitmore, R.; Zelon, H.; Stroebel, C. Author Information

Research Triangle Institute, Research Triangle Park, NC; L Sheldon, US EPA, Research Triangle Park, NC; J Morgan, M Berry, J Shoemaker, US EPA, Cincinnati, OH

Bonanno, L.; Freeman, N.; Lioy, P.; Pellizzari, E.; Quackenboss, J.; Paschal, D. Author Information

gamma-Glutamyl transpeptidase (gamma-GTP) activity in Sertoli cells can be stimulated by FSH. This cAMP-dependent metabolic event can be enhanced when Sertoli cells are co-cultured with germ cells, suggesting that different signal transduction pathways may be involved in the regulation of gamma-GTP activity. In this study we examined the participation of Ca(2+)- and protein kinase C (pkC)-dependent signal transduction pathways in the regulation of basal and FSH-stimulated gamma-GTP activity. Under basal conditions, the increase in extracellular Ca2+ concentration or the addition of the Ca2+ ionophore 4Br-A23187 produced a decrease in gamma-GTP activity. Conversely, blockage of voltage-dependent Ca2+ channels with verapamil or nifedipine or inhibition of Ca(2+)-calmodulin dependent processes with trifluoperazine resulted in an increase in gamma-GTP activity. To study the role of a pkC-dependent pathway the effects of low doses of staurosporine were evaluated. Under these experimental conditions an increase in gamma-GTP activity was observed. It was then investigated whether these signal transduction pathways could interact with the FSH-stimulated cAMP-dependent pathway to regulate gamma-GTP activity. Increase in extracellular Ca2+ concentration, the addition of 4Br-A23187 or the blockage of voltage-dependent Ca2+ channels did not modify FSH-stimulated gamma-GTP activity. However, staurosporine produced an additional increase in FSH-stimulated gamma-GTP activity and this effect was also observed when cells were stimulated with dbcAMP. In summary, our data are consistent with an inhibitory role of Ca(2+)-calmodulin- and pkC-dependent pathways in the regulation of basal gamma-GTP activity. Similar to what has been shown for other Sertoli cell parameters, a pkC-dependent pathway can interact with the FSH-stimulated cAMP-dependent pathway. The precise steps involved in this interaction are still unknown.

A brief review of the uses of breath analysis in studies of environmental exposure to volatile organic compounds (VOCs) is provided. The U.S. Environmental Protection Agency's large-scale Total Exposure Assessment Methodology Studies have measured concentrations of 32 target VOCs in the exhaled breath of about 800 residents of various U.S. cities. Since the previous 12-hr integrated personal air exposures to the same chemicals were also measured, the relation between exposure and body burden is illuminated. Another major use of the breath measurements has been to detect unmeasured pathways of exposure; the major impact of active smoking on exposure to benzene and styrene was detected in this way. Following the earlier field studies, a series of chamber studies have provided estimates of several important physiological parameters. Among these are the fraction, f, of the inhaled chemical that is exhaled under steady-state conditions and the residence times, tau(i) in several body compartments, which may be associated with the blood (or liver), organs, muscle, and fat. Most of the targeted VOCs appear to have similar residence times of a few minutes. 30 min, several hours, and several days in the respective tissue groups. Knowledge of these parameters can be helpful in estimating body burden from exposure or vice versa and in planning environmental studies, particularly in setting times to monitor breath in studies of the variation with time of body burden. Improvements in breath methods have made it possible to study short-term peak exposure situations such as filling a gas tank or taking a shower in contaminated water.

The PTEAM Study was the first large-scale probability-based study of personal exposure to particles. Sponsored by the U.S. Environmental Protection Agency (EPA) and the Air Resources Board of California, it was carried out by the Research Triangle Institute (RTI) and the Harvard University School of Public Health (HSPH). HSPH designed and constructed a 4-lpm, battery-operated personal monitor for inhalable particles (PM10) that could be worn comfortably for up to 14 hours by persons from 10 to 70 years old. The monitor was worn for two consecutive 12-hour periods (day and night) during the fall of 1990 by 178 participants representing 139,000 nonsmoking residents of Riverside, California. Nearly identical monitors were employed to collect concurrent indoor and outdoor samples. The monitors were equipped with a different sampling nozzle to collect fine particles (PM2.5). Population-weighted daytime personal PM10 exposures averaged 150 +/- 9 (SE) micrograms/m3, compared to concurrent indoor and outdoor concentrations of 95 +/- 6 micrograms/m3. This suggested the existence of excess mass near the person, a "personal cloud" that appeared related to personal activities. Fourteen of 15 prevalent elements also were evaluated in the personal samples. The two major indoor sources of indoor particles were smoking and cooking; even in these homes, however, more than half of the indoor particles came from outdoors, and a substantial portion of the indoor particles were of undetermined indoor origin. Outdoor concentrations near the homes were well correlated with outdoor concentrations at the central site, supporting the idea of using the central site as an indicator of of ambient concentrations over a wider area. Indoor concentrations were only weakly correlated with outdoor concentrations, however, and personal exposures were even more poorly correlated with outdoor concentrations. Elemental profiles were obtained for environmental tobacco smoke (ETS) (major contributions from potassium and chlorine) and cooking emissions (aluminum, iron, calcium, and chlorine). These profiles can be used in future source apportionment studies.

In male rats androgens are involved in the regulation of follicle-stimulating hormone (FSH) synthesis and secretion. Two nonsteroidal antiandrogens, flutamide and Casodex, were used to study the influence of androgens on the carbohydrate structure of FSH isoforms and the relationship with their bioactivity in prepubertal male rats. Different doses of flutamide or Casodex (vehicle, 1, 5, or 10 mg/rat/day) were administered subcutaneously for 10 days to 23-day-old rats. Immunological FSH was determined by radioimmunoassay and the bioactivity by in vitro Sertoli cell bioassay. Concanavalin A affinity chromatography was used to study the distribution of immunoactive and bioactive pituitary FSH isoforms. A significant depletion of immunological and biological pituitary FSH contents was observed even at the lowest dose of flutamide or Casodex used. The bioactive/immunoactive ratio of pituitary FSH was reduced at the highest dose of flutamide; however, no change was observed in Casodex-treated rats, suggesting a differential effect of the antiandrogens on the FSH bioactivity. Flutamide treatment provoked a significant decrease in proportion and bioactivity of FSH isoforms bearing biantennary and truncated hybrid oligosaccharide side chains and an increase in the proportion but a decrease in bioactivity of FSH isoforms bearing high-mannose oligosaccharides. Conversely, Casodex administration did not modify the proportions of FSH isoforms, although those bearing biantennary and truncated hybrid structures were less bioactive, while those bearing high-mannose oligosaccharides were more bioactive. The highest dose of flutamide decreased the bioactive/immunoactive ratio of FSH isoforms with a high degree of branching in their carbohydrate chains. Our results suggest that androgens, acting directly and indirectly at the pituitary, regulate the selective incorporation of sugar residues to the FSH molecule, thus modulating its biological activity.