Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice.

Welding fumes were reclassified as a Group 1 carcinogen by the International Agency for Research on Cancer in 2017. Gas metal arc welding (GMAW) is a process widely used in industry. Fume generated from GMAW-mild steel (MS) is abundant in iron with some manganese, while GMAW-stainless steel (SS) fume also contains significant amounts of chromium and nickel, known carcinogenic metals. It has been shown that exposure to GMAW-SS fume in A/J mice promotes lung tumors. The objective was to determine if GMAW-MS fume, which lacks known carcinogenic metals, also promotes lung tumors in mice. Male A/J mice received a single intraperitoneal injection of corn oil or the initiator 3-methylcholanthrene (MCA; 10 µg/g) and, one week later, were exposed by whole-body inhalation to GMAW-MS aerosols for 4 hours/day x 4 days/week x 8 weeks at a mean concentration of 34.5 mg/m3. Lung nodules were enumerated by gross examination at 30 weeks post-initiation. GMAW-MS fume significantly increased lung tumor multiplicity in mice initiated with MCA (21.86 ± 1.50) compared to MCA/air-exposed mice (8.34 ± 0.59). Histopathological analysis confirmed these findings and also revealed an absence of inflammation. Bronchoalveolar lavage analysis also indicated a lack of lung inflammation and toxicity after short-term inhalation exposure to GMAW-MS fume. In conclusion, this study demonstrates that inhalation of GMAW-MS fume promotes lung tumors in vivo and aligns with epidemiologic evidence that shows MS welders, despite less exposure to carcinogenic metals, are at an increased risk for lung cancer.

Changes in the expression of calcitonin gene-related peptide after exposure to injurious stretch-shortening contractions.

UNLABELLED: One of the factors that can result in musculoskeletal injuries, and time off work, is exposure to repetitive motion. The goal of this study was to determine if skeletal muscle injury induced by exposure to injurious stretch-shortening cycles (iSSCs), resulted in hyperalgesia in the hind limb and changes in calcitonin-gene related peptide (CGRP) immunolabeling in the dorsal root ganglia (DRG) in young and old male rats. METHODS: Young (3months) and old (30months) male Fisher 344×BN F1 rats were anesthetized with isoflurane and the left hind limbs were exposed to 15 sets of 10 SSCs. Control animals were exposed to a single bout of SSCs of equal intensity. Sensitivity to mechanical stimulation was assessed using von Frey filaments prior to beginning the experiment, and on days 2 and 9 following exposure to iSSCs. Rats were euthanized one, 3 or 10days after the exposure. The ipsilateral DRG were dissected from the L4-5 region of the spine, along with the left tibialis anterior (LTA) muscle. RESULTS: Rats exposed to iSSCs were more sensitive to mechanical stimulation than control rats 2days after the exposure, and showed a reduction in peak force 3days after exposure. Changes in sensitivity to pressure were not associated with increases in CGRP labeling in the DRG at 3days. However, 9days after exposure to iSSCs, old rats still displayed an increased sensitivity to mechanical stimulation, and this hyperalgesia was associated with an increase in CGRP immunolabeling in the DRG. Young rats exposed to iSSC did not display a change in CGRP immunolabeling and sensitivity to mechanical stimulation returned to control levels at 10days. CONCLUSIONS: These findings suggest that hyperalgesia seen shortly after exposure to iSSC is not influenced by CGRP levels. However, in cases where recovery from injury may be slower, as it is in older rats, CGRP may contribute to the maintenance of hyperalgesia.

Subchronic exposures to fungal bioaerosols promotes allergic pulmonary inflammation in naïve mice.

BACKGROUND: Epidemiological surveys indicate that occupants of mold contaminated environments are at increased risk of respiratory symptoms. The immunological mechanisms associated with these responses require further characterization. OBJECTIVE: The aim of this study was to characterize the immunotoxicological outcomes following repeated inhalation of dry Aspergillus fumigatus spores aerosolized at concentrations potentially encountered in contaminated indoor environments. METHODS: Aspergillus fumigatus spores were delivered to the lungs of naïve BALB/cJ mice housed in a multi-animal nose-only chamber twice a week for a period of 13 weeks. Mice were evaluated at 24 and 48 h post-exposure for histopathological changes in lung architecture, recruitment of specific immune cells to the airways, and serum antibody responses. RESULT: Germinating A. fumigatus spores were observed in lungs along with persistent fungal debris in the perivascular regions of the lungs. Repeated exposures promoted pleocellular infiltration with concomitant epithelial mucus hypersecretion, goblet cell metaplasia, subepithelial fibrosis and enhanced airway hyperreactivity. Cellular infiltration in airways was predominated by CD4(+) T cells expressing the pro-allergic cytokine IL-13. Furthermore, our studies show that antifungal T cell responses (IFN-γ(+) or IL-17A(+) ) co-expressed IL-13, revealing a novel mechanism for the dysregulated immune response to inhaled fungi. Total IgE production was augmented in animals repeatedly exposed to A. fumigatus. CONCLUSIONS & CLINICAL RELEVANCE: Repeated inhalation of fungal aerosols resulted in significant pulmonary pathology mediated by dynamic shifts in specific immune populations and their cytokines. These studies provide novel insights into the immunological mechanisms and targets that govern the health outcomes that result from repeated inhalation of fungal bioaerosols in contaminated environments.

Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis.

Damage to the CNS results in a complex series of molecular and cellular changes involving the affected targets and the ensuing glial reaction. The initial gene expression events that underlie these cellular responses may serve as early biomarkers of neurotoxicity. Here, we examined gene expression profiles during the initial phase of hippocampal damage resulting from systemic exposure of rats to the organometallic neurotoxicant, trimethyltin (TMT, 8.0 mg/kg, i.p.). Using TMT as a neurodegeneration tool confers several advantages for evaluating molecular events associated with neural damage: 1) regional and cellular targets and time course of damage are known, 2) the blood-brain barrier is not compromised, which limits the contribution of blood-borne factors, e.g. immune, to neural injury responses and 3) known protein and mRNA signatures of TMT-induced neurotoxicity can be used as positive controls to validate novel expression events associated with exposure to this neurotoxicant. Using Affymetrix Gene Chip® to assess gene expression after TMT, combined with Ingenuity Pathway Analysis®, we observed changes consistent for genes known to be affected in hippocampus, while corresponding changes were not detected in cerebellum, a non-target region. In agreement with previous observations, limited changes in expression of inflammation-related genes were observed. Correlated expression profiles were found after exposure to TMT, including changes in gene ontologies associated with neurological disease, cellular assembly and maintenance, as well as signaling pathways associated with cellular stress, energy metabolism and glial activation. Selected gene changes were confirmed from each category by q-RT-PCR and immunoblot analysis. The canonical relationships identified implicate molecular pathways and processes relevant to detection of early stages of hippocampal damage in the TMT model. These observations provide new insight into early events associated with neuronal degeneration and associated glial activation that may serve as the basis for discovery and development of biomarkers of neurotoxicity.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

An investigation on the biodynamic foundation of a rat tail vibration model.

The objectives of this study are to examine the fundamental characteristics of the biodynamic responses of a rat tail to vibration and to compare them with those of human fingers. Vibration transmission through tails exposed to three vibration magnitudes (1 g, 5 g, and 10 g r.m.s.) at six frequencies (32 Hz, 63 Hz, 125 Hz, 160 Hz, 250 Hz, and 500 Hz) was measured using a laser vibrometer. A mechanical-equivalent model of the tail was established on the basis of the transmissibility data, which was used to estimate the biodynamic deformation and vibration power absorption at several representative locations on the tail. They were compared with those derived from a mechanical-equivalent model of human fingers reported in the literature. This study found that, similar to human fingers, the biodynamic responses of the rat tail depends on the vibration magnitude, frequency, and measurement location. With the restraint method used in this study, the natural frequency of the rat tail is in the range 161-368 Hz, which is mostly within the general range of human finger resonant frequencies (100-350 Hz). However, the damping ratios of the rat tail at the unconstrained locations are from 0.094 to 0.394, which are lower than those of human fingers (0.708-0.725). Whereas the biodynamic responses of human fingers at frequencies lower than 100 Hz could be significantly influenced by the biodynamics of the entire hand-arm system, the rat tail biodynamic responses can be considered independent of the rat body in the frequency range used in this study. Based on these findings it is concluded that, although there are some differences between the frequency dependences of the biodynamic responses of the rat tail and human fingers, the rat tail model can provide a practical and reasonable approach to examine the relationships between the biodynamic and biological responses at midrange to high frequencies, and to understand the mechanisms underlying vibration-induced finger disorders.

Genetic susceptibility to progressive massive fibrosis in coal miners.

Progressive massive fibrosis (PMF) is a chronic interstitial lung disease with a complex aetiology that can occur after cumulative dust exposure. A case-control study was conducted to test the hypothesis that single nucleotide polymorphisms (SNPs) within genes involved in inflammatory and fibrotic processes modulate the risk of PMF development. The study population consisted of 648 underground coal miners participating in the National Coal Workers Autopsy Study, of which 304 were diagnosed with PMF. SNPs that influence the regulation of interleukin (IL)-1, IL-6, tumour necrosis factor-alpha, transforming growth factor-beta1, vascular endothelial growth factor (VEGF), epidermal growth factor intercellular cell adhesion molecule (ICAM)-1 and matrix metalloproteinase-2 genes were determined using a 5'-nuclease real-time PCR assay. There were no significant differences in the distribution of any individual SNP or haplotype between the PMF and control groups. However, the polygenotype of VEGF +405/ICAM-1 +241/IL-6 -174 (C-A-G) conferred an increased risk for PMF (odds ratio 3.4, 95% confidence interval 1.3-8.8). The present study suggests that the examined genetic variations that help regulate inflammatory and fibrotic processes are unlikely to strongly influence susceptibility to this interstitial lung disease, although the role of vascular endothelial growth factor, intercellular cell adhesion molecule-1 and interleukin-6 polymorphisms in the development of progressive massive fibrosis may require further investigation.

Occupational and other risk factors for hand-grip strength: the Honolulu-Asia Aging Study.

BACKGROUND: In certain occupations, including farm work, workers are exposed to hazardous substances, some of which are known to be toxic to the nervous system and may adversely affect muscle strength. Measurement of hand-grip strength may be useful for detecting neurotoxic exposure. METHODS: The authors studied 3522 participants of the Honolulu Heart Program and the Honolulu-Asia Aging Study to determine whether occupational exposures to pesticides, solvents, and metals assessed at exam I (1965-68) are associated with hand-grip strength at exam IV (1991-93) and change in hand-grip strength over 25 years. Correlation, analysis of variance and covariance, and linear regression were used to evaluate the associations. RESULTS: At exam IV, participants ranged in age from 71-93 years; mean hand-grip strength was 39.6 kg at exam I and 30.3 kg at exam IV. Over 25 years, the decline in hand-grip strength was an average of 8-9 kg for all exposures. Hand-grip strength was inversely associated with age and glucose but directly associated with cognitive function, BMI, and haemoglobin level. No other exposures were associated with hand-grip strength. CONCLUSION: This study did not provide evidence that occupational exposure to pesticides, solvents, and metals adversely affected hand-grip strength in this population, but confirmed other important associations with hand-grip strength.

Lack of association between antioxidant gene polymorphisms and progressive massive fibrosis in coal miners.

BACKGROUND: Oxidative stress plays a major role in the pathogenesis of interstitial lung diseases. The antioxidant enzymes glutathione S-transferases (GST) and manganese superoxide dismutase (MnSOD) are important components of lung defence against oxidative stress, and polymorphisms in the genes which regulate their expression may represent important disease modifiers. METHODS: A matched case-control study was conducted to determine the influence of the GSTP1, GSTT1 and MnSOD polymorphisms on susceptibility to progressive massive fibrosis (PMF). Seven hundred ex-coal miners were included in the study; 350 were classified as PMF cases while 350 with a similar underground mining tenure but no clinical or histological evidence of lung disease served as controls. Genotype analysis was performed on genomic DNA, using a 5' nuclease PCR assay. RESULTS: None of the individual investigated polymorphisms and two-way gene-gene interactions had a statistically significant association with PMF. CONCLUSION: The results of this study suggest that polymorphic genotypes within the GST gene cluster and MnSOD do not affect individual susceptibility to PMF.

Impact of muscle length during stretch-shortening contractions on real-time and temporal muscle performance measures in rats in vivo.

The objective of the present study was to investigate the impact of muscle length during stretch-shortening cycles on static and dynamic muscle performance. Animals were randomly assigned to an isometric (control, Con, n = 12), a short-muscle-length (S-Inj, 1.22-2.09 rad, n = 12), or a long-muscle-length (L-Inj, 1.57-2.44 rad, n = 12) group. The dorsiflexor muscles were exposed in vivo to 7 sets of 10 stretch-shortening contractions (conducted at 8.72 rad/s) or 7 sets of isometric contractions of the same stimulation duration by using a custom-designed dynamometer. Performance was characterized by multipositional isometric exertions and positive, negative, and net work before exposure, 6 h after exposure, and 48 h after exposure to contractions. Real-time muscle performance during the stretch-shortening cycles was characterized by stretch-shortening parameters and negative, positive, and net work. The S-Inj group recovery (force difference) was similar to the Con group force difference at 48 h, whereas the L-Inj group force difference was statistically greater at 1.39, 1.57, and 1.74 rad than the Con group force difference (P < 0.05). Negative work (P < 0.05) and net work (P < 0.05) were statistically lower in the S-Inj and L-Inj groups than in the Con group 48 h after exposure to contractions. Of the real-time parameters, there was a difference in cyclic force with treatment during the stretch-shortening cycles (P < 0.0001), with the L-Inj group being the most affected. Thus longer ranges of motion result in a more profound isometric force decrement 48 h after exposure to contractions and in real-time changes in eccentric forces.

Dynamic force responses of skeletal muscle during stretch-shortening cycles.

Muscle damage due to stretch-shortening cycles (i.e., cyclic eccentric/concentric muscle actions) is one of the major concerns in sports and occupational related activities. Mechanical responses of whole muscle have been associated with damage in neural motor units, in connective tissues, and the force generation mechanism. The objective of this study was to introduce a new method to quantify the real-time changes in skeletal muscle forces of rats during injurious stretch-shortening cycles. Male Sprague Dawley rats ( n=24) were selected for use in this study. The dorsi flexor muscle group was exposed to either 150 stretch-shortening cycles ( n=12) or 15 isometric contractions ( n=12) in vivo using a dynamometer and electrical stimulation. Muscle damage after exposure to stretch-shortening cycles was verified by the non-recoverable force deficit at 48 h and the presence of myofiber necrosis. Variations of the dynamic forces during stretch-shortening cycles were analyzed by decomposing the dynamic force signature into peak force ( F(peak)), minimum force ( F(min)), average force ( F(mean)), and cyclic force ( F(a)). After the 15th set of stretch-shortening cycles, the decrease in the stretch-shortening parameters, F(peak), F(min), F(mean), and F(a), was 50% ( P<0.0001), 26% ( P=0.0055), 68% ( P<0.0001), and 50% ( P<0.0001), respectively. Our results showed that both isometric contractions and stretch-shortening cycles induce a reduction in the isometric force. However, the force reduction induced by isometric contractions fully recovered after a break of 48 h while that induced by stretch-shortening cycles did not. Histopathologic assessment of the tibialis anterior exposed to stretch-shortening cycles showed significant myofiber degeneration and necrosis with associated inflammation, while muscles exposed to isometric contractions showed no myofiber degeneration and necrosis, and limited inflammation. Our results suggest that muscle damage can be identified by the non-recoverable isometric force decrement and also by the variations in the dynamic force signature during stretch-shortening cycles.

A method for reducing adaptor misalignment when testing gloves using ISO 10819.

OBJECTIVES: International standard ISO 10819 was established in order to quantify the vibration attenuation characteristics of anti-vibration gloves. One problem that exists with the standard is possible misalignment of the palm adaptor that is placed underneath the test glove. If the adaptor becomes misaligned, the measured glove transmissibility will be lower than the actual value. A tri-axial accelerometer was installed in the adaptor and was used as the basis for providing visual feedback of the adaptor alignment to the test subjects. The objective of this study was to test the hypothesis that adaptor misalignment could be reduced by providing feedback to the test subjects. METHODS: Eight male volunteers (mean age 24.8 yr) were used in the study. Each subject performed two sets of tests: the standard ISO 10819 glove test and the modified version. Three different anti-vibration gloves were tested. Glove transmissibility and adaptor misalignment were calculated for each glove. A three-way analysis of variance was used to analyze the results. RESULTS: A comparison of the two testing methods showed that the modified glove testing method did reduce misalignment significantly, which, in turn, resulted in an increase in the measured glove transmissibility. CONCLUSIONS: The proposed method greatly improved the standard deviation of transmissibility and made the test results more consistent.

Chronic smoking enhances tachykinin synthesis and airway responsiveness in guinea pigs.

This study tests the hypothesis that the bronchial hyperreactivity induced by chronic cigarette smoke (CS) exposure involves the increased expression and release of tachykinins and calcitonin gene-related peptide (CGRP) from afferent nerve fibers innervating the airways. In guinea pigs chronically exposed to CS (20 min twice daily for 14-17 d), peak response in total lung resistance to capsaicin (1.68 microg/kg, intravenously) was significantly greater than that evoked by the same dose of capsaicin in control (air-exposed) animals. This augmented response in CS-exposed animals was abolished after treatment with CP-99994 and SR-48968, the neurokinin (NK)-1 and NK-2 receptor antagonists, suggesting the involvement of tachykinins in chronic CS-induced airway hyperresponsiveness (AHR). Further, substance P (SP)-like immunoreactivity (LI) and CGRP-LI in the airway tissue were significantly greater in the CS animals than in the control animals. Finally, beta-preprotachykinin (PPT, a splice variant from the PPT A gene encoding tachykinins including SP and NKA) messenger RNA levels as measured by in situ hybridization histochemistry displayed a significant increase in jugular ganglion neurons but not in dorsal root or nodose ganglion neurons. These data suggest that chronic CS-induced AHR is related to an increase in SP synthesis and release in jugular ganglion neurons innervating the lungs and airways.

Quantitative relationship between arsenic exposure and AP-1 activity in mouse urinary bladder epithelium.

Because of the potential of arsenic for causing cancer in humans, and of the fact of widespread environmental and occupational exposure, deriving acceptable human-limit values has been of major concern to industry as well as to regulatory agencies. Based upon epidemiological evidence and mechanistic studies, it has been argued that a non-linear dose-response model at low-level exposures is more appropriate for calculating risk than the more commonly employed linear-response models. In the present studies, dose-response relationships and recovery studies employing a cancer precursor marker, i.e., activating protein (AP)-1 DNA-binding activity, were examined in bladders of mice exposed to arsenic in drinking water and compared to histopathological changes and arsenic tissue levels in the same tissue. While AP-1 is a functionally pleomorphic transcription factor regulating diverse gene activities, numerous studies have indicated that activation of the MAP kinase pathway and subsequently increased AP-1 binding activities, is a precursor for arsenic-induced cancers of internal organs as well as the skin. We observed previously that within 8 weeks of exposure AP-1 activation occurs in urinary bladder tissue of mice exposed to arsenic in the drinking water. In the present studies, C57BL/6 mice were exposed to sodium arsenite at various concentrations in the drinking water for 8 consecutive weeks. Minimal but observable AP-1 activity occurred in bladder tissue at exposure levels below which histopathological changes or arsenic tissue accumulation was detected. Marked AP-1 DNA-binding activity only occurred at exposure levels of sodium arsenite above 20 microg/ml, where histopathological changes and accumulation of arsenic in the urinary bladder epithelium occurred. Although the experimental design did not allow statistical modeling of the entire dose-response curve, the general shape of the dose-response curve is not inconsistent with the previously proposed hypothesis that arsenic-induced cancer follows a non-linear dose-response model.

Estradiol protects against ischemic injury.

Clinical studies demonstrate that estrogen replacement therapy in postmenopausal women may enhance cognitive function and reduce neurodegeneration associated with Alzheimer's disease and stroke. This study assesses whether physiologic levels of estradiol prevent brain injury in an in vivo model of permanent focal ischemia. Sprague-Dawley rats were ovariectomized; they then were implanted, immediately or at the onset of ischemia, with capsules that produced physiologically low or physiologically high 17beta-estradiol levels in serum (10 or 60 pg/mL, respectively). One week after ovariectomy, ischemia was induced. Estradiol pretreatment significantly reduced overall infarct volume compared with oil-pretreated controls (mean+/-SD: oil = 241+/-88; low = 139+/-91; high = 132+/-88 mm3); this protective effect was regionally specific to the cortex, since no protection was observed in the striatum. Baseline and ischemic regional CBF did not differ between oil and estradiol pretreated rats, as measured by laser Doppler flowmetry. Acute estradiol treatment did not protect against ischemic injury. Our finding that estradiol pretreatment reduces injury demonstrates that physiologic levels of estradiol can protect against neurodegeneration.

Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei.

The timing of the preovulatory surge of LH in female rodents is tightly coupled to the environmental light/dark cycle. This coupling is mediated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). Studies indicate that vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), which are synthesized in the SCN, transmit circadian information from the SCN to GnRH neurons, thereby regulating the timing of the LH surge. However, to date, the rhythmic expression of these two peptides in the SCN has only been examined in males. The pattern of VIP expression in males is difficult to reconcile with its role in the LH surge. The purpose of the present study was to assess the rhythm of VIP messenger RNA (mRNA) levels in the SCN of female rats under several endocrine conditions. We compared this rhythm to that in males and to AVP mRNA rhythms in all experimental groups. In all groups of females, VIP mRNA levels were rhythmic, with peak expression occurring during the light phase and a nadir occurring during the dark phase. The rhythm was approximately 12 h out of phase compared with that in males. The rhythmic expression of AVP mRNA in the SCN was virtually identical in all groups of animals. Based on these results, we conclude that 1) the rhythm of VIP seen in the SCN of females during the day may serve as a facilitory signal from the SCN to GnRH neurons; 2) the sex-specific pattern of VIP mRNA does not depend on estradiol; and 3) AVP gene expression within the SCN is not sexually differentiated or altered by estradiol.

Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats.

Our laboratory has shown that the ability of the suprachiasmatic nuclei (SCN) to regulate a number of rhythmic processes may be compromised by the time females reach middle age. Therefore, we examined the effects of aging on the rhythmic expression of two neuropeptides synthesized in the SCN, vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), using in situ hybridization. Because both VIP and AVP are outputs of the SCN, we hypothesized that age-related changes in rhythmicity are associated with alterations in the patterns of expression of these peptides. We found that VIP mRNA levels exhibited a 24 hr rhythm in young females, but by the time animals were middle-aged, this rhythm was gone. The attenuation of rhythmicity was associated with a decline in the level of mRNA per cell and in the number of cells in the SCN producing detectable VIP mRNA. AVP mRNA also showed a robust 24 hr rhythm in young females. However, in contrast to VIP, the AVP rhythm was not altered in the aging animals. The amount of mRNA per cell and the number of cells expressing AVP mRNA also was not affected with age. Based on these results we conclude that (1) various components of the SCN are differentially affected by aging; and (2) age-related changes in various rhythms may be attributable to changes in the ability of the SCN to transmit timing information to target sites. This may explain why the deterioration of various rhythmic processes occurs at different rates and at different times during the aging process.

Brain region-specific regulation of luteinizing hormone-releasing hormone messenger ribonucleic acid in the male ferret: interactions between pubertal maturation and testosterone.

This study examined the regulation of LHRH messenger RNA (mRNA) during pubertal maturation and by testosterone in male ferrets. Prepubertal and postpubertal ferrets were either intact or were castrated and treated with daily injections of oil or 5 mg/kg testosterone propionate for 14 days. In situ hybridization for LHRH mRNA was performed using an 35S-labeled 48-base oligonucleotide complementary to the human LHRH-coding region. Computerized image analysis was performed on cells in the preoptic area, retrochiasmatic area, arcuate nucleus (ARC), and median eminence; cells were classified as labeled if the number of pixels representing silver grains over the cell was 5 or more times the number of background silver grain pixels. Both pubertal maturation of intact males and castration of prepubertal males resulted in an increase in the number of labeled cells in the ARC. These effects were not observed in any of the other three brain regions, suggesting that ARC LHRH-producing neurons are of primary importance in the presumed increase in LHRH release that occurs as a consequence of either pubertal maturation or castration of prepubertal males. Castration of adults did not increase the number of labeled cells in any brain area, but resulted in an increase in silver grains per labeled cell only in the preoptic area. Thus, LHRH mRNA is regulated during puberty primarily in the ARC, and the particular cell group in which LHRH mRNA is most strongly regulated by testosterone changes with pubertal maturation.

Aging of the female reproductive system: a window into brain aging.

The menopause marks the permanent end of fertility in women. It was once thought that the exhaustion of ovarian follicles was the single, most important explanation for the transition to the menopause. Over the past decade, this perception has gradually changed with the realization that there are multiple pacemakers of reproductive senescence. We will present evidence that lends credence to the hypothesis that the central nervous system is a critical pacemaker of reproductive aging and that changes at this level contribute to the timing of the menopause. Studies demonstrate that an increasing de-synchronization of the temporal order of neuroendocrine signals may contribute to the accelerated rate of follicular loss that occurs during middle age. We suggest that the dampening and destabilization of the precisely orchestrated ultradian, circadian, and infradian neural signals lead to miscommunication between the brain and the pituitary-ovarian axis. This constellation of hypothalamic-pituitary-ovarian events leads to the inexorable decline of regular cyclicity and heralds menopausal transition.