Evaluation of four α-diketones for toll-like receptor-4 (TLR-4) activation in a human transfected cell line.

Toll-like receptor-4 (TLR-4) activity is upregulated in persons with fibrotic lung diseases secondary to chronic inflammatory conditions like Crohn's disease and rheumatoid arthritis. We hypothesized that α-diketones associated with fixed obstructive lung disease may activate TLR-4. We utilized a human embryonic kidney cell assay (HEK293) with human TLR-4 receptors to test for potential activation effects of 2,3-butandeione, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at test concentrations of 1, 10, 100, and 1000 µM. The assay detects NF-κB-induced expression of secreted alkaline phosphatase measured after 16 h incubation by a UV-VIS Spectrometer at 650 nm. Escherichia coli K12 lipopolysaccharide (LPS) at 0.5 ng/mL served as a positive control and was added with each test compound to evaluate combined effects. None of the tested α-diketones were found to exhibit cytotoxicity, agonism, or synergistic effects with LPS in the human TLR-4 assay up to 1000 µM. Screening of 2,3-butanedione for agonist activity using the HEK assay with mouse TLR-4 receptors exhibited cytotoxicity at 1000 µM, but no agonist activity. We conclude that the tested α-diketones at relatively high concentrations in vitro do not exhibit TLR-4 agonist or synergistic activity and, therefore, apparently do not directly induce inflammasome activation through this pathway in humans or mice.

History and evolution of warning labels for automotive friction products.

There have been claims over the years that asbestos-containing product manufacturers did not sufficiently warn end users early enough regarding the potential health hazards associated with their products (1930s-1990s). To address this issue, we compared the content of the warnings associated with asbestos-containing friction products (brakes, clutches, and gaskets) manufactured by the US automotive industries to what was expected by regulatory agencies during the time period in which an understanding of asbestos health hazards was being developed. We ended our evaluation around 1990, since asbestos-containing manufacturer supplied automotive products were functionally removed from commerce by 1985 in the United States. We assessed the warnings issued in users' manuals, technical service bulletins, product packaging materials, and labels placed on products themselves. Based on our evaluation, regulatory agencies had no guidelines regarding specific warning language for finished friction products, particularly when a product contained encapsulated asbestos fibers (i.e., modified by a bonding agent). Even today, federal regulations do not require labeling on encapsulated products when, based on professional judgment or sampling, user exposure is not expected to exceed the OSHA PEL. We concluded that, despite limited regulatory guidance, the US automotive industry provided adequate warnings with regards to its friction products.

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice.

Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury.

Inorganic cobalt supplementation: prediction of cobalt levels in whole blood and urine using a biokinetic model.

Soluble cobalt (Co) supplements with recommended daily doses up to 1000 µg Co/day are increasingly being marketed to consumers interested in healthy living practices. For example, some athletes may consider using Co supplements as blood doping agents, as Co is known to stimulate erythropoesis. However, the distribution and excretion kinetics of ingested Co are understood in a limited fashion. We used a Co-specific biokinetic model to estimate whole blood and urine Co levels resulting from oral exposure or ingestion of Co in amounts exceeding typical dietary intake rates. Following 10 days of Co supplementation at a rate of 400 to 1000 µg/day, predicted adult Co concentrations range from 1.7 to 10 µg/L in whole blood, and from 20 to 120 µg/L in urine. Chronic supplementation (≥ 1 year) at a rate of 1000 µg Co/day is predicted to result in blood levels of 5.7 to 13 µg/L, and in urine levels from 65 to 150 µg/L. The model predictions are within those measured in humans following ingestion of known doses. The methodology presented in this paper can be used to predict urinary or blood Co levels following acute or chronic occupational incidental ingestion, medicinal therapy, supplemental intake, or other non-occupational exposures.

Evaluation of tremolite asbestos exposures associated with the use of commercial products.

Tremolite is a noncommercial form of amphibole mineral that is present in some chrysotile, talc, and vermiculite deposits. Inhalation of asbestiform tremolite is suspected to have caused or contributed to an increased incidence of mesothelioma in certain mining settings; however, very little is known about the magnitude of tremolite exposure that occurred at these locations, and even less is known regarding tremolite exposures that might have occurred during consumer use of chrysotile, talc, and vermiculite containing products. The purpose of this analysis is to evaluate the exposure-response relationship for tremolite asbestos and mesothelioma in high exposure settings (mining) and to develop estimates of tremolite asbestos exposure for various product use scenarios. Our interpretation of the tremolite asbestos exposure metrics reported for the Thetford chrysotile mines and the Libby vermiculite deposits suggests a lowest-observed-adverse-effect level (LOAEL) for mesothelioma of 35-73 f/cc-year. Using measured and estimated airborne tremolite asbestos concentrations for simulated and actual product use, we conservatively estimated the following cumulative tremolite asbestos exposures: career auto mechanic: 0.028 f/cc-year; non-occupational use of joint compound: 0.0006 f/cc-year; non-occupational use of vermiculite-containing gardening products: 0.034 f/cc-year; home-owner removal of Zonolite insulation: 0.0002 f/cc-year. While the estimated consumer tremolite exposures are far below the tremolite LOAELs derived herein, this analysis examines only a few of the hundreds of chrysotile- and talc-containing products.

Chronic, low-dose prenatal exposure to methylmercury impairs motor and mnemonic function in adult C57/B6 mice.

Methylmercury (MeHg) has cytotoxic effects on animals and humans, and a major target organ for MeHg is the central nervous system (CNS). It is well known that the developing CNS is extremely vulnerable to MeHg-induced changes in comparison to the mature brain. Most studies have concentrated on the direct effects of high levels of prenatal MeHg exposure. Surprisingly, behavioral outcomes found in adult offspring exposed developmentally to the neurotoxic effects of chronic, low-dose mercury more akin to ingestion in humans are not well characterized. The objective of this study was to determine whether such exposure produces deleterious effects on behavior in adult mice, including motor/coordination abilities, overall activity and mnemonic function. Developing mouse fetuses were exposed in utero during gestational days 8-18 by giving pregnant C57Bl/6J female mice food containing MeHg at a daily dose of 0.01 mg/kg body weight. Adult mice prenatally exposed to MeHg exhibited significant deficits in motor abilities, coordination, and overall activity, as measured by rotarod, footprint analysis and open field. In addition, MeHg-exposed mice were impaired with respect to reference memory but not in a visible, cued version of the Morris water maze task. These results indicate that prenatal exposure to the lowest dose of MeHg examined to date can have long-lasting motor and cognitive consequences on adult offspring. These findings have far reaching implications related to putative safe levels of MeHg ingestion, particularly during pregnancy, and increasing rates of cognitive and psychological disorders (e.g. attention hyperactivity deficit disorder, autism) in our society.

Changes in biochemical processes in cerebellar granule cells of mice exposed to methylmercury.

At postnatal day 34, male and female C57BL/6J mice were exposed orally once a day to a total of five doses totaling 1.0 or 5.0 mg/kg of methylmercuric chloride or sterile deionized water in moistened rodent chow. Eleven days after the last dose cerebellar granule cells were acutely isolated to measure reactive oxygen species (ROS) levels and mitochondrial membrane potential using CM-H(2)DCFDA and TMRM dyes, respectively. For visualizing intracellular calcium ion distribution using transmission electron microscopy, mice were perfused 11 days after the last dose of methylmercury (MeHg) using the oxalate-pyroantimonate method. Cytosolic and mitochondrial protein fractions from acutely isolated granule cells were analyzed for cytochrome c content using Western blot analysis. Histochemistry (Fluoro-Jade dye) and immunohistochemistry (activated caspase 3) was performed on frozen serial cerebellar sections to label granule cell death and activation of caspase 3, respectively. Granule cells isolated from MeHg-treated mice showed elevated ROS levels and decreased mitochondrial membrane potential when compared to granule cells from control mice. Electron photomicrographs of MeHg-treated granule cells showed altered intracellular calcium ion homeostasis ([Ca(2+)](i)) when compared to control granule cells. However, in spite of these subcellular changes and moderate relocalization of cytochrome c into the cytosol, the concentrations of MeHg used in this study did not produce significant neuronal cell death/apoptosis at the time point examined, as evidenced by Fluoro-Jade and activated caspase 3 immunostaining, respectively. These results demonstrate that short-term in vivo exposure to total doses of 1.0 and 5.0 mg/kg MeHg through the most common exposure route (oral) can result in significant subcellular changes that are not accompanied by overt neuronal cell death.

Coordination deficits induced in young adult mice treated with methylmercury.

Male and female C57BL/6J mice starting at postnatal (P) day 34 were exposed orally to five divided doses totaling 1.0 or 5.0 mg/kg of methylmercury (MeHg; given as methylmercuric chloride) or sterile deionized water in moistened rodent chow. After a 5-day waiting period, control and MeHg-treated mice were subjected to a standard battery of behavior tests for balance and motor coordination. Latency to falling on the accelerating rota-rod was significantly decreased in 5.0 mg/kg MeHg-exposed mice when compared to control mice. In the open field, horizontal exploration with respect to total distance traveled during the first 5 min on the first test day was significantly reduced in 1.0 mg/kg MeHg-exposed mice when compared to control mice. Rearing activity was not affected by MeHg treatment. In the footprint analysis, angle of foot placement measured in 1.0 mg/kg MeHg-treated mice was significantly greater compared to control mice. Base stance and stride length were unaffected by MeHg treatment. On the vertical pole test, 10 mice from each treatment group fell off the pole during the time the pole was shifted from a horizontal position to a vertical position, whereas none of the control mice fell. These results indicate that short-term, low to moderate doses of MeHg in young adult mice can be detrimental to motor coordination and balance.

Assessment of mercury concentrations in mouse brain using different routes of administration and different tissue preparations.

ABSTRACT The objective of this study was to determine the best method for preparing brain tissue for mercury analysis from mice exposed to methylmercury either through subcutaneous (SC) injection or through ingestion. C57BL/6 J mice at postnatal day 29 were exposed to 0.0 or 5.0 mg/kg methylmercuric chloride (MMC) given SC or through food containing MMC. Eighteen mice received vehicle (sodium bicarbonate; SC) and 18 additional mice received 5.0 mg/kg MMC (SC). Whole brain tissue was prepared using one of four tissue preparation methods: rapid freezing, saline perfusion, 4% paraformaldehyde perfusion fixation, or 4% paraformaldehyde immersion fixation. Brains from vehicle-treated mice exhibited minimal levels of mercury (0.0007 to 0.0018 ppm) in all preparation methods. Mercury content in rapidly frozen control brains differed statistically from immersion-fixed control brain tissue. There was no significant difference in mercury content from mice given 5.0 mg/kg MMC (SC) in all preparation methods (0.2660 to 0.3650 ppm). Additional mice were divided into groups of six mice each: single oral dose of 5.0 mg/kg MMC; total oral dose of 5.0 mg/kg MMC divided into five doses; and vehicle only. Forebrain (0.3243 ppm) and hindbrain (0.1908 ppm) mercury content in MMC-treated mice given multiple doses was 10 times higher than in brain tissue from mice given a single 5.0 mg/kg dose. Brain mercury content following administration of 5.0 mg/kg MMC via the oral route (0.5354 ppm) differed statistically from the SC route (0.3430 ppm). In conclusion, different tissue preparation methods do not significantly affect brain mercury content, but route of administration and dosing regimen can influence total brain mercury content.

Response of larval Xenopus laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal morphology.

Larval Xenopus laevis were exposed to one of four concentrations of atrazine (0, 1, 10, or 25 microg/L, 11 replicate tanks per treatment, 60-65 larvae per replicate) dissolved in an artificial pond water (frog embryo teratogenesis assay- Xenopus [FETAX]) medium beginning 48 h after hatching until the completion of metamorphosis. Separate groups of larvae (six replicate tanks per treatment, 60-65 larvae per replicate) were exposed to estradiol (100 microg/L), dihydrotestosterone (100 microg/L), or ethanol vehicle control dissolved in FETAX medium. None of the treatments affected posthatch mortality, larval growth, or metamorphosis. There were no treatment effects on sex ratios except for estradiol, which produced a greater percentage of female offspring. Exposure to either estradiol or 25 microg atrazine/L increased the incidence of intersex animals based on assessment of gonadal morphology. Atrazine did not reduce the size of the laryngeal dilator muscle, a sexually dimorphic muscle in this species. We conclude that environmentally relevant concentrations of atrazine do not influence metamorphosis or sex ratios and do not inhibit sexually dimorphic larynx growth in X. laevis. The incidence of atrazine-induced intersex animals was small (<5%) and occurred only at the greatest concentration of atrazine tested, a concentration that is rarely observed in surface waters in the United States.

In utero and lactational exposure to ammonium perchlorate in drinking water: effects on developing deer mice at postnatal day 21.

The effects of in utero and lactational exposure to ammonium perchlorate (AP), a component of rocket fuel and a thyroid toxicant, on developing deer mice (Peromyscus maniculatus) were evaluated. Breeding pairs were dosed continuously with 0, 1 nM, 1 micro M, or 1 mM AP in drinking water, from cohabitation until pups were euthanized at postnatal day (PND) 21. Pups from the second litter were used for evaluation in this study. No significant differences were observed in any analysis performed when litter means were used in statistical analysis. All reported significant differences occurred when statistical analysis was performed on individual pup data. Body weights were significantly different between treatments at PND 5 and PND 20, with the 1- micro M body weights being lower than that of controls. Body weight and liver weight in the 1-mM group were significantly higher than the 1- micro M weights at PND 21 when analyzed by analysis of variance (ANOVA). However, there were no significant differences in liver weights when analyzed with body weight as the covariate. Heart weights were significantly different between males and females. Male heart weights in the 1- microM and 1-mM groups were significantly lower than in controls when analyzed by analysis of covariance (ANCOVA) with body weight as the covariate. Litter size and survival percentage were not significantly different among treatments. Although significant differences were observed only when the individual pup was used as the experimental unit, these data suggest that AP exposure at different concentrations may variably alter body weight and male heart weight during mammalian development.

Effects of in utero and lactational ammonium perchlorate exposure on thyroid gland histology and thyroid and sex hormones in developing deer mice (peromyscus maniculatus) through postnatal day 21.

Thyroid gland hormone levels and histology and sex hormone levels in developing deer mice (Peromyscus maniculatus) were measured following in utero and lactational exposure to ammonium perchlorate (AP), a component of rocket fuel and a thyroid toxicant. Breeding pairs were dosed continuously with 0, 1 nM, 1 micro M, or 1 mM concentrations of AP in drinking water from the time of cohabitation until pups from the third litter were weaned. Pups from the second litter were used for evaluation in this study. The active (colloid-containing) thyroid follicle number per unit area was significantly different between treatment groups. The 1-nM and 1-mM treatment groups had significantly fewer active follicles per unit area than did controls. The 1-mM treatment group also had significantly fewer active follicles than the 1- micro M and the 1-nM treatment groups. Total T(4) concentrations were significantly increased in the 1-nM and 1- micro M groups compared to the controls. No significant difference was observed in total T(3) concentrations. None of the 1-mM plasma had concentrations of total testosterone above the detection limit, and only one of the 1- micro M samples was above the detection limit of the assay. All estradiol concentrations were below the detection limits of the assay. In contrast to the situation in adult rodents, it appears that AP increases thyroid hormone production in developing deer mice and produces variable effects with increasing concentrations.