Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30170/1/0000555.pd

Tetrachlorodibenzo-p-dioxin alters rat hypothalamic endorphin and mu opioid receptors

The present study was undertaken to assess if hypothalamic [beta]-endorphin ([beta]E) and/or brain mu opioid receptors are associated with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (50 [mu]g/kg)-induced hypophagia and body weight decline in rats. Hypothalamic [beta]E concentrations were initially increased to 166% of controls on day 1, and then were depressed to 39% and 49% of control values on days 2 and 3, respectively. Brain mu opioid receptor number was increased 60% in TCDD-treated rats at day 3 without a change in the binding affinity. Food-restricted rats did not exhibit changes in hypothalamic [beta]E concentrations or brain mu opioid receptor number. These results indicate that TCDD causes early perturbations in hypothalamic [beta]E concentrations and brain mu receptor number, which may contribute to the mechanisms by which TCDD leads to decreased food intake and progressive weight loss.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29160/1/0000205.pd

Chronic ethanol consumption depresses hypothalamic-pituitary-adrenal function in aged rats

In separate experiments, nine (n=20) and fifteen (n=12) month old rats were treated with either 6% ethanol or 12% sucrose (to balance caloric intake) in the drinking water to examine the effect of chronic ethanol consumption on the hypothalamic-pituitary-adrenal axis of aged rats. Rats were maintained on these treatment regimens for thirty days and were killed by decapitation. Blood was collected and plasma concentrations of adrenocorticotropin (ACTH) and corticosterone were determined by radioimmunoassay. Adrenal glands were cleaned, quartered and used to test in vitro responsiveness to ACTH. Anterior pituitary glands from all 15 month old rats and one half of the nine month old rats were collected, frozen and extracted for measurement of tissue ACTH concentration. The remaining anterior pituitary glands from the nine month old rats were challenged with corticotropin releasing hormone (CRH) to test in vitro responsiveness. In nine month old rats, chronic ethanol consumption decreased plasma ACTH and corticosterone (P In vitro responsiveness of the adrenal gland to ACTH in nine month old rats consuming ethanol was unchanged (P > 0.05). Plasma ACTH and corticosterone concentrations were also decreased in 15 month old rats chronically consuming ethanol (P 0.05). The results of these experiments indicate that chronic ethanol consumption decreases hypothalamic-pituitary-adrenal function in aged rats.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29673/1/0000762.pd

Stimulation by voluntary exercise of adrenal glucocorticoid secretion in mature female hamsters

The possibility that habitual voluntary running induces a chronic change in adrenal glucocorticoid synthesis and secretion was examined in freely running mature female hamsters, in whom this behavior accelerates growth, reduces body fat levels, and elevates core temperature. Hamsters were free to run on horizontal discs or in vertical wheels between 32 and 80 days, in 14L:10D or in 10L:14D photoperiods, and at the end of this period, corticosterone and cortisol steroidogenesis and serial plasma corticosterone concentrations during day and night were used as measures of the chronic stimulation of adrenal cortical activity. Habitual voluntary running significantly increased steroidogenesis of both glucocorticoids and plasma corticosterone concentrations and alone accounted for all the variance in enhanced synthesis and secretion of corticosterone. Acute exercise and/or the nocturnal phase of circadian period enhanced the chronic stimulatory effects of exercise on cortiol. Despite its voluntary and apparently stress-free nature, running induces chronic increases in basal glucocorticoid secretion in mature female hamsters. Putative oversecretion of corticotropin releasing factor in freely running hamsters could account for increased steriodogenesis, acceleration of growth, reduced body fat levels, and core temperature elevation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30139/1/0000516.pd

Peroxidative reactions of diversozymes1

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154504/1/fsb2010004005.pd

Selective enumeration strategies for Brevundimonas diminuta from drinking water

Brevundimonas diminuta is used as a control organism for validating the efficiency of water filtration systems. Since these protocols use nonselective growth media, heterotrophic plate count bacteria (HPCs) indigenous to the water distribution system may interfere with B. diminuta enumeration, thus leading to inaccurate assessment of the filter\u27s microbial reduction capability. This could negatively impact public health as unsafe drinking water may be produced. This study was conducted to evaluate different potential routes for selective enumeration of B. diminuta in drinking water. B. diminuta\u27s biochemical and molecular relationships to HPCs recovered from a laboratory drinking-water system were investigated. Of the 24 HPC morphotypes recovered, members of the Alpha- and Betaproteobacteria were most commonly identified. Based on comparisons of catabolic profiles (generated by the Biolog system) using principal component analysis, B. diminuta possessed similar metabolic patterns to several of the Alphaproteobacteria (Sphingomonas and Caulobacter), indicating that development of a selective medium based solely on carbon source was not feasible. Antibiotic susceptibility profiles revealed that the HPCs were least resistant to kanamycin, making it a candidate for future selective applications. Sequence comparisons of partial 16S rRNA sequences did not reveal any distinct similarities. However, basic local alignment search tool (BLAST) alignments of the gyrB and rpoD sequences for B. diminuta did show uniqueness, with the next closest match being to Caulobacter (88% and 79% similarity, respectively). Future investigation will focus on applying molecular assays, such as fluorescent in situ hybridization and quantitative real-time polymerase chain reaction (PCR), and incorporating an antibiotic marker or expressed fluorescent protein into the wild-type strain of B. diminuta for selective enumeration of B. diminuta. © 2010 Society for Industrial Microbiology

TCDD alters pituitary-adrenal function II: Evidence for decreased bioactivity of ACTH

The present study assessed the ability of primary cultures of rat anterior pituitary cells to secrete bioactive ACTH in the presence of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The bioactivity of the secreted pituitary cell ACTH was determined by its ability to stimulate secretion of corticosterone from primary cultures of rat adrenal cells. ACTH from basal or CRH stimulated pituitary cells treated with TCDD was found to be less capable of stimulating corticosterone secretion from primary rat adrenal cell cultures than equimolar concentrations of ACTH purchased from a commercial supplier. Corticosterone secretion from adrenal cell cultures treated with ACTH from basal or CRH stimulated pituitary cell cultures exposed to TCDD was decreased by 60 and 70%, respectively. The decreased ability to stimulate corticosterone secretion can be overcome when extracts of ACTH from pituitary cell cultures treated with TCDD are supplemented with commercial ACTH. These findings indicate that TCDD may alter the bioactivity of secreted ACTH from the anterior pituitary gland.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30482/1/0000110.pd

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating Brevundimonas diminuta in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100-200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 103 colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5†6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid ( \u3c 4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems. © 2010 Society for Industrial Microbiology

TCDD alters pituitary-adrenal function I: Adrenal responsiveness to exogenous ACTH

Plasma ACTH concentrations in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats (50 [mu]g/kg; single, oral dose) were 2.1-, 2.1-, 2.9-, 1.7-, 1.5-, 2.0- and 3.0-fold greater than control values, respectively, at days 1, 3, 5, 7, 10, and 14. At days 1 and 5 plasma corticosterone concentrations were increased 5.1- and 8.0-fold, respectively; whereas, at days 10 and 14 they were depressed to values of 50% and 39% of controls, respectively. Adrenal glands were exised from rats treated with TCDD and corticosterone production was assessed. Basal corticosterone concentrations produced by treated adrenals were depressed to 81%, 72%, and 71% of control values at days 5, 7, and 14, respectively. Corticosterone secretion by ACTH stimulated adrenals was equivalent to controls. These findings suggest that TCDD exposure decreases the bioactivity of the ACTH secreted by the anterior pituitary.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30481/1/0000109.pd