Associations between air pollution and cardio-respiratory physiological measures in older adults exercising outdoors.

We examined whether exercising indoors vs. outdoors reduced the cardio-respiratory effects of outdoor air pollution. Adults ≥55 were randomly assigned to exercise indoors when the Air Quality Health Index was ≥5 and outdoors on other days (intervention group, n = 37), or outdoors everyday (control group, n = 35). Both groups completed cardio-respiratory measurements before and after exercise for up to 10 weeks. Data were analyzed using linear mixed effect regression models. In the control group, an interquartile range increase in fine particulate matter (PM2.5) was associated with increases of 1.4% in heart rate (standard error (SE) = 0.7%) and 5.6% (SE = 2.6%) in malondialdehyde, and decreases of 5.6% (SE = 2.5%) to 16.5% (SE = 7.5%) in heart rate variability measures. While the hypothesized benefit of indoor vs. outdoor exercise could not be demonstrated due to an insufficient number of intervention days (n = 2), the study provides evidence of short-term effects of air pollution in older adults. ISRCTN #26552763.

Exposure to Low Doses of Dechlorane Plus Promotes Adipose Tissue Dysfunction and Glucose Intolerance in Male Mice.

The prevalence of type 2 diabetes (T2D) continues to increase worldwide. It is well established that genetic susceptibility, obesity, overnutrition and a sedentary life style are risk factors for the development of T2D. However, more recently, studies have also proposed links between exposure to endocrine-disrupting chemicals (EDCs) and altered glucose metabolism. Human exposure to environmental pollutants that are suspected to have endocrine disruptor activity is ubiquitous. One such chemical is Dechlorane Plus (DP), a flame retardant, that is now detected in humans and the environment. Here we show that exposure of mice to low, environmentally relevant doses of DP promoted glucose intolerance in mice fed a high-fat diet independent of weight gain. Furthermore, DP had pronounced effects on the adipose tissue, where it induced the development of hypertrophied white adipose tissue (WAT), and increased serum levels of resistin, leptin, and plasminogen activator inhibitor-1. In addition, DP exposure induced "whitening" of brown adipose tissue (BAT), and reduced BAT uncoupling protein 1 expression. Importantly, some of these effects occurred even when the mice were fed a regular, low-fat, diet. Finally, WAT adipogenic markers were reduced with DP treatment in the WAT. We also show that DP directly inhibited insulin signaling in murine adipocytes and human primary subcutaneous adipocytes in vitro. Taken together, our results show that the exposure to low and environmentally relevant levels of DP may contribute to the development of T2D.

Effect of industrial point-source air pollutants on fractional exhaled nitric oxide in healthy volunteers.

BACKGROUND: Few studies have examined the effects of industrial, fixed-site sources of air pollution on lung inflammation in nearby residents. We investigated the effects of short-term exposure to ambient air near a steel plant on the fractional exhaled concentration of nitric oxide (FeNO), a measure of airway inflammation, in healthy volunteers. METHODS: A cross-over study design was used. Fifty-nine non-smoking participants (mean age 24 years) were randomly assigned to each of two 5-day exposure scenarios: breathing ambient air adjacent to a steel plant or 5 km away at a college campus site. FeNO and on-site air pollutants were measured daily. Mixed effects linear regression models were used for data analysis, adjusting for sex, temperature, humidity and day of week. RESULTS: Compared with the college site, PM 2.5, ultrafine PM, SO2, NO2 and CO levels were significantly greater near the steel plant. FeNO was 15.3% (95% CI, 6.6%, 24.8%) higher near the plant compared to the college site. CONCLUSIONS: Exposure to ambient air near a steel plant was associated with increased airway inflammation as measured by exhaled nitric oxide.

Comparison of tris(2-ethylhexyl) phosphate and di(2-ethylhexyl) phosphoric acid toxicities in a rat 28-day oral exposure study.

Tris(2-ethylhexyl) phosphate (TEHP, CAS no. 78-42-2) is a plasticizer and a flame retardant, while di(2-ethylhexyl) phosphoric acid (DEHPA, CAS no. 298-07-7) is an oil additive and extraction solvent. Publicly-available information on repeated exposure to these two related organophosphate compounds is fragmentary. Hence, adult male and female Fischer rats were exposed to TEHP (300, 1000 and 3000 mg/kg body weight [BW]/day) or DEHPA (20, 60 and 180 mg/kg BW/day) by gavage for 28 consecutive days, to assess and compare their toxicities. Although significantly impaired BW gains and evidence of TEHP enzymatic hydrolysis to DEHPA were observed only in males, exposures to the highest TEHP and DEHPA doses often resulted in similar alterations of hematology, serum clinical chemistry and liver enzymatic activities in both males and females. The squamous epithelial hyperplasia and hyperkeratosis observed in the non-glandular forestomach of rats exposed to the middle and high DEHPA doses were most likely caused by the slightly corrosive nature of this chemical. Although tubular degeneration and spermatid retention were observed only in the testes of males exposed to the highest TEHP dose, numerous periodic acid-Schiff stained crystalline inclusions were observed in testis interstitial cells at all TEHP dose levels. No-observed-adverse-effect levels for TEHP and DEHPA are proposed, but the lower serum pituitary hormone levels resulting from TEHP and DEHPA exposures and the perturbations of testicular histology observed in TEHP-treated males deserve further investigation. Improved characterization of the toxicity of flame retardants will contribute to better informed substitution choices for legacy flame retardants phased-out over health concerns.

Toxicity of Flame Retardant Isopropylated Triphenyl Phosphate: Liver, Adrenal, and Metabolic Effects.

The use of organophosphates phosphate flame retardants, particularly isopropylated triphenyl phosphate (IPTPP), has increased in recent years as replacements for polybrominated diphenyl ethers. This is despite limited understanding of the hazards of IPTPP. To examine the general and endocrine toxicity of IPTPP, adult Wistar rats were fed for 90 days on diets containing IPTPP estimated to deliver daily doses of 5 to 140 mg/kg/d. Exposure to IPTPP caused a dose-related increase in liver and adrenal gland weight in both sexes. Cells in the zona fasciculate (ZF) of the adrenal cortex were observed to be filled with droplets that stained with Nile red, suggesting they contained neutral lipid. Despite marked structural changes, there was no change in basal or stress-induced serum levels of their major secreted ZF product corticosterone (B), suggesting cell function was not altered. There were no effects on responses to glucose or insulin challenge, but serum levels of fructosamine were elevated by IPTPP exposure, suggesting a slight tendency of exposed animals to be hyperglycemic. Serum levels of total cholesterol and high-density lipoprotein cholesterol were significantly elevated in both sexes at the 2 highest doses. This study demonstrates that IPTPP exposure causes hypertrophy and neutral lipid accumulation in adrenal cortex ZF cells but does not result in impaired B production.

Cardiorespiratory Effects of Air Pollution in a Panel Study of Winter Outdoor Physical Activity in Older Adults.

OBJECTIVE: The aim of this study was to assess cardiorespiratory effects of air pollution in older adults exercising outdoors in winter. METHODS: Adults 55 years of age and older completed daily measurements of blood pressure, peak expiratory flow and oximetry, and weekly measurements of heart rate variability, endothelial function, spirometry, fraction of exhaled nitric oxide and urinary oxidative stress markers, before and after outdoor exercise, for 10 weeks. Data were analyzed using linear mixed effect models. RESULTS: Pooled estimates combining 2014 (n = 36 participants) and 2015 (n = 34) indicated that an interquartile increase in the Air Quality Health Index was associated with a significant (P < 0.05) increase in heart rate (0.33%) and significant decreases in forced expiratory volume (0.30%), and systolic (0.28%) and diastolic blood pressure (0.39%). CONCLUSION: Acute subclinical effects of air pollution were observed in older adults exercising outdoors in winter.

Cardio-Respiratory Effects of Air Pollution in a Panel Study of Outdoor Physical Activity and Health in Rural Older Adults.

OBJECTIVE: To examine cardio-respiratory effects of air pollution in rural older adults exercising outdoors. METHODS: Adults 55 and over completed measurements of blood pressure, peak expiratory flow and oximetry daily, and of heart rate variability, endothelial function, spirometry, fraction of exhaled nitric oxide and urinary oxidative stress markers weekly, before and after outdoor exercise, for 10 weeks. Data were analyzed using linear mixed effect models. RESULTS: Pooled estimates combining 2013 (n = 36 participants) and 2014 (n = 41) indicated that an interquartile increase in the air quality health index (AQHI) was associated with a significant (P < 0.05) increase in heart rate (2.1%) and significant decreases in high frequency power (-19.1%), root mean square of successive differences (-9.5%), and reactive hyperemia index (-6.5%). CONCLUSIONS: We observed acute subclinical adverse effects of air pollution in rural older adults exercising outdoors.

Gestational and Lactational Exposure to an Environmentally-Relevant Mixture of Brominated Flame Retardants: Effects on Neurodevelopment and Metabolism.

BACKGROUND: Developmental exposure to brominated flame retardants (BFRs), including polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCDD), has been associated with impaired neurodevelopment and some symptoms of metabolic syndrome. However, there are inconsistencies in studies reporting neurodevelopmental effects with studies of pure substances more likely to report effects than studies of technical products. In addition, the influence of early BFR exposures on later development of metabolic disease-like symptoms has not been investigated. This study examined the effects of perinatal exposure to an environmentally relevant mixture of BFRs based on relative levels observed in house dust, on several markers of neurodevelopment and metabolism in offspring. METHODS: Sprague-Dawley female rats were fed a diet estimated to deliver daily doses of 0, 0.06, 20, or 60 mg/kg of a mixture of PBDEs and HBCDD from before mating to weaning. Offspring were weaned to control diet and subjected to neurobehavioral and metabolic assessments. RESULTS: Exposure to BFRs decreased vertical movement in at postnatal day (PND) 32 and increased time to emerge to a lighted area on PND 105 in offspring of both sexes. Although early life exposure to the BFR mixture did not impact measures of glucose or insulin action, male offspring had significantly decreased fat pad weights at PND 46. Total cholesterol was increased in male and female offspring exposed to the highest dose at PND 21. CONCLUSIONS: These results suggest that gestational and lactational exposure to an environmentally relevant BFR mixture may induce changes in neurodevelopment and lipid metabolism in offspring. Birth Defects Research 109:497-512, 2017.© 2017 The Authors Birth Defects Research Published by Wiley Periodicals, Inc.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Associations between urinary biomarkers of oxidative stress and air pollutants observed in a randomized crossover exposure to steel mill emissions.

The effects of industrial air pollution on human health have not been as thoroughly investigated as those of urban air pollution which originates mostly from automotive transport. To better assess the health impacts of point sources of industrial air pollution, a randomized crossover exposure study was conducted. Sixty one young and healthy volunteers were randomly assigned to spend five consecutive eight-hour days near a steel mill or at a location five kilometres away. After a nine or sixteen-day washout period, volunteers spent another five consecutive days at the second site. Meteorological conditions and air pollutants were monitored at both exposure sites. On each exposure day, the first morning urine was collected along with a second urine sample obtained immediately before leaving the exposure site at the end of the day. Urinary levels of biomarkers of oxidative stress 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage), malondialdehyde (MDA, a biomarker of lipid peroxidation), 8-isoprostane (8-IsoP, a bioactive metabolite resulting from the peroxidation of arachidonic acid) and Vascular Endothelial Growth Factor (VEGF, involved in response to oxidative stress) were measured. According to mixed-effects linear regression models, intra-individual variations in 8-OHdG urinary levels were significantly associated with exposure site, but surprisingly, lower levels were observed at the steel mill site. Delayed, temporally-defined associations with specific air pollutants were observed for 8-OHdG, 8-IsoP and VEGF. However, these associations were subtle, presented complex patterns and their biological consequences remain unclear.

Gestational and Early Postnatal Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants: General Toxicity and Skeletal Variations.

Brominated flame retardants (BFRs) are stable environmental contaminants known to exert endocrine-disrupting effects. Developmental exposure to polybrominated diphenyl ethers (PBDEs) is correlated with impaired thyroid hormone signaling, as well as estrogenic and anti-androgenic effects. As previous studies have focused on a single congener or technical mixture, the purpose of the current study was to examine the effects of gestational and early postnatal exposure to an environmentally relevant mixture of BFRs designed to reflect house dust levels of PBDEs and hexabromocyclododecane on postnatal developmental outcomes. Pregnant Sprague-Dawley rats were exposed to the PBDE mixture from preconception to weaning (PND 21) through the diet containing 0, 0.75, 250, and 750 mg mixture/kg diet. BFR exposure induced transient reductions in body weight at PND 35 in male and from PND 30-45 in female offspring (250 and 750 mg/kg). Liver weights (PND 21) and xenobiotic metabolizing enzyme activities (PND 21 and 46) were increased in both male and female offspring exposed to 250 and 750 mg/kg diets. Furthermore, serum T4 levels were reduced at PND 21 in both,male and female offspring (250 and 750 mg/kg). At PND 21, Serum alkaline phosphatase (ALP) was decreased in males exposed to 750 mg/kg dietat, and females exposed to 250 and 750 mg/kg diets. At PND 46 ALP was significantly elevated in males (250 and 750 mg/kg). Variations in the cervical vertebrae and phalanges were observed in pups at PND 4 (250 and 750 mg/kg). Therefore, BFR exposure during gestation through to weaning alters developmental programming in the offspring. The persistence of BFRs in the environment remains a cause for concern with regards to developmental toxicity.

Assessment of urinary metabolite excretion after rat acute exposure to perfluorooctanoic acid and other peroxisomal proliferators.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Activation of the peroxisome proliferator activated receptor alpha (PPARα) resulting from exposure to PFOA has been extensively studied in rodents. However, marked differences in response to peroxisome proliferators prevent extrapolation of rodent PPARα activation to human health risks and additional molecular mechanisms may also be involved in the biological response to PFOA exposure. To further explore the potential involvement of such additional pathways, the effects of PFOA exposure on urinary metabolites were directly compared with those of other well-known PPARα agonists. Male rats were administered PFOA (10, 33, or 100 mg/kg/d), fenofibrate (100 mg/kg/d), or di(2-ethylhexyl) phthalate (100 mg/kg/d) by gavage for 3 consecutive days and allowed to recover for 4 days, and overnight urine was collected. Greater urinary output was observed exclusively in PFOA-treated rats as the total fraction of PFOA excreted in urine increased with the dose administered. Assessment of urinary metabolites (ascorbic acid, quinolinic acid, 8-hydroxy-2'-deoxyguanosine, and malondialdehyde) provided additional information on PFOA's effects on hepatic glucuronic acid and tryptophan-nicotinamide adenine dinucleotide (NAD) pathways and on oxidative stress, whereas increased liver weight and palmitoyl-CoA oxidase activity indicative of PPARα activation and peroxisomal proliferation persisted up to day five after the last exposure.

Effects of a 28-day oral exposure to a 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one biocide formulation in Sprague-Dawley rats.

Biocides are added to biodiesels to prevent degradation resulting from microbial growth. A 28-day repeated oral dose study was conducted to assess a potential risk arising from ingestion of isothiazolinone biocides in biodiesels. A mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT) diluted in corn oil was administered by gavage to male and female rats at 0, 0.26, 0.78, 2.33 and 7.0 mg/kg body weight per day. Rat water and food consumption was monitored. At the end of the dosing period, organs were weighed and histological examinations performed. Hematology, serum clinical chemistry and biomarkers of inflammation were assessed. Reduction of serum triglyceride levels in males and induction of hepatic phase 1 xenobiotic metabolizing enzymes in females accompanied by subtle histological changes in the liver were observed at the highest CMIT/MIT exposure. These changes were more indicative of an adaptive, reversible response than overt toxicity. Based on recommended levels for the control of microbial growth in fuels, CMIT/MIT contained in accidentally ingested biodiesels is not expected to represent a significant health risk.

Effects of Jatropha oil on rats following 28-day oral treatment.

Jatropha oil is an emerging feedstock for the production of biodiesels. The increasing use of this nonedible, toxic oil will result in higher potential for accidental exposures. A repeated-dose 28-day oral toxicity study was conducted to provide data for risk assessment. Jatropha oil diluted in corn oil was administered by gavage to male and female rats at 0.5, 5, 50 and 500 mg kg(-1) body weight per day for 28 consecutive days. Control rats were administered corn oil only. The growth rates and consumption of food and water were monitored. At necropsy, organs were weighed and hematological parameters assessed. Serum clinical chemistry and C-reactive protein were measured and histological examinations of organs and tissues were performed. Markedly depressed growth rate was observed in males and females receiving Jatropha oil at 500 mg kg(-1) per day. Decreased white blood cell and lymphocyte counts were detected in females at 50 and 500 mg kg(-1) per day and in males at 500 mg kg(-1) per day. These changes were correlated to mild and reversible histological changes in male and female spleens. In the liver, a mild increase in portal hepatocytes cytoplasm density was observed in males and females, while periportal vacuolation was observed exclusively in females. Mild acinar proliferation was observed in the female mammary glands at all dose levels. It is concluded that Jatropha oil produces adverse effects on female rats starting at 50 mg kg(-1) per day with decreased white blood cell and lymphocyte counts and at 500 mg kg(-1) per day in both genders in term of depressed growth rates.

Diesel and biodiesels induce hepatic palmitoyl-CoA oxidase enzymatic activity through different molecular mechanisms in rats.

Induction of palmitoyl-CoA oxidase enzymatic activity in rat liver suggests that ingestion of diesel and biodiesels can cause mild hepatic peroxisomal proliferation. Surprisingly, quantification by immunochemistry of the enzyme itself (ACOX1) revealed that palmitoyl-CoA oxidase enzymatic activity correlates with ACOX1 protein level following exposure to diesel, but not following exposure to biodiesels. Quantification of CYP4A1, another biomarker of peroxisomal proliferation, further indicates that contrary to diesel, the effects of biodiesels appear to be independent of this pathway. There are two ACOX1 protein isoforms that exhibit different enzymatic activities depending on the substrate. The results of our enzymatic assays performed on substrates presenting different carbon chain lengths (octanoyl-CoA and palmitoyl-CoA) are compatible with the hypothesis of a differential regulation of the ACOX1 isoforms by diesel and biodiesels. Further studies will be required to precisely determine the molecular mechanisms by which diesel and biodiesels induce palmitoyl-CoA oxidase activity in rat liver.

Expression, purification, and structural characterization of CfrA, a putative iron transporter from Campylobacter jejuni.

The gene for the Campylobacter ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen Campylobacter jejuni, was cloned, and the membrane protein was expressed in Escherichia coli, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a beta-sheet protein (approximately 44% beta-sheet and approximately 10% alpha-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to (2)H(2)O and yet is resistant to thermal denaturation at temperatures up to 95 degrees C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane beta-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the E. coli enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.

Identification of the coiled-coil domains of Enterococcus faecalis DivIVA that mediate oligomerization and their importance for biological function.

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.

Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth and chromosome segregation.

Enterococcus faecalis divIVA (divIVAEf) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVAEf was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVAEf mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.

A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays.

A region in the cell division site determinant MinD required for stimulation by MinE and which determines MinD topological specificity along coil-like structures has been identified. Structural modeling of dimeric MinD and sequence alignment of 24 MinD proteins revealed a conserved polar region in Gram-negative bacterial MinD proteins, corresponding to residues 92-94 of Neisseria gonorrhoeae MinD (MinD(Ng)). Using MinD(Ng) as a paradigm for MinD functionality in Gram-negative organisms, mutation of these conserved residues did not abrogate MinD(Ng) self-association, nor its interaction with MinE(Ng) and the cell division inhibitor MinC. Although the MinD(Ng) mutant dimerized in the presence of ATP, its ATPase activity was not stimulated by MinE(Ng), unlike wild-type MinD(Ng). GFP fusions to either MinD(Ng) or to Escherichia coli MinD bearing simultaneous or individual mutations to residues 92-94 localized within coiled arrays along the E. coli inner cell periphery, similar to wild-type GFP-MinD. However, unlike wild-type GFP-fusions, the mutant proteins were distributed uniformly throughout the array, despite the presence of MinE, which normally imparts topological specificity to MinD by inducing the latter to oscillate from pole-to-pole and away from midcell. Hence, despite localizing along the inner cell periphery as a polymeric structure, the mutant MinD proteins in this study have lost the ability to be efficiently stimulated by MinE(Ng), resulting in a loss of distinct pole-to-pole oscillation.

Short-term oral toxicity of pentyl ether, 1,4-diethoxybutane, and 1,6-dimethoxyhexane in male rats.

Pentyl ether (PE) and two newly synthesized polyoxy ethers, 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been proposed as candidate diesel fuel additives. To characterize and compare their toxicity and to provide information for risk assessment, a 4-week oral study was conducted on these compounds. Male Sprague-Dawley rats (288 +/- 20 g) were divided into groups of seven animals each, and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg body weight), or high (200 mg/kg body weight) doses of PE, DEB, or DMH, respectively, 5 days/week for 4 weeks. Animals in the control group received the vehicle (corn oil, 1 ml/100 g body weight) only. At the end of the exposure period, relative testis and thymus weights were reduced by 30 and 46%, respectively, in animals treated with the high dose of DMH. Significant reductions in serum lactate dehydrogenase (LDH), serum uric acid, and blood platelet counts were also observed in the high dose of DMH. Serum corticosterone was significantly depressed in the high doses of PE and DEB and in the low dose of DMH. Serum thiobarbituric acid-reactive substances (TBARS) were decreased (p < 0.05) in all DMH treatment groups and in the medium and high dose PE and DEB groups, while liver TBARS were unaffected by treatment. In the liver, increased glutathione (GSH) level and glutathione-S-transferases activity were detected in the high dose DMH group. Urinary ascorbic acid levels were markedly increased in animals receiving the high doses of PE, DEB, and DMH. Urinary formic acid was increased by 13 times in the high dose PE and DEB groups. Testes of all animals receiving the high dose of DMH showed a moderate to marked degree of degeneration of the seminiferous tubules, including a mild degree of vacuolation. At the same time, the epididymis of these animals had substantially reduced sperm density with prominent presence of spermatid giant cells. Mild histological changes were seen in the liver at all dose levels for all three chemicals. Thyroid effects were also observed in the high dose PE and DEB groups and in the medium and high dose DMH groups. It was concluded that DMH is the most toxic of the three ethers tested, with testicular, epidiymal, and thymic effects being the most prominent at 200 mg/kg. Other significant changes included depressed platelet counts and serum biochemical changes. Increased production of formic acid, an ocular toxin, from PE and DEB treatments may also be of toxicological concern.