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Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii.

Respiration, oxidative phosphorylation, and the mitochondrial membrane potential (DeltaPsi) of tachyzoites of the apicomplexan parasite Toxoplasma gondii were assayed in situ using very low concentrations of digitonin to render their plasma membrane permeable to succinate, ADP, safranin O, and other small molecules. The rate of basal respiration was slightly increased by digitonin when the cells were incubated in medium containing succinate. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was oxidized by antimycin A-poisoned mitochondria. The addition of ADP after TMPD/ascorbate also resulted in phosphorylating respiration. The antitoxoplasmosis drug atovaquone, at a very low concentration (0.03 microM), totally inhibited respiration and disrupted the mitochondrial membrane potential. Atovaquone was shown to inhibit the respiratory chain of T. gondii and mammalian mitochondria between cytochrome b and c1 as occurs with antimycin A1. Phosphorylation of ADP could not be obtained in permeabilized tachyzoites in the presence of either pyruvate, 3-oxo-glutarate, glutamate, isocitrate, dihydroorotate, alpha-glycerophosphate, or endogenous substrates. Although ADP phosphorylation was detected in the presence of malate, this activity was rotenone-insensitive and was probably due to the conversion of malate into succinate through a fumarate reductase activity that was detected in mitochondrial extracts. Together these results provide the first direct biochemical evidence that the respiratory chain and oxidative phosphorylation are functional in apicomplexan parasites, although the terminal respiratory pathway is different from that in the mammalian host.

Animals↗

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria.

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.

Hydroxamic Acids↗

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state.

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.

Electron Transport↗

In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate.

Mitochondria play a central role in neuronal death during pathological exposure to glutamate (excitotoxicity). To investigate the detailed bioenergetics of the in situ mitochondria, a method is described to monitor continuously the respiration of primary cerebellar granule neuron cultures while simultaneously imaging cytoplasmic Ca(2+) and mitochondrial membrane potential. Coverslip-attached cells were perfused in an imaging chamber with upstream and downstream flow-through oxygen electrodes. The bioenergetic consequences of chronic glutamate exposure were investigated, including ATP supply and demand, proton leak, and mitochondrial respiratory capacity during chronic glutamate exposure. In 25 mM K(+) medium supplemented with 10% dialyzed serum, cells utilized 54% of their respiratory capacity in the absence of receptor activation (37% for ATP generation, 12% to drive the mitochondrial proton leak, and the residual 5% was nonmitochondrial). glutamate initially increased mitochondrial respiration from 51 to 68% of capacity, followed by a slow decline. It was estimated that 85% of this increased respiration was because of increased ATP demand, whereas 15% was attributable to a transient mitochondrial proton leak. N-Methyl-D-aspartate receptor activation was only responsible for 62% of the increased respiration. When adjusted for cell death over 3 h of glutamate exposure, respiration of the viable cells remained near basal and protonophore stimulated respiration to the same extent as control cells. Pyruvate-supplemented media protected cells from glutamate excitotoxicity, although this was associated with mitochondrial dysfunction. We conclude that excitotoxicity under these conditions is not because of an ATP deficit or uncoupling. Furthermore, mitochondria maintain the same respiratory capacity as in control cells.

Adenosine Triphosphate↗

Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located in both the cytosol and the mitochondrial matrix: effects of respiration, anoxia, and the mitochondrial genome on its intracellular level and distribution.

Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in both the oxidative and nitrosative stress responses in Saccharomyces cerevisiae. Previous studies have shown that the expression of YHB1 is optimal under normoxic or hyperoxic conditions, yet respiring yeast cells have low levels of reduced YHb pigment as detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells. Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships between respiration, YHb level, and intracellular location. We have found that although intact respiration-proficient cells lack a YHb CO spectral signature, cell extracts from these cells have both a YHb CO spectral signature and nitric oxide (NO) consuming activity. This suggests either that YHb cannot be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells. By using an anti-YHb antibody and CO difference spectroscopy and by measuring NO consumption, we have found that YHb localizes to two distinct intracellular compartments in respiring cells, the mitochondrial matrix and the cytosol. Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome. The findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function.

Blotting, Northern↗

Manganese dioxide exposures and respirator performance at an alkaline battery plant.

Two industrial hygiene studies were conducted at an alkaline battery plant to evaluate worker exposures to manganese dioxide particulate and the effectiveness of filtering facepiece respirators. The work areas studied included the plant's powder-processing tower and press rooms where manganese was blended, compacted with graphite, and inserted into battery cans. Full-shift personal breathing zone monitoring was conducted to estimate manganese dust exposures of press operators, mechanics, and material handlers. In-facepiece and personal breathing zone air sampling pairs were also collected using a program protection factor protocol to estimate the protection provided by the respirators. Particle size evaluations were made using nylon cyclones and Marple personal multi-stage impactors. All samples were analyzed for manganese by inductively coupled argon plasma, atomic emission spectroscopy via NIOSH analytical method 7300 utilizing a modified acid digestion procedure. Fifty-four, full-shift, time-weighted average (TWA) exposures to total manganese ranged from 0.1 to 5.4 milligrams per cubic meter (mg/m3); worker exposures were substantially lower during a follow-up study due to engineering control improvements. Concurrent area sample comparisons of total and respirable manganese revealed that the respirable particulate mass fractions ranged from 6 to 32 percent, and mass median aerodynamic diameters determined from personal breathing zone air samples were mostly greater than 10 micrometers. Fifteen respirator performance evaluations were conducted using Moldex 2200 respirators fitted with 25 millimeter cassettes and light weight sampling probes. Protection factors ranged from 5 to 220, with a geometric mean and standard deviation of 31 and 2.97, respectively. The 5th percentile protection factor estimate was 5, as calculated from the protection factor distribution for this sample set. In 1995, the American Conference of Governmental Industrial Hygienists (ACGIH) lowered the elemental and inorganic manganese dust Threshold Limit Value (TLV) from 5 mg/m3 to 0.2 mg/m3 to address adverse pulmonary and central nervous system effects and male infertility. Although most personal breathing zone concentrations were above 0.2 mg/m3, none of the in-facepiece concentrations exceeded this concentration. Parkinson's-like symptoms have been reported in the literature for high manganese dust and fume exposures, but the importance of low dust exposures for producing neurological effects is uncertain.

Adult↗

Determination of known exhalation valve damage using a negative pressure user seal check method on full facepiece respirators.

A negative pressure user seal check (NPUSC) method was evaluated for its ability to adequately detect known exhalation valve leakage into a respirator. Three valves with different types of damage were included. Twenty-six test subjects, wearing full facepiece respirators, were asked to perform a NPUSC. Their responses as to whether they passed or failed the user seal check were compared to fit testing results from two quantitative fit test methods: ambient aerosol and controlled negative pressure. In addition, equipment developed at the University of Cincinnati was used to measure in-mask pressures that are generated during the performance of NPUSCs. This technique was employed to assess the ability of respirator wearers to properly conduct user seal checks. The data were analyzed to determine if the user seal check procedure is an effective method for detecting known exhalation valve damage. All test subjects reported passing the user seal check with the undamaged valve. With the warped valve installed, 95 percent of test subjects reported passing the user seal check. With the slit valve installed, 73 percent of test subjects reported passing. With the dirty valve installed, 65 percent reported passing. All fit factors, measured with the damaged valves, were below the Occupational Safety and Health Administration-recognized pass/fail criteria except one fit test with the respirator equipped with the slit valve. Results from the in-mask pressure measurements confirmed whether or not the subject properly conducted a user seal check, but did not detect respirator leakage. In conclusion, the performance of a NPUSC rarely helped to identify damaged exhalation valves. These results support the need for respirator inspection prior to donning with periodic fit testing and the performance of user seal checks as necessary components of an adequate respiratory protection program.

Equipment Failure↗

How protective are respirator assigned protection factors: an uncertainty analysis.

This investigation evaluated the risk of overexposure for a selected assigned protection factor by performing Monte Carlo simulations. A model was constructed to assess respirator performance by calculating the concentration inside the respirator. Estimates of the factors that affect respirator performance were described as distributions. The distributions used a worst case estimate for concentration in the workplace, the worst case for respirator performance (the fifth percentile person), and the worst case for exhalation valve leakage. A Monte Carlo analysis then provided estimates of the percentage of time that concentration inside the respirator exceeded the occupational exposure limit (OEL). For a half-facepiece respirator with an APF of 10, the calculations indicated a low risk of being exposed above an OEL, with mean exposures being controlled well below an OEL.

Humans↗

Telephone communications with several commercial respirators.

Previous work showed that telephone communications while wearing military respirators degraded both word comprehension and recognition speed. In addition, electronic amplification of the speech diaphragm signal had shown no advantage to the extra hardware. This experiment was performed to test effects of different configurations of commercially available respirators on telephone communications accuracy and speed. Twelve pairs of subjects were separated into different rooms and communicated by telephone. Modified rhyme-test words were presented by computer to the speaker, who transmitted the word by telephone to the listener. During the first replication, subjects were given no instruction about telephone communications procedure. During the second replication subjects followed a communications protocol that instructed them when to move the telephone handset from their ears to their mouths. Results showed that the protocol uniformly improved communications accuracy without incurring any extra time penalty. Word comprehension was still twice as fast without a respirator as with a respirator. Accuracy with the protocol nearly equaled the no respirator control value for most respirators tested.

Adolescent↗

Performance of dust respirators with facial seal leaks: I. Experimental.

The ability of representative half-mask and single-use respirators with facial seal leaks to provide protection against aerosols was evaluated by experimental measurement. Respirators were mounted on a manikin in a test chamber and operated at seven steady flow rates over the range of 2 to 150 L/min. Samples of polydisperse and monodisperse aerosols were taken from inside and outside the respirator and analyzed by a calibrated optical particle counter over the particle-size range 0.1 to 11.3 microns. Measurements were made separately for filter performance as a function of particle size and flow rate, and simulated leak performance (penetration) as a function of particle size, pressure drop, and leak size. Flow rate vs. pressure drop measurements were made for all filters and leaks tested. For a given leak condition the percentage of the total flow traversing the leak varied several fold over the usual range of airflow rates through a respirator. Aerosol penetration was found to depend strongly on particle size and flow rate for filters, and to depend strongly on particle size and less strongly on pressure drop for leaks. One can conclude from these measurements that the aerosol-size distribution inside a respirator will nearly always be significantly different from that outside the respirator.

Aerosols↗

Distribution of faceseal leak sites on a half-mask respirator and their association with facial dimensions.

Faceseal leaks on one brand of half-mask respirator worn by 73 human subjects were identified by deposition of a fluorescent tracer aerosol during a standard quantitative fit test. The identified leaks were categorized according to their location and shape. It was found that about 89% of all observed leaks occurred at the nose or chin or were multiple leaks which included these sites. Fit factors for these types of leaks were significantly lower than for other types of leaks. About 73% of all leaks approximated the shape of a slit rather than a round orifice, and the prevalence of these leaks was affected by gender. Significant association of facial dimensions and leak sites were found. Most of these were attributed to differences in gender, and only a very small percentage were for the facial dimensions used to define the Los Alamos respirator test panel. Significant correlation of facial dimensions and fit factor were found for only three facial dimensions; none of which are used to define the test panel. Evidence of airflow streamlining within the facepiece was observed on 22% of the subjects. Results of this study indicate that respirator leakage is strongly affected by nose and chin leaks, that gender is a factor in how a respirator fits, and that consideration should be given to including nasal dimensions when defining a respirator test panel and selecting a respirator for an individual wearer.

Adult↗

Effect of work load and respirator wear on postural stability, heart rate, and perceived exertion.

The effects on postural stability (sway) were investigated for different work loads under conditions of wearing a full facepiece respirator and not wearing any respiratory protection device. Fifteen subjects accomplished light (40 W), moderate (85 W), and heavy (125 W) work loads under the two conditions. Measurements of postural sway were made immediately after each load by using a multicomponent, strain gage-type force platform. Changes in each subject's movement pattern of the center of pressure were quantitated and compared to their initial baseline sway tests. Each subject's heart rate and perceived exertion were also recorded during each condition. A statistically significant effect (p = 0.007) caused by work load was observed for total length of sway with or without a respirator. An interaction approaching statistical significance (p = 0.056) between work load and respirator use was found. This indicated that sway increased more quickly and in a more consistently linear fashion with increasing work load under the respirator condition (p = 0.02) compared to the nonrespirator condition. The results from the respirator condition showed linear increases in postural sway length across the entire range of work loads, but sway length in the nonrespirator condition showed an increase only at 125-W work loads. The greater increase in sway during the postural balance test could be attributable to the increasing work load-induced proprioceptive fatigue effect on the nervous system's ability to process signals from proprioception systems incongruent with body sway. The heart rate was significantly higher during respirator wear (an increase in heart rate averaging 5.62 beats/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

A probability model for assessing exposure among respirator wearers: Part II-Overexposure to chronic versus acute toxicants.

A model describing the lognormal total distribution of contaminant levels inside a respirator (CI) is applied to assessing the probability of toxicant overexposure among a population of respirator wearers; the model accounts for between-wearer and within-wearer variability in ambient exposure levels (CO) and decimal fraction respirator penetration (P) values. The three exceedance probabilities are defined as PrI, the proportion of all CI levels over the permissible exposure limit (PEL); PrII, the proportion of wearers with an arithmetic mean CI level over the PEL; and PrIII, the proportion of wearers with a 95th percentile CI value over the PEL. PrII is considered that fraction of the population overexposed to a chronic toxicant; PrIII is considered that fraction overexposed to an acute toxicant. The behavior of PrII and PrIII over a range of exposure parameters is explored. An important observation is that a respirator-wearing population can have a substantial fraction of toxicant overexposure even though two conditions are met: (1) the P values for the population satisfy the criterion for the assigned protection factor (APF); and (2) the population arithmetic mean CO level is at or below the maximum use concentration (MUC), defined as APF x PEL. The authors recommend that the current MUCs for air-purifying respirators be reduced by one-half to reduce the potential respirator-wearing population fraction of overexposure and that appropriate exposure surveillance programs for all wearers be mandated.

Acute Disease↗

Review of respirator performance testing in the workplace: issues and concerns.

Performance capability of respirators has traditionally been evaluated by testing components of the respirator (e.g., filter efficiency), facepiece fit, total inward leakage, or some other measure of performance evaluated under laboratory conditions. In recent years, increased emphasis has been placed on development of test methods suitable for evaluating respirator performance in the workplace. The goal of such testing is to evaluate the level of protection provided by respirators in the work environment. The AIHA Respiratory Protection Committee believes that workplace testing of respirators has the potential to be an excellent tool for increasing knowledge about the effectiveness of respiratory protection. However, a number of technical issues remain to be addressed before optimal test protocols and data analysis methods can be defined. The progress made to date in workplace testing will be reviewed, and broader discussion about key elements that must be considered when developing guidelines for testing respirators in the workplace will be initiated.

Air Pollution, Indoor↗

Exposure to respirable crystalline silica in eastern North Carolina farm workers.

Occupational exposure to crystalline silica has been linked to silicosis, some forms of cancer, and certain autoimmune diseases. Little information exists on exposure levels of respirable silica in the agricultural industry. This study assessed respirable silica exposure of farm workers in eastern North Carolina. Sandy soils in this region have been shown to contain high levels of respirable silica. Personal breathing zone samples (n = 37) were collected from 27 workers at seven farms during various agricultural activities. The highest respirable silica concentrations were measured during sweet potato transplanting (3.91 +/- 2.07 mg/m3). Respirable silica exposure was observed to be associated with agricultural activity, soil moisture, relative humidity, and wind speed. Most of the variation in exposure (79%) was explained by agricultural activity and soil moisture. The observed percentage of silica levels (mean 34.7%) were almost twice as high as was reported in studies of California agriculture. This may be due to the loamy sand and sandy loam soil types in the regions in this study. In agriculture, respirable silica exposure is highly variable, but the potential for exposures above the threshold limit value of 0.05 mg/m3 exists during particular agricultural activities.

Agricultural Workers' Diseases↗

Exposure of taxi drivers and office workers to total and respirable manganese in an urban environment.

This research measured the exposure of two groups of workers to respirable and total manganese (Mn) and characterized the Mn particles emitted from an automobile tailpipe. The exposure of 20 office workers and 9 taxi drivers in Toronto to total airborne Mn and respirable Mn was measured over a 7-day period, 24 hours per day. Subjects were asked to wear two pumps (one included a size-selective cyclone that collected the respirable particles), and two battery chargers were supplied to each person so that the pump batteries could be recharged overnight while sampling continued. All filters were analyzed by neutron activation. In addition, Mn particles emitted from a car were collected directly at the exhaust. Particles were observed using secondary electron images in a scanning electron microscope (SEM), and their elemental composition was determined by energy dispersive x-ray spectrometry. The Mn concentrations obtained for the group of office workers ranged from 0.001 to 0.034 microgram/m3 for respirable Mn and from 0.002 to 0.044 microgram/m3 for total Mn. For the taxi drivers the Mn concentrations ranged from 0.007 to 0.032 microgram/m3 for respirable Mn and from 0.008 to 0.073 microgram/m3 for total Mn. There was a significant difference (p < 0.05) between the two groups for both respirable and total Mn. SEM analysis showed that the particles were mostly heterogeneous agglomerates varying from 1 to 100 microns. Even if the specific exposure to Mn from automobiles has not been directly established, these results suggest that the related increase of exposure may be limited.

Air Pollutants, Occupational↗

Cognitive performance during long-term respirator wear while at rest.

Cognitive performance was studied in six male and three female subjects exposed to two randomly administered 10-hour measurement periods, a control condition without a respirator and a respirator wear trial requiring continuous wear, under nonexercise conditions. Reaction time and decision-making speed were assessed using a series of simple and choice reaction time tasks at the start of each test iteration and after hours 2, 4, 6, 8, and 10 of testing. Subject anxiety levels were assessed along with reaction time measures. Visual tracking ability was measured after each hour of testing. Reaction time and decision-making speed did not differ significantly between control and respirator conditions at any time throughout the 10 hours of testing. Female volunteers exhibited significantly faster reaction times and decision-making speeds than males independent of respirator wear conditions and time of measurement. Subject anxiety increased significantly from initial measurements after 8 hours of testing for each condition, but no differences were observed between conditions at any time. Respirator wear did not detrimentally influence visual tracking ability. These findings suggest that respirator wear over a relatively long time period under nonexercise conditions should not significantly inhibit cognitive function.

Adult↗

Evaluation of respirator filters for asbestos fibers.

Fiber aerosols are known to have different aerodynamic behaviors than spherical particles and usually carry higher electrostatic charges. We investigated the effects of flow rate and charge status of filter cartridges on the penetration of spherical and fiber aerosols. Four types of test respirator filters were selected: two for passive respirators, one for a powered respirator, and one disposable respirator. Surface charges on respirator filters were determined using a noncontact field electrostatic field meter. Penetration tests were performed for filter cartridges before and after charge neutralization. The surface charge measurements on the respirator filters showed that some filters, including those used in disposable face masks, are charged to enhance the collection efficiency. Only high-efficiency particulate air filters performed consistently for both spherical test aerosols and the three types of asbestos fibers. The surface charge potential of filter cartridges and flow rate did not appear to affect the performance of these filters. In contrast to the high-efficiency filters, the aerosol penetration performance of low-efficiency filters and face masks deteriorated when the charge potential on the filter was removed. Our data also showed that the surface charges decreased in a high-temperature, high-humidity environment and disappeared after 1 week. Deposition of spherical particles and fibers in the charged disposable facemask filter was enhanced. For charged-neutralized, low-efficiency filter cartridges, asbestos fibers may penetrate more than spherical particles with a mean particle size of 0.3 microm diameter.

Asbestos↗