Hepatic microsomal phospholipids in rats exposed intratracheally to coal fly ash

The effects of intratracheal administration of fly ash (50 mg/kg body weight, daily for 7 days) on hepatic microsomal phospholipid metabolism has been studied in rats using various phospholipid precursors, viz NaH₂ ³²PO₄, (methyl-¹⁴C)-choline, and (methyl-¹⁴C)-methionine. Fly ash administration significantly increased microsomal phosphatidylcholine (PC), and lysophosphatidylcholine (LPC). The incorporation of NaH₂ ³²PO₄ into total liver phospholipids, PC and Phosphatidyl ethanolamine (PE) was significantly increased in fly ash-treated rats as compared to the control. Fly ash administration also increased the incorporation of (methyl-¹⁴C)-choline into microsomal PC. Incorporation of (methyl-¹⁴C)-methionine into microsomal PC was not affected. Fly ash administration decreased the per cent distribution of arachidonic acid in PC and PE and increased that of oleic acid in PC and of linoleic acid in PE.     

Fetal translocation and metabolism of PAH obtained from coal fly ash given intratracheally to pregnant rats

Polycyclic aromatic hydrocarbons (PAH) were extracted from coal fly ash collected from the electrostatic precipitator of a thermal power plant. The PAH extract was given intratracheally daily to pregnant rats (2 mg/100 g body weight) on d 18 and 19 of gestation. In addition on d 19 of gestation rats were also given [3H]benzo[a]pyrene intratracheally. Rats were sacrificed on d 20 of gestation, and the distribution of [3H]benzo[a]pyrene radioactivity and PAH of coal fly ash was studied in maternal lung, liver, and placenta, as well as in the liver and lung of the fetus. The radioactivity of intratracheally given benzo[a]pyrene was found in liver (68%), placenta (4%), fetal lung (1.9%), and fetal liver (1.4%) of maternal lung. Intratracheally administered PAH of coal fly ash were translocated to maternal liver and placenta, as well as to the liver and lung of the fetus. PAH of coal fly ash were also metabolized to several minor and major metabolites by maternal lung, liver, and placenta, as well as by the maternal fetal liver and lung. Some of the PAH metabolites in lung and liver were common; however, the major metabolite of liver, M-16, was different from the major metabolite M-16 of lung. The major PAH metabolite of placenta, M-5, and fetal liver, F-12, were common PAH metabolites. M-2 and M-6 of the placenta and F-5 and F-10 of the fetal lung were also common.     

Elevation of rat pulmonary, hepatic and lung surfactant lipids by fly ash inhalation

Fly ash contains many polycyclic aromatic hydrocarbons and genotoxic trace elements. In rats, fly ash exposure profoundly affects lung and liver histology. In the present study, the effect of fly ash inhalation on lung and liver lipids of rats was examined. Male Wistar strain rats were exposed daily to fly ash (0.27 +/- 0.01 mg/L air) in an inhalation chamber, 6 hr daily over a period of 15 days, and were killed on various days, i.e. 16, 30, 60, and 120. Fly ash inhalation significantly (P less than 0.05) increased total phospholipids (PL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in lungs. PC and dipalmitoylphosphatidylcholine (DPPC) contents in microsomes and lung surfactant also were significantly (P less than 0.05) higher in rats exposed to fly ash compared to control group animals. Radiolabeled precursor incorporation studies indicated that fly ash induced the synthesis of PC and DPPC by both CDP-choline pathway and N-methylation of PE in lung microsomes and enhanced their secretion into lung surfactant. In liver, PC and PE contents were elevated significantly (P less than 0.05) by fly ash exposure on days 16 and 30 respectively. A similar elevation of PC was observed in hepatic microsomes; this increase was due to its increased synthesis. However, the increased synthesis of PC in liver occurred to a greater extent by the N-methylation pathway than by the CDP-choline pathway.     

Induction of pulmonary drug metabolizing enzymes by coal fly ash in rats

The effect of intratracheal administration of fly ash, benzene extracted fly ash residue and benzene extract of fly ash has been studied on the activity of pulmonary mixed function oxidase. Fly ash, its benzene extract and benzene extracted residue significantly increased the levels of cytochrome P-450, cytochrome b5 and the activities of NADPH-cytochrome c reductase, NADH cytochrome b5 reductase, aminopyrine N-demethylase and glutathione S-transferase in a dose dependent manner. Phenobarbital or 3-methylcholanthrene treatment along with administration of fly ash, its benzene extracted residue and benzene extract of fly ash showed a synergistic effect on the activity of mixed function oxidase. The observed effects were due to chemical causes, i.e. organic and inorganic fractions of fly ash and not, due to its particulate nature. This was shown by the administration of glass beads which caused no alteration in the activity of pulmonary mixed function oxidase.     

Induction of pulmonary and hepatic cytochrome P-450 species by coal fly ash inhalation in rats

The effect of fly ash inhalation on xenobiotic metabolizing enzymes and heme metabolism in lung and liver has been studied in rats. Fly ash inhalation induced pulmonary and hepatic cytochrome P-450 content, aryl hydrocarbon hydroxylase and glutathione S-transferase activity. Induction of cytochrome P-450 was accompanied by induction of delta-amino levulinic acid synthetase in lung and inhibition of heme oxygenase in both lung and liver. Fly ash inhalation induced those species of cytochrome P-450 which closely resembled cytochrome P-448 in spectral properties and electrophoretic mobility.     

Acute pulmonary and systemic effects of inhaled coal fly ash in rats: comparison to ambient environmental particles.

Although primary particle emissions of ash from coal-fired power plants are well controlled, coal fly ash (CFA) can still remain a significant fraction of the overall particle exposure for some plant workers and highly impacted communities. The effect of CFA on pulmonary and systemic inflammation and injury was measured in male Sprague-Dawley rats exposed to filtered air or CFA for 4 h/day for 3 days. The average concentration of CFA particulate matter less than 2.5 microm (PM(2.5)) was 1400 microg/m(3), of which 600 microg/m(3) was PM(1). Animals were examined 18 and 36 h postexposure. Chemical analysis of CFA detected silicon, calcium, aluminum, and iron as major components. Total number of neutrophils in bronchoalveolar lavage fluid (BALF) following exposure to CFA was significantly increased along with significantly elevated blood neutrophils. Exposure to CFA caused slight increases in macrophage inflammatory protein-2, and marked increases in transferrin in BALF. Interleukin-1beta and total antioxidant potential in lung tissues were also increased in rats exposed to CFA. Histological examination of lung tissue demonstrated focal alveolar septal thickening and increased cellularity in select alveoli immediately beyond terminal bronchioles. These responses are consistent with the ability of CFA to induce mild neutrophilic inflammation in the lung and blood following short-term exposure at levels that could be occupationally relevant. However, when comparing the effects of CFA with those of concentrated ambient particles, CFA does not appear to have greater potency to cause pulmonary alterations. This study furthers our understanding of possible mechanisms by which specific sources of particulate air pollution affect human health.     

Enhanced lung toxicity of intratracheally instilled cadmium chloride in selenium-deficient rats

In order to further characterize biochemical correlates of CdCl₂-induced lung injury, CdCl₂ lung toxicity was studied in Se-deficient rats. A solution of 0.5 μmol/kg of CdCl₂ was intratracheally instilled into chronic respiratory disease-free 70-day-old rats fed a basal Se-deficient diet or a basal diet supplemented with 0.5 or 2.0 ppm of Se for 40 days. Within 3 days Se-deficient rats showed signs of marked respiratory distress and 25% had died, whereas rats from Se-supplemented groups all survived this time period. As evidenced by gross examination and lung water contents, Se-deficient rats exhibited a greater degree of inflammatory pulmonary edema than did rats from Se-supplemented groups. The results of the various enzymatic and biochemical determinations may be interpreted as being consistent with greater damage occurring in the Se-deficient rats.     

Antibody forming cell response to nickel and nickel-coated fly ash in rats.

The potential of nickel as nickel chloride, native fly ash and Ni-coated fly ash to alter pulmonary and systemic immune response was evaluated upon intratracheal (I/T) exposure of rats. The animals were sensitised with sheep red blood cells (SRBC) through I/T and intraperitoneal (I/P) routes. Nickel exposure resulted in a decrease in the number of antibody forming cells (AFC) in lung associated lymph nodes (LALN) and spleen. In rats exposed to native fly ash there was a reduction in the number of AFC in LALN but not in spleen. The results did not demonstrate any significant difference in the immunosuppression of fly ash and Ni-coated fly ash exposed rats. The decrease in AFC formation in Ni-coated fly ash exposed animals was of a lesser magnitude than in rats exposed to Ni-alone.     

Development of alterations in hamster distal lung following exposure to fly ash from fluidized bed coal combustion: a morphometric study

Fly ash (30 mg) from an experimental fluidized bed coal combustor (FBC) was intratracheally instilled once into male Syrian golden hamsters. At 1, 3, 6, 9, or 30 days postadministration, lungs were fixed by intratracheal perfusion and tissues were processed for light and electron microscopic evaluation. Standard point count morphometry, used to analyze distal lung structures, revealed: an increase in volume density (Vv) of noncellular interstitial space at Day 1 which remained elevated at Day 3 but returned to control values subsequently; increased Vv for polymorphonuclear leukocytes (PMNs) and alveolar macrophages in alveolar air space at Day 6; increased Vv of cellular interstitium at Day 9; and increased Vv of noncellular interstitium at Day 30 following exposure. These observations are consistent with the hypothesis that the primary response of the distal lung to particle exposure is recruitment of PMNs and macrophages. When activated, these cells may release substances affecting the cellular and noncellular composition of the interstitial space, leading eventually to increased connective tissue in the interstitium.     

Metabolism of 1,3-butadiene by lung and liver microsomes of rats and mice repeatedly exposed by inhalation to 1,3-butadiene

1,3-Butadiene, a colorless gas widely used as an intermediate in the production of synthetic rubber, is carcinogenic in rats and mice. Species differences exist in the sensitivity to inhaled 1,3-butadiene and the target tissue specificity for tumor formation. We examined whether repeated inhalation exposure of rats and mice to 1,3-butadiene would affect the rate of metabolism of 1,3-butadiene by lung and liver microsomes in these species. Male Sprague-Dawley rats and B6C3F1 mice were exposed nose-only to air (control) or 7600 +/- 170 ppm 1,3-butadiene (13,600 +/- 300 micrograms/l) and 740 +/- 10 ppm 1,3-butadiene (1300 +/- 20 micrograms/l), respectively, for 6 h/day for 5 days. After the last exposure, nasal tissue (rats only), lungs and livers were removed from the animals and microsomes were prepared. Microsomes from the different tissues were incubated with 6 mumol 1,3-butadiene and 10 mumol NADPH for 30 min and the rate of disappearance of 1,3-butadiene from the reaction flasks was quantitated. There was a statistically significant (P less than 0.05) depression in the rate of 1,3-butadiene metabolism (50%) in microsomes from lungs of both rats and mice that were exposed repeatedly to 1,3-butadiene compared to control animals. There was no effect of repeated 1,3-butadiene exposure on liver or nasal tissue (rats only) metabolism of 1,3-butadiene in rats or mice. The data from these studies indicate that it is unlikely that species differences in sensitivity or tissue susceptibility are due to an inductive or inhibitory effect of 1,3-butadiene on its own metabolism in the tissues examined.     

Isolation and characterization of fly ash from rat lung tissue

Inhaled fly ash may be leached by lung fluids, making potentially toxic trace elements in the fly ash bioavailable. We studied the composition and morphology of fly ash particles recovered from lungs of rats exposed to fly ash from a power plant burning pulverized eastern coal. Animals were sacrificed 1, 3, 6, and 12 mo after the commencement of the 4-wk exposures. Particles isolated from lungs of exposed animals, control fly ash samples, and samples recovered from control lungs spiked with fly ash were characterized by computer-controlled scanning electron microscopy (CCSEM) and thin window energy dispersive X-ray spectroscopy (EDS). EDS spectra of fly ash and ashed lung residues were distinct. Thus, fly ash particles could be distinguished from ashed lung residues. A majority of the fly ash particles recovered from lungs of exposed animals had similar morphology and composition to the exposure material. However, the number of silicon-rich particles decreased with time. After 6 mo, about 1% by number of the particles had been transformed, producing numerous "needles" associated with residues of fly ash particles. Particles that looked like diatoms were observed. This demonstrated that the sample preparation procedures used did not destroy delicate structures. Fly ash particles from a spiked control lung subjected to the same separation procedures did not have these structures. The structures may be the result of leaching of particles by lung fluids, which suggests that the glassy matrix components of fly ash particles may be bioavailable.     

Mutagenicity and cytotoxicity of coal fly ash from fluidized-bed and conventional combustion

In summary, fly-ash samples from a pressurized fluidized-bed combustion miniplant were found to consist of submicron, irregular particles that were cytotoxic and contained bioavailable mutagens. The fly-ash emission sample from a conventional coal-fired power plant was found to consist of spherical particles that were also cytotoxic but less mutagenic. The FBC fly ash investigated here was collected from an experimental miniplant and should not be considered representative of fly ash that may be obtained in the future from larger commercial-scale FBC plants. Further health and environmental assessment studies of coal fly-ash samples collected at multiple sites, including commercial-scale fluidized-bed and other conventional combustors, are needed to evaluate the potential health effects of coal fly ash from both types of combustion technology.     

Oxidative metabolism of lung macrophages exposed to sodium arsenite

Arsenic pollution has become increasingly severe. It occurs as the result of geological processes and different human activities. Arsenic toxicity at the respiratory level occurs mainly by inhalation of products of coal combustion. The aim of this study was to evaluate sodium arsenite (As³⁺) toxicity in murine alveolar macrophages (AMs) in vitro and its association with the alterations in cell metabolism.

No changes in viability, apoptosis or cell area were detected in AMs treated with As³⁺ concentrations up to 2 μM for 24–96 h. A marked decrease in these end-points was observed for As³⁺ concentrations ranging from 2.5 μM to 10 μM.

Regarding the dynamics of the endo-exocytic process triggered by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell incorporation, no variations were detected for As³⁺ concentrations lower than 2 μM while higher concentrations markedly modified this response.

MTT specific activity, as a measure of cell metabolic activity, was not modified irrespective of the As³⁺ concentration assayed. However, nitroblue tetrazolium (NBT) specific activity, as a measure of superoxide anion generation, is responsive but only to low As³⁺ doses.

Although this study focuses on lung macrophages, the effects of As³⁺ described herein may also apply to the response of macrophages residing in other organs.

Arsenite modifies the metabolic and the oxidative status of AMs in vitro. When macrophages are in an As³⁺ rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants.     

Application of leaching tests for toxicity evaluation of coal fly ash

The toxic properties of coal fly ash samples obtained from various coal combustion power plants were evaluated in this work using physicochemical analyses and bioassays. Physicochemical analyses showed that heavy metals present in solid samples included Cr, Cu, Mn, Ni, Pb, and Zn. The results of the chemical analysis of eluates deduced by the application of standard leaching tests according to EN 12457-2 and Toxicity Characteristic Leaching Procedure (TCLP) methods indicated that the compounds contained in fly ashes could potentially be transferred to the liquid phase depending upon the leaching method used. Heavy metal concentrations were higher in TCLP eluates, indicating that the initial pH value of the leaching medium significantly affected the transfer of these elements to the liquid phase. Tests conducted with the photobacterium Vibrio fischeri (Microtox test), the crustacean Daphnia magna, and the rotifer Brachionus calyciflorus were used to assess toxicity of eluates obtained by both leaching tests. Daphnia magna was the most sensitive test organism. The EN 12457-2 method proved to be more reliable for toxicity evaluation of eluates. In contrast, the TCLP method showed some interference owing to acetic acid toxicity, and precipitation occurred after pH adjustment of eluates from acid to neutral range. The toxicity of both fly ashes and the corresponding solid leaching residues of EN 12457-2 and TCLP leaching tests was also measured using the Microtox Basic Solid phase Test. The results generated with this bioassay indicated that toxicity was greatly influenced by the pH status of the solid samples.     

Pulmonary effects of ultrafine coal fly ash inhaled by guinea pigs

Guinea pigs were exposed to ultrafine coal fly ash produced in a laboratory furnace. The average mass median aerodynamic diameter and the average mass concentration of Illinois no. 6 fly ash produced in all exposure conditions were 0.21 microns and 5.8 mg/m3, respectively. In guinea pigs exposed to Illinois no. 6 fly ash, total lung capacity (TLC), vital capacity (VC), and diffusing capacity for carbon monoxide (DLco) were significantly reduced below the control values immediately, 2 h, and 8 h postexposure. The diffusing capacity was still 10% below the control 96 h after exposure. The total sulfate in the Illinois no. 6 fly ash as determined using ion chromatography is 1105 +/- 120 micrograms/m3. Animals exposed to the Montana lignite fly ash at comparable concentration and particle size did not show alteration in diffusing capacity. The data suggest that part of the sulfate present in the fly ash of Illinois no. 6 could be in the form of sulfuric acid and is responsible for the adverse effects observed in the exposed animals. The sulfuric acid in the fly ash of Montana lignite is neutralized by its high alkali content and produces no change in lung functions.     

Soluble metals associated with residual oil fly ash increase morbidity and lung injury after bacterial infection in rats

Inhalation of residual oil fly ash (ROFA) has been shown to impair lung defense mechanisms in laboratory animals and susceptible populations. Bioavailability of soluble transition metals has been shown to play a key role in lung injury caused by ROFA exposure. The goal of this study was to evaluate the effect of soluble metals on lung defense and injury in animals preexposed to ROFA followed by pulmonary challenge with a bacterial pathogen. ROFA was suspended in saline (ROFA-TOTAL), incubated overnight at 37 degrees C, and separated by centrifugation into soluble (ROFA-SOL) and insoluble (ROFA-INSOL) fractions. A portion of the soluble sample was treated with the metal-binding resin Chelex for 24 h at 37 degrees C. Sprague-Dawley rats were intratracheally dosed at d 0 with ROFA-TOTAL (1.0 mg/100 g body weight), ROFA-INSOL, ROFA-SOL, saline, saline + Chelex, or ROFA-SOL + Chelex. At d 3, 5 x 10(5) Listeria monocytogenes were intratracheally instilled into rats from each treatment group. At d 6, 8, and 10, left lungs were removed, homogenized, and cultured to assess bacterial clearance. Histopathological analysis was performed on the right lungs. Pulmonary exposure of ROFA-TOTAL or ROFA-SOL before infection led to a marked increase in lung injury and inflammation at all three time points after inoculation, and an increase in morbidity in comparison to saline control rats. Treatment with ROFA-INSOL, saline + Chelex, or ROFA-SOL + Chelex caused no significant increases in lung damage and morbidity when compared to control. By d 10, the ROFA-SOL and ROFA-TOTAL groups had approximately 200-fold more bacteria in the lung than saline control, indicating the inability of these groups to effectively respond to the infection. None of the other treatment groups had significant impairments in bacterial clearance when compared to saline. In conclusion, exposure to ROFA-TOTAL and ROFA-SOL significantly suppressed the lung response to infection. These results suggest that soluble metals present in ROFA may play a key role in increased susceptibility to pulmonary infection in exposed populations.     

Acute lung injury from intratracheal exposure to fugitive residual oil fly ash and its constituent metals in normo- and spontaneously hypertensive rats

We have recently shown that the spontaneously hypertensive (SH) rats with underlying cardiovascular disease exhibited greater pulmonary vascular leakage and oxidative stress than healthy normotensive (Wistar Kyoto, WKY) rats after a 3-day inhalation exposure to residual oil fly ash (ROFA) particles (Kodavanti et al., 2000). Since host responsiveness to a 3-day episodic ROFA inhalation could be different from a single acute exposure, we examined ROFA and its constituent metal (vanadium, V; nickel, Ni)-induced lung injury after a single intratracheal (IT) exposure. Male SH and WKY rats (12-13 wk) were IT instilled with either saline or ROFA (0.0, 0.83 or 3.33 mg/kg). The bronchoalveolar lavage fluid (BALF) was analyzed for lung injury markers at 24 and 96 h post-IT. Rats were also IT instilled with 0.0 or 1.5 micromol/kg of either VSO(4) or NiSO(4).6H(2)O in saline (equivalent to a dose of 2-3 mg ROFA), and assessed at 6 and 24 h post-IT. Basal levels of BALF protein, macrophages, and neutrophils, but not lactate dehydrogenase (LDH), were higher in control SH compared to control WKY rats. Lung histology of control SH rats exhibited mild focal alveolitis and perivascular inflammation; these changes were minimal in control WKY rats. ROFA-induced increases in BALF protein, and to a lesser extent in LDH, were greater in SH compared to WKY rats. ROFA IT was associated with the increases in BALF total cells in both strains (SH > WKY). BALF neutrophils increased at 24 h and macrophages at 96 h in a dose-dependent manner (SH > WKY). The increase in BALF neutrophils was largely reversed by 96 h in both rat strains. The V-induced increases in BALF protein and LDH peaked at 6 h post-IT and returned to control by 24 h in WKY rats. In SH rats, BALF protein and LDH were not affected by V. Ni caused BALF protein to increase in both strains at 6 and 24 h; however, the control values at 24 h were high in SH rats, and were not distinguishable from exposed rats. The Ni-induced increase in LDH activity was progressive over a 24-h time period (WKY > SH). The number of macrophages decreased following V and Ni exposure at 6 h, and this decrease was reversed by 24 h in both strains. V caused BALF neutrophils to increase only in WKY rats. The Ni-induced increase in BALF neutrophils was more dramatic and progressive than that of V, but was similar in both strains. Lung histology similarly revealed more severe and persistent edema, perivascular and peribronchiolar inflammation, and hemorrhage in Ni- than in V-exposed rats. This effect of Ni appeared slightly more severe in SH than in WKY rats. In summary, the acute single IT exposure to ROFA resulted in greater pulmonary protein leakage and inflammation in SH rats than in WKY rats. The metallic constituents of ROFA produced these effects in a strain-specific manner such that, at the dose level used, V caused pulmonary injury only in WKY rats, whereas Ni was toxic to both strains.     

Bone metabolism of male rats chronically exposed to cadmium

Recently, based on a female rat model of human exposure, we have reported that low-level chronic exposure to cadmium (Cd) has an injurious effect on the skeleton. The purpose of the current study was to investigate whether the exposure may also affect bone metabolism in a male rat model and to estimate the gender-related differences in the bone effect of Cd. Young male Wistar rats received drinking water containing 0, 1, 5, or 50 mg Cd/l for 12 months. The bone effect of Cd was evaluated using bone densitometry and biochemical markers of bone turnover. Renal handling of calcium (Ca) and phosphate, and serum concentrations of vitamin D metabolites, calcitonin, and parathormone were estimated as well. At treatment with 1 mg Cd/l, corresponding to the low environmental exposure in non-Cd-polluted areas, the bone mineral content (BMC) and density (BMD) at the femur and lumbar spine (L1-L5) and the total skeleton BMD did not differ compared to control. However, from the 6th month of the exposure, the Z score BMD indicated osteopenia in some animals and after 12 months the bone resorption very clearly tended to an increase. The rats' exposure corresponding to human moderate (5 mg Cd/l) and especially relatively high (50 mg Cd/l) exposure dose- and duration-dependently disturbed the processes of bone turnover and bone mass accumulation leading to formation of less dense than normal bone tissue. The effects were accompanied by changes in the serum concentration of calciotropic hormones and disorders in Ca and phosphate metabolism. It can be concluded that low environmental exposure to Cd may be only a subtle risk factor for skeletal demineralization in men. The results together with our previous findings based on an analogous model using female rats give clear evidence that males are less vulnerable to the bone effects of Cd compared to females.