Role of metal-induced reactive oxygen species generation in lung responses caused by residual oil fly ash

Inhalation of residual oil fly ash (ROFA) increases pulmonary morbidity in exposed workers. We examined the role of reactive oxygen species (ROS) in ROFA-induced lung injury. ROFA was collected from a precipitator at Boston Edison Co., Everett, MA, USA. ROFA (ROFA-total) was suspended in saline, incubated for 24 h at 37 degrees C, centrifuged, and separated into its soluble (ROFA-sol.) and insoluble (ROFA-insol.) fractions. Sprague-Dawley rats were intratracheally instilled with saline or ROFA-total or ROFA-sol. or ROFA-insol. (1 mg/100 g body wt.). Lung tissue and bronchoalveolar lavage cells were harvested at 4, 24, and 72 h after instillation. Chemiluminescence (CL) of recovered cells was measured as an index of ROS production, and tissue-lipid-peroxidation was assessed to determine oxidative injury. Significant amounts of Al, Fe, and Ni were present in ROFA-sol., whereas ROFA-insol. contained Fe, V, and Al. Using electron spin resonance (ESR), significantly more hydroxyl radicals were measured in ROFA-sol. as compared to ROFA-insol. None of the ROFA samples had an effect on CL or lipid peroxidation at 4 h. Treatment with ROFA-total and ROFA-insol. caused significant increases in both CL (at 24 h) and lipid peroxidation (at 24 and 72 h) when compared to saline control value. ROFA-sol. significantly reduced CL production at 72 h after treatment and had no effect on lipid peroxidation at any time point. In summary, ROFA, particularly its soluble fraction, generated a metal-dependent hydroxyl radical as measured by a cell-free ESR assay. However, cellular oxidant production and tissue injury were observed mostly with the ROFA-total and ROFA-insol. particulate forms. ROS generated by ROFA-sol. as measured by ESR appear not to play a major role in the lung injury caused after ROFA exposure.     

Acetaldehyde (CH3CHO) production in rodent lung after exposure to metal-rich particles.

Epidemiological reports demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter (PM). Metal-catalyzed oxidative stress has been postulated to contribute to lung injury in response to PM exposure. We studied the effects of residual oil fly ash (ROFA), a component of ambient air PM, on the formation of lung carbonyls that are indicators of lipid peroxidation. Rats were instilled intratracheally with ROFA (62.5-1000 micrograms) and underwent lung lavage. Lavage fluid carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and measured by high performance liquid chromatography with UV detection. Dose-dependent increases in a peak that eluted with the same retention time as the acetaldehyde (CH3CHO) derivative was observed in rats treated with ROFA 15 min after instillation (up to 25-fold greater than saline treated controls). The identification of CH3CHO was confirmed using gas chromatography-mass spectroscopy. ROFA-induced increases in other lavage fluid carbonyls were not seen. Increased CH3CHO in lavage fluid was observed as late as 8 h later. No increase in CH3CHO was observed in plasma from ROFA-treated rats. An increased formation of CH3CHO was observed in a human airway epithelial cell line incubated with ROFA suggesting a pulmonary source of CH3CHO production. Instillation of solutions of metals (iron, vanadium, nickel) contained in ROFA, or instillation of another ROFA-type particle containing primarily iron, also induced a specific increase in CH3CHO. These data support the hypothesis that metals were involved in the increased CH3CHO formation. Thus metals on PM may mediate lung responses through induction of lipid peroxidation and carbonyl formation.     

Acetaldehyde (CH3CHO) production in rodent lung after exposure to metal-rich particles

Epidemiological reports demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter (PM). Metal-catalyzed oxidative stress has been postulated to contribute to lung injury in response to PM exposure. We studied the effects of residual oil fly ash (ROFA), a component of ambient air PM, on the formation of lung carbonyls that are indicators of lipid peroxidation. Rats were instilled intratracheally with ROFA (62.5–1000 μg) and underwent lung lavage. Lavage fluid carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and measured by high performance liquid chromatography with UV detection. Dose-dependent increases in a peak that eluted with the same retention time as the acetaldehyde (CH₃CHO) derivative was observed in rats treated with ROFA 15 min after instillation (up to 25-fold greater than saline treated controls). The identification of CH₃CHO was confirmed using gas chromatography-mass spectroscopy. ROFA-induced increases in other lavage fluid carbonyls were not seen. Increased CH₃CHO in lavage fluid was observed as late as 8 h later. No increase in CH₃CHO was observed in plasma from ROFA-treated rats. An increased formation of CH₃CHO was observed in a human airway epithelial cell line incubated with ROFA suggesting a pulmonary source of CH₃CHO production. Instillation of solutions of metals (iron, vanadium, nickel) contained in ROFA, or instillation of another ROFA-type particle containing primarily iron, also induced a specific increase in CH₃CHO. These data support the hypothesis that metals were involved in the increased CH₃CHO formation. Thus metals on PM may mediate lung responses through induction of lipid peroxidation and carbonyl formation.     

Manganese and iron transport across pulmonary epithelium

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with (54)Mn or (59)Fe. Unlike (59)Fe, interactions between (54)Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled (54)Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of (54)Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca(2+) channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.     

Overexpression of extracellular superoxide dismutase decreases lung injury after exposure to oil fly ash

The mechanism of tissue injury after exposure to air pollution particles is not known. The biological effect has been postulated to be mediated via an oxidative stress catalyzed by metals present in particulate matter (PM). We utilized a transgenic (Tg) mouse model that overexpresses extracellular superoxide dismutase (EC-SOD) to test the hypothesis that lung injury after exposure to PM results from an oxidative stress in the lower respiratory tract. Wild-type (Wt) and Tg mice were intratracheally instilled with either saline or 50 microg of residual oil fly ash (ROFA). Twenty-four hours later, specimens were obtained and included bronchoalveolar lavage (BAL) and lung for both homogenization and light histopathology. After ROFA exposure, EC-SOD Tg mice showed a significant reduction in BAL total cell counts (composed primarily of neutrophils) and BAL total protein compared with Wt. EC-SOD animals also demonstrated diminished concentrations of inflammatory mediators in BAL. There was no statistically significant difference in BAL lipid peroxidation; however, EC-SOD mice had lower concentrations of oxidized glutathione in the BAL. We conclude that enhanced EC-SOD expression decreased both lung inflammation and damage after exposure to ROFA. This supports a participation of oxidative stress in the inflammatory injury after PM exposure rather than reflecting a response to metals alone.     

Immunotoxicity of soluble and insoluble salts of cadmium instilled intratracheally

Rats were intratracheally (i.t.) exposed to 36.5 or 27.5 microg of cadmium (Cd) as soluble cadmium chloride (CdCl2) and insoluble cadmium oxide (CdO) salts. The retention of metal in lungs, liver and kidney was assessed by atomic adsorption spectrophotometer. The animals were intraperitoneally (i.p.) primed with sheep red blood cells (SRBC) and assessed for the number of antibody forming cells in lung associated lymph nodes (LALN) and spleen. Both the compounds had similar retention of metal in lungs but CdO induced more pulmonary inflammatory and degradative changes than CdCl2. The larger influx of polymorphonuclear cells (PMNs) following CdO exposure appears to be due to the absence of protection afforded by Cd induced metallothionein cytoplasmic protein while the Cd metallothionein complex formed in the case of CdCl2 is more protective. However both forms of Cd had similar local immunosuppressive potential but CdO had more prolonged suppressive effect.     

In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively.

On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.     

ESR measurement of radical clearance in lung of whole mouse.

Clearance of the nitroxide radicals, hydroxy-TEMPO and carboxy-PROXYL, in whole-mouse lung was directly measured by in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the lung was confirmed by ESR imaging. The ESR signal of hydroxy-TEMPO was reduced in the lung and the clearance obeyed first-order kinetics, whereas the signal of carboxy-PROXYL remained constant. Comparison of the clearance rates of live and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the blood circulation system.     

Effect of stainless steel manual metal arc welding fume on free radical production, DNA damage, and apoptosis induction

Questions exist concerning the potential carcinogenic effects after welding fume exposure. Welding processes that use stainless steel (SS) materials can produce fumes that may contain metals (e.g., Cr, Ni) known to be carcinogenic to humans. The objective was to determine the effect of in vitro and in vivo welding fume treatment on free radical generation, DNA damage, cytotoxicity and apoptosis induction, all factors possibly involved with the pathogenesis of lung cancer. SS welding fume was collected during manual metal arc welding (MMA). Elemental analysis indicated that the MMA-SS sample was highly soluble in water, and a majority (87%) of the soluble metal was Cr. Using electron spin resonance (ESR), the SS welding fume had the ability to produce the biologically reactive hydroxyl radical (^•OH), likely as a result of the reduction of Cr(VI) to Cr(V). In vitro treatment with the MMA-SS sample caused a concentration-dependent increase in DNA damage and lung macrophage death. In addition, a time-dependent increase in the number of apoptotic cells in lung tissue was observed after in vivo treatment with the welding fume. In summary, a soluble MMA-SS welding fume was found to generate reactive oxygen species and cause DNA damage, lung macrophage cytotoxicity and in vivo lung cell apoptosis. These responses have been shown to be involved in various toxicological and carcinogenic processes. The effects observed appear to be related to the soluble component of the MMA-SS sample that is predominately Cr. A more comprehensive in vivo animal study is ongoing in the laboratory that is continuing these experiments to try to elucidate the potential mechanisms that may be involved with welding fume-induced lung disease.     

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.     

Hyperoxic inhibition of newborn rat lung development: protection by deferoxamine.

Prolonged exposure to hyperoxia markedly inhibits normal lung development (alveolarization and respiratory surface area expansion) in immature animals. Since (a) hyperoxia results in excess hydroxyl radical (OH.) formation, (b) (OH.) is implicated in O2-induced lipid peroxidation and DNA alterations, and (c) both OH. formation and its interaction with DNA are Fe++ dependent; chelation of Fe++ should act to protect against pulmonary O2 toxicity and hyperoxic inhibition of lung development. We therefore treated litters of newborn rats with the iron chelator Deferoxamine mesylate (DES) (150 mg/kg/day) during a 10-day exposure to greater than 95% O2. Morphometric analysis demonstrated that compared to the mean airspace size in air control rat pups (Lm = 44.5 microns), hyperoxic exposure resulted in a 34% larger mean air space diameter in O2-saline rat lungs (59.5 microns) versus only an 11% enlargement in O2-DES lungs (51.1 microns*). Lung internal surface area (cm2) per 100-g body weight were air control = 4480, O2-saline = 3570 (decreases 20.3%), and O2-DES = 4125* (decreases 7.9%) (*p less than 0.05 versus O2-saline group). DES-treated animals also had significantly decreased lung conjugated diene levels during hyperoxic exposure and increased lung elastin content (reflective of preserved lung alveolar formation) compared to O2-saline rats. These results indicate that DES treatment substantially ameliorated the inhibitory effects of neonatal hyperoxic exposure on normal lung development.     

NUCLEAR RIBONUCLEASE ACTIVITIES OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— The activities of alkaline and acidic RNAses were determined in soluble and insoluble fractions from nuclei of brain hemispheres of rats, aged from 1 day to adult. The activities increased rapidly and reached a maximum, at 30 days, of about 10 times (alkaline RNAsel or 5 times (acidic RNAse) that seen at day 1.     

Aerobic training reduces oxidative stress in skeletal muscle of rats exposed to air pollution and supplemented with chromium picolinate

Objective: The purpose of this study was to investigate the effects of chromium picolinate (CrPic) supplementation associated with aerobic exercise using measures of oxidative stress in rats exposed to air pollution.

Methods: Sixty-one male Wistar rats were divided into eight groups: residual oil fly ash (ROFA) exposure and sedentary (ROFA-SED); ROFA exposure, sedentary and supplemented (ROFA-SED-CrPic); ROFA exposure and trained (ROFA-AT); ROFA exposure, supplemented and trained (ROFA-AT-CrPic); sedentary (Sal-SED); sedentary and supplemented (Sal-SED-CrPic); trained (Sal-AT); and supplemented and trained (Sal-AT-CrPic). Rats exposed to ROFA (air pollution) received 50?µg of ROFA daily via intranasal instillation. Supplemented rats received CrPic (1?mg/kg/day) daily by oral gavage. Exercise training was performed on a rat treadmill (5×/week). Oxidative parameters were evaluated at the end of protocols.

Results: Trained groups demonstrated lower gain of body mass (P?P?P?P?=?.0014), although CAT activity did not differ among groups (P?=?.4487).

Conclusion: Air pollution exposure did not lead to alterations in oxidative markers in lungs and heart, and exercise training was responsible for decreasing oxidative stress of the gastrocnemius.     

In vivo bioassays of acute asbestosis and its correlation with ESR spectroscopy and imaging in redox status

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.     

Esr Spin Trapping and Cytotoxicity Investigations of Freshly Fractured Quartz: Mechanism of Acute Silicosis

Electron spin resonance (ESR) measurements show that grinding of quartz particles in air produces silicon-based (Si· and SiO·) radicals which decay with aging in air. ESR spin trapping measurements provide evidence for the generation of hydroxyl and possibly superoxide radicals from a suspension of fresh quartz particles. The hydroxyl radical generation potential of the fresh quartz particles decreases on storing in ambient air and on the addition of catalase, superoxide dismutase, desferroxamine. or DMSO. Silica-induced lipid peroxidation also decreases on storing the fresh particles in ambient air. These findings suggest that oxygenated radicals play a role in the biochemical mechanism of pneumoconiosis in general and acute silicosis in particular.     

Esr Spin Trapping and Cytotoxicity Investigations of Freshly Fractured Quartz: Mechanism of Acute Silicosis

Electron spin resonance (ESR) measurements show that grinding of quartz particles in air produces silicon-based (Si· and SiO·) radicals which decay with aging in air. ESR spin trapping measurements provide evidence for the generation of hydroxyl and possibly superoxide radicals from a suspension of fresh quartz particles. The hydroxyl radical generation potential of the fresh quartz particles decreases on storing in ambient air and on the addition of catalase, superoxide dismutase, desferroxamine. or DMSO. Silica-induced lipid peroxidation also decreases on storing the fresh particles in ambient air. These findings suggest that oxygenated radicals play a role in the biochemical mechanism of pneumoconiosis in general and acute silicosis in particular.     

Beta-adrenergic agonist therapy accelerates the resolution of hydrostatic pulmonary edema in sheep and rats.

To determine whether beta-adrenergic agonist therapy increases alveolar liquid clearance during the resolution phase of hydrostatic pulmonary edema, we studied alveolar and lung liquid clearance in two animal models of hydrostatic pulmonary edema. Hydrostatic pulmonary edema was induced in sheep by acutely elevating left atrial pressure to 25 cmH(2)O and instilling 6 ml/kg body wt isotonic 5% albumin (prepared from bovine albumin) in normal saline into the distal air spaces of each lung. After 1 h, sheep were treated with a nebulized beta-agonist (salmeterol) or nebulized saline (controls), and left atrial pressure was then returned to normal. beta-Agonist therapy resulted in a 60% increase in alveolar liquid clearance over 3 h (P < 0.001). Because the rate of alveolar fluid clearance in rats is closer to human rates, we studied beta-agonist therapy in rats, with hydrostatic pulmonary edema induced by volume overload (40% body wt infusion of Ringer lactate). beta-Agonist therapy resulted in a significant decrease in excess lung water (P < 0.01) and significant improvement in arterial blood gases by 2 h (P < 0.03). These preclinical experimental studies support the need for controlled clinical trials to determine whether beta-adrenergic agonist therapy would be of value in accelerating the resolution of hydrostatic pulmonary edema in patients.     

Quantitative and qualitative variations in the glycoproteins in the chick embryo brain

Sugar content was examined in soluble and insoluble glycoproteins extracted from the chick embryo brain at different developmental stages. The content of hexosamines and uronic acids in the soluble fraction is higher during the whole period examined. The difference between the two fractions reaches a maximum at the 15th day. The insoluble fraction shows the highest content of sialic acid and fucose in comparison with the soluble one, especially toward hatching. The sialic acid/fucose ratio shows a different pattern in the two fractions examined, particularly in the soluble glycoproteins. The patterns of sialic acid and fucose indicate that quantitative and qualitative developmental changes occur in the soluble and insoluble glycoproteins. All sugars examined show significant changes on the 15th day, suggesting that this stage may represent a critical period in the development of the chick embryo brain.     

Synthesis and degradation rates of collagens in vivo in whole skin of rats, studied with 1802 labelling.

Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.