Identification of active gaseous-alkane degraders at natural gas seeps.

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using 13C-propane and 13C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70-99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain ANDR5 and MAG19a, highly abundant in incubations with 13C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.

Horizontal Gene Transfer of Genes Encoding Copper-Containing Membrane-Bound Monooxygenase (CuMMO) and Soluble Di-iron Monooxygenase (SDIMO) in Ethane- and Propane-Oxidizing Rhodococcus Bacteria.

The families of copper-containing membrane-bound monooxygenases (CuMMOs) and soluble di-iron monooxygenases (SDIMOs) are involved not only in methane oxidation but also in short-chain alkane oxidation. Here, we describe Rhodococcus sp. strain ZPP, a bacterium able to grow with ethane or propane as the sole carbon and energy source, and report on the horizontal gene transfer (HGT) of actinobacterial hydrocarbon monooxygenases (HMOs) of the CuMMO family and the sMMO (soluble methane monooxygenase)-like SDIMO in the genus Rhodococcus. The key function of HMO in strain ZPP for propane oxidation was verified by allylthiourea inhibition. The HMO genes (designated hmoCAB) and those encoding sMMO-like SDIMO (designated smoXYB1C1Z) are located on a linear megaplasmid (pRZP1) of strain ZPP. Comparative genomic analysis of similar plasmids indicated the mobility of these plasmids within the genus Rhodococcus. The plasmid pRZP1 in strain ZPP could be conjugatively transferred to a recipient Rhodococcus erythropolis strain in a mating experiment and showed similar ethane- and propane-consuming activities. Finally, our findings demonstrate that the horizontal transfer of plasmid-based CuMMO and SDIMO genes confers the ability to use ethane and propane on the recipient. IMPORTANCE CuMMOs and SDIMOs initiate the aerobic oxidation of alkanes in bacteria. Here, the supposition that horizontally transferred plasmid-based CuMMO and SDIMO genes confer on the recipient similar abilities to use ethane and propane was proposed and confirmed in Rhodococcus. This study is a living example of HGT of CuMMOs and SDIMOs and outlines the plasmid-borne properties responsible for gaseous alkane degradation. Our results indicate that plasmids can support the rapid evolution of enzyme-mediated biogeochemical processes.

Optimized Gas Chromatography-Tandem Mass Spectrometry for 1,1'-sulfonylbis[2-(methylthio) ethane] Quantification in Human Urine.

Sulfur mustard (SM) which is a bifunctional alkylating vesicant is one of the mostly used chemical warfare agent in First World War and the Iran-Iraq War. ß-Lyase metabolites of SM especially 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) is an unequivocal biomarker of the exposure. An optimized gas chromatography-tandem mass spectrometry method was developed and validated for the retrospective detection of SBMTE in human urine. Urine samples were treated with acidic titanium trichloride to reduce ß-lyase metabolites to the single analyte SBMTE. After neutralization and precipitation, SBMTE was extracted from urine by C8 solid-phase extraction cartridge and analyzed in the multiple-reaction monitoring mode. The lower limit of quantification was 1 ng/mL with relative standard deviation of <10%. Acceptable intra-day and inter-day precisions and accuracies were obtained. The developed method was successfully measured various levels of SBMTE which could be used as the forensic evidence of such a chemical attack.

Rapid and complete dehalogenation of halonitromethanes in simulated gastrointestinal tract and its influence on toxicity.

Halonitromethanes (HNMs) as one typical class of nitrogenous disinfection byproducts in drinking water and wastewater are receiving attentions due to their high toxicity. This study applied a simulator of the human gastrointestinal tract to determine the dehalogenation processes of trichloronitromethane, bromonitromethane and bromochloronitromethane for the first time. Influence of digestion process of HNMs on gut microbiota and hepatotoxicity was further analyzed. Results showed that the three HNMs were rapidly and completely dehalogenated in the gastrointestinal tract, especially in the stomach (2 h retention Time) and small intestine (4 h retention Time). Mucin, cysteine, pancreatin and bile salts in the digestive juice played major roles in the dehalogenation process. HNMs and their dehalogenation products in the resulting fluids of stomach induced the highest toxicity followed by those in intestine and colon, exhibiting dose-dependent effects. Although most HNMs were degraded in the stomach and small intestine, residual HNMs entered into colon changed the microbial community. Abundance of several genera, such as Bacteroides, Lachnospiraceae_unassigned and Lactobacillus had high correlation with exposure concentration of HNMs. This study sheds new light on dehalogenation and toxic processes of HNMs by oral exposure, which provides basic data for their human health risk assessment.

Enhancing aerobic biodegradation of 1,2-dibromoethane in groundwater using ethane or propane and inorganic nutrients.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was previously used as both a soil fumigant and a scavenger in leaded gasoline. EDB has been observed to persist in soils and groundwater, particularly under oxic conditions. The objective of this study was to evaluate options to enhance the aerobic degradation of EDB in groundwater, with a particular focus on possible in situ remediation strategies. Propane gas and ethane gas were observed to significantly stimulate the biodegradation of EDB in microcosms constructed with aquifer solids and groundwater from the FS-12 EDB plume at Joint Base Cape Cod (Cape Cod, MA), but only after inorganic nutrients were added. Ethene gas was also effective, but rates were appreciably slower than for ethane and propane. EDB was reduced to <0.02 µg/L, the Massachusetts state Maximum Contaminant Level (MCL), in microcosms that received ethane gas and inorganic nutrients. An enrichment culture (BE-3R) that grew on ethane or propane gas but not EDB was obtained from the site materials. The degradation of EDB by this culture was inhibited by acetylene gas, suggesting that degradation is catalyzed by a monooxygenase enzyme. The BE-3R culture was also observed to biodegrade 1,2-dichloroethane (DCA), a compound commonly used in conjunction with EDB as a lead scavenger in gasoline. The data suggest that addition of ethane or propane gas with inorganic nutrients may be a viable option to enhance degradation of EDB in groundwater aquifers to below current state or federal MCL values.

Selective ethanolysis of sunflower oil with Lipozyme RM IM, an immobilized Rhizomucor miehei lipase, to obtain a biodiesel-like biofuel, which avoids glycerol production through the monoglyceride formation.

The obtaining of Ecodiesel, a biofuel applicable to diesel engines which keeps the glycerin as monoglyceride (MG), was achieved through a selective ethanolysis process of sunflower oil, by application of Lipozyme RM IM, a Rhizomucor miehei lipase immobilized on macroporous anion exchange resins. This biocatalyst that was already described in the synthesis of conventional biodiesel has also shown its efficiency in the present selective enzymatic process, after optimization of the influence of various reaction parameters. Thus, an adequate activity is obtained that is maintained throughout five successive reuses. Quantitative conversions of triglycerides (TG) with high yields to fatty acid ethyl esters (FAEE) were obtained under mild reaction conditions that correspond to the transformation of TG in a mixture of two moles of FAEE and a mole of MG, thus avoiding the glycerol production. Thus, the selective transesterification reaction of sunflower oil with absolute ethanol can be carried out under standard conditions with oil/ethanol volume ratio 12/3.5 (mL), at constant pH obtained by the addition of 50 µl of aqueous solution of 10 N NaOH, reaction temperature of 40 °C and 40 mg of Lipozyme RM IM. Under these experimental conditions six successive reactions can be efficiently carried out.

Propane respiration jump-starts microbial response to a deep oil spill.

The Deepwater Horizon event resulted in suspension of oil in the Gulf of Mexico water column because the leakage occurred at great depth. The distribution and fate of other abundant hydrocarbon constituents, such as natural gases, are also important in determining the impact of the leakage but are not yet well understood. From 11 to 21 June 2010, we investigated dissolved hydrocarbon gases at depth using chemical and isotopic surveys and on-site biodegradation studies. Propane and ethane were the primary drivers of microbial respiration, accounting for up to 70% of the observed oxygen depletion in fresh plumes. Propane and ethane trapped in the deep water may therefore promote rapid hydrocarbon respiration by low-diversity bacterial blooms, priming bacterial populations for degradation of other hydrocarbons in the aging plume.

Vanadium nitrogenase reduces CO.

Vanadium nitrogenase not only reduces dinitrogen to ammonia but also reduces carbon monoxide to ethylene, ethane, and propane. The parallelism between the two reactions suggests a potential link in mechanism and evolution between the carbon and nitrogen cycles on Earth.

Repair of O4-alkylthymine by O6-alkylguanine-DNA alkyltransferases.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) plays a major role in repair of the cytotoxic and mutagenic lesion O(6)-methylguanine (m(6)G) in DNA. Unlike the Escherichia coli alkyltransferase Ogt that also repairs O(4)-methylthymine (m(4)T) efficiently, the human AGT (hAGT) acts poorly on m(4)T. Here we made several hAGT mutants in which residues near the cysteine acceptor site were replaced by corresponding residues from Ogt to investigate the basis for the inefficiency of hAGT in repair of m(4)T. Construct hAGT-03 (where hAGT sequence -V(149)CSSGAVGN(157)- was replaced with the corresponding Ogt -I(143)GRNGTMTG(151)-) exhibited enhanced m(4)T repair activity in vitro compared with hAGT. Three AGT proteins (hAGT, hAGT-03, and Ogt) exhibited similar protection from killing by N-methyl-N'-nitro-N-nitrosoguanidine and caused a reduction in m(6)G-induced G:C to A:T mutations in both nucleotide excision repair (NER)-proficient and -deficient Escherichia coli strains that lack endogenous AGTs. hAGT-03 resembled Ogt in totally reducing the m(4)T-induced T:A to C:G mutations in NER-proficient and -deficient strains. Surprisingly, wild type hAGT expression caused a significant but incomplete decrease in NER-deficient strains but a slight increase in T:A to C:G mutation frequency in NER-proficient strains. The T:A to C:G mutations due to O(4)-alkylthymine formed by ethylating and propylating agents were also efficiently reduced by either hAGT-03 or Ogt, whereas hAGT had little effect irrespective of NER status. These results show that specific alterations in the hAGT active site facilitate efficient recognition and repair of O(4)-alkylthymines and reveal damage-dependent interactions of base and nucleotide excision repair.

Ultraviolet radiation drives methane emissions from terrestrial plant pectins.

Recent studies demonstrating an in situ formation of methane (CH(4)) within foliage and separate observations that soil-derived CH(4) can be released from the stems of trees have continued the debate about the role of vegetation in CH(4) emissions to the atmosphere. Here, a study of the role of ultraviolet (UV) radiation in the formation of CH(4) and other trace gases from plant pectins in vitro and from leaves of tobacco (Nicotiana tabacum) in planta is reported. Plant pectins were investigated for CH(4 )production under UV irradiation before and after de-methylesterification and with and without the singlet oxygen scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO). Leaves of tobacco were also investigated under UV irradiation and following leaf infiltration with the singlet oxygen generator rose bengal or the bacterial pathogen Pseudomonas syringae. Results demonstrated production of CH(4), ethane and ethylene from pectins and from tobacco leaves following all treatments, that methyl-ester groups of pectin are a source of CH(4), and that reactive oxygen species (ROS) arising from environmental stresses have a potential role in mechanisms of CH(4) formation. Rates of CH(4 )production were lower than those previously reported for intact plants in sunlight but the results clearly show that foliage can emit CH(4) under aerobic conditions.

Intercomparison of infrared cavity leak-out spectroscopy and gas chromatography-flame ionization for trace analysis of ethane.

Comparison of two different methods for the measurement of ethane at the parts-per-billion (ppb) level is reported. We used cavity leak-out spectroscopy (CALOS) in the 3 microm wavelength region and gas chromatography-flame ionization detection (GC-FID) for the analysis of various gas samples containing ethane fractions in synthetic air. Intraday and interday reproducibilities were studied. Intercomparing the results of two series involving seven samples with ethane mixing ratios ranging from 0.5 to 100 ppb, we found a reasonable agreement between both methods. The scatter plot of GC-FID data versus CALOS data yields a linear regression slope of 1.07 +/- 0.03. Furthermore, some of the ethane mixtures were checked over the course of 1 year, which proved the long-term stability of the ethane mixing ratio. We conclude that CALOS shows equivalent ethane analysis precision compared to GC-FID, with the significant advantage of a much higher time resolution (<1 s) since there is no requirement for sample preconcentration. This opens new analytical possibilities, e.g., for real-time monitoring of ethane traces in exhaled human breath.

Volatile organic compounds in the exhaled breath of young patients with cystic fibrosis.

Inflammatory mediators in the exhaled breath are receiving growing medical interest as noninvasive disease markers. Volatile organic compounds have been investigated in this context, but clinical information and methodological standards are limited. The levels of ethane, propane, n-pentane, methanol, ethanol, 2-propanol, acetone, isoprene, benzene, toluene, dimethyl sulphide (DMS) and limonene were measured in repeated breath samples from 20 cystic fibrosis patients and 20 healthy controls (aged 8-29 yrs). Three end-exhaled and one ambient air sample were collected per person and analysed on a customised gas chromatography system. Intra-subject coefficients of variation ranged between 9 and 34%, and hydrocarbon breath levels were influenced by their inspired concentrations. The alveolar gradient for pentane was higher in cystic fibrosis patients than in healthy controls (0.36 versus 0.21 ppb) and inversely proportional to forced expiratory volume in one second; highest values were observed in patients with pulmonary exacerbations (0.73 versus 0.24 ppb). Cystic fibrosis patients also exhibited a lower output of DMS (3.9 versus 7.6 ppb). Group differences were not significant for ethane and the remaining substances. It was concluded that chemical breath analysis for volatile organic compounds is feasible and may hold potential for the noninvasive diagnosis and follow-up of inflammatory processes in cystic fibrosis lung disease.

Kinetics of trichloroethene dechlorination with iron powder.

The dechlorination of trichloroethene (TCE) with metallic iron is an advantageous method for the remediation of contaminated groundwater and soil. The toxic reaction intermediates such as dichloroethenes (DCEs) and vinyl chloride (VC), however, occasionally accumulate in the pathway of the reaction. We have been trying to suppress these intermediates by using metallic iron powder containing impurities. In order to investigate the reaction pathways, we measured the production rates of the intermediates and the final products of the dechlorination of TCE such as DCEs, VC, ethyne or ethene. Ethyne, ethene, ethane and cis-DCE were observed as the major products, and trans-DCE, 1,1-DCE, VC, C3-hydrocarbons (such as propane, propylene), C4-hydrocarbons (such as n-butane) and methane were observed as the minors. Also the rate constants of TCE to ethyne and ethyne to ethene were larger than any other constants. These fact show the production of ethene/ethane via ethyne is the main pathway of the dechlorination of TCE using the metallic iron powder.

Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco.

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin under high light and UV stress we have increased the capacity for its biosynthesis in tobacco plants (Nicotiana tabacum L. cv. Samsun) by transformation with a heterologous carotenoid gene encoding beta-carotene hydroxylase (crtZ) from Erwinia uredovora under constitutive promoter control. This enzyme is responsible for the conversion of beta-carotene into zeaxanthin. Although the total pigment content of the transgenics was similar to control plants, the transformants synthesized zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light. Low-light-adapted tobacco plants were shown to be susceptible to UV exposure and therefore chosen for comparative analysis of wild-type and transgenics. Overall effects of UV irradiation were studied by measuring bioproductivity and pigment content. The UV exposed transformed plants maintained a higher biomass and a greater amount of photosynthetic pigments than controls. For revelation of direct effects, photosynthesis, pigment composition and chlorophyll fluorescence were examined immediately after UV treatment. Low-light-adapted plants of the crtZ transgenics showed less reduction in photosynthetic oxygen evolution and had higher chlorophyll fluorescence levels in comparison to control plants. After 1 h of high-light pre-illumination and subsequent UV exposure a greater amount of xanthophyll cycle pigments was retained in the transformants. In addition, the transgenic plants suffered less lipid peroxidation than the wild-type after treatment with the singlet-oxygen generator rose bengal. Our results indicate that an enhancement of zeaxanthin formation in the presence of a functional xanthophyll cycle contributes to UV stress protection and prevention of UV damage.

Reductive dehalogenation of chlorinated ethenes with elemental iron: the role of microorganisms.

Trichloroethene (TCE) transformation and the product distribution in an aqueous medium containing zero-valent iron (Fe(0)) was investigated in the presence of an anaerobic mixed culture to assess the potential role of microorganisms in permeable iron barriers. The presence of the culture increased the rate of TCE disappearance and changed the product distribution. Rapid formation and degradation of cis-dichloroethene (cis-DCE) was observed in reactors containing cells plus Fe(0) or H2 as a bulk reducing agent. High levels of vinyl chloride (VC) were formed and very similar profiles were obtained in the Fe(0) plus cell and H2 plus cell reactors, but not in Fe(0)-only reactors. The similar trends observed in Fe(0)-cell and H2-cell reactors suggest that most cis-DCE and VC in the Fe(0)-cell reactors were produced and transformed biologically rather than abiotically. Accumulation of methane in the Fe(0)-cell system indicates that hydrogen gas generated during anaerobic iron corrosion could support a methanogenic culture. Digital confocal images showed that the microorganisms were able to colonize the iron surface. The results suggest that potential development of dechlorinating populations in Fe(0) barriers may alter the TCE reduction pathway and produce VC, which would have significant impact on the performance of Fe(0) barriers.

Substrate interactions during aerobic biodegradation of methane, ethene, vinyl chloride and 1,2-dichloroethenes.

Intrinsic biodegradation of trichloroethene and 1,1,1-trichloroethane in groundwater at a Superfund site in California has been observed. An anaerobic zone exists in the area closest to the source location, yielding the expected complement of reductive dechlorination daughter products, including cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Significant levels of methane and ethene were also generated in the anaerobic zone. The groundwater returns to aerobic conditions downgradient of the source, with methane, ethene, VC, and several other compounds still present. Attenuation of VC in the aerobic zone suggests that it is being biodegraded. In this study microcosms were used to evaluate the role of methane and ethene as primary substrates for aerobic biodegradation of VC. Biodegradation of VC was fastest in the bottles containing ethene, with 40 mumol of VC consumed over a 150 day period, compared to approximately 15-20 mumol with methane or a mixture of methane and ethene. VC did not noticeably inhibit ethene biodegradation but did slow the rate of methane use. Methane inhibited ethene metabolism, which apparently caused a reduction in VC biodegradation when methane was present with ethene. These results suggest that ethene plays an important role during in situ natural attenuation of VC under aerobic conditions. Microcosms were also set up with VC alone. Following a 75 day lag period. VC consumption began and subsequent additions were consumed without a lag, suggesting the presence of organisms capable of using VC as a growth substrate. After providing VC alone for nearly 400 days, aliquots of the enrichment culture were used to evaluate its ability to biodegrade cis- and trans-DCE. Both compounds were readily consumed, although addition of VC as the primary substrate was needed to sustain biodegradation of repeated additions. This result suggests that organisms capable of using VC as a sole substrate may play an active role in aerobic natural attenuation of DCEs.

Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation.

Perchloroethene (PER) was a widely used solvent and is an environmental contaminant. In bioassays for carcinogenicity, PER was found to increase the incidence of liver tumors in mice and of renal tumors in male rats. Toxic effects of PER after repeated administration are likely caused by bioactivation. PER bioactivation occurs by two pathways. Oxidation by cytochrome P450 results in trichloroacetyl chloride, which binds to lipids and proteins. Glutathione S-conjugate formation from PER and further processing of the formed S-(trichlorovinyl)glutathione to S-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyase catalyzed cleavage, resulted in the reactive dichlorothioketene, which binds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine in proteins. The objective of this study was to comparatively quantify the dose-dependent formation of protein adducts from PER in rats and humans using antibodies with high specificity for either N epsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysine in proteins. Male and female rats (n = 2, per concentration and time point) were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various time points. Formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was comparatively quantified in subcellular fractions from liver and kidney and in blood. In addition, three male and three female human volunteers were exposed to 10 and 40 ppm PER, and formation of protein adducts in blood was analyzed using the antibodies and GC/MS after immunoaffinity enrichment of modified proteins. In liver and kidney subcellular fractions and blood of PER-exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed. Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were formed in kidney mitochondria, followed by kidney cytosol. Only low concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were present in liver proteins; blood concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower than in kidney mitochondria. Highest concentrations of N epsilon-(trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteins from the liver of rats exposed to PER. A higher protein adduct formation was seen in PER-exposed-male than -female rats for N epsilon-(dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposure to 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 h exposures to PER, N epsilon-(trichloroacetyl)-L-lysine-containing proteins were present in low concentrations. N epsilon-(Dichloroacetyl)-L-lysine-containing proteins were not detected either by Western blotting or GC/MS after immunoaffinity chromatography. The obtained results indicate a dose-dependent covalent binding of PER metabolites to proteins in rat liver, kidney, and blood and suggest that the concentration of covalent protein adducts is much lower in blood of humans as compared to the blood of rats exposed under identical conditions.

Effect of vitamin E on exhaled ethane in cigarette smokers.

STUDY OBJECTIVES: We hypothesized that micronutrient antioxidant intake may be one factor determining the development of significant COPD. Vitamin E was administered to smokers to determine if exhaled ethane was reduced and if ethane correlated with measures of lung function. STUDY DESIGN: Longitudinal placebo lead-in trial with posttreatment observation period. SETTING: Tucson Veterans Affairs Medical Center. PARTICIPANTS: Twenty-nine current stable smokers having no interest in smoking cessation. INTERVENTIONS: Spirometry, exhaled breath ethane measurements, and vitamin E and [-carotene plasma levels followed by 3 weeks of placebo with repeat plasma vitamin levels and ethane measurements; next, 3 weeks of vitamin E (dl-a-tocopherol), 400 IU po bid followed by plasma vitamin levels and breath ethane measurements; finally, 3 weeks without vitamins followed by breath ethane and plasma vitamin levels. RESULTS: Vitamin E treatment did not reduce ethane significantly. Exhaled ethane levels (mean + SD: pm/min/kg) were as follows: baseline, 7.39 + 5.39; after run-in period, 6.86 + 4.09; after vitamin E, 6.36+/-3.02; and final, 7.23+/-4.63. After vitamin E therapy, a significant negative correlation existed between exhaled ethane and FEV1/FVC. Pack-years of smoking at baseline and after vitamin E were significantly associated with ethane exhaled. Initial lung function was not significantly negatively associated with vitamin E-induced changes in exhaled ethane but a negative trend was found. CONCLUSIONS: Vitamin E alone, unlike the combination of vitamins C, E, and beta-carotene, failed to reduced exhaled ethane in cigarette smokers. Exhaled ethane was correlated with pack-years of smoking. Smokers whose ethane values were found to fall the most tended to have better preserved lung function.

Effects of deferoxamine on H2O2-induced oxidative stress in isolated rat heart.

During myocardial reperfusion injury, iron has been implicated in the Fenton based generation of hydroxyl radical, .OH, leading to further organ injury. Although previous studies have investigated the protective effect of iron chelators including deferoxamine (DFX) in myocardial reperfusion injury, there is little information regarding the role of iron chelation during oxidative stress produced by H2O2 on the heart. Isolated hearts from male Sprague-Dawley rats were retrograde-perfused with Krebs-Henseleit solution at 5 ml/min. After a 60-min equilibration, oxyradical challenge was instituted by the addition of H2O2 (200-600 microM) to the perfusate for 60 min. A subgroup of animals received DFX (400 microM) in the perfusate prior to challenge with 400 microM H2O2. Contractility was continuously monitored; perfusate samples for glutathione (GSH) and lactate dehydrogenase (LDH) estimations were collected at 30-min intervals. Headspace ethane, an indicator of lipid peroxidation, was estimated at 30-min intervals by gas chromatography. Control hearts maintained contractility during the perfusion period. H2O2 perfusion caused a dose dependent decrease in myocardial contractility; DFX pretreatment was partially protective. Headspace ethane slowly accumulated in control hearts; perfusion with H2O2 caused dose dependent increase in ethane accumulation indicative of enhanced lipid peroxidation. GSH and LDH in the perfusate remained low in control hearts. In contrast, H2O2 treated hearts had a dose dependent increase in the efflux of GSH and LDH which was markedly increased by perfusion with 600 microM H2O2. Pretreatment with DFX did not significantly reduce GSH or LDH efflux from hearts perfused with peroxide. While H2O2 perfusion causes a dose dependent decrease in myocardial contractility with a corresponding increase in headspace ethane release with GSH & LDH efflux indicative of oxidative stress, concurrent treatment with DFX reduces myocardial dysfunction and ethane generation. However, sublethal damage of plasma membrane still continues as reflected by continuous enhancement of LDH efflux, possibly indicating involvement of other reactive species besides hydroxyl radical.