Biodegradation of low concentrations of 1,2-dibromoethane in groundwater is enhanced by phenol.

The lead scavenger 1,2-dibromoethane (EDB), a former additive to leaded gasoline, is a common groundwater contaminant, yet not much knowledge is available for its targeted bioremediation, especially under in situ conditions. The study site was an aviation gas spill site, which, although all hydrocarbons and most of the EDB were remediated in the mid-1990s, still exhibits low levels of EDB remaining in the groundwater (about 11 µg EDB/l). To evaluate the effect of phenol on biostimulation of low concentration of EDB, microcosms were established from an EDB-contaminated aquifer. After 300 days at environmentally relevant conditions (12 ± 2 °C, static incubation), EDB was not significantly removed from unamended microcosms compared to the abiotic control. However, in treatments amended with phenol, up to 80 % of the initial EDB concentration had been degraded, while added phenol was removed completely. Microbial community composition in unamended and phenol-amended microcosms remained unchanged, and Polaromonas sp. dominated both types of microcosms, but total bacterial abundance and numbers of the gene for phenol hydroxylase were higher in phenol-amended microcosms. Dehalogenase, an indicator suggesting targeted aerobic biodegradation of EDB, was not detected in either treatment. This finding suggests phenol hydroxylase, rather than a dehalogenation reaction, may be responsible for 1,2-dibromoethane oxidation under in situ conditions. In addition, biostimulation of EDB is possible through the addition of low levels of phenol in aerobic groundwater sites.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (µ), and half-saturation coefficients (K(S)) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while µ's were similar for the two compounds, ranging from 0.19 to 0.52 day(-1) with 1,2-DCA to 0.28 to 0.36 day(-1) for EDB. K(S) was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 µg/liter for 1,2-DCA, 0.05 µg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.

Differences in crystallization of two LinB variants from Sphingobium japonicum UT26.

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

Aerobic and Anaerobic Biodegradation of 1,2-Dibromoethane by a Microbial Consortium under Simulated Groundwater Conditions.

This study was conducted to explore the potential for 1,2-Dibromoethane (EDB) biodegradation by an acclimated microbial consortium under simulated dynamic groundwater conditions. The enriched EDB-degrading consortium consisted of anaerobic bacteria Desulfovibrio, facultative anaerobe Chromobacterium, and other potential EDB degraders. The results showed that the biodegradation efficiency of EDB was more than 61% at 15 °C, and the EDB biodegradation can be best described by the apparent pseudo-first-order kinetics. EDB biodegradation occurred at a relatively broad range of initial dissolved oxygen (DO) from 1.2 to 5.1 mg/L, indicating that the microbial consortium had a strong ability to adapt. The addition of 40 mg/L of rhamnolipid and 0.3 mM of sodium lactate increased the biodegradation. A two-phase biodegradation scheme was proposed for the EDB biodegradation in this study: an aerobic biodegradation to carbon dioxide and an anaerobic biodegradation via a two-electron transfer pathway of dihaloelimination. To our knowledge, this is the first study that reported EDB biodegradation by an acclimated consortium under both aerobic and anaerobic conditions, a dynamic DO condition often encountered during enhanced biodegradation of EDB in the field.

In situ bioremediation of 1,2-dibromoethane (EDB) in groundwater to part-per-trillion concentrations using cometabolism.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was historically added to leaded gasoline as a scavenger to prevent the build-up of lead oxide deposits in engines. Studies indicate that EDB is present at thousands of past fuel spill sites above its stringent EPA Maximum Contaminant Level (MCL) of 0.05 µg/L. There are currently no proven in situ options to enhance EDB degradation in groundwater to meet this requirement. Based on successful laboratory studies showing that ethane can be used as a primary substrate to stimulate the aerobic, cometabolic biodegradation of EDB to <0.015 µg/L (Hatzinger et al., 2015), a groundwater recirculation system was installed at the FS-12 EDB plume on Joint Base Cape Cod (JBCC), MA to facilitate in situ treatment. Groundwater was taken from an existing extraction well, amended with ethane, oxygen, and inorganic nutrients and then recharged into the aquifer upgradient of the extraction well creating an in situ reactive zone. The concentrations of EDB, ethane, oxygen, and anions in groundwater were measured with time in a series of nested monitoring wells installed between the extraction and injection well. EDB concentrations in the six monitoring wells that were hydraulically well-connected to the pumping system declined from ~ 0.3 µg/L (the average concentration in the recirculation cell after 3 months of operation without amendment addition) to <0.02 µg/L during the 4-month amendment period, meeting both the federal MCL and the more stringent Massachusetts MCL (0.02 µg/L). The data indicate that cometabolic treatment is a promising in situ technology for EDB, and that low regulatory levels can be achieved with this biological approach.

Variable dual carbon-bromine stable isotope fractionation during enzyme-catalyzed reductive dehalogenation of brominated ethenes.

The potential of compound-specific stable isotope analysis (CSIA) to characterize biotransformation of brominated organic compounds (BOCs) was assessed and compared to chlorinated analogues. Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S catalyzed the dehalogenation of tribromoethene (TBE) to either vinyl bromide (VB) or ethene, respectively. Significantly lower isotope fractionation was observed for TBE dehalogenation by S. multivorans (εC = -1.3 ± 0.2‰) compared to D. hafniense (εC = -7.7 ± 1.5‰). However, higher fractionation was observed for dibromoethene (DBE) dehalogenation by S. multivorans (εC = -16.8 ± 1.8‰ and -21.2 ± 1.6‰ for trans- and cis-1,2- (DBE) respectively), compared to D. hafniense PCE-S (εC = -9.5 ± 1.2‰ and -14.5 ± 0.7‰ for trans-1,2-DBE and cis-1,2-DBE, respectively). Significant, but similar, bromine fractionation was observed for for S. multivorans (εBr = -0.53 ± 0.15‰, -1.03 ± 0.26‰, and -1.18 ± 0.13‰ for trans-1,2-DBE, cis-1,2-DBE and TBE, respectively) and D. hafniense PCE-S (εBr = -0.97 ± 0.28‰, -1.16 ± 0.36‰, and -1.34 ± 0.32‰ for cis-1,2-DBE, TBE and trans-1,2-DBE, respectively). Variable CBr dual-element slopes were estimated at Λ (εC/εBr) = 1.03 ± 0.2, 17.9 ± 5.8, and 29.9 ± 11.0 for S. multivorans debrominating TBE, cis-1,2-DBE and trans-1,2-DBE, respectively, and at 7.14 ± 1.6, 8.27 ± 3.7, and 8.92 ± 2.4 for D. hafniense PCE-S debrominating trans-1,2-DBE, TBE and cis-1,2-DBE, respectively. A high variability in isotope fractionation, which was substrate property related, was observed for S. multivorans but not D. hafniense, similar as observed for chlorinated ethenes, and may be due to rate-limiting steps preceding the bond-cleavage or differences in the reaction mechanism. Overall, significant isotope fractionation was observed and, therefore, CSIA can be applied to monitor the fate of brominated ethenes in the environment. Isotope effects differences, however, are not systematically comparable to chlorinated ethenes.

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.

Binding of carcinogenic halogenated hydrocarbons to cell macromolecules.

Ethylene dibromide (EDB), a known stomach carcinogen, and ethylene dichloride (EDC), which is carcinogenic to the liver, have been shown in in vitro experiments to bind covalently to stomach and hepatic microsomal proteins and to salmon sperm DNA. The binding of EDB or EDC with proteins was not significant when denatured microsomes were used or when DNA was used in the absence of microsomes. The binding of EDB to these macromolecules was augmented with increasing concentrations of microsomes. SKF-525A, an inhibitor of the microsomal metabolism of various substrates, significantly inhibited the binding of EDB to protein and DNA. These findings suggest that metabolic activation of EDB and EDC is required for their covalent binding to macromolecules. Glutathione and 1-methyl-2-mercaptolmidazole markedly decreased the binding of EDB, which indicated that a reactive electrophilic intermediate(s) of EDB is (are) involved in the binding. The binding of EDC to liver proteins of (C57BL/6 X C3//He)F1 mice, which are susceptible to liver tumor induction by EDC and to DNA, was significantly higher than the corresponding binding for Osborne-Mendel rats, a species not susceptible to liver tumor induction by this compound.

Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes.

The major DNA adduct formed by the carcinogen ethylene dibromide (EDB) is S-[2-(N7-guanyl)ethyl]glutathione. This adduct results from the glutathione S-transferase (GST)-catalyzed conjugation of EDB with glutathione (GSH), which generates an episulfonium ion capable of reacting with cellular nucleophiles. Purified rat and human GST enzymes were compared for their ability to conjugate EDB with GSH and displayed high selectivity. Of the six forms of rat GST tested, conjugation was catalyzed by the alpha class enzyme 2-2 and, to a lesser extent, by the mu class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the alpha class enzymes. Three dimers of the human alpha class (alpha x-alpha x, alpha x-alpha y, and alpha y-alpha y) were separated by chromatofocusing. The alpha x-alpha x preparation demonstrated the highest specific activity. Rat microsomal GST had negligible activity for the conjugation of EDB with GSH. The levels of EDB-DNA adducts formed in rat and human hepatocytes were compared. DNA was isolated from both rat and human hepatocytes incubated with 0.5 mM EDB, and the level of DNA adduct formation in the human samples was about 40% of that in the rat hepatocytes. EDB concentration-dependent unscheduled DNA synthesis was demonstrated in isolated human hepatocytes. Concurrent treatment of the hepatocytes with diethylmaleate to deplete intracellular GSH inhibited EDB-induced unscheduled DNA synthesis. These results indicate that EDB alkylates DNA in human hepatocytes and that enzymatic repair of adducts may occur. The results of experiments done in rat and human systems using both purified GST enzymes and intact hepatocytes imply that the genotoxic pathway of EDB metabolism in rats and humans is similar.

Epithelial binding of 1,2-dibromoethane in the respiratory and upper alimentary tracts of mice and rats.

The metabolism and binding of the volatile carcinogen 1,2-dibromo[14C]ethane (DBE) were studied in C57BL mice, Sprague-Dawley rats, and Fischer rats. As shown by the whole-body and light microscopic autoradiography with heated and/or extracted sections, a selective accumulation of metabolites occurred in a number of tissues, preferentially in the reported target tissues for DBE-induced lesions [i.e., in the nasal cavity, lung, forestomach, and liver (tumors) and the adrenal, testicle, liver, and kidney (nonneoplastic lesions)]. High levels of nonextractable metabolites were registered in the epithelia of the entire respiratory tract, the upper alimentary tract, the vagina, and the subepithelial glands of the olfactory mucosa. Lower levels of metabolites were observed in the liver, adrenal cortex, testicular interstitium, and kidney. Autoradiography of slices from various extrahepatic tissues incubated in vitro with DBE showed that most epithelia of the respiratory tract, upper alimentary tract, vagina, and the testicular interstitium have a marked ability to activate DBE to metabolites that become bound to the tissue. Further in vitro experiments, performed with S-1 fractions prepared from various tissues, indicated that the nasal mucosa was most active in transforming DBE to products which could not be extracted from the protein precipitate. It is proposed that tissue-selective metabolism and activation of DBE in the epithelia of the respiratory and upper alimentary tract are responsible for the observed DBE-induced lesions in these organ systems.

Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione.

The major DNA adduct formed from the carcinogen ethylene dibromide (1,2-dibromoethane, EDB) is S-[2-(N7-guanyl)ethyl]glutathione, resulting from the reaction of guanyl residues with the half-mustard S-(2-bromoethyl)glutathione, which is generated by glutathione S-transferase-catalyzed conjugation of EDB with glutathione. The half-life of the alkylating species [putative S-(2-bromoethyl)glutathione or the derived episulfonium ion] was estimated to be less than 10 s. However, the stability was enough for approximately half of the alkylating metabolites to leave isolated rat hepatocytes before reacting with nucleic acids. Treatment of isolated rat hepatocytes with diethylmaleate decreased covalent binding of EDB to DNA, but treatment with 1-phenylimidazole did not, consistent with the view that conjugative metabolism is of greater importance than oxidation with regard to DNA binding. When EDB was administered to rats in vivo, only one major adduct, S-[2-(N7-guanyl)ethyl]glutathione, was formed in liver or kidney. S-[2-(N7-Guanyl)ethyl]glutathione was found in liver and kidney DNA of rats treated with 1,2-dichloroethane, but other adducts were also present. The gamma-glutamyl transpeptidase inhibitor AT-125 [L-(alpha-(5S)-alpha-amino-S-chloro-4,5-dihydro-5-isoxazoleacetic acid] did not affect the level of EDB bound to DNA by glutathione-fortified rat kidney homogenates or bound to liver or kidney DNA in vivo. The in vitro half-life of S-[2-(N7-guanyl)ethyl]glutathione in calf thymus DNA was 150 h; the half-life of the adduct in rat liver, kidney, stomach, and lung was between 70 and 100 h. Isolated S-[2-(N7-guanyl)ethyl]glutathione did not react with DNA to form new adducts. These results provide a further basis for understanding the carcinogenic action of 1,2-dihaloethanes.

Excretion of the mercapturic acid S-[2-(N7-guanyl)ethyl]-N-acetylcysteine in urine following administration of ethylene dibromide to rats.

Administration of the carcinogen ethylene dibromide (EDB) to rats resulted in the urinary excretion of S-[2-(N7-guanyl)ethyl]-N-acetylcysteine, which is derived from the nucleic acid adduct S-[2-(N7-guanyl)ethyl]glutathione. This mercapturic acid was isolated from urine by reversed-phase and propylamino high-performance liquid chromatography and was quantitated by measurement of fluorescence intensity. The urinary mercapturic acid was identified as S-[2-(N7-guanyl)ethyl]-N-acetylcysteine on the basis of cochromatography and UV, fluorescence, 1H nuclear magnetic resonance, and fast atom bombardment mass spectra, all of which were identical with the authentic synthesized material. The excretion of mercapturic acid into urine of rats given injections of various doses of EDB occurred in a dose-dependent, linear manner over the range of 0.5-37 mg EDB/kg body weight. A good correlation was found between the excretion of mercapturic acid and the (in vivo) formation of DNA adducts in liver and kidney DNA. The higher level of urinary mercapturic acid compared to the level of hepatic DNA adduct indicates that extra-hepatic DNA adducts and RNA adducts may contribute to the mercapturic acid production. The measurement of the mercapturic acid may provide a means of noninvasive estimation of DNA adducts derived from EDB exposure.

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.

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes.

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using 13C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.

Molecular mechanisms of dibromoalkane cytotoxicity in isolated rat hepatocytes.

The cytotoxicity of dibromoalkanes to isolated hepatocytes was proportional to the dibromoalkane concentration and increasing chain length of the dibromoalkane (C2-C6). The rapid hepatocyte glutathione (GSH) depletion which occurred upon addition of the dibromoalkanes was also dependent on the concentration and chain length of the dibromoalkane. When added to hepatocytes, dibromoalkanes also caused a loss in protein sulfhydryl groups. After a lag period, lipid peroxidation occurred before the onset of cytotoxicity. Antioxidants or removing the oxygen from the medium markedly delayed dibromoalkane cytotoxicity. Bromoaldehydic metabolites formed by cytochrome P450-dependent mixed-function oxidases were probably responsible for lipid peroxidation as deuterated 1,2-dibromoethane (d4-DBE) induced less lipid peroxidation and was less cytotoxic even though GSH was depleted as rapidly and as effectively. Hepatocytes were also more resistant to dibromoalkanes if cytochrome P450 isoenzymes were inactivated with SKF 525A or methyl pyrazole. Furthermore, hepatocyte susceptibility to dibromoalkanes was increased markedly if aldehyde dehydrogenase was inactivated with disulfiram, cyanamide or chloral hydrate. Cytochrome P450-induced hepatocytes isolated from pyrazole-, phenobarbital- or 3-methylcholanthrene-pretreated rats were also more susceptible to dibromoalkanes. These results suggest that dibromoalkane-induced cell lysis is due to lipid peroxidation as well as cytochrome P450-dependent formation of toxic bromoaldehydic metabolites which can bind with cellular macromolecules. Dibromoethane GSH conjugates also contribute to DBE cytotoxicity as depleting hepatocyte GSH beforehand increased hepatocyte resistance to DBE but not other dibromoalkanes.

Conjugative metabolism of 1,2-dibromoethane in mitochondria: disruption of oxidative phosphorylation and alkylation of mitochondrial DNA.

1,2-Dibromoethane (DBE) is an environmental contaminant that is metabolized by glutathione S-transferases to a haloethane-glutathione conjugate. Since haloethane-glutathione conjugates are known to alkylate nuclear DNA and cytoplasmic proteins, these effects were investigated in isolated rat liver mitochondria exposed to DBE by measuring guanine adducts and several aspects of oxidative phosphorylation including respiratory control ratios, respiratory enzyme activity, and ATP levels. Mitochondrial large-amplitude swelling and glutathione status were assessed to evaluate mitochondrial membrane integrity and function. When exposed to DBE, mitochondria became uncoupled rapidly, yet no large-amplitude swelling or extramitochondrial glutathione was observed. Mitochondrial GSH was depleted to 2-53% of controls after a 60-min exposure to micromolar quantities of DBE; however, no extramitochondrial GSH or GSSG was detected. The depletion of mitochondrial glutathione corresponded to an increase of an intramitochondrial GSH-conjugate which, based on HPLC elution profiles and retention times, appeared to be S,S'-(1,2-ethanediyl)bis(glutathione). Activities of the NADH oxidase and succinate oxidase respiratory enzyme systems were inhibited 10-74% at micromolar levels of DBE, with succinate oxidase inactivation occurring at lower doses. ATP concentrations in DBE-exposed mitochondria in the presence of succinate were 5-90% lower than in the controls. The DNA adduct S-[2-(N(7)-guanyl)ethyl]glutathione was detected by HPLC in mtDNA isolated from DBE-exposed mitochondria. The results suggest that respiratory enzyme inhibition, glutathione depletion, decreased ATP levels, and DNA alkylation in DBE-exposed mitochondria occur via the formation of an S-(2-bromoethyl)glutathione conjugate, the precursor of the episulfonium ion alkylating species of DBE.

Biochemical studies on the metabolic activation of halogenated alkanes.

This paper reviews recent investigations by Slater and colleagues into the metabolic activation of halogenated alkanes in general and carbon tetrachloride in particular. It is becoming increasingly accepted that free radical intermediates are involved in the toxicity of many such compounds through mechanisms including lipid peroxidation, covalent binding, and cofactor depletion. Here we describe the experimental approaches that are used to establish that halogenated alkanes are metabolized in animal tissues to reactive free radicals. Electron spin resonance spectroscopy is used to identify free-radical products, often using spin-trapping compounds. The generation of specific free radicals by radiolytic methods is useful in the determination of the precise reactivity of radical intermediates postulated to be injurious to the cell. The enzymic mechanism of the production of such free radicals and their subsequent reactions with biological molecules is studied with specific metabolic inhibitors and free-radical scavengers. These combined techniques provide considerable insight into the process of metabolic activation of halogenated compounds. It is readily apparent, for instance, that the local oxygen concentration at the site of activation is of crucial importance to the subsequent reactions; the formation of peroxy radical derivatives from the primary free-radical product is shown to be of great significance in relation to carbon tetrachloride and may be of general importance. However, while these studies have provided much information on the biochemical mechanisms of halogenated alkane toxicity, it is clear that many problems remain to be solved.

Biodehalogenation.

Haloorganic biocides are widely employed as soil fumigants to combat the destructive action of plant parasitic nematodes and fungi. These substances are dehalogenated by soil organisms, principally species of Pseudomonas and Flavobacteria, to nontoxic metabolities. The paths of metabolism of a vareity of simply alkyl halides are described with emphasis upon the biodehalogenation step.

Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts.

Ethylene dibromide (1,2-dibromoethane, EDB) can be activated to electrophilic species by either oxidative metabolism or conjugation with glutathione. Although conjugation is generally a route of detoxication, in this case it leads to genetic damage. The major DNA adduct has been identified as S-[2-(N7-guanyl)ethyl]glutathione, which is believed to arise via half-mustard and episulfonium ion intermediates. The adduct has a half-life of about 70 to 100 hr and does not appear to migrate to other DNA sites. Glutathione-dependent DNA damage by EDB was also demonstrated in human hepatocyte preparations. The possible relevance of this DNA adduct to genetic damage is discussed.

In vivo and in vitro binding of 1,2-dibromoethane and 1,2-dichloroethane to macromolecules in rat and mouse organs.

The comparative interaction of equimolar amounts of 1,2-dichloroethane and 1,2-dibromoethane with rat and mouse nucleic acids was studied in both in vivo (liver, lung, kidney and stomach) and in vitro (liver microsomal and/or cytosolic fractions) systems. In vivo, liver and kidney DNA showed the highest labeling, whereas the binding to lung DNA was barely detectable. Dibromoethane was more highly reactive than dichloroethane in both species. With dichloroethane, mouse DNA labeling was higher than rat DNA labeling whatever the organ considered: the opposite was seen for the bioactivation of dibromoethane. RNA and protein labelings were higher than DNA labeling, with no particular pattern in terms of organ or species involvement. In vitro, in addition to a low chemical reactivity towards nucleic acids shown by haloethanes per se, both compounds were bioactivated by either liver microsomes and cytosolic fractions to reactive forms capable of binding to DNA and polynucleotides. UV irradiation did not photoactivate dibromoethane and dichloroethane. The in vitro interaction with DNA mediated by enzymatic fractions was PB-inducible (one order of magnitude, using rat microsomes). In vitro bioactivation of haloethanes was mainly performed by microsomes in the case of dichloroethane and by cytosolic fractions in the case of dibromoethane. When microsomes plus cytosol were used, rat enzymes were more efficient than mouse enzymes in inducing a dibromoethane-DNA interaction: the opposite situation occurred for dichloroethane-DNA interaction, and this is in agreement with the in vivo pattern. In the presence of both metabolic pathways, addition or synergism occurred. Dibromoethane was always more reactive than dichloroethane. An indication of the presence of a microsomal GSH transferase was achieved for the activation of dibromoethane. No preferential binding in vitro to a specific polynucleotide was found. Polynucleotide labeling was higher than (or equal to) DNA binding. The labeling of microsomal RNA and proteins and of cytosolic proteins was many times lower than that of DNA or polynucleotides. The in vivo and in vitro data reported above give an unequivocal indication of the relative reactivity of the haloethanes examined with liver macromolecules from the two species and agree, on the whole, with the relative genotoxicity (DNA repair induction ability, mutagenicity and carcinogenicity) of the chemicals.