Two distinct Dehalobacter strains sequentially dechlorinate 1,1,1-trichloroethane and 1,1-dichloroethane at a field site treated with granular zero valent iron and guar gum.

Chlorinated ethanes are environmental pollutants found frequently at many contaminated industrial sites. 1,1,1-Trichloroethane (1,1,1-TCA) can be dechlorinated and detoxified via abiotic transformation or biologically by the action of dechlorinating microorganisms such as Dehalobacter (Dhb). At a field site, it is challenging to distinguish abiotic vs. biotic mechanisms as both processes share common transformation products. In this study, we evaluated using the Dhb 16S rRNA gene and specific reductive dehalogenase genes as biomarkers for 1,1,1-TCA and 1,1-dichloroethane (1,1-DCA) dechlorination. We analyzed samples from laboratory groundwater microcosms and from an industrial site where a mixture of granular zero valent iron (ZVI) and guar gum was injected for 1,1,1-TCA remediation. Abiotic and biotic transformation products were monitored and the changes in dechlorinating organisms were tracked using quantitative PCR (qPCR) with primers targeting the Dhb 16S rRNA gene and two functional genes cfrA and dcrA encoding enzymes that dechlorinate 1,1,1-TCA to 1,1-DCA and 1,1-DCA to chloroethane (CA), respectively. The abundance of the cfrA- and dcrA-like genes confirmed that the two dechlorination steps were carried out by two distinct Dhb populations at the site. The biomarkers used in this study proved useful for monitoring different Dhb populations responsible for step-wise dechlorination and tracking biodegradation of 1,1,1-TCA and 1,1-DCA where both abiotic (e.g., with ZVI) and biotic processes co-occur.

Microbial reductive dehalogenation of trihalomethanes by a Dehalobacter-containing co-culture.

Trihalomethanes such as chloroform and bromoform, although well-known as a prominent class of disinfection by-products, are ubiquitously distributed in the environment due to widespread industrial usage in the past decades. Chloroform and bromoform are particularly concerning, of high concentrations detected and with long half-lives up to several hundred days in soils and groundwater. In this study, we report a Dehalobacter- and Desulfovibrio-containing co-culture that exhibits dehalogenation of chloroform (~0.61 mM) to dichloromethane and bromoform (~0.67 mM) to dibromomethane within 10-15 days. This co-culture was further found to dechlorinate 1,1,1-trichloroethane (1,1,1-TCA) (~0.65 mM) to 1,1-dichloroethane within 12 days. The Dehalobacter species present in this co-culture, designated Dehalobacter sp. THM1, was found to couple growth with dehalogenation of chloroform, bromoform, and 1,1,1-TCA. Strain THM1 harbors a newly identified reductive dehalogenase (RDase), ThmA, which catalyzes chloroform, bromoform, and 1,1,1-TCA dehalogenation. Additionally, based on the sequences of thmA and other identified chloroform RDase genes, ctrA, cfrA, and tmrA, a pair of chloroform RDase gene-specific primers were designed and successfully applied to investigate the chloroform dechlorinating potential of microbial communities. The comparative analysis of chloroform RDases with tetrachloroethene RDases suggests a possible approach in predicting the substrate specificity of uncharacterized RDases in the future.

Rough-interval-based multicriteria decision analysis for remediation of 1,1-dichloroethane contaminated groundwater.

A rough-interval-based multicriteria decision analysis method (RI-MCDA) is developed for supporting the selection of remediation strategies for 1,1-dichloroethane contaminated sites. The concept of ''rough interval'' is introduced in the design framework to represent dual-uncertain parameters. Three rough-interval scenarios generated through pair-wise combining the values under three confidence levels (i.e. 68.3%, 95.4% and 99.7%) and one deterministic scenario adopted crisp numbers for parameters are introduced into the framework. The proposed method is then applied to a contaminated site in the Pudong district of Shanghai, China. Fifty remediation alternatives under four duration options (i.e. 5, 10, 15, and 20 years) and ten criteria, including daily total pumping rate, total cost and rough-interval risk information in light of uncertainty parameter (e.g. slope factor), are taken into consideration to compare different alternatives through RI-MCDA. Results indicated that the most desirable remediation strategy lied in A25 for the 5-year, A10 for the 10-year, A15 for the 15-year, and A11 for the 20-year remediation. Compared to the traditional MCDA, the proposed RI-MCDA shows the uniqueness in addressing the interaction between dual intervals of highly uncertain parameters, as well as their joint impact on the decision results, which reduces the subjectivity as much as possible.

Fragment Coupling and the Construction of Quaternary Carbons Using Tertiary Radicals Generated From tert-Alkyl N-Phthalimidoyl Oxalates By Visible-Light Photocatalysis.

The coupling of tertiary carbon radicals with alkene acceptors is an underdeveloped strategy for uniting complex carbon fragments and forming new quaternary carbons. The scope and limitations of a new approach for generating nucleophilic tertiary radicals from tertiary alcohols and utilizing these intermediates in fragment coupling reactions is described. In this method, the tertiary alcohol is first acylated to give the tert-alkyl N-phthalimidoyl oxalate, which in the presence of visible-light, catalytic Ru(bpy)3(PF6)2, and a reductant fragments to form the corresponding tertiary carbon radical. In addition to reductive coupling with alkenes, substitution reactions of tertiary radicals with allylic and vinylic halides is described. A mechanism for the generation of tertiary carbon radicals from tert-alkyl N-phthalimidoyl oxalates is proposed that is based on earlier pioneering investigations of Okada and Barton. Deuterium labeling and competition experiments reveal that the reductive radical coupling of tert-alkyl N-phthalimidoyl oxalates with electron-deficient alkenes is terminated by hydrogen-atom transfer.

A microscopic view of phospholipid insertion into biological membranes.

Understanding the process of membrane insertion is an essential step in developing a detailed mechanism, for example, for peripheral membrane protein association and membrane fusion. The highly mobile membrane mimetic (HMMM) has been used to accelerate the membrane association and binding of peripheral membrane proteins in simulations by increasing the lateral diffusion of phospholipid headgroups while retaining an atomistic description of the interface. Through a comparative study, we assess the difference in insertion rate of a free phospholipid into an HMMM as well as into a conventional phospholipid bilayer and develop a detailed mechanistic model of free phospholipid insertion into biological membranes. The mechanistic insertion model shows that successful irreversible association of the free phospholipid to the membrane interface, which results in its insertion, is the rate-limiting step. Association is followed by independent, sequential insertion of the acyl tails of the free phospholipid into the membrane, with splayed acyl tail intermediates. Use of the HMMM is found to replicate the same intermediate insertion states as in the full phospholipid bilayer; however, it accelerates overall insertion by approximately a factor of 3, with the probability of successful association of phospholipid to the membrane being significantly enhanced.

Identification of Dehalobacter reductive dehalogenases that catalyse dechlorination of chloroform, 1,1,1-trichloroethane and 1,1-dichloroethane.

Two novel reductive dehalogenases (RDases) that are highly similar to each other but catalyse distinct dechlorination reactions were identified from Dehalobacter-containing mixed cultures. These two RDases were partially purified from crude protein extracts of anaerobic dechlorinating enrichment cultures using blue native polyacrylamide gel electrophoresis. Gel slices were assayed for dechlorinating activity, and associated proteins were identified using liquid chromatography tandem mass spectrometry with the metagenome of the parent culture as the reference database. The two RDases identified, annotated as CfrA and DcrA, share an amino acid identity of 95.2 per cent, but use different substrates: CfrA dechlorinates chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA), but not 1,1-dichloroethane; DcrA dechlorinates 1,1-dichloroethane, but not CF or 1,1,1-TCA. These two novel RDases share no more than 40 per cent amino acid identity to any other known or putative RDases, but both have a twin-arginine motif and two iron-sulfur binding motifs conserved in most RDases. Peptides specific to two putative membrane anchor proteins, annotated as CfrB and DcrB, were also detected in gel slices.

Abiotic degradation of chlorinated ethanes and ethenes in water.

INTRODUCTION: Chlorinated ethanes and ethenes are among the most frequently detected organic pollutants of water. Their physicochemical properties are such that they can contaminate aquifers for decades. In favourable conditions, they can undergo degradation. In anaerobic conditions, chlorinated solvents can undergo reductive dechlorination. DEGRADATION PATHWAYS: Abiotic dechlorination is usually slower than microbial but abiotic dechlorination is usually complete. In favourable conditions, abiotic reactions bring significant contribution to natural attenuation processes. Abiotic agents that may enhance the reductive dechlorination of chlorinated ethanes and ethenes are zero-valent metals, sulphide minerals or green rusts. OXIDATION: At some sites, permanganate and Fenton's reagent can be used as remediation tool for oxidation of chlorinated ethanes and ethenes. SUMMARY: Nanoscale iron or bimetallic particles, due to high efficiency in degradation of chlorinated ethanes and ethenes, have gained much interest. They allow for rapid degradation of chlorinated ethanes and ethenes in water phase, but they also give benefit of treating dense non-aqueous phase liquid.

Quantifying the effects of 1,1,1-trichloroethane and 1,1-dichloroethane on chlorinated ethene reductive dehalogenases.

Mixtures of chlorinated ethenes and ethanes are often found at contaminated sites. In this study, we undertook a systematic investigation of the inhibitory effects of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) on chlorinated ethene dechlorination in three distinct Dehalococcoides-containing consortia. To focus on inhibition acting directly on the reductive dehalogenases, dechlorination assays used cell-free extracts prepared from cultures actively dechlorinating trichloroethene (TCE) to ethene. The dechlorination assays were initiated with TCE, cis-1,2-dichloroethene (cDCE), or vinyl chloride (VC) as substrates and either 1,1,1-TCA or 1,1-DCA as potential inhibitors. 1,1,1-TCA inhibited VC dechlorination similarly in cell suspension and cell-free extract assays, implicating an effect on the VC reductases associated with the dechlorination of VC to nontoxic ethene. Concentrations of 1,1,1-TCA in the range of 30-270 µg/L reduced VC dechlorination rates by approximately 50% relative to conditions without 1,1,1-TCA. 1,1,1-TCA also inhibited reductive dehalogenases involved in TCE and cDCE dechlorination. In contrast, 1,1-DCA had no pronounced inhibitory effects on chlorinated ethene reductive dehalogenases, indicating that removal of 1,1,1-TCA via reductive dechlorination to 1,1-DCA is a viable strategy to relieve inhibition.

Simultaneous counter-flow of chlorinated volatile organic compounds across the saturated-unsaturated interface region of an aquifer.

Concentrations of chlorinated volatile organic compounds (Cl-VOCs) at the saturated-unsaturated interface region (SUIR; depth of approximately 18m) of a sandy phreatic aquifer were measured in two monitoring wells located 25m apart. The concentrations of the Cl-VOCs obtained above and below the water table along a 413-day period are interpreted to depict variable, simultaneous and independent movement of trichlorothene, tetrachloroethene, 1,1-dichloroethene, cis-1,2-dichloroethene, 1,1,1-trichloroethane, chloroform and 1,1-dichloroethane vapors in opposite directions across the SUIR.

1,1,1-trichloroethane and 1,1-dichloroethane reductive dechlorination kinetics and co-contaminant effects in a Dehalobacter-containing mixed culture.

1,1,1-Trichloroethane (1,1,1-TCA) is a common groundwater contaminant that can be reductively dechlorinated to 1,1-dichloroethane (1,1-DCA) and monochloroethane, and can support the growth of certain dehalorespiring strains of Dehalobacter We used reductive dehalogenase cell-free extract assays (with reduced methyl viologen) and whole cell suspension dechlorination assays (with hydrogen) and a Dehalobacter-containing enrichment culture to explore the kinetics of l,1,1-TCA and 1,1-DCA reductive dechlorination in the presence of the common co-contaminants trichloroethene (TCE), cis-dichloroethene (cDCE), and vinyl chloride (VC). These chlorinated ethenes were most significant inhibitors of 1,1,1-TCA dechlorination in cell-free extracts, indicating direct effects on the reductive dehalogenase enzyme(s). The inhibition was present but less pronounced in whole cell suspension assays. None of the chlorinated ethenes inhibited 1,1-DCA dechlorination in cell-free extract assays, yet cDCE and particularly VC were inhibitors in whole cell assays, indicating an effect on Dehalobacter, but not on the dehalogenase enzyme(s). Marked differences in kinetic parameters for 1,1,1-TCA and 1,1-DCA, and an uncoupling of these two activities in cultures grown on 1,1-DCA compared to those grown on 1,1,1-TCA was strong evidence for the existence of distinct 1,1,1-TCA and 1,1-DCA reductive dehalogenase enzymes.

Bioaugmentation with butane-utilizing microorganisms to promote in situ cometabolic treatment of 1,1,1-trichloroethane and 1,1-dichloroethene.

A field study was performed to evaluate the potential for in-situ aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) through bioaugmentation with a butane enrichment culture containing predominantly two Rhodococcus sp. strains named 179BP and 183BP that could cometabolize 1,1,1-TCA and 1,1-dicholoroethene (1,1-DCE). Batch tests indicated that 1,1-DCE was more rapidly transformed than 1,1,1-TCA by both strains with 183BP being the most effective organism. This second in a series of bioaugmentation field studies was conducted in the saturated zone at the Moffett Field In Situ Test Facility in California. In the previous test, bioaugmentation with an enrichment culture containing the 183BP strain achieved short term in situ treatment of 1,1-DCE, 1,1,1-TCA, and 1,1-dichloroethane (1,1-DCA). However, transformation activity towards 1,1,1-TCA was lost over the course of the study. The goal of this second study was to determine if more effective and long-term treatment of 1,1,1-TCA could be achieved through bioaugmentation with a highly enriched culture containing 179BP and 183BP strains. Upon bioaugmentation and continuous addition of butane and dissolved oxygen and or hydrogen peroxide as sources of dissolved oxygen, about 70% removal of 1,1,1-TCA was initially achieved. 1,1-DCE that was present as a trace contaminant was also effectively removed (approximately 80%). No removal of 1,1,1-TCA resulted in a control test leg that was not bioaugmented, although butane and oxygen consumption by the indigenous populations was similar to that in the bioaugmented test leg. However, with prolonged treatment, removal of 1,1,1-TCA in the bioaugmented leg decreased to about 50 to 60%. Hydrogen pexoxide (H2O2) injection increased dissolved oxygen concentration, thus permitting more butane addition into the test zone, but more effective 1,1,1-TCA treatment did not result. The results showed bioaugmentation with the enrichment cultures was effective in enhancing the cometabolic treatment of 1,1,1-TCA and low concentrations of 1,1-DCE over the entire period of the 50-day test. Compared to the first season of testing, cometabolic treatment of 1,1,1-TCA was not lost. The better performance achieved in the second season of testing may be attributed to less 1,1-DCE transformation product toxicity, more effective addition of butane, and bioaugmentation with the highly enriched dual culture.

Measurement of residual solvents in a drug substance by a purge-and-trap method.

The purge-and-trap (P&T) gas extraction method combined with gas chromatography was studied for its suitability for quantitative residual solvents determination in a water-soluble active pharmaceutical ingredient (API). Some analytical method performance characteristics were investigated, namely, the repeatability, the accuracy and the detection limit of determination. The results show that the P&T technique is--as expected--more sensitive than the static headspace, thus it can be used for the determination of residual solvents pertaining to the ICH Class 1 group. It was found that it could be an alternative sample preparation method besides the static headspace (HS) method.

Potential for identifying abiotic chloroalkane degradation mechanisms using carbon isotopic fractionation.

Degradation of 1,1- and 1,2-dichloroethane (1,1-DCA, 1,2-DCA) and carbon tetrachloride (CCl4) on Zn0 was investigated using compound specific isotope analysis (CSIA) to measure isotopic fractionation factors for chloroalkane degradation by hydrogenolysis, by alpha-elimination, and by beta-elimination. Significant differences in enrichment factors (epsilon) and associated apparent kinetic isotope effects (AKIE) were measured for these different reaction pathways, suggesting that carbon isotope fractionation by beta-elimination is substantially larger than fractionation by hydrogenolysis or by alpha-elimination. Specifically, for 1,1-DCA, the isotopic composition of the reductive alpha-elimination product (ethane) and the hydrogenolysis product (chloroethane) were the same, indicating that cleavage of a single C-Cl bond was the rate-limiting step in both cases. In contrast, for 1,2-DCA, epsilon = epsilon(reactive position) = -29.7 +/- 1.5% per hundred, and the calculated AKIE (1.03) indicated that beta-elimination was likely concerted, possibly involving two C-Cl bonds simultaneously. Compared to 1,1-DCA hydrogenolysis, the AKIE of 1.01 for hydrogenolysis of CCl4 was much lower, indicating that, for this highly reactive organohalide, mass transfer to the surface was likely partially rate-limiting. These findings are a first step toward delineating the relative contribution of these competing pathways in other abiotic systems such as the degradation of chlorinated ethenes on zerovalent iron (ZVI), iron sulfide, pyrite, or magnetite, and, potentially, toward distinguishing between degradation of chlorinated ethenes by abiotic versus biotic processes.

Enzymatic removal of carboxyl protecting groups. III. Fast removal of allyl and chloroethyl esters by Bacillus subtilis esterase (BS2).

An esterase from Bacillus subtilis (BS2) allows the fast and selective removal of allyl, 2-chloroethyl, and 2,2,2-chloroethyl esters under mild conditions in high yields. In addition, BS2 easily hydrolyzes phenacyl esters, while the hydrolysis of sterically hindered diphenylmethyl esters is slow, requiring longer reaction time and higher enzyme/substrate ratio.

De novo formation of chloroethyne in soil.

To date, chloroethyne in the environment has been proposed to occur as a reactive intermediate during the reductive dechlorination of tri- and tetrachloroethene with zerovalent metals. Such artificial conditions might possibly be found at organohalide-contaminated sites that are surrounded by remediation barriers made of metallic iron. In this paper, it is shown that the highly reactive chloroethyne is also a product of natural processes in soil. Soil air samples from three differentterrestrial ecosystems of Northern Germany showed significant chloroethyne concentrations, besides other naturally produced monochlorinated compounds, such as chloromethane, chloroethane and chloroethene. Measured amounts range from 5 to 540 pg chloroethyne in air purged from 1 L of soil. A possible route of chloroethyne formation in soil is discussed, where chloroethyne is probably produced as a byproduct of the oxidative halogenation of aromatic compounds in soil. A series of laboratory studies, using the redox-sensitive catechol as a discrete organic model compound, showed the formation of chloroethyne when Fe3+ and hydrogen peroxide were added to the system. We therefore propose that the natural formation of chloroethyne in soil proceeds via oxidative cleavage of a quinonic system in the presence of the ubiquitous soil component chloride.

Radiolysis of aqueous solutions of 1,1- and 1,2-dichloroethane.

The yields of chloride ion and molecular hydrogen were determined in the gamma, the fast electron, and the 5 MeV helium ion radiolysis of deaerated and aerated aqueous solutions of 1,1- and 1,2-dichloroethane. In deaerated solutions irradiated with gamma-rays or fast electrons, the yield of chloride ion increases while the yield of molecular hydrogen decreases with increasing dichloroethane concentration. These results are due to the quantitative reaction of both the hydrated electron and the hydrogen atom with the dichloroethane to produce chloride ions. The yield of chloride ions is significantly larger in aerobic than in anaerobic conditions and is dependent upon the dose rate. Formation of peroxyl radicals by the reaction of molecular oxygen with chlorinated hydrocarbon radicals and their subsequent chemistry are responsible for the observed increase in chloride ions. The yield of chloride ion with 5 MeV helium ions is smaller than with gamma irradiation, while the yield of molecular hydrogen is larger reflecting the higher density of reactive species and consequent increase in intratrack reactions in a helium ion track compared to a gamma-ray track.

Cytogenetic effects of 1,1-dichloroethane in mice bone marrow cells.

The major concern for the halogenated compounds is their widespread distribution, in addition to occupational exposures. Several chlorinated alkanes and alkenes were found to induce toxic effects. In this study, we investigated the genotoxic potential of 1,1-dichloroethane in the bone marrow cells obtained from Swiss-Webster mice, using chromosomal aberrations (CA), mitotic index (MI), and micronuclei (MN) formation as toxicological endpoints. Five groups of three male mice each, weighing an average of 24 +/- 2 g, were injected intraperitoneally, once with doses of 100, 200, 300, 400, 500 mg/kg body weight (BW) of 1,1-dichloroethane dissolved in ethanol. A control group was also made of three animals injected with ethanol (1%) without the chemical. All animals were sacrificed 24 hours after the treatment. Chromosome and micronuclei preparations were obtained from bone marrow cells following standard protocols. Chromatid and chromosome aberrations were investigated in 100 metaphase cells per animal and percent micronuclei frequencies were investigated in 1,000 metaphase cells per animal. 1,1-dichloroethane exposures significantly increased the number of chromosomal aberrations and the frequency of micronucleated cells in the bone marrow cells of Swiss-Webster mice. Percent chromosomal aberrations of 2.67 +/- 0.577, 7.66 +/- 2.89, 8.33 +/- 2.08, 14.67 +/- 2.51, 20.3 +/- 3.21, 28 +/- 3.61; mitotic index of 9.4%, 7.9%, 6.2%, 4.3%, 3.0%, 2.6% and micronuclei frequencies of 3.33 +/- 0.7, 7.33 +/- 0.9, 8.00 +/- 1.0, 11.67 +/- 1.2, 15.33 +/- 0.7, 18.00 +/- 1.7 were recorded for the control, 100, 200, 300, 400, and 500 mg/kg BW respectively; indicating a gradual increase in number of chromosomal aberrations and micronuclei formation, with increasing dose of 1,1,-dichloroethane. Our results indicate that 1,1-dichloroethane has a genotoxic potential as measured by the bone marrow CA and MN tests in Swiss-Webster mice.

Acute, subacute, and subchronic oral toxicity studies of 1,1-dichloroethane in rats: application to risk evaluation.

1,1-Dichloroethane (DCE) is a solvent that is often found as a contaminant of drinking water and a pollutant at hazardous waste sites. Information on its short- and long-term toxicity is so limited that the U.S. EPA and ATSDR have not established oral reference doses or minimal risk levels for the volatile organic chemical (VOC). The acute oral LD(50) in male Sprague-Dawley (S-D) rats was estimated in the present study to be 8.2 g/kg of body weight (bw). Deaths appeared to be due to CNS depression and respiratory failure. In an acute/subacute experiment, male S-D rats were given 0, 1, 2, 4, or 8 g DCE/kg in corn oil by gavage for 1, 5, or 10 consecutive days. The animals were housed in metabolism cages for collection of urine and sacrificed for blood and tissue sampling 24 h after their last dose. There were decreases in body weight gain and relative liver weight at all dosage levels, as well as increased renal nonprotein sulfhydryl levels at 2 and 4 g/kg after 5 and 10 days. Elevated serum enzyme levels, histopathological changes, and abnormal urinalyses were not manifest. For the subchronic study, adult male S-D rats were gavaged with 0.5, 1, 2, or 4 g DCE/kg 5 times weekly for up to 13 weeks. Animals receiving 4 g/kg exhibited pronounced CNS depression, with more than one-half dying by week 11. The 2-g/kg rats exhibited moderate CNS depression. One 2-g/kg rat died during week 6. There were very few manifestations of organ damage in animals that succumbed or in survivors at any dosage level. Decreases in bw gain and transient increases in enzymuria were noted at 2 and 4 g/kg. Serum enzyme levels and blood urea nitrogen were not elevated, nor were glycosuria or proteinuria present. Chemically induced histological changes were not seen in the liver, kidney, lung, brain, adrenal, spleen, stomach, epididymis, or testis. Hepatic microsomal cytochrome P450 experiments revealed that single, high oral doses of DCE did not alter total P450 levels, but did induce CYP2E1 levels and activity and inhibit CYP1A1 activity. These effects were reversible and regressed with repeated DCE exposure. There was no apparent progression of organ damage during the 13-week subchronic study, nor appearance of adverse effects not seen in the short-term exposures. One g/kg orally (po) was found to be the acute, subacute, and subchronic LOAEL for DCE, under the conditions of this investigation. In each instance, 0.5 g/kg was the NOAEL.

Commensal interactions in a dual-species biofilm exposed to mixed organic compounds.

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.

Measurements of DNA damage in Chinese hamster cells treated with equitoxic and equimutagenic doses of nitrosoureas.

The DNA of V-79 Chinese hamster cells was examined by alkaline elution following treatment of cultures with eight different nitrosoureas. Drug incubations were performed under consistent biological conditions of equal toxicity and equal mutation induction at the hypoxanthineguanine phosphoribosyltransferase locus. The goals of this study were to determine whether DNA damage could be detected in cells treated with biologically relevant doses of nitrosoureas and to determine whether the type and number of observed DNA lesions could be correlated with the cytotoxic and mutagenic effects of the drugs. All of the compounds tested produced, to some degree, lesions that were observed as DNA strand breaks upon exposure of the DNA to alkali. The levels of DNA strand breaks and/or alkali-labile lesions were comparable for all of the drugs at the equimutagenic doses. DNA cross-linking was observed at both the equitoxic and the equimutagenic concentrations of the haloethylnitrosoureas, but cross-linking was not observed with methylnitrosourea or streptozotocin. Methylnitrosourea and streptozotocin required approximately 40 times the drug concentration to produce toxicity equal to the haloethylnitrosoureas. These data suggest that the ability to cross-link DNA confers increased cytotoxicity to the haloethylnitrosoureas.