Detoxification of hydroxylated polychlorobiphenyls by Sphingomonas sp. strain N-9 isolated from forest soil.

To examine the biodegradation of hydroxylated polychlorobiphenyls (OH-PCBs), we isolated Sphingomonas sp. strain N-9 from forest soil using mineral salt medium containing 4-hydroxy-3-chlorobiphenyl (4OH-3CB) at the concentration of 10 mg/L. Following incubation with strain N-9, the concentration of 4OH-3CB decreased in inverse proportion to strain N-9 proliferation, and it was converted to 3-chloro-4-hydroxybenzoic acid (4OH-3CBA) after 1 day. We observed that strain N-9 efficiently degraded lowly chlorinated OH-PCBs (1-4 Cl), while highly chlorinated OH-PCBs (5-6 Cl) were less efficiently transformed. Additionally, strain N-9 degraded PCBs and OH-PCBs with similar efficiencies, and the efficiency of OH-PCB degradation was dependent upon the positional relationships between OH-PCB hydroxyl groups and chlorinated rings. OH-PCB biodegradation may result in highly toxic products, therefore, we evaluated the cytotoxicity of two OH-PCBs [4OH-3CB and 4-hydroxy-3,5-dichlorobiphenyl (4OH-3,5CB)] and their metabolites [4OH-3CBA and 3,5-chloro-4-hydroxybenzoic acid (4OH-3,5CBA)] using PC12 rat pheochromocytoma cells. Our results revealed that both OH-PCBs induced cell membrane damage and caused neuron-like elongations in a dose-dependent manner, while similar results were not observed for their metabolites. These results indicated that strain N-9 can convert OH-PCBs into chloro-hydroxybenzoic acids having lower toxicity.

A putative porin gene of Burkholderia sp. NK8 involved in chemotaxis toward ß-ketoadipate.

Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and ß-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward ß-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward ß-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.

Aerobic degradation of 3-chlorobenzoic acid by an indigenous strain isolated from a polluted river.

An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l(-1) 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l(-1). Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.

Biodegradation of chlorobenzoic acids by ligninolytic fungi.

We investigated the abilities of several perspective ligninolytic fungal strains to degrade 12 mono-, di- and trichloro representatives of chlorobenzoic acids (CBAs) under model liquid conditions and in contaminated soil. Attention was also paid to toxicity changes during the degradation, estimated using two luminescent assay variations with Vibrio fischeri. The results show that almost all the fungi were able to efficiently degrade CBAs in liquid media, where Irpex lacteus, Pycnoporus cinnabarinus and Dichomitus squalens appeared to be the most effective in the main factors: degradation and toxicity removal. Analysis of the degradation products revealed that methoxy and hydroxy derivatives were produced together with reduced forms of the original acids. The findings suggest that probably more than one mechanism is involved in the process. Generally, the tested fungal strains were able to degrade CBAs in soil in the 85-99% range within 60 days. Analysis of ergosterol showed that active colonization is an important factor for degradation of CBAs by fungi. The most efficient strains in terms of degradation were I. lacteus, Pleurotus ostreatus, Bjerkandera adusta in soil, which were also able to actively colonize the soil. However, in contrast to P. ostreatus and I. lacteus, B. adusta was not able to significantly reduce the measured toxicity.

A survey of the cellular responses in Pseudomonas putida KT2440 growing in sterilized soil by microarray analysis.

Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both the triplicate-microarray analysis using amplified RNA and the single-microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB. Genes specifically involved in the transport of 3CB were upregulated, including a K(+)/H(+) antiporter complex, a universal stress protein, two cytochrome P450 proteins and an efflux transporter. The downregulated expression of several genes involved in carbon metabolism and the genes belonging to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis of gene expression in bacteria growing in sterilized soil.

Removal of 4-chlorobenzoic acid from spiked hydroponic solution by willow trees (Salix viminalis).

BACKGROUND: Chlorobenzoic acids (CBA) are intermediate products of the aerobic microbial degradation of PCB and several pesticides. This study explores the feasibility of using basket willows, Salix viminalis, to remove 4-CBA from polluted sites, which also might stimulate PCB degradation. METHODS: The removal of 4-CBA by willow trees was investigated with intact, septic willow trees growing in hydroponic solution and with sterile cell suspensions at concentrations of 5 mg/L and 50 mg/L 4-CBA. Nutrient solutions with different levels of ammonium and nitrate were prepared to achieve different pH levels. The concentration of 4-CBA was tracked over time and quantified by HPLC. RESULTS AND DISCUSSION: At the low level of 4-CBA (5 mg/L), willows removed 70% (pH 4.2) to 90% (pH 6.8), while 48% (pH 4.2) to 52% (pH 6.8) of the water was transpired. At the high 4-CBA level (50 mg/L), the pH varied between 4.4 and 4.6, and 10% to 30% of 4-CBA was removed, but only 5% to 9% of the water. In sterile cell suspensions, removal of 4-CBA by fresh biomass was much higher than removal by dead biomass. CONCLUSIONS: The results indicate that 4-CBA is toxic to willow trees at 50 mg/L. The removal of 4-CBA from solution is by both passive processes (uptake with water, sorption to plant tissue) and metabolic processes of the plants. RECOMMENDATIONS AND OUTLOOK: Plants, such as willow trees, might assist in the degradation of PCB and their degradation products CBA.

Genomic damage induced in tobacco plants by chlorobenzoic acids--metabolic products of polychlorinated biphenyls.

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.

Effects of cyanobacterium Fischerella ambigua isolates and cell free culture media on zebrafish (Danio rerio) embryo development.

The toxic effects of several species of fresh water cyanobacteria, notably Microcystis species and associated toxins, the microcystins, Anabaena species (anatoxin), Nodularia sp. (nodularin), and Cylindrospermopsis raciborskii (cylindrospermopsin), are well known. Little, however, is known about the effects of secondary metabolites other than alkaloids. Early life stage tests with zebrafish (Danio rerio) were used to detect bioactive properties of compounds released by healthy cyanobacteria (Fischerella ambigua), particularly on the early developmental stages of fish. This approach, using F. ambigua is probably most valuable as it shows the toxicity of healthy growing cyanobacteria. The effects of cyanobacterial secondary metabolites on the embryonic stages of fish are of considerable interest as many aquatic creatures, particularly fish, are unable to avoid the potential toxins that may be released by undesirable algal blooms or as a result of allelopathic effects. In the current study, the zebrafish (D. rerio) was used as a model experimental system to investigate the effects of ambigols A and C, tjipanazole D and C, 2,4-dichlorobenzoic acid, cell free culture media, and media extracts of a terrestrial/fresh water strain of the cyanobacterium F. ambigua on embryo development. Fish embryo tests performed with the cell free culture medium showed that after 3h of exposure to undiluted culture medium all fish embryos died. At a tenfold dilution the process of epiboly (formation of the gastrula) was retarded in all embryos, lesions were observed, and their general development was significantly arrested, finally followed by death. The same tests performed with extracts (dichloromethane, n-butanol, and residual cell free culture medium) of the cell free culture medium, ambigol A, ambigol C, 2,4-dichlorobenzoic acid and tjipanazole D showed only ambigol A to have an influence on zebrafish development at concentrations>or=1 mg/l (2.06 microM). After 55 h all embryos showed pectoral oedema, irregularly shaped fin folds, bent tails, and unusual circular neoplasms in the dorsal tail fin fold. Due to the high concentration of ambigol A used in this assay these effects were considered to be of minor importance when compared to those of the culture medium.

Physicochemical and toxicological studies on 4-chloro-3,5-dinitrobenzoic acid in aqueous solutions.

Physicochemical characterization of hazardous compounds often is required for the development of structure-reactivity correlations. Physical, chemical, and toxicological properties of target pollutants require determination for an efficient application of wastewater treatments. In the present work, we chose a chloro-nitro-aromatic derivative (4-chloro-3,5-dinitrobenzoic acid [CDNBA]), as a model compound on which to perform physicochemical and toxicological studies. Several properties of CDNBA are not available in the literature, although many aromatic nitro-compounds are considered hazardous materials. Measurements of solubility in water, acid dissociation constant, and kinetic parameters for the nucleophilic substitution of chlorine atom in alkaline media are reported. We also performed cytotoxicity studies of CDNBA and ultraviolet-irradiated CDNBA solutions. From the analysis of CDNBA solubility in water at different temperatures, an enthalpy of solution of 23.2 +/- 2.5 kJ/mol was found. The study of the acid dissociation constant Kc by using conductivity measurements and the modified Gran's method yielded values for the equilibrium constant Ka of 2.36 x 10(-3) and 2.26 x 10(-3), respectively. The bimolecular rate constant for the reaction of CDNB- and hydroxyl ion (HO) measured at room temperature and 0.1 M of ionic strength was 5.92/M x s, and the activation energy for this process was 70.7 +/- 3.4 kJ/mol. Cytotoxicity assays with aqueous suspensions of Tetrahymena pyriformis showed lethal effects due to the pH change induced by CDNBA. On the other hand, in buffered solutions, a value of 104.47 microM was observed for the median effective concentration, that is, the concentration of CDNBA at which the proliferation was restricted to one half of the blank. Irradiation of CDNBA solutions increased the toxicity, suggesting the formation of intermediate products with higher cytotoxicity effects.

Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in vivo.

m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks 36 h after a single CPBA treatment but is maximal 5 h after two applications of CPBA at a 48-h interval. The ODC-inducing activity of CPBA is dose dependent and sustained after chronic treatment. In contrast to TPA, two CPBA treatments at 12-24 h intervals produce no refractory state against ODC induction. The mechanism of ODC induction by CPBA is iron dependent. Various hydrolyzable tannins, condensed tannins (CTs) and their monomeric units remarkably inhibit the ODC response to multiple CPBA treatments. At 12 mg, gallic acid, Aleppo gall tannic acid (TA), catechin, and loblolly pine bark CT inhibit the most CPBA-induced ODC activity. Aleppo gall TA is even effective when applied several hours before CPBA. The tumor-promoting activity of CPBA and its inhibition by plant tannins remain to be evaluated.

Oral toxicity of trichlorobenzyl chloride and its acid derivative.

The acute rat oral LD50 of 2,4,6-Trichlorobenzyl chloride (TCBC) was determined to be 3075 mg/kg. When male and female rats were administered 1500 and 3000 ppm TCBC in the diet for 3 weeks, marked retardation in weight gain was observed. Reductions in food consumption were noted in both groups of treated male, but not female, rats. All blood chemistry and urinalysis parameters including those indicative of liver injury, were within the range of normal limits. Gross necropsy revealed pale livers in all high-dose rats and the majority of low-dose animals. Gross observations correlated with observation of degenerative hepatic changes (irregularity of cell and nuclear size, cord pattern, edema and cytoplasmic alterations) following microscopic evaluation. Rats and dogs administered up to 100 ppm trichlorobenzoic acid (TCBA) in the diet for 90 days exhibited no effects considered related to treatment.

Studies on the derivatives of aziridine. II. Synthesis and immunopharmacological analysis of substituted amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid.

Several new amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid were synthetized. The beta-/p-chlorobenzoyl/-acrylic acid 2 was used as the substrate. This compound was converted by reaction with appropriate amine into amides and anilides of beta-/p-chlorobenzoyl/-acrylic acid (3-10). These compounds react with ethylenoimine giving appropriate amides and anilides of alpha-aziridinyl-beta-/p-chlorobenzoyl/-propionic acid (11-18). When pharmacologically analyzed, they appeared to possess marked immunotropic activity. The derivatives in question modulated both humoral as well as cellular immune response, the effect being related to the type of substitutent in the amide group.

Studies on the derivatives of aziridine. I. synthesis and immunopharmacological analysis of amides of alpha-aziridinyl- beta-/p-chlorobenzoyl/-propionic acid.

Several new aziridinyl derivatives of beta-/p-chlorobenzoyl/propionic acid were synthesized (3, 5, 7, 14-18). The gamma-/p-chlorophenyl/-dihydrofuran-2-one 2 and beta-/p-chlorobenzoyl/acrylic acid 4 were used as the substrates. Compound 2 reacts with ethylenimine yielding aziridinylamide of beta-/p-chlorobenzoyl/-propionic acid 3. The sodium salt of acid 4 and methyl ester 6 were converted by reactions with ethylenimine into appropriate alpha-aziridinyl derivatives 5 and 7. The acid 4 in the reaction with phosphorus pentachloride gives the acid chloride 8 which is transformed under the influence of appropriate amines into corresponding amides 9-13. These amides react with ethylenimine giving the appropriate alpha-aziridinyl derivatives of beta-/p-chlorobenzoyl/-propionic acid 14-18. Pharmacological analysis revealed that the compounds studied possessed immunotropic activity; they modulate both humoral as well as cellular immune response. Their effect has been shown to be related to chemical structure and to substituents of the carboxyl group in particular.

[Evaluation of the hepatotoxic activity of several chlor-nitro derivatives of benzoic acid]. / Otsenka gepatotoksicheskogo deistviia nekotorykh khlor- nitroproizvodnykh benzoinoi kisloty.

Hepatotoxic effects of 4-chloro-3-nitrobenzoic acid (x-NBA), 3-nitrobenzoic acid (NBA) and 4-chlorobenzoic acid (CBA) were studied at the doses corresponding to LD50, 1/10 LD50 and 1/50 LD50. The toxic effects were estimated by monitoring alterations in activity of protein-synthesizing system in liver tissue and by the analyses for the presence in blood serum of two tissue-specific cytoplasmic enzymes of liver cells--urokaninase (EC 4.2.1.49) and histidase (EC 4.3.1.3). Single peroral administration of the toxic substances at a dose equal to LD50 led to dissimilar alterations in synthesis of liver proteins: activation within 0.5-5 hrs, distinct increase up to the maximal value within 60 hrs, with a gradual decrease approaching the control values within 8 days. Urokaninase and histidase activities were found in blood serum within 8 hrs after the intoxication, reaching the maximum within 15 hrs and exhibited the constant level during 25 hrs. Then the enzymatic activity was gradually decreased but it did not reach the control values within 70 hrs. After daily peroral administration of these toxic substances at the doses corresponding to 1/10 LD50 and 1/50 LD50, only the first dose inhibited the protein synthesis within 2 weeks. Within this period the enzymes studied were not found in blood serum. Distinct inhibition of the protein synthesis was observed within 4 weeks after administration of both these doses. In this case enzymatic activity was present in blood serum (up to 3 mumole/I L blood serum). Within 2 months after administration of these preparations the alterations studied were the most distinct.

The pathophysiology of myotonia produced by aromatic carboxylic acids.

A series of nine related aromatic monocarboxylic acids (ACAs) previously shown to inhibit muscle membrane chloride conductance (GCl) selectively in the rat were studied for their ability to produce myotonia. All nine induced characteristic repetitive electrical activity and delayed relaxation in isolated muscle, although the concentrations required for this action varied widely. In each case, myotonia was observed at concentrations that correlated closely with previously determined half-maximal concentrations for inhibition of GCl. Intracellular recordings from muscle made myotonic with ACA revealed prolonged latencies at rheobase, multiple driven spikes, and self-sustaining repetitive activity similar to that previously reported in hereditary goat myotonia. Phase-plane diagrams of membrane action potentials recorded after exposure to the most effective of these compounds suggested little effect on the voltage-dependent sodium system. The changes seen could be duplicated by simple removal of chloride ion. The expression of repetitive electrical activity in the presence of low membrane GCl depends on ambient temperature and on the concentration of calcium ion. Increasing temperature and decreasing Ca++ predispose toward myotonic activity; converse conditions inhibit myotonia. Myotonia induced by ACA is inhibited by concentrations of diphenylhydantoin that are clinically effective in controlling hereditary myotonia in humans.