Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria. (25/31)

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.  (+info)

Bacterial metabolism of carbofuran. (26/31)

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.  (+info)

Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains. (27/31)

A cytochrome P-450 system in Rhodococcus strains, encoded by thcB, thcC, and thcD, participates in the degradation of thiocarbamates and several other pesticides. The regulation of the system was investigated by fusing a truncated lacZ in frame to thcB, the structural gene for the cytochrome P-450 monooxygenase. Analysis of the thcB-lacZ fusion showed that the expression of thcB was 10-fold higher in the presence of the herbicide EPTC (s-ethyl dipropylthiocarbamate). Similar enhancement of the thcB-lacZ expression was found with other thiocarbamate pesticides. Atrazine, simazine, or carbofuran, although metabolized by the system, had no effect on the thcB-lacZ expression. The presence of glucose slightly increased the expression of thcB-lacZ, indicating no catabolic repression of the thcB-lacZ expression. The expression of thcB-lacZ was decreased more than twofold in Luria-Bertani medium. This was due in part to cysteine, which repressed thcB-lacZ expression. It was confirmed that the thcR gene, which is transcribed divergently from thcB, codes for a positive regulatory protein which is essential for the thcB-lacZ expression. Studies of the thcR-lacZ protein fusion showed that the thcR gene is expressed constitutively.  (+info)

Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. strain CF06. (28/31)

A bacterial strain (CF06) that mineralized both the carbonyl group and the aromatic ring of the insecticide carbofuran and that is capable of using carbofuran as a sole source of carbon and nitrogen was isolated from a soil in Washington state. Phospholipid fatty acid and 16S rRNA sequencing analysis indicate that CF06 is a Sphingomonas sp. CF06 contains five plasmids, at least some of which are required for metabolism of carbofuran. Loss of the plasmids induced by growth at 42 degrees C resulted in the inability of the cured strain to grow on carbofuran as a sole source of carbon. Introduction of the plasmids confers on Pseudomonas fluorescens M480R the ability to use carbofuran as a sole source of carbon for growth and energy. Of the five plasmids, four are rich in insertion sequence elements and contain large regions of overlap. Rearrangements, deletions, and loss of individual plasmids that resulted in the loss of the carbofuran-degrading phenotype were observed following introduction of Tn5.  (+info)

Reproductive efficiency in mink (Mustela vison) treated with the pesticides lindane, carbofuran and pentachlorophenol. (29/31)

Mink are carnivores of agroforestry fringe habitats and are exposed to pesticides that biomagnify within the food chain. Some pesticides are thought to disrupt reproductive and endocrine functions. In Expt 1, four groups of mink (n = 10) were fed either a control diet, or diets treated with lindane (1 mg kg-1 day-1), carbofuran (0.05 mg kg-1 day-1) or pentachlorophenol (1 mg kg-1 day-1) from before breeding until weaning. Mink were mated twice, at 7-8 day intervals. The treatments had no effect on the proportion of mink accepting the first mating; however, lindane and pentachlorophenol caused a decrease in the percentage of females accepting the second mating. Lindane and pentachlorophenol caused a decrease in whelping rate, although litter size was not affected. Carbofuran had no effect on fertility. Mink that mated only once had a lower whelping rate than mink that mated twice; therefore, it could not be determined whether the decreased whelping rates were due to the lack of a second mating or to increased embryo loss. In Expt 2, two groups of mink (n = 15) were fed a control diet or a diet treated with lindane (1 mg kg-1 day-1) from before mating until weaning. Mink were mated twice on two consecutive days. Lindane did not affect mating response at either mating. Whelping rate, but not implantation rate, was decreased by the lindane treatment. The proportion of embryos lost after implantation (implantation scars not represented by kits at whelping) was increased by the lindane treatment. In conclusion, both lindane and pentachlorophenol decreased fertility in mink, and the lindane effect was primarily a result of embryo mortality after implantation.  (+info)

Reproductive effects in mink (Mustela vison) exposed to the pesticides Lindane, Carbofuran and Pentachlorophenol in a multigeneration study. (30/31)

The mammalian reproductive system is sensitive to exposure to endocrine disrupting chemicals, particularly during sexual maturation. The purpose of this study was to examine reproductive function in second and third generation male and female mink exposed to pesticides from conception to maturity. The mink were fed untreated feed or feed treated with Lindane (1 mg kg-1 day-1), Carbofuran (0.05 mg kg-1 day-1) or Pentachlorophenol (1 mg kg-1 day-1) from the time they were weaned. The second generation mink had also been exposed to the pesticides in utero and from their mother's milk as their mothers were similarly fed pesticides, from 3 weeks before breeding. The third generation mink were the offspring of mink (second generation females) who had themselves undergone long-term exposure to pesticides from conception onwards. Blood samples and endocrine tissues were obtained at necropsy from both generations of mink. No overt signs of toxicity were seen. The pesticides did not affect the percentage of mink mated. Lindane treatment reduced the proportion of mated mink that subsequently whelped (P < 0.1) and the litter size of mink that whelped (P < 0.05). Testis size was reduced in the Lindane-treated, third generation males (P < 0.05). Serum concentrations of cortisol, testosterone and oestradiol were not affected by any pesticide treatment; however, thyroxine concentration was reduced by Pentachlorophenol (P < 0.05). In conclusion, exposure of mink to Lindane from conception resulted in a decrease in reproductive efficiency when they were subsequently mated, leading to a 60% reduction in the number of kits born.  (+info)

Genetic toxicity of N-methylcarbamate insecticides and their N-nitroso derivatives. (31/31)

N-Methylcarbamate esters are an important group of insecticides. They have lower acute toxicity to vertebrates than organophosphates, although their genotoxicity has not been adequately studied. Here we investigate the cytotoxicity and genotoxicity of N-methylcarbamate insecticides and their N-nitroso derivatives in Chinese hamster V79 cells, using the hprt locus as a marker, and also assess inhibition of gap junctional intercellular communication. N-Methylcarbamate insecticides were chemically N-nitrosated to obtain the N-nitroso derivatives. N-Nitrosation greatly increased the cytotoxicity and mutagenicity of N-methylcarbamates at the hprt locus in Chinese hamster V79 cells. The mutagenic potential of N-nitroso-N-methylcarbamates was much higher than those of many other known mutagenic nitroso compounds, as well as some non-nitroso mutagenic alkylating agents. Parental N-methylcarbamates themselves were not mutagenic, however, they inhibited gap junctional intercellular communication half as effectively as the well-studied tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The findings show that N-methylcarbamate insecticides and their N-nitroso derivatives have the potential to act through mediation of epigenetic and genotoxic mechanisms respectively in the multiple stages of chemical carcinogenesis.  (+info)