Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.     

Metabolism of organophosphorus insecticides

Summary The metabolism of ³²P-Malathion in Rhizobium leguminosarum and Rhizobium trifolii has been investigated. In addition to inorganic phosphates and/or thiophosphates, 5 hydrolytic metabolites could be identified. The carboxylic acid derivatives constituted the major portion (35–40% of the total metabolites output) suggesting the presence of powerful carboxyesterases in both Rhizobium spp. Malaoxon could not be detected in the media of both organisms.     

Inhibition of DNA methylation by chemical carcinogens in vitro

A diverse range of ultimate chemical carcinogens inhibited the transfer of methyl groups from S-adenosylmethionine to hemimethylated DNA in a reaction catalyzed by mouse spleen methyltransferase. The formation of alkali-labile sites in DNA lessened its ability to accept methyl groups in vitro, but the methylation reaction was much less sensitive to thymine dimers or double-strand breaks. Carcinogens induced the formation of alkali-labile DNA lesions, but the degree of methyltransferase inhibition observed was greater than that expected for this damage alone. Certain carcinogens were also capable of direct modification and inactivation of the methyltransferase enzyme. Benzo(a)pyrene treatment of living BALB/3T3 A31 clone 1-13 but not C3H/10T1/2 clone 8 cells resulted in a 12% decrease in total 5-methylcytosine content of cellular DNA. Carcinogenic agents may therefore cause heritable changes in 5-methylcytosine patterns in certain cell types by a variety of mechanisms, including adduct formation, induction of apurinic sites and single-strand breaks and direct inactivation of DNA methyltransferase.     

Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase

The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl-d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent [sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10-80% and 3.6-45% in enzyme reaction cycles after immobilization on Ca-alginate and agar-agar, respectively.     

Cytogenetic activity of various organophosphorus insecticides

The action of certain insecticides belonging to the group of organophosphorous compounds (chlorophos, metaphos, etaphos, phosphamid, phosalone, carbophos, selecron) has been studied by the test of chromosome aberrations in Crepis capillaris cells. It is shown that these insecticides possess mutagenic activity in all phases of the cell cycle and are mutagens of the undelayed action.     

Polyamines inhibit DNA methylation in vitro.

DNA methylase from rat liver differs from RNA methylase in response to polyamines, being inhibited rather than stimulated. With methyl-deficient DNA from rat liver as substrate, the DNA methylase is inhibited 97%, 59% and 42% respectively, by spermine, spermidine and putrescine at 1 mM concentration.     

Effects of DNA binding proteins on DNA methylation in vitro

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.     

Mutagen influence with different mechanisms of action on DNA global methylation in human whole-blood lymphocytes in vitro

Data that support the evidence of mutagens known to cause epigenetic abnormalities that could potentially result in genomic instability and the development of cancer rather than to modifications in the human genome at the gene and chromosomal levels only. The level of global methylation in human lymphocytes in vitro caused by exposure to two mutagens with different mechanisms of action, i.e., dioxidine and methyl methanesulphonate (MMS), was demonstrated in the present study. Global methylation was assessed by methyl-sensitive comet assay. An increase in the level of global methylation to 45.64% was revealed during culturing with dioxidine in a concentration of 0.01 mg/mL (p < 0.001), while the addition of dioxidine in a concentration of 0.1 mg/mL resulted in a decreased level of methylation up to 42.31% (p < 0.001). The addition of MMS in concentrations of 0.0025 and 0.01 mg/mL resulted in minor but significant modifications (p < 0.05) of the global methylation level ranged within natural variations in global methylation. Accordingly, the addition of dioxidine in the concentration of 0.1 mg/mL might cause genomic instability and might be considered a potential carcinogen.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.