Reaction mechanism of dicofol removal by cellulase.

It remains unclear whether dicofol should be defined as a persistent organic pollutant. Its environmental persistence has gained attention. This study focused on its degradation by cellulase. Cellulase was separated using a gel chromatogram, and its degradation activity towards dicofol involved its endoglucanase activity. By analyzing the kinetic parameters of cellulase reacting with mixed substrates, it was shown that cellulase reacted on dicofol and carboxyl methyl cellulose through two different active centers. Thus, the degradation of dicofol was shown to be an oxidative process by cellulase. Next, by comparing the impacts of tert-butyl alcohol (a typical OH free-radical inhibitor) on the removal efficiencies of dicofol under both cellulase and Fenton reagent systems, it was shown that the removal of dicofol was initiated by OH free radicals produced by cellulase. Finally, 4,4'-dichloro-dibenzophenone and chloride were detected using gas chromatography mass spectrometry and ion chromatography analysis, which supported our hypothesis. The reaction mechanism was analyzed and involved an attack by OH free radicals at the orthocarbon of dicofol, resulting in the degradation product 4,4'-dichloro-dibenzophenone.

Effects of metal ions on the catalytic degradation of dicofol by cellulase.

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment.

Biodegradation of 14C-dicofol in wastewater aerobic treatment and sludge anaerobic biodigestion.

Organic micropollutants are often found in domestic and industrial effluents. Thus, it is important to learn their fate, the metabolites generated and their sorption during biological treatment processes. This work investigated the biodegradation of 14C-dicofol organochloride during wastewater aerobic treatment and sludge anaerobic biodigestion. The performance of these processes was evaluated by physical-chemical parameters. Radioactivity levels were monitored in both treatments, and residues of dicofol (DCF) and dichlorobenzophenone (DBP) were quantified by HPLC/UV. The efficiency of the aerobic and anaerobic processes was slightly reduced in the presence of DCF and DBP. After aerobic treatment, only 0.1% of DCF was mineralized, and 57% of radioactivity remained sorbed on biological sludge as DBP. After 18 days of anaerobiosis, only 3% of DCF and 5% of DBP were detected in the sludge. However, 70% of radioactivity remained in the sludge, probably as other metabolites. Dicofol was biodegraded in the investigated process, but not mineralized.

Removal of dicofol from water by immobilized cellulase and its reaction kinetics.

Researches on the removal of dicofol catalyzed by immobilized cellulase were conducted. Factors, such as acidity, temperature, enzyme activity, and initial concentration of dicofol, which could influence the removal were studied. The optimal pH for dicofol removal by immobilized cellulase was approximately 4-7, broader than that for free enzymes. The removal efficiencies for immobilized and free cellulase both decreased with increasing initial concentration of dicofol. The K(m) for immobilized cellulase was slightly lower than that of free cellulase, suggesting that substrate affinity may be enhanced by immobilization. The optimum temperatures for immobilized and free cellulase were 45 °C and 50 °C. The removal reaction for immobilized cellulase was found to be a first-order reaction. The activation energy was 64.3 kJ mol(-1). The continuous oxidation of dicofol carried out in the static system of immobilized cellulase showed that the removal efficiency of immobilized cellulase remained after six cycles of operation. Thus, the catalytic efficiency of cellulase was improved greatly. As evidenced by infrared and gas chromatography-mass spectrometry data, the mechanism of reaction might involve an attack by the OH free radical of cellulase at a weak location of the dicofol molecule, resulting in the removal of three chlorine atoms from dicofol, thus oxygenizing dicofol and producing 4,4'-dichloro-dibenzophenone.

Enhanced disappearance of dicofol by water hyacinth in water.

The efficiency and primary mechanism of phytoremediation of water contaminated with dicofol, an organochlorine pesticide, by water hyacinth (Eichhornia crassipes) was investigated. After 10 days of incubation in nutrient solution at 25 +/- 1 degree C, the remaining dicofol which was spiked initially at 1 mg l(-1) was 0.05 and 0.26 mg l(-1) in the non-sterile planted and non-sterile unplanted, 0.07 and 0.31 mg l(-1) in the sterile planted and sterile unplanted treatments, respectively. The half-life of dicofol in nutrient solutions was reduced to 59-68 h in the planted treatments from 118-137 h in the unplanted treatments. The accumulated dicofol in water hyacinth plant decreased by 41-53% after the plant had grown for 7 days in dicofol-free nutrient solution. Two phytoprocesses were found to be most important in the remediation of dicofol contaminated water: (i) uptake, accumulation and phytodegradation of dicofol by the plant, accounted for 50% of the removal of the spiked dicofol, and (ii) microbial degradation associated with the rhizosphere, contributed about 7% to dicofol removal. Water hyacinth may thus be a good candidate for development as a phytoremediation system for dicofol-contaminated water.

Metabolic and mutagenicity studies on DDT and 15 derivatives. Detection of 1,1-bis(p-chlorophenyl)-2,2-dichloroethane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (kelthane acetate) as mutagens in Salmonella typhimurium and of 1,1-bis(p-chlorophenyl) ethylene oxide, a likely metabolite, as an alkylating agent.

Using a novel in vitro technique, whereby microsomal enzymes were embedded in an agar layer to prolong their viability, 1,1-bis(p-chlorophenyl) ethylene(DDNU), a mammalian metabolite of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), was converted by microsomal mono-oxygenases of mouse liver into 1,1-bis(p-chlorophenyl)-1,2-ethanediol (DDNU-diol). The putative epoxide intermediate, 1,1-bis(p-chlorophenyl)ethylene oxide (DDNU-oxide), a new compound, was synthesized; it showed weak alkylating activity with 4-(4-nitrobenzyl)pyridine but was not mutagenic in Salmonella typhimurium strains TA100 and TA98. DDT and 13 of its metabolites or putative synthetic derivatives, including 1,1-bis(p-chlorophenyl)-2,2-dichloroethylene (DDE), 1 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS)-DDNU, 2,2-bis(p-chlorophenyl)ethanol (DDOH), bis(p-chlorophenyl)acetic acid (DDA) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethanol (Kethane), caused no mutagenic effects in S. typhimurium strains TA100 or TA98, either in the presence or absence of a mouse-liver microsomal fraction. 1,1-Bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (Kelthane acetate) was a direct-acting mutagen in strain TA100, whereas 1,1-bis(p-chlorophenyl)-2,2-dichloroethane (DDD) was mutagenic in TA98, only in the presence of a mouse-liver microsomal system. The results are discussed in relation to possible pathways whereby DDT is activated to mutagenic and/or carcinogenic metabolites.

Assessing the potential toxic effect of one persistent organic pollutant: non-covalent interaction of dicofol with the enzyme trypsin.

Because of the widespread concern that persistent organic pollutants (POPs) may be adversely affecting the health of humans, reliable assessing their toxic effects is urgently needed. We selectively study the interaction between dicofol (DCF) and trypsin by steady state and time resolved fluorescence quenching measurements and UV-visible absorption spectroscopy under physiological conditions as well as applying molecular docking method to establish the interaction model. The fluorescence results indicate DCF can spontaneously form a complex with trypsin mainly by hydrogen bond with only one binding site, which had been validated in molecular docking. The conformational change of trypsin was proved by UV-visible absorption and synchronous fluorescence spectroscopy indicating a red shift of carbonyl absorption peak. All the results indicated DCF had potential toxic effects on both the structure and activity of the enzyme trypsin and the effects enhanced with the increasing concentration of DCF.

Detection of dicofol and related pesticides in human breast milk from China, Korea and Japan.

Previously, we demonstrated that the concentrations of DDTs were greater in breast milk collected from Chinese mothers than from Japanese and Korean mothers. To investigate dicofol as a possible source of the DDTs in human breast milk, we collected breast milk samples from 2007 to 2009 in China (Beijing), Korea (Seoul, Busan) and Japan (Sendai, Takarazuka and Takayama). Using these breast milk samples, we quantified the concentrations of dichlorobenzophenone, a pyrolysis product of dicofol (simply referred to as dicofol hereafter), dichlorodiphenyltrichloroethane and its metabolites (DDTs) using GC-MS. Overall, 12 of 14 pooled breast milk samples from 210 mothers contained detectable levels of dicofol (>0.1 ng g⁻¹ lipid). The geometric mean concentration of dicofol in the Japanese breast milk samples was 0.3 ng g⁻¹ lipid and significantly lower than that in Chinese (9.6 ng g⁻¹ lipid) or Korean breast milk samples (1.9 ng g⁻¹ lipid) (p<0.05 for each). Furthermore, the ΣDDT levels in breast milk from China were 10-fold higher than those from Korea and Japan. The present results strongly suggest the presence of extensive emission sources of both dicofol and DDTs in China. However, exposure to dicofol cannot explain the large exposure of Chinese mothers to DDTs because of the trace levels of dicofol in the ΣDDTs. In the present study, dicofol was confirmed to be detectable in human breast milk. This is the first report to identify dicofol in human samples.

Dicofol degradation to p,p'-dichlorobenzophenone - a potential antiandrogen.

In the present investigation, the degradation of the acaricide dicofol (also known as kelthane) was investigated with special emphasis on generation of p,p'-dichlorobenzophenone (DCBP) under alkaline conditions as well as induced by UV-light. Dicofol was also incubated in the presence and absence of microsomal preparations to measure potential metabolic formation of DCBP. The results indicate that the degradation of dicofol to DCBP primarily proceeds as an abiotic process via hydroxide ion catalysed elimination of a trichloromethyl anion. The generated anion picks up a proton from the solvent to generate chloroform. Microsomal metabolism does not appear to play a major role in the degradation of dicofol. DCBP is structurally analogous to the antiandrogen p,p'-dichlorodiphenylethene (DDE). We therefore investigated whether DCBP displays antiandrogenic properties. In an in vitro transactivation system utilising transiently transfected African green monkey kidney (COS-7) cells, DCBP showed potent antiandrogenic efficacy. This finding was confirmed by further studies in T47D human mammary carcinoma cells by measuring mRNA and protein expression of androgen dependent genes i.e. TRMP-2 (testosterone-repressed prostate message-2) mRNA and PSA (prostate-specific antigen) protein.

Chromatographic determination of dicofol and metabolites in egg yolks.

Egg yolk was spiked with p,p'-dicofol (p,p'-DCF) (0.1-2.0 micrograms/gm), p,p'-dichlorobenzophenone (p,p'-DCBP) (0.1-2.0 micrograms/gm), and 1,1-bis(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE) (0.05-1.0 micrograms/gm). The fortified egg yolk (2-5 g) was mixed with acetonitrile to extract non-fat organic materials. After removal of acetonitrile, the spiked chemicals were separated with a column chromatograph packed with acid alumina. Recovery efficiencies for p,p'-DCBP and p,p'-DDE were determined by gas chromatography, and for p,p'-dicofol by high performance liquid chromatography. The recovery efficiencies for p,p'-dicofol, p,p'-DCBP and p,p'-DDE were 77.2-93.8%, 84.1-101.1%, and 88.5-96.0%, respectively.

Biodegradation of DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by the white rot fungus Phanerochaete chrysosporium was demonstrated by disappearance and mineralization of [14C]DDT in nutrient nitrogen-deficient cultures. Mass balance studies demonstrated the formation of polar and water-soluble metabolites during degradation. Hexane-extractable metabolites identified by gas chromatography-mass spectrometry included 1,1,-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol), 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol (FW-152), and 4,4'-dichlorobenzophenone (DBP). DDD was the first metabolite observed; it appeared after 3 days of incubation and disappeared from culture upon continued incubation. This, as well as the fact that [14C]dicofol was mineralized, demonstrates that intermediates formed during DDT degradation are also metabolized. These results demonstrate that the pathway for DDT degradation in P. chrysosporium is clearly different from the major pathway proposed for microbial or environmental degradation of DDT. Like P. chrysosporium ME-446 and BKM-F-1767, the white rot fungi Pleurotus ostreatus, Phellinus weirii, and Polyporus versicolor also mineralized DDT.

Metabolism of a dicofol impurity alpha-chloro-DDT, but not dicofol or dechlorodicofol, to DDE in mice and a liver microsomal system.

1. The important acaricide dicofol and two related compounds, alpha-chloro-DDT (an impurity in dicofol) and dechlorodicofol (DCD) (a photolysis product of dicofol), as [phenyl-14C]-labelled preparations, were administered i.p. to male mice (30 mg/kg) and incubated with rat-liver microsomes alone and with NADPH, both aerobically and anaerobically, and with rat-liver cytosol alone and with glutathione. 2. alpha-Chloro-DDT is metabolically dechlorinated to DDE in the following systems: in vivo, based on analyses of mouse brain, fat and liver; in vitro, with anaerobic rat-liver microsomes plus NADPH; and with reduced haematin. Trace amounts of DDT are also detected in vivo in mouse liver. 3. Dicofol is reductively dechlorinated to DCD, and both compounds are metabolized to dichlorobenzophenone and dichlorobenzhydrol in vivo in the mouse tissues examined, and also in vitro exclusively with anaerobic rat-liver microsomes in the presence of NADPH. Liver cytosol with glutathione is less effective or inactive. 4. The in vivo metabolic dechlorinations of alpha-chloro-DDT and dicofol probably involve a reduced porphyrin in liver microsomes.

Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.