White-rot fungus Phanerochaete chrysosporium metabolizes chloropyridinyl-type neonicotinoid insecticides by an N-dealkylation reaction catalyzed by two cytochrome P450s.

We previously identified a cytochrome P450 (CYP) derived from the white-rot fungus Phanerochaete chrysosporium as involved in degradation of acetamiprid, a neonicotinoid (NEO) insecticide. In the present study, we investigated biodegradation of other NEOs by P. chrysosporium, and attempted to identify the CYP enzyme responsible for NEO degradation. P. chrysosporium was able to degrade some NEOs (acetamiprid, clothianidin, imidacloprid, and thiacloprid) in nutrient-rich medium. Two CYPs in P. chrysosporium (PcCYPs), CYP5037B3 and CYP5147A3, were identified as major isozymes involved in metabolism of three neonicotinoids that have in common a chloropyridinyl moiety (acetamiprid, imidacloprid, and thiacloprid) by screening yeast that heterologously express PcCYPs. Both PcCYPs catalyzed cleavage of the chloropyridinyl moiety and side chain of the three NEOs by N-dealkylation, resulting in 6-chloro-3-pyridinemethanol and respective side chain fragments. In a culture of P. chrysosporium, 97 % and 74 % of imidacloprid and thiacloprid were modified to form degradation products, and one of these, 6-chloro-3-pyridinemethanol, was further degraded. These two PcCYPs catalyzed almost the same reaction but their substrate specificity and expression pattern are slightly different. Altogether, we found that P. chrysosporium degrades NEOs via the activity of at least two different CYP isozymes.

Biotransformation and detoxification of the neonicotinoid insecticides nitenpyram and dinotefuran by Phanerochaete sordida YK-624.

Neonicotinoid insecticides have been widely used throughout the world over the last two decades. In the present study, we investigated the degradation of neonicotinoid insecticides nitenpyram (NIT) and dinotefuran (DIN) by the white-rot fungus Phanerochaete sordida YK-624. While NIT was completely degraded by P. sordida YK-624 under ligninolytic conditions, only a 20% decrease was observed under nonligninolytic conditions. On the other hand, P. sordida YK-624 degraded 31% of DIN under ligninolytic conditions after a 20-day incubation, while it did not degrade DIN under nonligninolytic conditions. We found that cytochromes P450 played a key role in the biotransformation of NIT and DIN by P. sordida YK-624. A novel NIT metabolite (E)-N-((6-chloropyridin-3-yl)methyl)-N-ethyl-N'-hydroxy acetimidamide (CPMHA) and a novel DIN metabolite N-((4aS,7aS,E)-1-methylhexahydrofuro[2,3-d]pyrimidin-2(1H)-ylidene)nitramide (PHPF) were identified in this study. In addition, to evaluate neurotoxicity, the effects of NIT, DIN and their metabolites on the viability of human neuroblastoma cells SH-SY5Y were determined. PHPF showed higher neurological toxicity than DIN, whereas the metabolite of NIT, CPMHA, showed no toxic effect. Our results indicated that the neurological toxicity of NIT could be effectively removed by P. sordida YK-624.

Identification of the cytochrome P450 involved in the degradation of neonicotinoid insecticide acetamiprid in Phanerochaete chrysosporium.

We previously reported that cytochrome P450 s play critical roles in neonicotinoid insecticide biodegradation by white-rot fungi. Here, we investigated the biodegradation of acetamiprid (ACET) by Phanerochaete chrysosporium to identify the cytochrome P450 involved in this degradation process. During a 20-day incubation period, P. chrysosporium degraded 21% and 51% of ACET in ligninolytic and nonligninolytic media, respectively. The degradation rate of ACET was markedly decreased by the addition of cytochrome P450 inhibitors. Recombinant cytochrome P450s in P. chrysosporium (PcCYP) were heterologously expressed in Saccharomyces cerevisiae strain AH22, and the PcCYP involved in ACET degradation was identified. The results showed that CYP5147A3 can degrade ACET, and two ACET metabolites, N'-cyano-N-methyl acetamidine and 6-chloro-3-pyridinemethanol, were identified. To the best of our knowledge, this study provides the first characterization of the fungal cytochrome P450 that is responsible for the degradation and detoxification of ACET.

[Interlaboratory Study on Evaporation Residue Test for Food Contact Products (Report 1)].

An interlaboratory study was performed to evaluate the equivalence between an official method and a modified method of evaporation residue test using three food-simulating solvents (water, 4% acetic acid and 20% ethanol), based on the Japanese Food Sanitation Law for food contact products. Twenty-three laboratories participated, and tested the evaporation residues of nine test solutions as blind duplicates. For evaporation, a water bath was used in the official method, and a hot plate in the modified method. In most laboratories, the test solutions were heated until just prior to evaporation to dryness, and then allowed to dry under residual heat. Statistical analysis revealed that there was no significant difference between the two methods, regardless of the heating equipment used. Accordingly, the modified method provides performance equal to the official method, and is available as an alternative method.

[Interlaboratory Study on Evaporation Residue Test for Food Contact Products (Report 2)].

An interlaboratory study was performed to evaluate the equivalence between an official method and a modified method of evaporation residue test using heptane as a food-simulating solvent for oily or fatty foods, based on the Japanese Food Sanitation Law for food contact products. Twenty-three laboratories participated, and tested the evaporation residues of nine test solutions as blind duplicates. In the official method, heating for evaporation was done with a water bath. In the modified method, a hot plate was used for evaporation, and/or a vacuum concentration procedure was skipped. In most laboratories, the test solutions were heated until just prior to dryness, and then allowed to dry under residual heat. Statistical analysis revealed that there was no significant difference between the two methods. Accordingly, the modified method provides performance equal to the official method, and is available as an alternative method. Furthermore, an interlaboratory study was performed to evaluate and compare two leaching solutions (95% ethanol and isooctane) used as food-simulating solvents for oily or fatty foods in the EU. The results demonstrated that there was no significant difference between heptane and these two leaching solutions.

Interlaboratory Study on Caprolactam Test for Food-Contact Nylon Products.

The Japanese Food Sanitation Law sets a limit on the migration level of caprolactam for food-contacting nylon products. Here, we carried out an interlaboratory study in twenty laboratories to evaluate the performance of the official GC-FID test method and a GC-MS method as an alternative test method to the official method. Each laboratory quantified caprolactam in three test solutions in 20% ethanol as blind duplicates using GC-FID or GC-MS. The official method (GC-FID with absolute calibration) gave trueness, repeatability (RSDr) and reproducibility (RSDr) values of 96-97%, 3.3-5.4% and 4.0-6.7%, respectively. These values met the target criteria (trueness: 80-110%, RSDr: 10%, RSDr: 25%). The performance of the method was further improved by the introduction of heptalactam as an internal standard. As for GC-MS method, some values of the RSDr exceeded 10% when absolute calibration was used. However, when an internal standard was introduced, the trueness, RSDr and RSDr of GC-MS method were all acceptable at 94-96%, 2.0-4.4% and 7.0-9.4%, respectively. Therefore, GC-MS with an internal standard is available as an alternative test method to the official method.

Interlaboratory Study on Volatiles Test for Food-Contacting Polystyrene Products.

Using polystyrene, acrylonitrile-styrene resin and acrylonitrile-butadiene-styrene resin pellets as samples, an interlaboratory study was performed to evaluate the volatiles test method, based on the specifications described in the Japanese Food Sanitation Law for food-contacting polystyrene products. The study was conducted with the participation of twenty-one laboratories. Each laboratory quantified the contents of styrene, toluene, ethylbenzene, isopropylbenzene and propylbenzene in three test pellets using GC-FID, GC-MS or headspace-GC-FID. Statistical analysis revealed that the repeatability (RSDr) and reproducibility (RSDr) were 1.0-2.6 and 2.5-5.5% for the GC-FID method. The values of the performance parameters fulfilled the requirements (RSDr: 10%, RSDr: 25%), and the performance is sufficient for specifications testing. The RSDr and RSDr of results obtained using the GC-MS and HS-GC methods were 1.4-7.8 and 4.9-13%(GC-MS), and 2.0-2.6 and 3.3-6.9%(HS-GC-FID), respectively. The quantified levels were similar to those obtained with GC-FID. The study suggests that the GC-MS and HS-GC methods can be employed as alternative methods to the GC-FID method.

[Interlaboratory Study on Zinc Test for Food-Contact Rubber Products].

Using six kinds of zinc solution in water and 4% acetic acid as samples, an interlaboratory study was performed to evaluate a zinc (Zn) test method for food-contact rubber products, based on the Japanese Food Sanitation Law. Eighteen laboratories participated, and quantified Zn in six test solutions as blind duplicates using flame atomic absorption spectrometry, induced coupled plasma-optical emission spectrometry or induced coupled plasma-mass spectrometry. Statistical analysis revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 97-103%, 0.7-4.9% and 1.7-8.9% by all measuring methods. The values of the performance parameter fulfilled the target value (trueness: 80-110%, RSDr: 10%, RSDr: 25%). The performance of these methods is sufficient for testing the adherence of samples to the specifications.

[Interlaboratory study on migration test of antimony and germanium for food-contact polyethylene terephthalate].

An interlaboratory study was performed to evaluate a migration test method of antimony (Sb) and germanium (Ge), based on the Japanese Food Sanitation Law for food- contact polyethylene terephthalate. Eighteen laboratories participated, and quantified Sb and Ge in three test solutions as blind duplicates using graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma-optical emission spectrometry (ICP-OES) or induced coupled plasma-mass spectrometry (ICP-MS). Statistical analysis revealed that the trueness, repeatability and reproducibility were 98-107%, 1.7-7.5% and 2.0-18.8% by using GF-AAS and ICP-OES. The performance of these methods is sufficient for testing the specifications. The performance parameters of ICP-MS were 99-106%, 0.7-2.2% and 2.2-10.5%, respectively. ICP-MS is available as an alternative measuring method. However, in some laboratories, the quantitative values of Sb were higher than the addition levels. We found that Sb in working solutions is absorbed on glass vessels. Careful control of concentration in working solutions is required for Sb analysis.

Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor.

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5 days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6 days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3 days, but decreased slightly after 5 days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3-5 days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.

[Simultaneous determination of pesticide residues in agricultural products by LC-MS/MS].

A method for the simultaneous determination of multiple pesticide residues in agricultural products was developed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile. Co-extractives were removed by GPC/graphitized carbon column SPE, and silica gel/PSA cartridge column SPE. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. Recoveries of 124 pesticides from spinach, brown rice, soybean, orange and tomato were tested at the level of 0.1 µg/g, and those of 121 pesticides ranged from 70 to 120% (RSD≤15%). Pesticide residues in 239 agricultural products were investigated by this method, and residues of 49 pesticides were detected in 98 agricultural products.

[Analysis of spinosad in animal and fishery products by LC-MS].

We investigated the determination of spinosyn A and spinosyn D, the active ingredients of spinosad, in animal and fishery products by liquid chromatography with mass spectrometry (LC-MS). The sample was homogenized with 1 mol/L dipotassium hydrogenphosphate aqueous solution and extracted with acetone-n-hexane under mildly alkaline conditions. After n-hexane-acetonitrile partitioning using an EXtrelut(®) column, the extract was cleaned up on a tandem SAX/PSA mini-column, and examined by means of fragmenter-voltage-switching ESI-SIM mode LC-MS. Mean recoveries (n=5) of spinosyn A and spinosyn D from eleven kinds of fortified samples at the analyte concentration of 0.01 µg/g and 0.05 µg/g ranged from 76.1% to 93.8% (RSD≤8.7%) and from 75.1% to 104.1% (RSD≤8.6%), respectively.

[Multiresidue analysis of PCB, organochlorine pesticides and chlordanes in marine products by GC-micro ECD].

A multiresidue method using dual-injection, dual-column, and dual-micro electron capture detection gas chromatography (dual-column GC-µECD) was developed for the determination of PCB, organochlorine pesticides and chlordanes in marine products. The sample was extracted with hexane-acetone (2 : 1), and the extract was cleaned up by gel permeation chromatography(GPC)/solid-phase extraction (SPE). The GPC fraction was selectively collected, and loaded directly onto a graphitized carbon/PSA 2-layered column. After fractionation by 4% hydrated silica-gel column chromatography, each fraction was determined by dual-column GC-µECD. Recoveries of PCB, organochlorine pesticides and chlordanes were in the ranges of 84-109% (RSD ≤ 21.6%), 74-117% (RSD ≤ 14.6%) and 69-114% (RSD ≤ 12.9%), respectively. This method is superior to single chromatography for the determination of total PCB, and should be useful for monitoring of these pollutants in marine products.

[Analysis of acephate, methamidophos and omethoate in animal and fishery products, and honey by LC-MS].

We studied the simultaneous determination of acephate, methamidophos, and omethoate in animal and fishery products, their processed foods, and honey by means of liquid chromatography coupled with mass spectrometry (LC-MS). The sample was extracted with ethyl acetate in the presence of anhydrous Na(2)SO(4) (and diatomaceous earth for honey). An aliquot of the crude sample extract was loaded into the GPC system, and the pesticide fraction was selectively collected. The extract was cleaned up on a PSA mini-column, and determined by a column-switching ESI-SIM mode LC-MS. Mean recoveries (2 replicates x 5 days) of compounds from eleven kinds of samples, except honey, fortified at the analyte concentration of 0.05 microg/g were from 71.4% to 98.4%. The repeatability relative standard deviation values were < or =12.5%, and the intermediate reproducibility relative standard deviation values were < or =14.1%. In honey, the recoveries were improved to 97.6-98.6% by using highly purified surrogates.