Residue Distribution and Daily Exposure of Per- and Polyfluoroalkyl Substances in Indica and Japonica Rice.

Per- and polyfluoroalkyl substances (PFAS) have excellent chemical stability but have adverse environmental impacts of concern. Furthermore, bioaccumulation of PFAS in rice varieties─which is the essential staple food crop in Asia─has not been verified. Therefore, we cultivated Indica (Kasalath) and Japonica rice (Koshihikari) in the same Andosol (volcanic ash soil) paddy field and analyzed the air, rainwater, irrigated water, soil, and rice plants for 32 PFAS residues, throughout the cultivation to human consumption. During the rice cultivation period, the cultivation environment in atmospheric particulate matter (PM) constituted perfluoroalkyl carboxylic acids (PFCAs), with minimal perfluorinated sulfonic acids (PFSAs). Furthermore, perfluorooctanesulfonic acid (PFOS) migrates at a PM > 10 to drop in a cultivation field and was conducive to leakage and accumulation of PFCAs in air particles in the field environment. Moreover, precipitation was a sources of irrigation water contamination, and cultivated soil with a high carbon content could capture PFSAs and PFCAs (over C10). There were no major differences in residual PFAS trends in the rice varieties, but the distribution of PFAS in the growing soil, air, and rainwater differed. The edible white rice part was mainly affected by irrigation water in both varieties. Monte Carlo simulations of daily exposure assessments of PFOS, PFOA, and perfluorononanic acid showed similar results for Indians consuming Indica rice and Japanese consuming Japonica rice. The results indicate that the ultratrace PFAS residue concentrations and their daily exposure were not cultivar-specific.

Differences in Enantioselective Hydroxylation of 2,2',3,6-Tetrachlorobiphenyl (CB45) and 2,2',3,4',6-Pentachlorobiphenyl (CB91) by Human and Rat CYP2B Subfamilies.

Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.

Genome-wide identification and characterization of major latex-like protein genes responsible for crop contamination in Cucurbita pepo.

BACKGROUND: Zucchini plants (Cucurbita pepo) accumulate persistent organic pollutants (POPs) at high concentrations in their aerial parts, and major latex-like proteins (MLPs) play crucial roles in their accumulation. MLPs bind to POPs in root cells, MLP-POP complexes are then translocated into xylem vessels, and POPs are transported to the aerial parts. We previously identified three CpMLP genes (MLP-PG1, MLP-GR1, and MLP-GR3) as transporting factors for POPs; however, other studies have shown that the genomes of several plant species contain more than 10 MLP genes, thus, further MLP genes responsible for POP accumulation may have been overlooked. METHODS AND RESULTS: Here, we investigated the number of CpMLP genes by performing a hidden Markov model search against the C. pepo genome database and characterized their effects on POP accumulation by performing the expression analysis in the organs and in silico structural analysis. The C. pepo genome contained 21 CpMLP genes, and several CpMLP genes, including MLP-PG1 and MLP-GR3, were highly expressed in roots. 3D structural prediction showed that all examined CpMLPs contained a cavity with a hydrophobic region, which facilitated binding to POPs. CONCLUSIONS: The present study provides insights regarding CpMLP genes responsible for POP accumulation.

MLP-PG1, a major latex-like protein identified in Cucurbita pepo, confers resistance through the induction of pathogenesis-related genes.

MAIN CONCLUSION: MLP-PG1, identified in Cucurbita pepo, plays a crucial role in resistance against fungal pathogens through the induction of pathogenesis-related genes. ASTRACT: MLP-PG1, a major latex-like protein (MLP) from zucchini (Cucurbita pepo), was identified as a transporting factor for hydrophobic organic pollutants. MLPs are members of the Bet v 1 family, similar to pathogenesis-related class 10 proteins (PR-10s). However, the biological functions of MLPs remain unclear. Herein, we show that MLP-PG1 induces the expression of pathogenesis-related (PR) genes and indirectly promotes resistance against pathogens. The activity of the MLP-PG1 promoter in leaves of transgenic tobacco plants was significantly enhanced by inoculation with Pseudomonas syringae pv. tabaci. However, MLP-PG1 did not induce direct resistance through RNase activity. Therefore, we examined the possibility that MLP-PG1 is indirectly involved in resistance; indeed, we found that MLP-PG1 induced the expression of defense-related genes. Overexpression of MLP-PG1 highly upregulated PR-2 and PR-5 and decreased the area of lesions caused by Botrytis cinerea in the leaves of transgenic tobacco plants. Our results demonstrate that MLP-PG1 is involved in indirect resistance against plant diseases, especially caused by fungal pathogens, through the induction of PR genes. This study is the first report to show the induction of PR genes by the expression of MLP from the RNA sequencing analysis and the involvement of MLP-PG1 in the resistance.

Capillary zone electrophoresis determination of fluoride in seawater using transient isotachophoresis.

We developed capillary zone electrophoresis (CZE) with indirect UV detection for the determination of fluoride (F-) in seawater using transient isotachophoresis (tITP) as an on-line concentration procedure. A method of correcting sample salinity effects was also proposed so that F- concentrations were obtained using a calibration graph. The proposed method is simple: it requires no sample pretreatment aside from dilution. The following optimum conditions were established: background electrolyte (BGE), 5 mM 2,6-pyridinedicarboxylic acid (PDC) adjusted to pH 3.5 containing 0.03% m/v hydroxypropyl methylcellulose (HPMC); detection wavelength, 200 nm; vacuum (50 kPa) injection period of sample, 5 s (254 nL); and applied voltage, 23 kV with the sample inlet side as the cathode. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) for F- reached 0.024 and 0.070 mg/L, respectively. The respective values of the relative standard deviation (RSD) of the peak area, peak height, and migration time for F- were 2.5, 3.4, and 0.30%. The proposed method was applied for the determination of F- in seawater samples collected from coastal waters of western Japan during August 26-28, 2014. Both results obtained using standard addition method and a calibration graph agreed with those obtained using a conventional spectrophotometric method.

Molecular insights into the role of a distal F240A mutation that alters CYP1A1 activity towards persistent organic pollutants.

BACKGROUND: Cytochromes P450 are major drug-metabolizing enzymes involved in the biotransformation of diverse xenobiotics and endogenous chemicals. Persistent organic pollutants (POPs) are toxic hydrophobic compounds that cause serious environmental problems because of their poor degradability. This calls for rational design of enzymes capable of catalyzing their biotransformation. Cytochrome P450 1A1 isoforms catalyze the biotransformation of some POPs, and constitute good starting points for the design of biocatalysts with tailored substrate specificity. METHODS: We rationalized the activities of wild type and mutant forms of rat cytochrome P450 1A1 towards 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and 3,3',4,4'-tetrachlorobiphenyl (PCB77) using experiments and molecular dynamics simulations. RESULTS: We showed that the enhanced activity of the CYP1A1 mutant towards TCDD was due to more efficient binding of the substrate in the active site even though the mutated site was over 2.5nm away from the catalytic center. Moreover, this mutation reduced activity towards PCB77. GENERAL SIGNIFICANCE: Amino acids that affect substrate access channels can be viable targets for rational enzyme design even if they are located far from the catalytic site.

A major latex-like protein is a key factor in crop contamination by persistent organic pollutants.

This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.

Molecular mechanisms of herbicide-inducible gene expression of tobacco CYP71AH11 metabolizing the herbicide chlorotoluron.

Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter ß-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.

Mammalian cytochrome P450-dependent metabolism of polychlorinated dibenzo-p-dioxins and coplanar polychlorinated biphenyls.

Polychlorinated dibenzo-p-dioxins (PCDDs) and coplanar polychlorinated biphenyls (PCBs) contribute to dioxin toxicity in humans and wildlife after bioaccumulation through the food chain from the environment. The authors examined human and rat cytochrome P450 (CYP)-dependent metabolism of PCDDs and PCBs. A number of human CYP isoforms belonging to the CYP1 and CYP2 families showed remarkable activities toward low-chlorinated PCDDs. In particular, human CYP1A1, CYP1A2, and CYP1B1 showed high activities toward monoCDDs, diCDDs, and triCDDs but no detectable activity toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD). Large amino acids located at putative substrate-recognition sites and the F-G loop in rat CYP1A1 contributed to the successful metabolism of 2,3,7,8-tetraCDD. Rat, but not human, CYP1A1 metabolized 3,3',4,4',5-pentachlorobiphenyl (CB126) to two hydroxylated metabolites. These metabolites are probably less toxic than is CB126, due to their higher solubility. Homology models of human and rat CYP1A1s and CB126 docking studies indicated that two amino acid differences in the CB126-binding cavity were important for CB126 metabolism. In this review, the importance of CYPs in the metabolism of dioxins and PCBs in mammals and the species-based differences between humans and rats are described. In addition, the authors reveal the molecular mechanism behind the binding modes of dioxins and PCBs in the heme pocket of CYPs.

Major latex-like proteins show pH dependency in their binding to hydrophobic organic pollutants.

The Cucurbitaceae family accumulates hydrophobic organic pollutants in its aerial parts at high concentrations. Major latex-like proteins (MLPs) were identified in zucchini (Cucurbita pepo) as a transporting factor for hydrophobic organic pollutants. MLPs bind to hydrophobic organic pollutants in the roots, are secreted to xylem vessels as complexes, and are transported to the aerial parts. However, the suitable conditions for binding MLPs to hydrophobic organic pollutants remain elusive. In the present study, we show that MLPs bind to the hydrophobic organic pollutant pyrene with higher affinity under acidic conditions. Our results demonstrated that pH regulates the binding of MLPs to hydrophobic organic pollutants.

Cucurbita pepo contains characteristic proteins without a signal peptide in the xylem sap.

Xylem sap is a fluid that transfers water and nutrients from the rhizosphere. This sap contains relatively low concentrations of proteins that originate from the extracellular space among the root cells. One of the characteristic proteins in the xylem sap of the Cucurbitaceae family, which includes cucumber and zucchini, is a major latex-like protein (MLP). MLPs are responsible for crop contamination through the transport of hydrophobic pollutants from the roots. However, detailed information on the content of MLPs in the xylem sap is not available. Proteomic analysis of root and xylem sap proteins from the Cucurbita pepo cultivars Patty Green (PG) and Raven (RA) showed that the xylem sap of cv. RA, a high accumulator of hydrophobic pollutants, contained four MLPs that accounted for over 85% of the total xylem sap proteins in this cultivar. The xylem sap of PG, a low accumulator, mainly contained an uncharacterized protein. The amount of each root protein between the PG and RA cultivars was significantly and positively correlated in spite of being with and without a signal peptide (SP). However, the amount of xylem sap proteins without an SP was not correlated. These results suggest that cv. RA is characterized by MLPs in the xylem sap.

Enhanced metabolism of 2,3',4,4',5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium.

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.

Assays of PCB congeners and organochlorine insecticides with the transgenic Arabidopsis and tobacco plants carrying recombinant guinea pig AhR and GUS reporter genes.

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of ß-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige-Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g(-1) PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated ß-glucuronidase reporter gene expression system.

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated ß-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated ß-glucuronidase reporter gene expression system.

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated ß-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Simple monitoring of endocrine-disrupting chemicals using transgenic Arabidopsis plants expressing medaka estrogen receptor.

Endocrine-disrupting chemicals (EDCs) are widespread contaminants that severely affect the endocrine systems of living organisms. In addition to the conventional instrument-based approaches for quantifying organic pollutants, a monitoring method using transgenic plants has also been proposed. Plants carrying a recombinant receptor gene combined with a reporter gene represent a system for the easy detection of ligands that specifically bind to the receptor molecule. Here, the EDC detection sensitivity of transgenic Arabidopsis plants expressing the medaka (Oryzias latipes) estrogen receptor (mER) and green fluorescent protein (GFP) genes, was assessed. Four transgenic Arabidopsis lines, obtained by transformation with expression plasmids constructed using combinations of two types of the ligand-binding domains of mER, the DNA-binding domain of LexA and the transactivation domain of VP16 in the chimeric receptors, showed significant induction of GFP when germinated on a medium contaminated with 1 ng/mL 4-t-octylphenol (OP). The most sensitive XmEV19-2 plants detected 0.1 ng/mL OP and 1 pg/mL 17ß-estradiol. GFP expression was suppressed by the insecticides imidacloprid and fipronil, whereas perfluorooctanesulfonic acid induced it at 0.1 ng/mL. Experiments with river water-based medium showed that XmEV19-2 can be used for monitoring polluted waters, detecting OP at concentrations as low as 5 ng/mL. Notably, XmEV19-2 showed a significant decrease in root length when grown on 0.1 ng/mL OP. mER transgenic plants can be a promising tool for simple monitoring of EDCs, without the need for extraction and concentration steps in sample preparation.

A20/AN1 zinc-finger proteins positively regulate major latex-like proteins, transporting factors toward dioxin-like compounds in Cucurbita pepo.

The Cucurbitaceae family accumulates dioxin-like compounds in its fruits. We previously showed that A20/AN1 zinc finger protein (ZFP) genes were highly expressed in the zucchini (Cucurbita pepo) subspecies pepo, which accumulates dioxin-like compounds at high concentrations. Transgenic tobacco (Nicotiana tabacum) plants overexpressing A20/AN1 ZFP genes show accumulation of dioxin-like compounds in their upper parts. However, the mechanisms underlying the accumulation of dioxin-like compounds regulated by the A20/AN1 ZFPs remain unclear. Here, we show that A20/AN1 ZFPs positively regulate the expression of the major latex-like protein (MLP) and its homolog genes in N. tabacum and C. pepo. MLPs are involved in the transport of dioxin-like compounds from the roots to the upper parts of C. pepo. Overexpression of A20/AN1 ZFP genes in N. tabacum leads to the upregulation of pathogenesis-related protein class-10 genes with the binding ability toward dioxin-like compounds. Our results demonstrated that A20/AN1 ZFPs upregulate MLP and its homolog genes in N. tabacum and C. pepo, resulting in the accumulation of dioxin-like compounds.

Hydroxylation and dechlorination of 3,3',4,4'-tetrachlorobiphenyl (CB77) by rat and human CYP1A1s and critical roles of amino acids composing their substrate-binding cavity.

Cytochrome P450 (CYP) monooxygenases play critical roles in determining the toxicity of polychlorinated biphenyls (PCBs) in mammals. Hydroxylation of PCBs by these enzymes leads to increased water solubility, promoting the elimination of PCBs from the body. The CYP1 family is mainly responsible for metabolizing PCBs that exhibit a dioxin-like toxicity. Although the dioxin-like PCB 3,3',4,4'-tetrachlorobiphenyl (CB77) is abundant in the environment and accumulates in organisms, information on CB77 metabolism by CYP1A1s is limited. In this study, recombinant rat CYP1A1 metabolized CB77 to 4'-hydroxy (OH)-3,3',4,5'-tetrachlorobiphenyl (CB79) and 4'-OH-3,3',4-trichlorobiphenyl (CB35), whereas human CYP1A1 produced only 4'-OH-CB79. Rat CYP1A1 exhibited much higher metabolizing activity than human CYP1A1 because CB77 was stably accommodated in the substrate-binding cavity of rat CYP1A1 and was close to its heme. In a rat CYP1A1 mutant with two human-type amino acids, the production of 4'-OH-CB79 decreased, whereas that of the dechlorinated metabolite 4'-OH-CB35 increased. These results are explained by a shift in the CB77 positions toward the heme. This study provides insight into the development of enzymes with high metabolizing activity and clarifies the structural basis of PCB metabolism, as dechlorination contributes to a drastic decrease in dioxin-like toxicity.

Enantioselective metabolism of chiral polychlorinated biphenyl 2,2',3,4,4',5',6-Heptachlorobiphenyl (CB183) by human and rat CYP2B subfamilies.

Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (-)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (-)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3'-OH-CB183 and 5-OH-CB183, were identified. The docking study showed that the 3'-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.

Congener specificity in the accumulation of dioxins and dioxin-like compounds in zucchini plants grown hydroponically.

Zucchini cultivars Cucurbita pepo subsp. ovifera cv. Patty Green and subsp. pepo cv. Gold Rush were cultivated hydroponically in a nutrient solution supplemented with a mixture of dioxins and dioxin-like compounds. Patty Green and Gold Rush showed low and high accumulation of these compounds in the aerial parts respectively. In both cultivars, the accumulation of each congener negatively depended on its hydrophobicity. This suggests that desorption and solubilization were partly responsible for congener specificity of accumulation, since this was not found in soil experiments. In contrast, no clear difference in accumulation in the roots was observed between the cultivars, whereas the translocation factors, which are indicators of efficient translocation from the roots to the aerial parts, differed among the congeners hydrophobicity-dependently. There were positive correlations between accumulation in the roots and the hydrophobicity of the polychlorinated biphenyl congeners in both cultivars. These results indicate that translocation was also partly responsible for the congener specificity and accumulation concentrations.