3,3'-Dichlorobiphenyl Is Metabolized to a Complex Mixture of Oxidative Metabolites, Including Novel Methoxylated Metabolites, by HepG2 Cells.

3,3'-Dichlorobiphenyl (PCB 11) is a byproduct of industrial processes and detected in environmental samples. PCB 11 and its metabolites are present in human serum, and emerging evidence demonstrates that PCB 11 is a developmental neurotoxicant. However, little is known about the metabolism of PCB 11 in humans. Here, we investigated the metabolism of PCB 11 and the associated metabolomics changes in HepG2 cells using untargeted high-resolution mass spectrometry. HepG2 cells were exposed for 24 h to PCB 11 in DMSO or DMSO alone. Cell culture media were analyzed with ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Thirty different metabolites were formed by HepG2 cells exposed to 10 µM PCB 11, including monohydroxylated, dihydroxylated, methoxylated-hydroxylated, and methoxylated-dihydroxylated metabolites and the corresponding sulfo and glucuronide conjugates. The methoxylated PCB metabolites were observed for the first time in a human-relevant model. 4-OH-PCB 11 (3,3'-dichlorobiphenyl-4-ol) and the corresponding catechol metabolite, 4,5-di-OH-PCB 11 (3',5-dichloro-3,4-dihydroxybiphenyl), were unambiguously identified based on liquid and gas chromatographic analyses. PCB 11 also altered several metabolic pathways, in particular vitamin B6 metabolism. These results demonstrate that complex PCB 11 metabolite profiles are formed in HepG2 cells that warrant further toxicological investigation, particularly since catechol metabolites are likely reactive and toxic.

Two-Step Sequence of Cycloadditions Gives Structurally Complex Tetracyclic 1,2,3,4-Tetrahydrocinnoline Products.

Intramolecular [4 + 2]-cycloaddition of heteroallene salts gives polycyclic tetrahydrocinnoline structures that contain an N-aminoiminium motif. Deprotonation with mild base gives the corresponding azomethine imines, which readily participate in 1,3-dipolar cycloaddition reactions. The structurally complex tetracyclic 1,2,3,4-tetrahydrocinnoline products typically form as a mixture of two separable diastereomers. The major diastereomer is generally formed by reaction of the dipolarophile with the convex face of the dipole.

Intramolecular (4 + 2) cycloaddition of aryl-1-aza-2-azoniaallene salts: A practical approach to highly sterically-congested polycyclic protonated azomethine imines.

We report an improved two-step reaction sequence that gives tricyclic protonated azomethine imine products containing a 1,2,3,4-tetrahydrocinnoline scaffold in high yield. This sequence involves the oxidation of aryl hydrazones with TFAA-activated DMSO to give the corresponding α-trifluoroacetoxyazo products, which react readily with TMSOTf to give 1-aza-2-azoniaallene salt intermediates that undergo intramolecular (4+2) cycloadditions with pendent alkenes. This reaction sequence is more general, more practical and more environmentally friendly than our initially reported method. The cycloaddition provides exceptionally sterically-hindered products in high yield.

Mechanism and Dynamics of Intramolecular C-H Insertion Reactions of 1-Aza-2-azoniaallene Salts.

The 1-aza-2-azoniaallene salts, generated from α-chloroazo compounds by treatment with halophilic Lewis acids, undergo intramolecular C-H amination reactions to form pyrazolines in good to excellent yields. This intramolecular amination occurs readily at both benzylic and tertiary aliphatic positions and proceeds at an enantioenriched chiral center with retention of stereochemistry. Competition experiments show that insertion occurs more readily at an electron-rich benzylic position than it does at an electron-deficient one. The C-H amination reaction occurs only with certain tethers connecting the heteroallene cation and the pendant aryl groups. With a longer tether or when the reaction is intermolecular, electrophilic aromatic substitution occurs instead of C-H amination. The mechanism and origins of stereospecificity and chemoselectivity were explored with density functional theory (B3LYP and M06-2X). The 1-aza-2-azoniaallene cation undergoes C-H amination through a hydride transfer transition state to form the N-H bond, and the subsequent C-N bond formation occurs spontaneously to generate the heterocyclic product. This concerted two-stage mechanism was shown by IRC and quasi-classical molecular dynamics trajectory studies.

Synthesis of mono- and dimethoxylated polychlorinated biphenyl derivatives starting from fluoroarene derivatives.

Polychlorinated biphenyls (PCBs) are environmental pollutants implicated in a variety of adverse health effects, including cancer and noncancer diseases in animals and humans. PCBs are metabolized to hydroxylated compounds, and some of these PCB metabolites are more toxic than the parent PCBs. Unfortunately, most PCB metabolites needed for toxicological studies are not available from commercial sources. Moreover, it is challenging to synthesize PCB metabolites because starting materials with suitable substitution patterns are not readily available. Here, we report the novel synthesis of a variety of mono- and dimethoxyarene derivatives from commercially available fluoroarenes via nucleophilic aromatic substitution with sodium methoxide. This reaction provided good to excellent yields of the desired methoxylated products. Suzuki coupling of selected mono- and dimethoxy haloarenes with chlorinated phenylboronic acids yielded methoxylated derivatives of PCB 11, 12, 25, 35, and 36 in low to good yields. Crystal structures of 3,3'-dichloro-2,5-dimethoxy-1,1'-biphenyl and 3',5-dichloro-2,3-dimethoxy-1,1'-biphenyl confirmed the substitution pattern of both compounds. This synthesis strategy provides straightforward access to a range of mono- and dimethoxylated PCB derivatives that were not readily accessible previously.

A semi-target analytical method for quantification of OH-PCBs in environmental samples.

Hydroxylated polychlorinated biphenyls (OH-PCBs) are oxidative metabolites of PCBs and residuals found in original Aroclors. OH-PCBs are known to play a role as genotoxicants, carcinogens, and hormone disruptors, and therefore it is important to quantify their presence in human tissues, organisms, and environmental matrices. Of 837 possible mono-OH-PCBs congeners, there are only ~ 70 methoxylated PCB (MeO-PCB) standards commercially available. Hence, a semi-target analytical method is needed for unknown OH-PCBs. The mass concentrations of these unknowns are sometimes determined by assuming the peak responses of other available compounds. This can bias the results due to the choices and availabilities of standards. To overcome this issue, we investigated the peak responses of all commercially available MeO-PCB standards with gas chromatography (GC) coupling with triple quadrupole (QqQ) mass spectrometry (MS) system, with positive electron impact (EI) ionization at 20-70 eV in selected ion monitoring (SIM) mode. We found correlations between the relative peak responses (RRFs) and the number of chlorine (#Cl) in the molecules of MeO-PCBs. Among the studied models, the quadratic regression of #Cl is the most suitable model in the RRF prediction (RRF = ß1 × #Cl^2 + ß0) when the peak responses are captured at 30 eV. We evaluated the performance of the model by analyzing 12 synthesized MeO-PCB standards and a PCB-contaminated sediment collected from a wastewater lagoon. We further demonstrate the utility of the model using a different chromatography column and GC-EI-MS system. We found the method and associated model to be sufficiently simple, accurate, and versatile for use in quantifying OH-PCBs in complex environmental samples.

Combined Maternal Exposure to Cypermethrin and Stress Affect Embryonic Brain and Placental Outcomes in Mice.

Prenatal exposure to cypermethrin is a risk factor for adverse neurodevelopmental outcomes in children. In addition, maternal psychological stress during pregnancy has significant effects on fetal neurodevelopment and may influence end-stage toxicity to offspring by altering maternal xenobiotic metabolism. As such, this study examined effects of maternal exposure to alpha-cypermethrin and stress, alone and in combination, on offspring development, with a focus on fetal neurotoxicity. CD1 mouse dams were administered 10 mg/kg alpha-cypermethrin or corn oil vehicle via oral gavage from embryonic day 11 (E11) to E14. In addition, dams from each treatment were subjected to a standard model of restraint stress from E12 to E14. Cypermethrin treatment impaired fetal growth, reduced fetal forebrain volume, and increased ventral forebrain proliferative zone volume, the latter effects driven by combined exposure with stress. Cypermethrin also impaired migration of GABAergic progenitors, with different transcriptional changes alone and in combination with stress. Stress and cypermethrin also interacted in effects on embryonic microglia morphology. In addition, levels of cypermethrin were elevated in the serum of stressed dams, which was accompanied by interacting effects of cypermethrin and stress on hepatic expression of cytochrome P450 enzymes. Levels of cypermethrin in amniotic fluid were below the limit of quantification, suggesting minimal transfer to fetal circulation. Despite this, cypermethrin increased placental malondialdehyde levels and increased placental expression of genes responsive to oxidative stress, effects significantly modified by stress exposure. These findings suggest a role for interaction between maternal exposures to cypermethrin and stress on offspring neurodevelopment, involving indirect mechanisms in the placenta and maternal liver.

3-(3,5-Dichlorophenyl)benzene-1,2-diol.

The title structure [systematic name: 4-(3,5-dichlorophenyl)benzene-1,2-diol], C12H8Cl2O2, is a putative metabolite of 3,5-dichlorobiphenyl (PCB 14). The dihedral angle between the two benzene rings of the title compounds is 58.86 (4)°. In the crystal, it displays intra and intermolecular O-H···O hydrogen bonding and intermolecular O-H···Cl hydrogen···chlorine interactions.

3,5-Dichloro-3',4'-dimethoxybiphenyl.

3,5-Dichloro-3',4'-dimethoxybiphenyl, C14H12Cl2O2, is a dihydroxylated metabolite of 3,4-dichlorobiphenyl (PCB 14). The title compound displays π-π stacking interactions between inversion related chlorinated benzene rings, with an inter-planar stacking distance of 3.3695 (17) Å. The dihedral angle between the two benzene rings was 42.49 (6)°. The methoxy groups on the non-chlorinated ring lie essentially in the plane of the benzene ring, with dihedral angles of 4.0 (2)° and -2.07 (19)°.

2,3-Dichloro-3',4'-dihydroxybiphenyl.

2,3-Dichloro-3',4'-dihydroxybiphenyl, C12H8Cl2O2, is a putative dihydroxylated metabolite of 2,3-dichlorobiphenyl (PCB 5). The title structure displays intramolecular O-H···O hydrogen bonding, and the π-π stacking distance between inversion related chlorinated benzene rings of the title compound is 3.371 (3) Å. The dihedral angle between two benzene rings is 59.39 (8)°.