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Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N 6-ethenodeoxyadenosine (1,N 6-edA), N ²,3-ethenoguanine (N ²,3-eG), 1,N ²-ethenodeoxyguanosine (1,N ²-edG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N 6-edA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 μg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 μg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N 6-edA, AcrdG, and M1dG. Exposure to PCB 153 (1000 μg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N 6-edA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N 6-edA, AcrdG, 1,N ²-edG, and N ²,3-eG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and carcinogenesis in female Sprague-Dawley rats. [ABSTRACT FROM AUTHOR]

Xenobiotic metabolism in the livercan give rise to reactive metabolitesthat covalently bind to proteins, and determining which proteins aretargeted is important in drug discovery and molecular toxicology.However, there are difficulties in the analysis of these modifiedproteins in complex biological matrices due to their low abundance.In this study, an analytical approach was developed to systematicallyidentify target proteins of acetaminophen (APAP) in rat liver microsomes(RLM) using two-dimensional chromatography and high-resolution tandemmass spectrometry. In vitromicrosomal incubations,with and without APAP, were digested and subjected to strong cationexchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Fourdata processing strategies were combined into an efficient label-freeworkflow meant to eliminate potential false positives, using peptidespectral matching, statistical differential analysis, product ionscreening, and a custom-built delta-mass filtering tool to pinpointpotential modified peptides. This study revealed four proteins, involvedin important cellular processes, to be covalently modified by APAP.Data are available via ProteomeXchange with identifier PXD002590. [ABSTRACT FROM AUTHOR]

Acetaminophen is known to cause hepatoxicityvia the formationof a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liverproteins. Serum albumin (SA) is known to be covalently modified byNAPQI and is present at high concentrations in the bloodstream andis therefore a potential biomarker to assess the levels of proteinmodification by NAPQI. A newly developed method for the absolute quantitationof serum albumin containing NAPQI covalently bound to its active sitecysteine (Cys34) is described. This optimized assay represents thefirst absolute quantitation of a modified protein, with very low stoichiometricabundance, using a protein-level standard combined with isotope dilution.The LC–MS/MS assay is based on a protein standard modifiedwith a custom-designed reagent, yielding a surrogate peptide (followingdigestion) that is a positional isomer to the target peptide modifiedby NAPQI. To illustrate the potential of this approach, the methodwas applied to quantify NAPQI-modified SA in plasma from rats dosedwith acetaminophen. The resulting method is highly sensitive (capableof quantifying down to 0.0006% of total RSA in its NAPQI-modifiedform) and yields excellent precision and accuracy statistics. A time-coursepharmacokinetic study was performed to test the usefulness of thismethod for following acetaminophen-induced covalent binding at fourdosing levels (75–600 mg/kg IP), showing the viability of thisapproach to directly monitor in vivosamples. Thisapproach can reliably quantify NAPQI-modified albumin, allowing directmonitoring of acetaminophen-related covalent binding. [ABSTRACT FROM AUTHOR]

Domoic acid (DA) causes neurological effects in multiplespeciesupon exposure, including status epilepticus in pregnant sea lionsand an epileptic disease state that commonly develops in juveniles.This study aims to define brain toxicokinetic parameters in the pregnantrat in the larger context of maternal-fetal toxin transfer. Specifically,Sprague–Dawley rats were exposed to a low observable effectlevel of 1.0 mg DA/kg intravenously at gestational day 20, and plasma,brain, and cerebrospinal fluid (CSF) samples were taken at discretetime points over 24 h. Domoic acid concentrations were determinedby a tandem LC/MS method recently optimized for brain tissue and CSF.Data showed that 6.6% of plasma DA reached the brain, 5.3% reachedthe CSF, and DA levels were nearly identical in both brain and CSFfor 12 h, remaining above the threshold to activate isolated hippocampalneurons for 2 h. The calculated terminal half-life of CSF was 4 h,consistent with the time for complete CSF regeneration, suggestingthat CSF acts as a mechanism to clear DA from the brain. [ABSTRACT FROM AUTHOR]

Polychlorinated biphenyls (PCBs) are legacy pollutantsthat exerttoxicities through various mechanisms. In recent years exposure toPCBs via inhalation has been recognized as a hazard. Those PCBs withlower numbers of chlorine atoms (LC-PCBs) are semivolatile and havebeen reported in urban air, as well as in the indoor air of olderbuildings. LC-PCBs are bioactivated to phenols and further to quinoneelectrophiles with genotoxic/carcinogenic potential. We hypothesizedthat phenolic LC-PCBs are subject to conjugation and excretion inthe urine. PCB3, often present in high concentrations in air, is aprototypical congener for the study of the metabolism and toxicityof LC-PCBs. Our objective was to identify metabolites of PCB3 in urinethat could be potentially employed in the estimation of exposure toLC-PCBs. Male Sprague–Dawley rats (150–175 g) were housedin metabolism cages and received a single intraperitoneal injectionof 600 μmol/kg body weight of PCB3. Urine was collected every4 h; rats were euthanized at 36 h; and serum was collected. LC/MSanalysis of urine before and after incubation with β-glucuronidaseand sulfatase showed that sulfate conjugates were in higher concentrationsthan glucuronide conjugates and free phenolic forms. At least twomajor metabolites and two minor metabolites were identified in urinethat could be attributed to mercapturic acid metabolites of PCB3.Quantitation by authentic standards confirmed that approximately 3%of the dose was excreted in the urine as sulfates over 36 h, withpeak excretion occurring at 10–20 h after exposure. The majormetabolites were 4′PCB3sulfate, 3′PCB3 sulfate, 2′PCB3sulfate, and presumably a catechol sulfate. The serum concentrationof 4′PCB3 sulfate was 6.18 ± 2.16 μg/mL. This isthe first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach forexposure assessment of LC-PCBs by analysis of phase II metabolitesin urine. [ABSTRACT FROM AUTHOR]

Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography-tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly. [ABSTRACT FROM AUTHOR]

With formaldehyde as the major sourceof endogenous N6-formyllysine proteinadducts, we quantified endogenousand exogenous N6-formyllysine in the nasalepithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm[13C2H2]-formaldehyde using liquidchromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium,with concentration-dependent formation in total as well as fractionated(cytoplasmic, membrane, nuclear) proteins, but was not detected inthe lung, liver, or bone marrow. Endogenous adducts dominated at allexposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resultingin one-third of the total load of N6-formyllysinebeing derived from exogenous sources. The results parallel previousstudies of formaldehyde-induced DNA adducts. [ABSTRACT FROM AUTHOR]