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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]

Opioid abuse involving emerging opioid compounds is a growing public health problem, which was highlighted recently by cases of human morbidity and mortality linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the toxicology and metabolism of acetyl fentanyl precludes its detection in human samples. The following study was conducted to test a new analytical procedure for the simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl norfentanyl, in human urine. Metabolic reference standards and deuterium-labeled internal standards were synthesized for use in an assay that coupled solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accuracy (% Relative Error <5%) and inter- and intrarun precision (%CV <20%) of this new method resulted in low levels of quantification (~1 ng/mL). Similar results were obtained using liquid chromatography columns manufactured with phenyl-hexyl and biphenyl stationary phases (r2 > 0.98). Preliminary human liver microsomal and in vivo rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450s to acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further toxicokinetic studies are required to fully elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and excretion. [ABSTRACT FROM AUTHOR]

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS² (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MSAll (also called MSE) techniques. We compared these three techniques in their capabilities to produce comprehensive MS² data by assessing both metabolite MS² acquisition hit rate and the quality of MS² spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MSAll) using an ultrahigh-performance liquid chromatography-qudrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS² acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS² acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS² acquisition with either the SWATH or MSAll method. However, the quality of the acquired MS² spectra decreased in the order of IDA, SWATH, and MSAll methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS² spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MSAll methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS² spectra but with a lower MS² acquisition hit rate than the other two methods. SWATH outperformed the MSAll method given its better quality of MS² spectra with an identical MS² acquisition hit rate. [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]

A radiolabeled tracer for imaging therapeutic targetsin the brain is a valuable tool for lead optimization in CNS drugdiscovery and for dose selection in clinical development. We reportthe rapid identification of a novel phosphodiesterase 10A (PDE10A)tracer candidate using a LC–MS/MS technology. This structurallydistinct PDE10A tracer, AMG-7980 (5), has been shownto have good uptake in the striatum (1.2% ID/g tissue), high specificity(striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (KD) and PDE10A targetdensity (Bmax) were determined to be 0.94nM and 2.3 pmol/mg protein, respectively, using [3H]5on rat striatum homogenate. Autoradiography on rat brainsections indicated that the tracer signal was consistent with knownPDE10A expression pattern. The specific binding of [3H]5to rat brain was blocked by another structurally distinct,published PDE10A inhibitor, MP-10. Lastly, our tracer was used tomeasure in vivo PDE10A target occupancy of a PDE10A inhibitor in ratsusing LC–MS/MS technology. [ABSTRACT FROM AUTHOR]

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nudeoside S'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn2+. When Mg is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble guanylyl cydase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nudeotide-mediated signal transduction. [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]