Protein-Labeling Reagents Selectively Activated by Copper(I).

Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu+) and cupric (Cu2+) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu+-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu+, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu+ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu+ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu+ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu+-responsive protein labeling in providing insights into the molecular mechanisms of Cu+ metabolism and homeostasis.

Subchronic toxicity study of indium-tin oxide nanoparticles following an intratracheal administration into the lungs of rats.

OBJECTIVES: We aimed to analyze the subchronic toxicity and tissue distribution of indium after the intratracheal administration of indium-tin oxide nanoparticles (ITO NPs) to the lungs of rats. METHODS: Male Wistar rats were administered a single intratracheal dose of 10 or 20 mg In/kg body weight (BW) of ITO NPs. The control rats received only an intratracheal dose of distilled water. A subset of rats was periodically euthanized throughout the study from 1 to 20 weeks after administration. Indium concentrations in the serum, lungs, mediastinal lymph nodes, kidneys, liver, and spleen as well as pathological changes in the lungs and kidneys were determined. Additionally, the distribution of ionic indium and indium NPs in the kidneys was analyzed using laser ablation-inductively coupled plasma mass spectrometry. RESULTS: Indium concentrations in the lungs of the two ITO NP groups gradually decreased over the 20-week observation period. Conversely, the indium concentrations in the mediastinal lymph nodes of the two ITO groups increased and were several hundred times higher than those in the kidneys, spleen, and liver. Pulmonary and renal toxicities were observed histopathologically in both the ITO groups. Both indium NPs and ionic indium were detected in the kidneys and their distributions were similar to the strong indium signals detected at the sites of inflammatory cell infiltration and tubular epithelial cells. CONCLUSIONS: Our results demonstrate that intratracheal administration of 10 or 20 mg In/kg body weight of ITO NPs in male rats produces pulmonary and renal toxicities.

Evaluation of organophosphorus pesticide tyrosine adducts for postmortem change by human serum albumin with liquid chromatography quadrupole Orbitrap mass spectrometry.

Organophosphorus pesticides (OPPs) having a phosphate ester moiety, such as malathion (MA) and methidathion (DMTP), are widely used and have been detected in many fatal cases of accidental exposure or suicide in Japan. In forensic toxicology, the accurate determination of blood OPP concentration is mandatory to prove death by OPP poisoning. However, fatal pesticide concentration in blood at autopsy varies depending on the circumstances surrounding the dead body. In this study, we found that 16 OPPs were degraded by human serum albumin (HSA) in a temperature-dependent fashion. The mechanism underlying MA, DMTP, azinphos-methyl, etrimfos, fenthion (MPP), pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, edifenphos (EDDP), fosthiazate, and pyraclofos degradation involves the formation of adducts with tyrosine residues in HSA. The mass spectra obtained by liquid chromatography quadrupole Orbitrap mass spectrometry revealed that phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5 were formed in HSA solution incubated with OPPs. These results indicate that the 16 OPPs are postmortem changed by HSA. The detection of phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5, instead of MA, DMTP, azinphos-methyl, etrimfos, MPP, pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, EDDP, fosthiazate, and pyraclofos per se, may be used to determine death by these OPPs poisoning.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

Determination of intracellular dopamine by liquid chromatography-fluorescence detection with post-column derivatization using the König reaction.

Dopamine is an important neurotransmitter, and the disruption of dopaminergic homeostasis causes various neurological diseases such as Parkinson's disease. Analysis of intracellular dopamine levels is important to understand the pathology of neurological diseases. We have developed a new method for the fluorometric detection of dopamine by adopting the König reaction, which is commonly used for the detection of cyanide, thiocyanate, and selenocyanate, and demonstrated that it can be applied to the determination of intracellular dopamine levels. The present method only requires a conventional LC system with isocratic elution and post-column derivatization and is simple to perform. The LOD, LOQ, and linearity range were 10.8 nM, 32.8 nM, and 0.05-10 µM, respectively, with accuracies of 101.8-106.3 % and precisions within 5 %, which are sufficient for the quantification of intracellular dopamine. We also determined dopamine levels in PC12 cells and found that the levels increased and decreased when the cells were exposed to L-dopa and cyanide, respectively, possibly because of the conversion of L-dopa into dopamine and the depletion of intracellular dopamine by exposing cells to cyanide, respectively. These results suggest the applicability of the present method, and that this new use of the König reaction offers a reliable and useful means of quantifying intracellular dopamine.

The C-terminal tail of Rad17, iVERGE, binds the 9‒1‒1 complex independently of AAA+ ATPase domains to provide another clamp-loader interface.

The ATR pathway plays a crucial role in maintaining genome integrity as the major DNA damage checkpoint. It also attracts attention as a therapeutic target in cancer treatment. The Rad17-RFC2-5 complex loads the Rad9-Hus1-Rad1 (9-1-1) DNA clamp complex onto damaged chromatin to activate the ATR pathway. We previously reported that phosphorylation of a polyanionic C-terminal tail of human Rad17, iVERGE, is essential for the interaction between Rad17 and the 9-1-1 complex. However, the molecular mechanism has remained unclear. Here, we show that iVERGE directly interacts with the Hus1 subunit of the 9-1-1 complex through Rad17-S667 phosphorylation independently of the AAA+ ATPase domains. An exogenous iVERGE peptide interacted with the 9-1-1 complex in vivo. The binding conformation of the iVERGE peptide was analyzed by de novo modeling with docking simulation, simulated annealing-molecular dynamics simulation, and the fragment molecular orbital method. The in silico analyses predicted the association of the iVERGE peptide with the hydrophobic and basic patches on the Hus1 protein, and the corresponding Hus1 mutants were deficient in the interaction with the iVERGE peptide in vivo. The iVERGE peptide occupied the same position as the C-terminus of Saccharomyces cerevisiae RAD24 on MEC3. The interaction energy calculation suggested that the Rad17 KYxxL motif and the iVERGE peptide are the primary and secondary interaction surfaces between the Rad17-RFC2-5 and 9-1-1 complexes. Our data reveal a novel molecular interface, iVERGE, between the Rad17-RFC2-5 and 9-1-1 complexes in vertebrates and implicate that Rad17 utilizes two distinct molecular interfaces to regulate the 9-1-1 complex.

Cadmium body burden and health effects after restoration of cadmium-polluted soils in cadmium-polluted areas in the Jinzu River basin.

BACKGROUND: Itai-itai disease is caused by environmental cadmium (Cd) pollution in the Jinzu River basin in Japan. To reduce the Cd contamination of rice, soil restoration of paddy fields was carried out. We evaluated the effect of soil restoration on the health status of residents of the former Cd-polluted area. METHODS: Participants were 1,030 men and 944 women who lived in the area of restoration of Cd-polluted rice paddies. First morning urine was collected and urinary Cd, ß2-microglobulin (ß2MG), and N-acetyl-ß-D-glucosaminidase (NAG) levels were measured. Associations among age, years of residence before and after soil restoration, and urinary Cd, ß2MG, and NAG levels were evaluated by multiple regression analysis. RESULTS: The geometric mean (interquartile range) of urinary Cd (µg/g Cr) was 1.00 (0.58-1.68) in men and 1.67 (1.02-2.91) in women. The geometric means of urinary ß2MG (µg/g Cr) and NAG (U/g Cr) were 174.6 (92.6-234.2) and 1.47 (0.72-3.14) in men, and 217.6 (115.3-28.7) and 1.48 (0.73-2.96) in women, respectively. Urinary Cd, ß2MG, and NAG were significantly positively correlated (p < 0.01 all). Age and duration of residence in the Cd-polluted area before soil restoration were independently associated with urinary Cd, ß2MG, and NAG. Among the 916 participants who had resided in the area before the soil restoration, urinary Cd concentrations were significantly higher, thus by 1.03-fold (95% CI, 1.01-1.04) in men and 1.03-fold (95% CI, 1.01-1.05) in women, when the years of residence before soil restoration by each 5-years increment. By contrast, urinary Cd concentrations were significantly lower, thus 0.97-fold (95% CI, 0.96-0.99) lower in men and 0.97-fold (95% CI, 0.95-0.99) lower in women, by each 5-year increment of residence after soil restoration. A similar association was observed for urinary ß2MG concentration, and no significant association was observed for urinary NAG levels in men or women. CONCLUSIONS: Cd exposure and associated renal tubular dysfunction in residents of a former Cd-polluted area were influenced by Cd exposure from the environment prior to soil restoration. Soil restoration in Cd-polluted areas reduced the Cd exposure of local residents.

Structural Basis of PML-RARA Oncoprotein Targeting by Arsenic Unravels a Cysteine Rheostat Controlling PML Body Assembly and Function.

PML nuclear bodies (NB) are disrupted in PML-RARA-driven acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) cures 70% of patients with APL, driving PML-RARA degradation and NB reformation. In non-APL cells, arsenic binding onto PML also amplifies NB formation. Yet, the actual molecular mechanism(s) involved remain(s) elusive. Here, we establish that PML NBs display some features of liquid-liquid phase separation and that ATO induces a gel-like transition. PML B-box-2 structure reveals an alpha helix driving B2 trimerization and positioning a cysteine trio to form an ideal arsenic-binding pocket. Altering either of the latter impedes ATO-driven NB assembly, PML sumoylation, and PML-RARA degradation, mechanistically explaining clinical ATO resistance. This B2 trimer and the C213 trio create an oxidation-sensitive rheostat that controls PML NB assembly dynamics and downstream signaling in both basal state and during stress response. These findings identify the structural basis for arsenic targeting of PML that could pave the way to novel cancer drugs. SIGNIFICANCE: Arsenic curative effects in APL rely on PML targeting. We report a PML B-box-2 structure that drives trimer assembly, positioning a cysteine trio to form an arsenic-binding pocket, which is disrupted in resistant patients. Identification of this ROS-sensitive triad controlling PML dynamics and functions could yield novel drugs. See related commentary by Salomoni, p. 2505. This article is featured in Selected Articles from This Issue, p. 2489.

Simultaneous determination of intracellular reduced and oxidized glutathiones by the König reaction.

The König reaction is commonly used for the detection of cyanide and its derivatives, including thiocyanate and selenocyanate. We found that this reaction can be used to quantify glutathione fluorometrically, and applied it to the simultaneous determination of reduced and oxidized glutathiones (GSH and GSSG) using a conventional LC system with isocratic elution. The limits of detection were 6.04 nM and 9.84 nM for GSH and GSSG, respectively, and the limits of quantification were 18.3 nM and 29.8 nM, respectively. We also determined GSH and GSSG levels in PC12 cells exposed to paraquat, an oxidative stressor, and observed a decrease in GSH/GSSG ratio, as expected. Total GSH levels quantified by this method and by the conventional colorimetric method with 5,5'-dithiobis(2-nitrobenzoic acid) were comparable. Our new application of the König reaction offers a reliable and useful method to simultaneously quantify intracellular GSH and GSSG.

Identification of postmortem paliperidone metabolite in human blood by LC-Q-Orbitrap-MS.

Paliperidone is a widely used antipsychotic agent detected in many fatal intoxications and suicide cases. In forensic toxicology, the accurate determination of blood paliperidone concentrations is required to prove death by paliperidone poisoning. However, the lethal concentration of paliperidone in blood at autopsy differs from that at the time of death. In this study, we found that paliperidone was decomposed by hemoglobin (Hb) through the Fenton reaction in a temperature-dependent fashion. The mechanism underlying paliperidone decomposition involves the cleavage of its C-N bond linker moiety. The mass spectra obtained by liquid chromatography-quadrupole orbitrap mass spectrometry revealed the formation of 6-fluoro-3-(4-piperidinyl)benzisoxazole (PM1) in Hb/H2O2 solution incubated with paliperidone, as well as in the blood of individuals who died from intentional ingestion of paliperidone. These results suggest that PM1 is the only metabolite produced from paliperidone as a result of temperature-dependent, postmortem changes induced by Hb via the Fenton reaction and may be useful as a biomarker to correct for the concentration of paliperidone in blood at the time of death in clinical cases.

Distribution, metabolism, and toxicity of antimony species in wistar rats. A bio-analytical approach.

This work studied the distribution, reactivity, and biological effects of pentavalent or trivalent antimony (Sb(V), Sb(III)) and N-methylglucamine antimonate (NMG-Sb(V)) in Wistar Rats. The expression of fibrosis genes such as α - SMA, PAI-1, and CTGF were determined in Liver, and Kidney tissues. Wistar rats were treated with different concentrations of Sb(V), Sb(III), As(V) and As(III), and MA via intra-peritoneal injections. The results indicated a noteworthy elevation in mRNA levels of plasminogen activator 1 (PAI-1) in the kidneys of rats that were injected. The main accumulation site for Sb(V) was observed to be the liver, from which it is primarily excreted in its reduced form (Sb(III)) through the urine. The generation of Sb(III) in the kidneys has been found to induce damage through the expression of α-SMA and CTGF, and also lead to a higher creatinine clearance compared to As(III).

Comparison of Nutritional Availability of Biogenic Selenium Nanoparticles and Chemically Synthesized Selenium Nanoparticles.

Selenium (Se) is an essential micronutrient, and animals biosynthesize selenoproteins from various selenocompounds such as inorganic salts and organic selenocompounds as a Se source. In addition to the inorganic and organic forms of Se, it is also known that elemental Se is biologically synthesized at the nanoscale in nature. Biologically synthesized Se nanoparticles (Se-NPs), i.e., biogenic Se-NPs (Se-BgNPs), have not been fully investigated as a Se source compared with the other forms of Se. In this study, we evaluated the nutritional availability of Se-BgNPs biosynthesized in E. coli and revealed that Se-BgNPs were less assimilated into selenoproteins in rats as a Se source than inorganic Se salt or chemically synthesized Se-NPs. Se-BgNPs showed tolerance toward digestion and low absorbability in gut, which resulted in the low nutritional availability. Se-BgNPs seem to be coated with a biomaterial that functions to reduce their toxicity toward E. coli and at the same time lowers their availability to animals.

Ropinirole involved in a fatal case: blood and urinary concentrations.

PURPOSE: Ropinirole is an antiparkinsonian  drug and has recently been suggested to be effective in amyotrophic lateral sclerosis. It is expected that ropinirole prescriptions will increase in the near future. However, the fatal concentration in blood is unclear at this time. Therefore, we report a fatal case involving ropinirole intoxication and discuss the fatal concentrations with reference to several autopsy cases involving ropinirole. METHODS: Ropinirole was quantified in femoral vein blood, cardiac blood, and urine from five autopsy cases in which ropinirole was detected by drug screening in our laboratory. One is a ropinirole intoxication case (this report) and the others  were non-intoxication cases. Their ropinirole concentrations were compared and discussed. RESULTS: The ropinirole concentration in this case was 100 ng/mL in femoral blood, 160 ng/mL in cardiac blood, and 1840 ng/mL in urine. The ropinirole concentrations in the four non-ropinirole poisoning cases were 7-35 ng/mL (mean: 24 ng/mL) in femoral blood, 13-100 ng/mL (mean: 60 ng/mL) in cardiac blood, and 140-1090 ng/mL (mean: 640 ng/mL) in urine. Cardiac/peripheral ratios were in the range of 1.6-2.1 (mean 1.8). CONCLUSIONS: There were no obvious signs of overdose, and the high cardiac/peripheral blood ratio suggested that postmortem redistribution may have occurred, but the  peripheral blood ropinirole concentration (100 ng/mL) was obviously higher than that reported in the previous fatal case of ropinirole poisoning (64 ng/mL). Based on these results, the cause of death in this case was considered to be shock and fatal arrhythmia due to ropinirole poisoning. This case provides important data on postmortem blood and urinary levels of ropinirole poisoning.

Formation of biogenic tellurium nanorods in unicellular green alga Chlamydomonas reinhardtii.

Tellurium (Te) is an industrially useful element but causes environmental contamination. The formation of biogenic Te nanorods (Te-BgNRs) in plants is one of the Te detoxification pathways associated with the phytoremediation of Te because Te-BgNRs contain low-toxicity Te at high densities. In this study, we investigated the mechanism of Te-BgNR formation in a common unicellular green alga, Chlamydomonas reinhardtii, on the basis of elemental analysis by inductively coupled plasma mass spectrometry (ICP-MS). After exposure to 1000 µM sodium tellurate (Na2TeO4) for 2 weeks, the alga accumulated 65.2 fg of Te per cell, and 55.8% of which was present in an insoluble form. Electron microscopic observations revealed that the insoluble Te was rod-shaped elemental Te, i.e. Te-BgNRs, and had a highly crystalline nanostructure. We determined the Te contents in Te-BgNRs by single-particle ICP-MS analysis and found that these nanorods were formed at tellurate exposure concentrations of 100 to 1000 µM. In contrast, soluble Te compounds were found in algal cells even at exposure concentrations lower than 100 µM. These findings suggest that the algal cells initially metabolized tellurate to form soluble Te compounds, and excess tellurate that could not be metabolized was then transformed to Te-BgNRs, which are less toxic than tellurate. Our findings provide a novel approach to Te remediation through the formation of BgNRs in C. reinhardtii.

Rad17 Translocates to Nucleolus upon UV Irradiation through Nucleolar Localization Signal in the Central Basic Domain.

The nucleolus is a non-membranous structure in the nucleus and forms around ribosomal DNA repeats. It plays a major role in ribosomal biogenesis through the transcription of ribosomal DNA and regulates mRNA translation in response to cellular stress including DNA damage. Rad17 is one of the proteins that initiate and maintain the activation of the ATR pathway, one of the major DNA damage checkpoints. We have recently reported that the central basic domain of Rad17 contains a nuclear localization signal and that the nuclear translocation of Rad17 promotes its proteasomal degradation. Here, we show that the central basic domain contains the nucleolar localization signal as well as the nuclear localization signal. The nucleolar localization signal overlaps with the nuclear localization signal and is capable of transporting an exogenous protein into the nucleolus. Phosphomimetic mutations of the central basic domain inhibit nucleolar accumulation, suggesting that the post-translational modification sites regulate the nucleolar localization. Nucleolar accumulation of Rad17 is promoted by proteasome inhibition and UV irradiation. Our data show the nucleolar localization of Rad17 and suggest a possible role of Rad17 in the nucleolus upon UV irradiation.

Recent advances in copper analyses by inorganic mass spectrometry.

Copper (Cu) participates in the biological redox reaction in the body, and its deficiency is fatal to the body. At the same time, Cu is extremely toxic when it exists in excess. Thus, the body has to tightly and spatiotemporally regulate the concentration of Cu within a physiological range by several groups of Cu-regulating proteins. However, entire mechanisms underlying the maintenance of Cu homeostasis in body and cells have not fully understood. It is necessary to analyze Cu itself in a body and in a cell to reveal the Cu homeostasis. In this review, recent advances in the analytical techniques to understand the Cu metabolism such as speciation, imaging and single-cell analysis of Cu were highlighted.

Presence of nano-sized mercury-containing particles in seafoods, and an estimate of dietary exposure.

The toxicity of nano-sized particles of mercury (NP-Hg), which are thought to be generated during the detoxification of methyl mercury (MeHg), may differ from that of MeHg, elemental Hg (Hg0), and inorganic Hg (I-Hg). From a human health perspective, it is important to evaluate the presence of NP-Hg in seafoods. We investigated the in vivo formation of NP-Hg in fish and shellfish, which are the main sources of Hg exposure in humans. NP-Hg was measured in 90 fish samples with single-particle inductively coupled plasma mass spectrometry (spICP-MS) after enzyme degradation with pancreatin and lipase. In addition to NP-Hg, total Hg (T-Hg), MeHg, and selenium (Se) concentrations were evaluated. Transient Hg signals were detected as nanoparticles from almost all samples by using spICP-MS. Higher particle number concentrations (CPN) were observed in the tuna-swordfish group than in the shellfish group (17.7 × 107 vs. 1.2 × 106 particles/g, respectively). Although the CPN and maximum particle mass increased significantly with increasing T-Hg concentration, the increase in CPN was greater than those in maximum particle mass. Assuming that the NP-Hg detected was HgSe (tiemannite) and spherical based on previous reports, the maximum particle diameter was estimated to be 89 nm. The mean dietary exposures to NP-Hg, T-Hg, and MeHg were estimated to be 0.067, 5.75, and 5.32 µg/person per day, respectively. Generation of NP-Hg was inferred to be widespread in marine animals, with a preferential increase in the number of particles rather than an increase in particle size. The mean dietary exposure to NP-Hg in Japanese people was estimated to be 1.2 ng/kg body weight (BW) per day. Compared to PTWI of 4 µg/kg BW per week (0.57 µg/kg BW per day) derived by JECFA (2011), the health risk from redissolved I-Hg from NP-Hg is small.

Evaluation of Post-Mortem Interaction between Hemoglobin and Oxime-Type Carbamate Pesticides.

Oxime-type carbamate pesticides having an oxime moiety such as aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are widely used and have been detected in many fatal cases of accidental exposure or suicide. In forensic toxicology, the accurate determination of blood pesticide concentration is obligatory to prove death by oxime-type carbamate pesticide poisoning. However, the fatal pesticide concentration in blood at autopsy differs from that at the time of death. In this study, we found that oxime-type carbamate pesticides were decomposed by Hb in a temperature-dependent fashion. The mechanism underlying methomyl, aldicarb, oxamyl, and thiofanox decomposition involves the formation of adducts with the amino acids in Hb. With regard to butocarboxim, its decomposition involves the oxidation of the free form and the formation of adducts with the amino acids in Hb. The mass spectra obtained by liquid chromatography quadrupole time-of-flight mass spectrometry revealed that carbamylated amino acid adducts such as Wcar-adduct and Vcar-adduct were formed in Hb solution incubated with methomyl, aldicarb, oxamyl, and thiofanox, whereas alkylated amino acid adducts such as Walkyl-adduct were formed in Hb solution incubated with butocarboxim. These results indicate that aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are post-mortem changed by Hb.

Detection of Histidine-Tagged Protein in Escherichia coli by Single-Cell Inductively Coupled Plasma-Mass Spectrometry.

We have developed a rapid and precise quantification method for a histidine (His)-tagged recombinant protein produced in Escherichia coli (E. coli) by single-cell inductively coupled plasma-mass spectrometry (SC-ICP-MS). Plasmid vector containing enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene fused with His-tag was transformed into E. coli. The transformed E. coli was exposed to nickel (Ni) chloride or cobalt (Co) chloride for labeling His-tag with the Ni or Co ion. Then, E. coli was analyzed by SC-ICP-MS to determine the amount of EGFP or mCherry protein on the basis of the signal of Ni or Co bound to His-tag. By comparing Ni and Co contents in E. coli expressing His-tagged mCherry with those in nontagged mCherry, the specific binding of Co to His-tag was more clearly detected than that of Ni. The Co contents were increased until 6 h after the protein induction, and this observation was coincident with the increases in fluorescence intensity of EGFP or mCherry measured by a flow cytometer. However, the Co contents were decreased for EGFP and kept at a constant level for mCherry from 6 to 24 h despite the continuous increase in the fluorescence intensity through incubation. The fluorescent proteins were mainly recovered in the insoluble fraction 24 h after the induction. This can be explained by the fact that the overexpressed fluorescent proteins with His-tag are transferred into inclusion bodies, which hampers the binding of the fluorescent proteins to the Co ion. SC-ICP-MS can be a useful technique to precisely quantify soluble recombinant proteins in E. coli without the extraction and purification process.