山岸 由和

Tellurium (Te) is a metalloid widely used in various industries. However, its toxicological impact on humans is poorly understood. In this study, we investigated the role of two methyltransferases, thiopurine S‑methyltransferase (TPMT) and indolethylamine N‑methyltransferase (INMT), in the methylation of tellurite, an inorganic Te oxyanion. The products of the reaction of Te compounds catalyzed by recombinant human TPMT and/or INMT were analyzed by liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (LC-ICP-MS) and gas chromatography mass spectrometry (GC-MS). We found that TPMT catalyzes the methylation of non-methylated Te and methanetellurol to generate dimethyltelluride. On the other hand, INMT catalyzes the methylation of methanetellurol and dimethyltelluride to produce trimethyltelluronium ion, a metabolite excreted into animal urine. We conclude that TPMT and INMT are cooperatively responsible for the detoxification of Te oxyanions through methylation to form trimethyltelluronium ions.

Tellurium (Te) is a chalcogen element like sulfur and selenium. Although it is unclear whether Te is an essential nutrient in organisms, unique Te metabolic pathways have been uncovered. We have previously reported that an unknown Te metabolite (UKTe) was observed in plants exposed to tellurate, a highly toxic Te oxyanion, by liquid chromatography-inductively coupled plasma mass spectrometer (LC-ICP-MS). In the present study, we detected UKTe in tellurate-exposed broccoli (Brassica oleracea var. italica) by LC-ICP-MS and identified it as gluconic acid-3-tellurate (GA-3Te) using electrospray ionization mass spectrometer with quadrupole-Orbitrap detector and tandem MS analysis, the high-sensitivity and high-resolution mass spectrometry for organic compounds. We also found that GA-3Te was produced from one gluconic acid and one tellurate molecule by direct complexation in an aqueous solution. GA-3Te was significantly less toxic than tellurate on plant growth. This study is the first to identify the Te metabolite GA-3Te in plants and will contribute to the investigation of tellurate detoxification pathways. Moreover, gluconic acid, a natural and biodegradable organic compound, is expected to be applicable to eco-friendly remediation strategies for tellurate contamination.

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 (LC-Q-Orbitrap-MS) 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 post-mortem 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.

Methicillin-resistant Staphylococcus aureus (MRSA) often causes serious infections in hospitals. Vancomycin is widely accepted as the standard therapy for MRSA infection, but its widespread use has resulted in the generation of vancomycin-resistant S. aureus (VRSA). To reduce the potential risk of MRSA and VRSA emergence in aquatic environments, we investigated the degradation of methicillin and vancomycin by cold atmospheric pressure plasma jet (APPJ) irradiation using N2, O2, and CO2 gases. The concentrations of methicillin and vancomycin in distilled water were decreased in a time-dependent manner by the plasma jet irradiation; that is, compared with the pre-treatment levels, the concentrations of methicillin and vancomycin were reduced by 20 to 50% after plasma jet irradiation for 10 s. No methicillin and vancomycin signals were detected after 300 s irradiation. Reactive species generated from the plasma jet electrophilically attacked and fragmented the antibiotic molecules. The present method realizes direct plasma ignition in a solution, and therefore, the reactive species can easily react with antibiotic molecules. In addition, plasma can be generated from various gas species that are abundant in the atmosphere. Therefore, cold APPJ irradiation can be a powerful, cost-effective, and environmentally friendly means for the treatment of antibiotics in aqueous samples.

Abstract 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.

Understanding the actual conditions of methamphetamine (MA)-related death is important from the perspectives of criminal justice and public health. In this report, we review 104 cases of MA-related death handled by our departments between January 2014 and December 2020. Based on information from police and autopsy examinations, we classified the cases into the following categories: "accidental intoxication" ("MA only" and "multiple drugs or alcohol"), "fatal disease" ("definitively MA-related," "possibly MA-related," and "unlikely MA-related"), "accident," "suicide," "homicide," and "undetermined." The total number and annual trends for each category and their respective femoral blood concentrations were investigated. "Fatal disease" was the most common category (48 cases), followed by "suicide" (25 cases), "accidental intoxication" (14 cases), and "accident" (11 cases). "Definitively MA-related" in which MA may have played a role in their onset or exacerbation accounted for the majority of "fatal disease": 12 cases of heart disease, 4 cases of aortic dissection, 12 cases of cerebral hemorrhage, and 4 cases of subarachnoid hemorrhage. Cases classified as "definitively MA-related" died with lower femoral blood concentrations of MA compared with "MA only." Cases with "fatal disease" might have been misdiagnosed as "death by natural causes" if a proper autopsy and toxicology examinations were not performed. In death investigations, it is necessary to keep in mind that there are some MA-related deaths, and efforts should be made to increase awareness about the risk of death in using this drug.

Malathion, diethyl 2-[(dimethoxyphosphinothioyl)thio]butanedioate, is one of most widely used organophosphoryl pesticide, and it has been detected in several clinical cases of accidental exposure and suicide. It is reported that the observed malathion concentration in blood of persons who suffer from malathion poisoning is smaller than the expected concentration. Because malathion is bound to human serum albumin (HSA), recovery of malathion in the free form is insufficient. We detected malathion adducts in HSA by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra showed that malathion was preferably bound to the lysine (K) and cysteinylproline (CP) residues of HSA. The K- and CP-adducts of malathion were increased in vitro with a dose-dependent fashion when its concentration was smaller than the lethal dose. Further, the K-adduct was also detected in post-mortem blood of an autopsied subject suffering from intentional malathion ingestion. These results suggest that the K-adduct seems to be available to use a biomarker of malathion poisoning, and the determination of the K-adduct could make possible to estimate the amount of malathion ingestion.

Methomyl, (E,Z)-methyl N-{[(methylamino)carbonyl]oxy}ethanimidothioate, is a widely used pesticide that has been detected in many fatal cases of accidental exposure or suicide. Forensic toxicologists have been baffled that the blood methomyl concentration in persons who have died of methomyl poisoning is much lower than the expected concentration in blood. In this study, we speculated two mechanisms underlying the insufficient recovery of methomyl in blood. First, methomyl is decomposed by serum albumin as esterase. Second, methomyl is bound to a specific blood protein, resulting in insufficient recovery in the free form. However, human serum albumin does not show esterase activity for the decomposition of methomyl. On the contrary, specific methomyl hemoglobin adducts have been detected by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra indicated that methomyl was specifically bound to tryptophan (W), tyrosine (Y), and valine (V) residues in hemoglobin. The amounts of W- and V-adducts dose-dependently increased in vitro when the methomyl concentration was lower than the lethal concentration. In addition, the W-adduct was detected in blood sampled from an autopsied subject who died of intentional methomyl ingestion, suggesting that the W-adduct could be used as a biomarker of methomyl poisoning. We were able to estimate the amount of methomyl ingested on the basis of the amount of the W-adduct.

日本においては、かつて薬学科あるいは薬学部に裁判化学講座があり、法医解剖事例の薬物検査を実施していた時期があったが、現在は法医学教室と同様に衰退し、法医学における薬物検査の実施状況は諸外国に比べ手薄となっている。法医解剖において薬物検査は死因判定上重要な位置を占め、また傷害や殺人未遂などの事件では、生体の薬物検査も必要とされるので、現状を放置することは問題であろう。一方で、薬物検査を警察の科学捜査研究所に依存してしまっては、医学診断上の妥当性や客観性を維持することは困難であると考えられる。今後は、薬学部出身者などの薬物分析の専門家が捜査の都合などに左右されず、死因や傷害度を判定する検案医、法医専門医などと密接に連携して薬物検査を実施できる体制を整備したうえで、検出法や致死濃度が未知である新規薬物による死亡や傷害事例に対応するために研究を行ったり、後継者を育成するために大学薬学部と連携をはかるべきである。(著者抄録)

In recent years, there has been an increase in the use of phosphodiesterase type 5 inhibitors (PDE5i) that are purchased from abroad without a doctor's diagnosis via the Internet or other means. We report six cases in which nonprescription use of PDE5i may have led to death. Among the four deceased individuals who were believed to have experienced sudden cardiac death, three (cases 1-3) had a history of cardiovascular disease, which is a contraindication, and the remaining case (case 4) involved combined use of multiple PDE5i. Sildenafil (0.063 µg/mL, 0.087 µg/mL) was detected in two of the four cases of sudden cardiac death. Tadalafil (0.096 µg/mL) was detected in one of the remaining two cases, and tadalafil (0.197 µg/mL) and vardenafil (0.011 µg/mL) were detected in the other case. Sildenafil (0.032 µg/mL), tadalafil (0.062 µg/mL), and ethanol were detected in a traffic accident case with a history of contraindications. In a case of asphyxiation by vomit aspiration, autopsy showed 90% stenosis in the anterior descending branch of the coronary artery, and sildenafil (0.063 µg/mL) was detected. To the best of our knowledge, this is the first report of postmortem blood levels of tadalafil and vardenafil likely contributing to the cause of death. Despite all the warnings about the dangers of using PDE5 inhibitors, cases of PDE5i contributing to death are still identified during autopsies. Therefore, raising public awareness of the risks of the risks associated with the imported drug use by individuals is necessary.

In order to elucidate the effect of chorioallantoic and yolk sac placenta on the embryonic/fetal toxicity in dibutyltin dichloride (DBTC1)-exposed rats, we examined the histopathological changes and the tissue distribution of dibutyltin in the placentas and embryos. DBTC1 was orally administered to the groups at doses of 0 mg/kg during gestation days (GD)s 7-9 (control group) and 20 mg/kg during GDs 7-9 (GD7-9 treated group), and GDs 10-12 (GD10-12 treated group). The total fetal mortality was increased, and malformations characterized by craniofacial dysmolphism were detected in the GD7-9 treated group. The embryonic/fetal weight and placental weight showed a decrease in both DBTC1-treated groups. Histologically, some embryos on GD 9.5 in.the GD7-9 treated group underwent apoptosis without any changes of yolk sac. In the laser ablation-inductively coupled plasma-mass spectrometry analysis (LA-ICP-MS), tin was detected in the embryo, allantois, yolk sac, ectoplacental cone and decidual mass surrounding the conceptus on GD 9.5 in the GD7-9 treated group. Thus, it is considered that the embryo in this period is specifically sensitive to DBTC1-induced apoptosis, compared with other parts. The chorioallantoic placentas in both DBTC1-treated groups showed the developmental delay and hypoplasia in the fetal parts of placenta, resulting from apoptosis and mitotic inhibition. Thus, it was speculated that the DBTC1-induced malformations and fetal resorption resulted from the apoptosis in the embryo caused by the direct effect of DBTC1. The DBTC1-induced lesions in the chorioallantoic placenta were a non-specific transient developmental retardation in the fetal parts of placenta, leading to intrauterine growth retardation.

In order to clarify the histological localization of cadmium (Cd) in the placenta, we analyzed paraffin sections of placentas from rats with a single Cd exposure on gestation day 18 by the LA-ICP-MS imaging method compared with the histopathological changes. The placentas were sampled at 1 hour, 2 hours, 3 hours, 6 hours, and 24 hours after treatment. Histopathologically, the trophoblasts in the labyrinth zone of the Cd group showed swelling at 1 hour. At 2 and 3 hours, the trophoblasts showed swelling and vacuolar degeneration. At 6 and 24 hours, the syncytiotrophoblasts selectively underwent necrosis/apoptosis, resulting in a decrease in number. Remarkable metallothionein expression was observed in the trophoblastic septa, particularly cytotrophoblasts at 24 hours. The LA-ICP-MS analysis detected the localization of Cd in the fetal part of the placenta from 1 hour onwards. In particular, the intensity of Cd was prominent in the labyrinth zone and tended to increase with the progression of trophoblastic septa damages. The LA-ICP-MS analysis using the paraffin sections detected the localization of Cd in the fetal part of the placenta, and this methodology will be one of the valuable tools to detect heavy metals in toxicological pathology.

The effects of chlorpromazine-treatment timing on the development of the placenta in pregnant rats were examined. Chlorpromazine was administered intraperitoneally at 100 mg/kg on gestation day (GD) 11 (GD11-treated group), GD 13 (GD13-treated group) or GD 15 (GD15-treated group) into pregnant rats. All treated dams exhibited decreased body weight, prone position, hypothermia, loss or decrease of locomotor activity, etc. The fetal mortality rates were increased up to 42.9% in the GD11- and GD13-treated groups and up to 16.7% in the GD15-treated group. The embryo/fetal weight was on a declining trend from GD 16 onward, and the intrauterine growth retardation (IUGR) rates on GD 21 were increased in all treated groups. The placental weight showed a declining trend from GD 15 onward in all treated groups. Histopathologically, apoptosis was detected 1 or 2 days after treatment, and led to hypoplasia in the labyrinth zone and metrial gland, and cystic degeneration in the basal zone on GD 21 in all treated groups. There was no difference in the histopathological lesions on GD 21 among the treated groups. Thus, it is considered that chlorpromazine-induced placental toxicity is characterized in that there is no obvious specific sensitive period from GD 11 to GD 15. Chlorpromazine induced a non-specific transient development retardation of the placenta by apoptosis independently of the cell proliferation period in each part/zone. (C) 2015 Elsevier GmbH. All rights reserved.

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMCFar-OCH3) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.