Ako Koreeda

Inhibitory activity of epigallocatechin gallate (EGCg) in paraquat-induced microsomal lipid peroxidation - A mechanism of protective effects of EGCg against paraquat toxicity

Recently we have reported that epigallocatechin gallate (EGCg), a major component of Japanese green tea, significantly increased the survival rate of paraquat (Pq) poisoned mice. This paper describes two biochemical activities of EGCg, which relate to its protective effects against Pq toxicity. EGCg inhibited Pq-induced microsomal malondialdehyde (MDA) productions in rat liver microsome system containing 40 microM FeSO(4). Forty micromolar EGCg inhibited MDA production significantly. EGCg may inhibit the Pq-induced MDA production by at least two mechanisms. One may be iron-chelating activity as the inhibition disappeared when excess amounts of FeSO(4) were added to the reaction mixture, which indicated that EGCg reduced iron driven lipid peroxidation by pulling out available irons in the reaction mixture. The other is radical scavenging activity. EGCg scavenged DMPO-OOH spin adducts generated by the microsome-Pq system. The dose response curve of EGCg was similar to that obtained by ascorbic acid which is a typical water-soluble radical scavenger. Although ascorbic acid had a potential activity of scavenging superoxide radicals, it can not be recommended to use for the treatment of Pq poisoning, because ascorbic acid acts as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction (Fe(2+)+H(2)O(2)-->Fe(3+)+OH(-)+OH*), which is responsible for lipid peroxidation. On the contrary, EGCg inhibited iron-driven lipid peroxidation presumably not only by chelating to Fe ions but also by scavenging superoxide radicals, which are responsible for the reduction of ferric (Fe(3+)) to ferrous (Fe(2+)) that catalyzes the Fenton reaction. Chelating and radical scavenging activity of EGCg can be expected simultaneously in the occurrence of Pq toxicity, which may explain the protective effects of EGCg against Pq toxicity.

Immunohistochemical demonstration of the distribution of chloroquine (CQ) and its metabolites in CQ-poisoned mice

Chloroquine (CQ) distribution in tissues of acutely poisoned mice was demonstrated by immunohistochemistry using anti-CQ polyclonal antibodies (PAC). PAC recognized 4-amino-7-chloro-quinoline structure and sufficiently reacted with CQ and CQ’s metabolite bisdesethyl-chloroquine. In the brain, CQ and its metabolites (CQs) localized in the region of the choroids plexus, indicating an important role in the blood–cerebrospinal barrier system. In the heart, most regions showed diffused positive staining, and relatively strong reaction was observed in Purkinje cells, indicating an important role in acute CQ toxicity. In the lungs, CQs were observed in the bronchial epithelium, type II pneumocytes, and on the surface of alveolar walls. It was suggested that CQs were excreted to the alveolar wall with surfactant phospholipids, which are produced by type II pneumocytes. In the liver, CQs were concentrated in the centrolobular area rather than in the periportal area, in agreement with CQ’s metabolic pathway. In the kidneys, tubular cells were strongly stained compared to glomerular capsules, and the distal part of renal tubules was better stained than the proximal tubules. These findings suggested that CQs were predominantly excreted or reabsorbed through the distal tubules and the collecting duct. Distribution of CQs in tissues presented here were mostly consistent with the physico-chemical properties of CQ and its metabolites. However, the elucidation of CQs’ localization in Purkinje cells remains open. Further experimental studies at the level of microorganella will be needed to clarify the present result.

Fatalities by inhalation of volcanic gas at Mt. Aso crater in Kumamoto, Japan

Volcanic gas is one of the hazards which tourists rarely meet in mountains. We present six fatalities due to volcanic gas inhalation on Mt. Aso, an active volcano in Kumamoto, Japan, over a period of nine years (1989-1997). One accident occurred at the lip of the crater and the other five within a distance of 250 m from the lip of the crater. Four of the six fatalities had a history of bronchial asthma. A forensic autopsy was performed for a fatality with a history of hypertensive heart disease; pulmonary emphysema was disclosed for this victim. These findings supported the idea that individuals with chronic lung diseases were at higher risk of death by volcanic gas inhalation. Safety precautions are effected through regular surveillance of the area and monitoring of volcanic gas levels. The criterion for sulfur dioxide levels has been changed to a more rigorous one (from >5.0 ppm to >0.2 ppm) according to our advice based on the autopsy. Further forensic analyses will help to design additional preventive measures so as to reduce mortality resulting from inhalation of toxic volcanic gas.

Clocapramine-related fatality: Postmortem drug levels in multiple psychoactive drug poisoning

A suicide caused by ingestion of multiple psychoactive drugs is reported. A 42-year-old man with a history of psychosis was found dead in a blood pool in his room. The forensic autopsy revealed two stab wounds on his chest. However, these wounds could not explain the cause of death. Eighty-six tablets were found in his stomach. Four psychoactive drugs; clocapramine (CC), chlorpromazine (CP), promethazine (PM) and clotiazepam (CT) were detected in blood and tissues. The concentrations of CC, CP, PM and CT in the femoral vein (FV) blood were 0.39, 0.61, 1.23 and 0.09 microg/ml, respectively. The cause and manner of death were attributed to suicidal multiple psychoactive drug poisoning. Postmortem drug redistribution showed great site-dependent variations with the lowest level in the FV blood. Remarkable variations were observed in CC, CP and PM, but not in CT compared to other three drugs. The variations were dependent on the volume of distribution (Vd) of the drugs. Our human case has demonstrated drugs with higher Vd values showed higher degree of postmortem redistribution of the drug and vice versa.

A simple dot enzyme-linked immunosorbent assay for ABO blood typing of biological fluid and stains: effects of heating samples

A simple dot enzyme-linked immunosorbent assay (Dot-ELISA) using commercially available monoclonal anti-A and anti-B antibodies and biotinylated anti-H lectin was developed for ABO blood typing of biological fluid and stains. Its application to forensic practice was examined with 117 saliva samples and their stains, and practical case samples of 8 seminal, 6 vaginal and 45 aged salivary stains. In the simple Dot-ELISA, a new step to heat biological samples was introduced in the system in order to block unfavorable non-specific reactions of the samples with secondary enzyme conjugate. The simple Dot-ELISA could determine accurately the ABO blood type of a small amount of secretors and non-secretors salivary samples. In practical tests of seminal, vaginal and salivary stains, all results were confirmed to be identical to those determined by the conventional absorption-inhibition test and the absorption-elution test. The simple Dot-ELISA is considered to be accurate, rapid, simple, sensitive and easy to perform in routine forensic practice. It is also a unique and helpful method to determine the ABO blood types of various biological samples.