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.

[Two cases of diphenhydramine related suicide].

Two suicidal cases associated with ingestion of diphenhydramine (DPH) were reported. Case 1 is a typical DPH overdose case of a young man with the blood DPH concentration of 12.2 microg/ mL. Case 2 is a double suicide of a man and a woman. They ingested DPH and fell asleep in a vehicle which had a cooking clay charcoal stove. Their blood DPH concentrations were 0.4 and 0.7 microg/mL, which were high enough to make them sleep. Their cause of death, however, was carbon monoxide poisoning with blood CO-Hb concentration of 14 and 19%. DPH is a low toxic agent and is available as an OTC drug in Japan. Similar fatal cases can be expected to happen in Japan.

An accidental death due to Freon 22 (monochlorodifluoromethane) inhalation in a fishing vessel.

A case of accidental Freon 22 (monochlorodifluoromethane) poisoning in a fishing vessel is reported. Forensic autopsy revealed severe pulmonary edema and congestion (left lung; 576 g, right lung; 740 g). GC-MS analysis clearly showed that the deceased inhaled Freon 22 gas prior to his death. Freon 22 concentration was 169+/-7.0 microg/ml in the heart blood. The distribution pattern of Freon 22 in tissue samples was similar to that in previously reported cases. The brain had the highest concentration of Freon 22 followed by the spleen, liver, kidney and lung, respectively. Histopathologically, Oil red O staining of the liver showed many small, positive red areas in the cytosol, which have been reported in other cases of Freon 22 poisoning. However, Schmorl staining revealed that most areas of Oil red O positivity were lipofuscin granules. Lipofuscin in the liver, which closely relates to aging and other cell stresses, could have a relevance to Freon 22 exposure, but further experimental studies are needed to confirm it.

HPLC analysis of anti-malaria agent, chloroquine in blood and tissue from forensic autopsy cases in Tanzania.

HPLC analysis of anti-malaria agent, chloroquine (CQ) in blood and tissues with a simple HCl back extraction method was applied to three forensic autopsy cases in Dar es Salaam, Tanzania. CQ concentrations in femoral vein blood were 8.5, 48.4 and 43.8 microg/ml in three cases, respectively, which were high enough to attribute the cause of deaths to an acute CQ poisoning. There were great site dependent variations in blood CQ levels. The right heart blood samples were very high, which may be explained by incomplete distribution of the drug before death or postmortem diffusion from liver and its surrounding blood, as high CQ levels were remarkable in the liver. Suicidal and accidental CQ poisonings are very common and CQ is a very important chemical in the field of forensic toxicology in Tanzania.

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.

[Ethanol concentrations in multi-site sampling blood in forensic autopsy cases--a retrospective analysis over a period of six years (1994-1999) in Kumamoto University].

Ethanol and n-propanol concentrations in forensic autopsy cases determined in Department of Forensic Medicine, Kumamoto University School of Medicine were reviewed retrospectively. Out of 388 autopsies in 6 years (1994-1999), ethanol was positive in 88 (22.7%) cases. Higher positive rates were observed in bleeding and burning cases compared to other cases. Histograms of the blood ethanol concentrations in all ethanol positive cases had two peaks at 0.1 mg/ml to 0.5 mg/ml and 1.5 mg/ml to 2.0 mg/ml ranges, which indicated that not only an intermediate but also a weak drunkenness level could be a risk factor of being involved in forensic fatalities. There were no differences in mean ethanol concentrations in the blood samples of the right, left and whole heart blood collected from each victim. The femoral blood, however, was slightly higher than those of heart blood. N-Propanol, an indicator for postmortem ethanol production, was detected in 14.7% of stomach contents samples as early as 6 to 12 hours of post mortem intervals, whereas it was not remarkable in urine and femoral vein blood.

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 secretor's and non-secretor's 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.