1、 Forensic Science International 168 (2007) 208211 An accidental death due to Freon 22 (monochlorodifluoromethane) inhalation in a fishing vesselCase reportAko Koreeda*, Kosei Yonemitsu, Sohtarou Mimasaka, Yuki Ohtsu, Shigeyuki TsunenariDepartment of Forensic Medicine, Graduate School of Medical Scie
2、nces, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, JapanReceived 15 June 2005; received in revised form 22 December 2005; accepted 12 January 2006Available online 21 February 2006AbstractA case of accidental Freon 22 (monochlorodifluoromethane) poisoning in a fishing vessel is reported. Fore
3、nsic autopsy revealed severe pulmonary edema and congestion (left lung; 576 g, right lung; 740 g). GCMS analysis clearly showed that the deceased inhaled Freon 22 gas prior to his death. Freon 22 concentration was 169 7.0 mg/ml in the heart blood. The distribution pattern of Freon 22 in tissue sampl
4、es 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
5、 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
6、needed to confirm it.1. IntroductionMonochlorodifluoromethane (Freon 22) is a chlorofluorocarbon, which has been widely used as a refrigerant for freezers, refrigerators and air conditioners 1. It is a colorless, non-flammable gas with a slightly ethereal odor at room temperature and has a high vapo
7、r density (3.03; air = 1) 2. According to the classification of solvent and gas toxicity by Underwriters Laboratories Inc., Freon 22 belongs to group 5a, and the toxicity is in the same class with Freon 11 (1,2dibromotetrafluoroethane) and carbon dioxide 3. Although Freon 22 is believed to have a lo
8、w toxicity compared to other chlorofluorocarbons, fatalities caused by accidental inhalation have been reported, especially in fishing vessels 48.We had a forensic autopsy case of a man who died accidentally while maintaining a freezing system in a fishing vessel. We present the details of the case
9、and a toxicological analysis of Freon 22 in the body. The cause and manner of death are discussed based on the toxicological and histopathological findings. 2. Case historyThe chief engineer and a deck-hand of a deep-sea trawler were found unconscious in their vessel at about 08:30 am. They had been
10、 working in the vessel from 07:30 that morning to prepare for the next voyage. Their colleagues rescued them. The deck hand, who was lying on the middle deck of the vessel, survived (Fig. 1B). The chief engineer who was on the bottom deck died (Fig. 1 A) at 10:22 am after hospitalization. The collea
11、gues did not notice any strange odor in the cabins, but they nearly fainted within a minute of entering the cabin.Three months before the fatal incident, the freezing system of the vessel had a gas-leaking accident. Although it was repaired, a further Freon gas-leak was expected because the system w
12、as too old. The police only investigated the scene the next day after ventilating the cabin space for their own safety, therefore, the oxygen and Freon 22 concentrations in the cabin at the time of the accident are not known.Fig. 1. Side view of the deep-sea fishing vessel. (A) Chief engineer (deced
13、ent); (B) deck hand (survived); (C) compressor of the refrigerator.38-year-old man weighing 64 kg and 174 cm tall. There was severe pulmonary edema and congestion (left lung; 740 g). Mucosal hemorrhages were noted in most parts of the small intestine. There were no specific findings in other organs
14、except for severe congestion. Tissues were formalin fixed and examined by HE and Schmorl stainings after an ammonia-ethanol treatment to remove formaldehyde-derived artifacts 9. Oil red O staining of the liver tissue was also performed using cryostat sections 10. For toxicological analysis, blood an
15、d tissue samples were collected in thick plastic bags (Unipack G-4, Seinichi Co. Ltd., Japan), vacuumsealed and immediately stored at 30 8C until analysis.4. Toxicological analysis4.1. ChemicalsStandard Freon 22 gas (99.9%) was purchased from Fukuhoteisan Co., Fukuoka, Japan and used for calibration
16、. All solvents and chemicals were of analytical grade and purchased through local suppliers. Standard Freon 22 gas was transferred into a glass vial by the water displacement method. Briefly, a glass vial was submerged in tap water and the water in the vial was displaced by the standard Freon 22 gas
17、. The vial was then sealed with a Teflon rubber under water. The exact volume of Freon 22 was taken from the vial by using gas-tight cyringes and used for calibration. The volume of Freon 22 was converted into weight according to the Avogadros law.4.2. Sample preparationsOne milliliter of defrosted
18、blood and 1 g each of sliced frozen tissue was sealed in a glass vial with a silicon septum. For quantitative calibrations, exact volumes of pure Freon 22 gas were transferred into sealed vials containing 1 ml of blanc blood using gas-tight syringes. Each vial was heated at 50 8C in an electric alum
19、inum block heater (Eira Co. Ltd., Tokyo, Japan) for 15 min and its 100 mL of the headspace were introduced onto the GCMS by using a gas-tight syringe. Each sample and calibration vial was prepared in triplicate.4.3. GCMS conditionsGCMS analysis was performed by Shimadzu QP-5000 (Shimadzu Co., Kyoto,
20、 Japan). The GC conditions were as follows: splitless injection mode; column, DB-WAX capillary column, 30 m 0.25 mm i.d., 250 nm film thickness; injection port temperature, 180 8C; carrier gas, helium; flow rate, 1.2 ml/ min; column temperature, 30 8C. The MS conditions were as follows: full scan mo
21、de for qualification (m/z 30150), selected ion monitoring (SIM) mode for quantification (m/z 51); ionization, EI; interface temperature, 230 8C.5. Results5.1. Histological findingsHE staining of the lung showed severe congestion and edema. There were no marked findings in other organs except for con
22、gestion. Oil red O staining of the liver showed many small positive red areas in the cytosol (Fig. 2A). Schmorl staining revealed that most portions of the Oil red O staining positive areas were lypofuscin granules (Fig. 2B).5.2. Toxicological analysisThe presence of Freon 22 in the blood and tissue
23、s was confirmed by the GCMS analysis. Freon 22 was eluted at a retention time of 1.4 min and the obtained mass chromatogram was the same as that obtained from authentic Freon 22 (Fig. 3). No other volatile substances, including fluorocarbons, were detected in any of the samples. Linearity of the cal
24、ibration curve was observed from 0 to 380 mg/ml of pure Freon 22 with a correlation coefficient of 0.995 (Fig. 4). Results of Freon 22 analysis are shown in Table 1. The concentration of Freon 22 in blood was 169 7.0 mg/ml. In tissue samples, the highest Freon 22 level was observed in.6. DiscussionI
25、t is well recognized that halogenated hydrocarbons including Freon 22 depress the central nervous system andFig. 4. (A) SIM (m/z 51) chromatograms of (1) heart blood, (2) standard Freon 22, (3) blanc blood. (B) A typical standard curve of Freon 22 in blood.induce both cardiac arrhythmias and excessi
26、ve secretion in the trachea 1113. These direct toxic effects of the chemical could be a cause of death. On the other hand, due to the heavy vapor density of Freon 22, it displaces air and results in insufficient oxygen concentration, which could also be a cause of death.In the present case, toxicolo
27、gical analysis clearly showed that the deceased inhaled Freon 22 gas prior to his death.The autopsy findings excluded suffocation due to excessive secretions in the trachea from the causes of death. However, toxicological and histological findings could not fully elucidate the manner or the direct c
28、ause of death. First of all, as in mostpreviously reported cases 48, both Freon 22 and oxygen concentrations at the scene were unknown, and asphyxia due to low oxygen concentrations can not be confirmed as a cause of death. Secondly, autopsy findings specific to Freon 22 poisoning have not yet been
29、reported. Granular fat droplets positive to Sudan III staining in the hepatocytes were reported in similar Freon 22 poisoning cases, but the mechanisms of the findings are controversial 47. Morita et al. reported two Freon 22 poisoning cases and suspected that the fat droplets in the liver were prod
30、uced by the effect of Freon 22. Other reports suggested that they were not specific to the toxicity of Freon 22 57. Kazama et al. showed in animal experiments that fine fat droplets appeared temporarily in the course of inhaling Freon 22, but it disappeared immediately after stopping the inhalation
31、and they were different from hepatic toxicity reported in chronic exposure to chloroform and other chlorinated hydrocarbons 5.In the present case, similar pathological findings were observed in the liver. However, most of them were positive to Schmorl staining, which is specific to lipofuscin pigmen
32、t. This has not been mentioned in similar previous case reports 48. Lipofuscin is located in lysosomes and contains products of the peroxidation of unsaturated fatty acids, which closely relate to aging and other cell stresses 14. It is reported that the existence of lipofuscin in tissues could be a marker of chronic exposures to low concentration of chemical substances 15. High Freon 22 concentration in the liver ma
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