32EPIDEMIOLOGICAL STUDIES OFANTICHOLINESTERASE PESTICIDEPOISONING IN JAPAN

TAKEMI YOSHIDA

Department of Biochemical Toxicology, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan

YUMIKO KUROKI

Japan Poison Information Center, Tsukuba, Ibaraki 305-0005, Japan

32.1 Introduction 457

32.2 Toxicological Data on AChE Poisoning in Japan 45832.2.1 General Features 45832.2.2 Annual Data on OP Poisonings 45832.2.3 Intentional OP Poisoning 459

32.2.4 Toxicokinetic Data on Fenitrothion(MEP) Poisonings 459

32.2.5 CM Insecticide Poisonings 461

32.3 Conclusions 461References 461

32.1 INTRODUCTION

Organophosphate insecticides (OPs), inhibitors of acetylcho-linesterase (AChE), were introduced to Japan followingWorld War II. One of the OPs, parathion, was applied torice fields to fight against insects; it is highly toxic and hasno selective toxicity. The Ministry of Agriculture, Forestry,and Fisheries (MAFF) of Japan began at that time to examinethe methods of handling and spraying parathion, and re-searchers evaluated its safety for both users and others from1951 to 1953 with the help of medical doctors. Accordingto the results of these surveys, the regulatory administrationdecided to permit the use of parathion as an insecticide, withspecial cautions to be used when preparing it and using it inthe field. Vast amounts of parathion began to be used nation-wide from around 1953, and many farmers, sprayers, andothers were soon after harmed by parathion, with the numberof deaths increasing to significant levels. In order to avoidparathion’s harmful and toxic effects, as well as deaths,

both the Ministry of Welfare and Health and MAFF madespecial efforts to regulate it.

Although extensive efforts were made to prevent intoxi-cation and the life-threatening effects resulting from OPssuch as parathion, many agricultural workers and otherscontinued to suffer from accidental and intentional poisoningincidents. Although OP insecticides such as tetra ethylpyrophosphate (TEPP), parathion, and methyl parathion hadproperties desirable for insecticidal activity, they exhibiteda marked unselective mammalian toxicity and towardsboth target and non-target biological species. Additionally,agricultural workers and others did not understand the reper-cussions of the toxicity at that time. Currently, OP poisoningsare still an important consideration in Japanese society,particularly in rural areas.

Following the Tokyo subway sarin disaster, Nagao andcolleagues (1997) showed that sarin residue (isopropylmethyl phosphonic moiety) bound to the inhibited AChEof erythrocytes from four victims could be detected by a

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

457

newly developed method. Namely, sarin-bound AChE wassolubilized from erythrocyte membranes from sarin victims,digested with trypsin; the sarin hydrolysis products boundto the AChE were then released by alkaline phosphatasedigestion, and the products were subjected to trimethylsilylderivatization and detected by gas chromatography-massspectrometry (GC-MS). They also applied this new methodto identifying the sarin hydrolysis product in formalin-fixedbrain tissues of sarin-affected victims. They were able toidentify the methyl phosphoric moiety of the brain tissue,but not the isopropyl methyl phosphoric moiety; lack ofidentification of the latter could be due to chemical hydrolysisduring long-term storage (two years) of the fixed brains(Matsuda et al., 1998). This would be the first case to identifythe OP-derived alkyl moiety in such aged AChE of erythro-cytes and formalin-fixed brains in humans. Therefore, thedata are very important in view of the fact that the inhibitedenzyme activity by nerve gas sarin is accurately demonstratedby detecting the esterified enzyme by its alkyl moiety. Thus,this method would be useful in the forensic diagnosis of acutesarin and other highly toxic OP poisonings.

Okumura and colleagues (1996, 2005) reported details for640 victims of the Tokyo subway sarin attack and alsoreported the lessons learned. In other words, the highlytoxic agents of OPs should be kept in mind, because theyare easy to manufacture in small amounts and have been aweapon of choice in terrorist attacks. There are also manyOP-derived accidental and suicidal poisonings, includingthose involving the highly toxic parathion, in Japan.

Many carbamate (CM) insecticides are also used nation-wide in Japan. The toxicity of CM insecticides is ratherweaker than that of OPs. Because of the attention paid inpreventing poisoning by the previously introduced OPs,health hazards from using CMs are rather low among fieldworkers and sprayers. However, intentional poisonings byCMs have occurred continuously every year in Japan todate. This Chapter describes the epidemiology of AChEpesticides poisoning in Japan.

32.2 TOXICOLOGICAL DATA ON AChEPOISONING IN JAPAN

32.2.1 General Features

In Japan, many accidental and suicidal OP poisonings occurevery year, probably because of the easy access to OPs.OP insecticides were introduced into Japan for agriculturalapplications, particularly in rice fields, following WorldWar II. Highly toxic insecticides such as TEPP and parathionhave effectively increased rice production and other cropsin Japan. Because of their toxicity and lack of knowledgeabout their safe handling, many farmers and citizens havebecome intoxicated either accidentally or intentionally from

the earliest use of OPs. OP poisonings have subsequentlybeen reduced after developing the selectively less toxicOPs such as malathion and fenitrothion, which are toxic totarget organisms but less so to non-target human and othermammals. Thus, they have the property of selective toxicity,and may be used safely, with proper handling, as insecticides.However, OP poisonings still occur in Japan. In the past,about 200 people have died by OP poisoning every year,mainly through committing suicide. It is rather difficult tocollect detailed data on the number of OP poisonings sentto emergency hospitals and others, because there is no obli-gation to report the status of recovered patients followingmedical treatment to the regulatory office. This may be oneof the reasons for the difficulty in establishing the exactnumbers of patients intoxicated by OPs.

From late 2007, food contamination and poisoning fromOPs in imported dried “gyoza” dumplings occurred inJapan. Methamidophos, which is not a registered insecticidein Japan, was detected in the “gyoza” dumplings. Severalpeople consumed the dumplings and suffered from OP intoxi-cation syndromes. Methamidophos is one of the metabolitesof acephate, and is very toxic when taken in orally, dermallyand via inhalation. The acute oral toxicity of methamidophosis 21 and 16 mg/kg in male and female rats, respectively.

In 2008, an incident occurred in a family when one adultand four children ate “gyoza” dumpling. Sumi and colleagues(2008) reported details of these methamidophos-poisonedpatients suffering from the outbreak of “gyoza” dumpling.They found a severely intoxicated 5-year-old girl presentingwith cholinergic symptoms, including miosis, bronchorrhea,and hypersalivation, and very low serum cholinesteraseactivity (9 U/L, compared to the normal range of 194–467 U/L). Thereafter, many imported “gyoza” dumplingswere found to be contaminated with OPs such as methamido-phos and DDVP. Although it has not yet been clearlyresolved how and why the imported “gyoza” dumplingswere contaminated with such toxic OPs, food contaminationwith such toxic chemicals is very serious. Analysis of OPsand other chemicals in imported food stuffs and ready-made foods is mandatory and has required the developmentof sophisticated analytical methods.

32.2.2 Annual Data on OP Poisonings

Table 32.1 presents data on accidental OP poisoningsreported to the Japan Poison Information Center (JPIC)from 1998 to 2007. However, accurate data regarding acci-dental OP poisonings, specifically the numbers of poisonedpatients admitted to emergency care, are not collected inJapan. Therefore, the data shown in Table 32.1 are mainlybased on the cases responded to by JPIC at Tsukuba andOsaka. As shown in the table, many OP insecticides commer-cially available and prohibited from use have been addressedby JPIC by phone or facsimile in the care of outpatients

458 EPIDEMIOLOGICAL STUDIES OF ANTICHOLINESTERASE PESTICIDE POISONING IN JAPAN

attending many hospitals in Japan. Of the OPs, fenitrothionand malathion are the most common insecticides to be repo-rted each year, followed by acephate, trichlorfon, DDVP,and so on. As described above, acephate is metabolized tomethamidophos, which is the contaminant compound in the“gyoza” dumplings and much more highly toxic than theparent compound. Parathion, which is one of the poisonsspecified under the Toxic and Deleterious Control Law ofJapan, also necessitates patient treatment, although rarely,irrespective of the strict control in its distribution and hand-ling. Recently, Koyama and colleagues (2006) reportedacute poisonings from OPs and fenitrothion toxicokineticsin Japanese patients; these are described in a later part ofthis chapter.

32.2.3 Intentional OP Poisoning

In Japan, data on deaths from intentional drug and chemicalpoisonings, mainly suicides, have been gathered by the pre-fectural Police Research Institute, and the data have beenreported to the National Institute of Police Science eachyear. Of the drug- and chemical-induced deaths (�5000every year), cases due to pesticide poisonings amounted to�500. Deaths from herbicide paraquat poisonings havebeen most common in Japan over the last 15 years or more.Over a thousand people commit suicide with paraquat everyyear. Thereafter, mixed preparations of paraquat and diquatwith emetics were distributed as a herbicide, leading to adecrease in the number of deaths, but several hundredpeople have died with this herbicide. Table 32.2 presentsfatal cases of OP poisoning in the period 2002–2006.Unfortunately, the data did not include OP-intoxicatedcases occurring in the Tokyo Metropolitan area. Therefore,OP-poisoned numbers may be greater than those shown inthe table. Of the OP insecticides, the number of fenitrothionpoisonings is greater than the others each year. DDVP,malathion, and other low toxicity OP insecticides are alsoused for committing suicide. Fenitrothion, which has the

property of selective toxicity, has been developed by theJapanese company Sumitomo Chemicals Ltd., and has beenused widely in various fields, so it may therefore be easy toaccess for such a purpose.

32.2.4 Toxicokinetic Data on Fenitrothion(MEP) Poisonings

Intentional fenitrothion poisonings occur every year, and withconsiderable numbers, as shown in Table 32.2. Koyamaand colleagues (2006) carried out a toxicokinetic study onfenitrothion poisonings based on the data obtained fromemergency hospitals. Although the number of patients inthis study may not be enough to analyze OP poisoningsstatistically, they reported some interesting findings in thesefenitrothione-intoxicated patients.

Table 32.3 shows serum ChE activity and toxic syndromesin patients. Data from nine female and six male patients ofvariable ages were analyzed for toxicokinetics, including

TABLE 32.1 OP-related Inquiries Received at JPIC in the Period 1998–2007

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Fenitrothion (MEP) 89 84 98 115 102 96 73 64 56 57Malathon 36 47 36 38 22 26 19 23 27 24Acephate 34 28 30 36 24 35 16 26 23 17Trichlorfon (DEP) 23 26 22 18 21 14 22 19 11 20Dichlorvos (DDVP) 16 23 24 7 14 24 13 21 12 8Fenitrothion (MEP)

Malathion15 9 24 17 8 5 3 3 9 5

Isoxathion 14 9 14 18 5 8 5 10 9 8Others 82 118 95 94 85 73 70 71 39 50

OPS (total) 309 344 343 343 281 281 221 237 186 189

OP, Organophosphate.

TABLE 32.2 Incidents of Intoxications with OP Insecticidesa

2002 2003 2004 2005 2006

MEP (fenitrothion) 46 34 31 27 36Malathion 38 29 31 24 26DDVP 34 33 22 17 20DEP (trichlorfon) 14 14 4 10 11EPN 7 10 2 6 4Acephate 3 2 5 4 4Parathion 3 3 4 3 4Diazinon 1 4 1 3 1Othersb 101 90 116 91 102

Total 247 219 216 185 208

aAdapted and modified from the annual case reports of drug and toxic poison-ing in Japan published by the National Research Institute of Police Science:data do not include Tokyo Metropolitan area.b“Others” indicate that the examined cadaver samples contained two or moreinsecticides including the compounds described above and other insecticides,and also OP herbicides.

32.2 TOXICOLOGICAL DATA ON AChE POISONING IN JAPAN 459

time before admission, pupil diameters, suspected doses,serum ChE activities and MEP concentrations, ICU stays,and toxic syndromes. In these cases, miosis was found inonly 60% of patients.

Figure 32.1 presents the toxicokinetics of fenitrothion poi-sonings in 15 patients. Most clinical manifestations of acutepoisonings described in Table 32.3 were resolved withindays after admission to hospital with standard treatment,and even in severe cases fortunately survived with intensivecare including dialysis, atropine, and 2-PAM antidote admin-istrations. Patients with higher serum fenitrothion concen-trations tended to unconsciousness.

TABLE 32.3 Serum Fenitrothion (MEP) Concentrations and Toxic Syndromes in Diagnosed Patients at the Time of Admission

No.Age

(years) SexSuspectedDose (g)

Time BeforeAdmission (h)

Pupil(mm)

ChE(U/L)

SerumMEP Conc.(mg/mL)

TrachealIntubation

(days)ICU Stay

(days) Toxic Symptoms

1 79 F 15 1 3 18 Low — Nausea2 50 F 50 11 4 19 0.08 3 Vomiting, HR403 24 F 25 6 3.5 99 0.10 — Vomiting4 33 M 15 1 3 141 0.11 3 Normal5 28 F 5 1 3 54 0.14 2 Normal6 57 F 50 1 1.5 67 0.44 4 30/JCS7 60 M 50 0.5 3 17 2.00 — Salivation8 73 M 35 1 2.5 15 2.60 3 Vomiting, 3/JCS9 47 F 35 1 2 — 2.71 7 10/JCS, nausea

10 39 M 37.5 7 4 — 3.20 3 Vomiting, HR3511 76 F 10 1 2.5 18 3.32 4 Hr40, 1/JCS12 62 F 50 2 3.5 35 4.70 4 Diarrhea, BP58/20,

HR58, 200/JCS13 65 M 25 0.5 3.5 83 5.83 2 3 Miosis, 10/JCS14 35 F 50 1 1 83 7.67 4 11 100/JCS, salivation15 51 M 50? 12 4 19 9.73 7 31 100/JCS

HR, heart rate (bpm); BP, blood pressure (mmHg); JCS, Japan coma scale.

0 100 200 300 400Low

0.10

1.00

10.00

Time after ingestion (h)

ME

P co

ncen

trat

ion

in s

erum

(µg

/mL

)

¨15

¨14

¨9

¨513ƨ4

≠3

6Æ11Æ

Figure 32.1 Toxicokinetics of fenitrothion elimination fromserum in intoxicated patients (cases 1–15). Numbers against datarepresent case numbers.

0 10 20 30 40 50 60

Time after ingestion (h)

DHP

Ø

t½ = 9.9 ± 7.7 h (mean ± s.d.)Range 3.2–18.6 h

ME

P co

ncen

trat

ion

in s

erum

(µg

/mL

)

Ø

¨4 ≠3

¨2

¨9

13Æ1

¨5

¨11

10Ø

12Ø

8Æ7Æ

≠6

10.00

1.00

0.10

Low

(a)

(b)

ME

P co

ncen

trat

ion

in s

erum

(µg

/mL

)

Time after ingestion (h)

DHPƨDHP

DHPÆ

≠DHP

14Æ

¨15

¨t1/2 = 6.7 h (a phase + b phase + DHP)

¨t1/2 = 52 h (b phase)

t1/2 = 35 hÆ(b phase)

0Low

0.10

1.00

10.00

100 200 300 400

TI (Case 15)

TI (Case 14)

t1/2 = 5.3 h(a phase + b phase + DHP)

≠

Figure 32.2 (a) Toxicokinetics of fenitrothion with one elimina-tion phase (cases 1–13); (b) toxicokinetics of fenitrothion with twoelimination phases (cases 14, 15). Numbers against data representcase numbers. DHP, direct hemoperfusion; TI, tracheal intubation.

460 EPIDEMIOLOGICAL STUDIES OF ANTICHOLINESTERASE PESTICIDE POISONING IN JAPAN

As shown in Figure 32.1, the toxicokinetics data of the15 patients divided into two toxic kinetics. Namely, in patientsshowing serum fenitrothion levels lower than 7 mg/mL, t1/2

was about 10 h and fenitrothion was not detected �1–2days after intoxication (Fig. 32.2a). Patients with serumlevels higher than 7 mg/mL appeared b phase (�35–53 h)with prolonged elimination from the serum over �10 days(Fig. 32.2b). As shown in Table 32.3, serum ChE activitiesdecreased to a lowest point within �1–2 days after intoxi-cation. Thereafter, ChE activity returned to control levelsdepending on the magnitudes of the early decrease. Insome intoxication cases, more than 10 days were necessaryto return to normal levels. Therefore, it will be necessary todetermine serum ChE activity at several time points evenafter admission to hospital when symptoms or other signsseem to be severe in an intoxicated patient. From thesedata, the decreases in ChE activities did not occur in parallelwith serum fenitrothion concentrations. It is worth notingthat patients with the higher serum fenitrothion levels of�7 mg/mL tended to present with an “intermediate syn-drome” such as tachycardia, hypertension, and other manifes-tations within 1 week of admission to hospital.

32.2.5 CM Insecticide Poisonings

32.2.5.1 General features Many CM insecticides havebeen used in various fields in Japan. Although full data oncases of accidental CM poisonings in Japan are not as avail-able as for OPs, data are accumulating gradually. Many inten-tional CM poisonings have been reported every year, withmethomyl used most often and causing most deaths.

32.2.5.2 Annual Data on CM Poisonings Table 32.4presents annual data on CM poisonings from 1998 to 2007in Japan. Reports of methomyl poisonings to JPIChave been recorded every year. However, the numbers, at�29–57 cases, are lower than those for OP poisonings(Table 32.2). The lower number of reports for CM (insecti-cides) poisonings may indicate that access to this kind ofinsecticide is rather difficult and/or its distribution is not sowide when compared with OP insecticides.

32.2.5.3 Intentional CM Poisoning Table 32.5 showsannual data regarding CM poisonings from 2002 to 2006 inJapan. As mentioned above in relation to OP poisonings,these data do not include CM poisoning cases in TokyoMetropolitan. There were �73–99 fatal cases, and methomylwas the most common CM used every year. Methomyl hasthe highest acute toxicity of the CM insecticides commer-cially available in Japan. Methomyl poisonings are the mostreported to JPIC by general citizens and emergency hospitals(Table 32.4).

32.3 CONCLUSIONS

Many OPs and CMs are used in agriculture and for domesticpurposes in Japan. OP and CM pesticides that are approvedand registered by the regulatory authority are generally lowin toxicity, unlike those in developing countries. However,OPs and CMs cause about 250 deaths per year in Japan.The numbers of fatalities arising from OP and CM intoxi-cations are lower compared to those caused by paraquatpoisonings. Therefore, OP- and CM-induced intoxicationsare not so serious when compared to paraquat poisoning inJapan. Although effective antidotes are available for OPsand CMs, a procedure to improve clinical outcomes remainsto be determined and developed.

REFERENCES

Koyama K, Suzuki R, Kikuno T, Kaziwara H, and Shinba T (2006)Serum fenitrothion concentration and toxic symptom in acuteintoxication patients. Jpn J. Toxicol. 19: 41–47.

Matsuda Y, Nagao M, Takatori T, Niijima H, Nakajima M, Iwase H,Kobayashi M, and Iwadate K (1998) Detection of the sarin

TABLE 32.4 CM-reports Received at JPIC in 1998–2007

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Methomyl 43 34 39 31 34 34 23 24 29 17Other 14 9 9 9 13 8 6 7 3 14

CMs (total) 57 43 48 40 47 42 29 31 32 31

TABLE 32.5 Incidents of Intoxications with CMsa

2002 2003 2004 2005 2006

Methomyl 77 93 85 62 80Caltap 3 2 5NAC, DCPA 11 6 1 1Othersb 2 4 5

Total 94 99 92 73 80

aAdapted and modified from the annual case reports of drug and toxic poison-ing in Japan published by the National Research Institute of Police Science:data do not include Tokyo Metropolitan area.b“Others” indicate that the examined cadaver samples contained two insecti-cides including the compounds described above and other CMs.

REFERENCES 461

hydrolysis product in formalin-fixed brain tissues of victims ofthe Tokyo subway terrorist attack. Toxicol. Appl. Pharmacol.150: 310–320.

Okumura T, Takasu N, Ishimatsu S, Miyanoki S, Mitsuhashi A,Kumada K, Tanaka K, and Hinohara S (1996) Reports on 640victims of the Tokyo subway sarin attack. Ann. Emerg. Med.28: 129–135.

Okumura T, Hisaoka T, Yamada A, Naito T, Isonuma H, OkumuraS, Miura K, Sakurada M, Maekawa H, Ishimatsu S, Takasu N,

and Suzuki K (2005) The Tokyo subway sarin attack—lessonslearned. Toxicol. Appl. Pharmacol. 207 (Suppl. 2): 471–476.

Nagao M, Takatori T, Matsuda Y, Nakajima M, Iwase H, andIwadate K (1997) Definitive evidence for the acute sarin poison-ing diagnosis in the Tokyo subway. Toxicol. Appl. Pharmacol.144: 198–203.

Sumi Y, Oode Y, and Tanaka H (2008). Chinese dumpling scare hitsJapan—a case of methamidophos food poisoning. J. Toxicol. Sci.33: 485–486.

462 EPIDEMIOLOGICAL STUDIES OF ANTICHOLINESTERASE PESTICIDE POISONING IN JAPAN