Chlorpyrifos metabolites in serum and urine of poisoned persons

Chem.-Biol. Interactions, 87 (1993) 315-322

315

Elsevier Scientific Publishers Ireland Ltd.

CHLORPYRIFOS METABOLITES IN SERUM AND URINE OF POISONED PERSONS

V. D R E V E N K A R a, Z. VASILI(~ a, B. STENGL a, Z. FROBE a A N D V. RUMENJAK b

alnstitute for Medical Research and Occupational Health, University of Zagreb, Ksaverska c. 2, 41000 Zagreb, and bEmergency Medical Centre, Dordiceva 26, 41000 Zagreb (Croatia)

SUMMARY

Concentrations of parent pesticide and corresponding diethylphosphorus metabolites in blood serum and urine were investigated in persons who had ingested a concentrated solution of organophosphorus pesticide chlorpyrifos. The organophosphate poisoning was indicated by a significant depression of blood cholinesterase (EC 3.1.1.7 and EC 3.1.1.8) activities. Blood and spot urine samples were collected daily after admission of the persons to hospital. Chlorpyrifos was detected only in serum samples in a period up to 15 days after posioning. In the same samples chlorpyrifos oxygen analogue, chlorpyrifos oxon, was not detected. The presence of diethylphosphorothioate in all serum and urine samples confirmed that part of chlorpyrifos was hydrolysed before its oxidation. The maximum concentrations of chlorpyrifos in serum and of metabolites in serum and urine were measured on the day of admission. The decrease in concentrations followed the first-order kinetics with the initial rate constant faster and the later one slower. In the faster elimination phase chlorpyrifos was eliminated from serum twice as fast (tl/2 = 1.1-3.3 h) as the total diethylphosphorus metabolites (tl/2 = 2 . 2 - 5 . 5 h). The total urinary diethylphosphorus metabolites in six chlorpyrifos posioned persons were excreted with an average elimination half-time of 6.10 ± 2.25 h (mean + S.D.) in the faster and of 80.35 ± 25.8 h in the slower elimination phase.

Key words: Chlorpyrifos -- Poisoning -- Chlorpyrifos oxon -- Diethylphosphate -- Diethylphosphorothioate -- Kinetic of elimination from blood and urine -Activities of cholinesterase and acetylcholinesterase in blood INTRODUCTION

Chlorpyrifos O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl]phosphorothioate) (Fig. 1) is a widely used, moderately toxic organophosphorus pesticide [1]. Its metabolic Correspondence to: Vlasta Drevenkar, Institute for Medical Research and Occupational Health, University of Zagreb, Ksaverska c. 2, 41000 Zagreb, Croatia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

316

CHLORPYR IFOS

CHLOR PYRIFOS OXON I

el-If

O

~-O-P (OC H2CH3)2

CI " ~ L C l

;.- el

FCI

H

I

s(oj

(c H3C H20)2 P-O (S)DETP

0

ii (CH3CH20)2P-ODEP

Fig. 1. Organophosphorus compounds analysed in serum and urine of persons poisoned by chlorpyrifos.

activation, occurring mainly in the liver, is based on oxidative desulfuration to chlorpyrifos oxon (O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl] phosphate) (Fig. 1), a potent inhibitor of cholinesterase and far more toxic than the parent thion. Biotransformation of both compounds results in the formation of the dephosphorylated metabolite 3,5,6-trichloro-2-pyridinol and of corresponding diethylphosphorus metabolites (Fig. 1). The content of the former metabolite in human blood or urine was used as parameter in estimation of occupational exposure to chlorpyrifos [2] and in pharmacokinetic investigations in human volunteers [3]. The urinary excretion of diethylphosphorus metabolites was shown to be a sensitive indicator of chlorpyrifos absorption and retention in acutely poisoned persons [4]. To provide more data on the fate of chlorpyrifos in humans after a severe intoxication the presence in and elimination from serum and urine of the parent pesticide, its oxygen analogue and their metabolites diethylphosphate (DEP) and diethylphosphorothioate (DETP) were studied. The subjects were three persons who were hospitalized for ingesting a concentrated pesticide solution. The metabolite levels, measured daily during hospitalization, were compared with changes in activities of serum cholinesterase (BChE; EC 3.1.1.8) and erythrocyte acetylcholinesterase (ACHE; EC 3.1.1.7). MATERIALS AND METHODS

Chemicals Standards for gas chromatographic analysis: chlorpyrifos, chlorpyrifos oxon, O,O-diethylphosphate (DEP) barium salt and O,O-diethylphosphorothioate (DETP) potassium salt were all obtained as standard reference compounds from

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each patient to the sum of two first-order kinetic equations Y = Yl" e-klt

+

Y2" e-k2t

The y value is the concentration of compound in serum or urine at time t and the sum of Yl and Yz corresponds to the calculated concentration of metabolites in the 'zero time' samples. The ka and k2 are the elimination rate constants in the faster and the slower elimination phase, respectively. RESULTS AND DISCUSSION

Acute organophosphate poisoning was indicated by a significant depression of BChE and AChE activities measured immediately after admission of the poisoned persons to hospital (Table I). After treatment with oximes a rapid AChE reactivation occurred within 24 h reaching 66 - 87% of the AChE activity measured at the end of hospitalization and taken as the baseline value. The BChE activity did not return to reference values (2.3 - 4.6 kU/1) [8] in any person during 15 days of hospitalization. In persons B and C the BChE activity began to increase gradually three days after poisoning. However, the increase of BChE activity in person A, having the highest 'zero time' concentrations of chlorpyrifos and metabolites, did not begin earlier than 27 days after poisoning, and after 10 more days reached only 33% of the median reference activity (3.45 kU/1) [8]. This delay in return of BChE activity could be related to the fact that traces of chlorpyrifos were detectable in the serum of person A for as long as 15 days after poisoning. This is a much longer period than in the other two patients: four days in person B and one day in person C.

TABLE I THE ACTIVITIES O F S E R U M C H O L I N E S T E R A S E (BChE) A N D E R Y T H R O C Y T E ACETYLC H O L I N E S T E R A S E (ACHE) A N D C O N C E N T R A T I O N S O F C H L O R P Y R I F O S A N D DIETHYLP H O S P H O R U S M E T A B O L I T E S DEP A N D DETP IN S E R U M A N D U R I N E D E T E R M I N E D I M M E D I A T E L Y A F T E R A D M I S S I O N O F POISONED PERSONS TO HOSPITAL (AT ' Z E R O TIME')

Person

Enzyme activity

Concentration in

(%)a

Serum (nmol/ml)

BChE

A B C

II 16

11

Urine (nmol/mg treat.)

AChE

18 28 19

Chlorpyrifos

DEP

DETP

DEP

DETP

7.09 0.42 0.15

10.00 9.92 3.23

16.47 9.32 2.24

594.92 303.40 336.51

423.09 618.60 282.76

aBChE percentages of the median reference value (3.45 kU/l) [ref. 8]: AChE percentages of the activity measured at the end of hospitalization.

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The intact pesticide was detected in all patients only in serum samples. The highest concentrations of chlorpyrifos in serum and of metabolites in serum and urine were determined on the day of patients admission to hospital ('zero time') (Table I). The initial concentrations of chlorpyrifos were 1 - 2 orders of magnitude lower than those of total diethylphosphorus metabolites measured in the same serum samples. The concentrations of total urinary diethylphosphorus metabolites were significantly higher than those in serum suggesting a rapid chlorpyrifos metabolic degradation and metabolite elimination from the body. The concentrations of chlorpyrifos found in the serum of the poisoned persons were in the range of those reported after suicidal intoxication with this pesticide [19]. In person C chlorpyrifos was detectable only in two samples collected at 'zero time' and 12 h later. In persons A and B the chlorpyrifos concentrations showed a biphasic log-linear decrease (Table II). The same time course was exhibited also by the concentrations of total diethylphosphorus metabolites in serum (Table II) and urine (Table III) of all poisoned persons. The semilogarithmic plots of chlorpyrifos concentrations in serum and of metabolites in serum and urine of person A as functions of time are shown as examples in Fig. 2. The duration of the initial faster kl elimination phase was 0.3 - 0.8 days for chlorpyrifos in serum and for total diethylphosphorus metabolites in serum and urine 0.5-1.3 days and 1.0-2.9 days, respectively. In that phase chlorpyrifos was eliminated from serum about twice as fast (t1/2 = 1.1- 3.3 h) as the total diethylphosphorus metabolites (tl/2 = 2.2-5.5 h). The elimination halftime of total diethylphosphorus metabolites from urine was about two times slower than that from serum and ranged from 5.1- 9.5 h. In the slower k2 phase

TABLE II THE E L I M I N A T I O N O F C H L O R P Y R I F O S A N D ITS T O T A L D I E T H Y L P H O S P H O R U S METABOLITES (DEP+DETP) F R O M SERUM: K I N E T I C D A T A F R O M T H E SUM OF T W O FIRSTO R D E R E Q U A T I O N S y = Yl "e-kit + Y2 "e-k2t Compound Person

Time period a (days)

Nb

nc

Kinetic data Yl (nmol/ml)

Chlorpyrifos A 0-15 B 0- 9 C 0 - 14

10 8 7

10 6 2

6.80 0.39

DEP + DETP A 0-15 B 0- 9 C 0-14

10 8 7

10 5 7

23.94 17.79 4.92

aDays after poisoning w h e n the metabolites were m e a s u r e d . bN= n u m b e r of analysed samples. Cn = n u m b e r of positive samples.

Y2

kl (days - 1)

k2

0.29 5.01 0.03 14.79 not calculated

0.17 0.41

2.53 1.45 0.55

0.21 0.76 0.01

3.06 7.66 3.03

320 TABLE III KINETIC DATA FOR URINARY EXCRETION OF TOTAL D I E T H Y L P H O S P H O R U S METABOLITES (DEP + DEPT) OF CHLORPYRIFOS FROM THE SUM OF TWO FIRST-ORDER KINETIC EQUATIONS y = Yl " e - k l t = Y2 " e-k2t Person

A B C

Time period a (days)

Nb

0 - 15 0- 9 0-3

9 7 5

nc

Kinetic data

9 7 5

Yl Y2 (nmol/mg ereat,)

kl (days -1)

k2

862.29 902.00 612.02

3.28 2.41 1.75

0.22 0.39 0.25

155.71 20.00 7.25

aDays after poisoning when the metabolites were measured. bN = number of analysed samples. Cn = number of positive samples.

.J 2.0~

SERUM

o! O z

A

o

URINE 2.0

E o E

A

0

0.0

5

DAYS

10

15

Fig. 2. Time course of chlorpyrifos and total diethylphosphorus metabolites (DEP+DETP) elimination from serum and urine in person A. Plots of the fitted functions. (-I¢) Chlorpyrifos (-A-) DEP+DETP.

321

the minimum elimination half-time for chlorpyrifos in serum was 40.6 h, for total diethylphosphorus metabolites in serum 21.9 h and for total diethylphosphorus metabolites in urine 38.8 h. Taking into account the values obtained in the present and previous studies [4], the total urinary diethylphosphorus metabolites in six chlorpyrifos poisoned persons were excreted with an average elimination half-time of 6 ± 2 h (mean + S.D.) in the initial elimination phase and of 80 ± 25 h in the slower elimination phase. The identity of chlorpyrifos in combined serum samples was unambiguously confirmed by gas chromatographic -- mass spectrometric analysis. In the mass spectrum of the chlorpyrifos extracted from serum the molecular ion was observed at m/z 349 and exhibited the characteristic intensity distribution of a species containing three chlorine atoms. It was followed by intensive ions typical of the chlorpyrifos decomposition: at m/z 314 formed by elimination of one chlorine atom and at m/z 286 and m/z 258 formed by further successive elimination of ethyl groups [10]. By the gas chromatographic -- mass spectrometric analysis is of the same serum extracts the chlorpyrifos oxygen analogue, chlorpyrifos oxon, was not detected. Experiments using mouse liver perfusion have shown that the capacity of mouse hepatic microsomes to hydrolyse chlorpyrifos oxon significantly exceeded their capacity to generate it from chlorpyrifos [11]. However, the perfusion of rat livers with chlorpyrifos resulted in the appearance of oxon in the effluent indicating that oxon could exit from the liver before its detoxification by hepatic A-esterases [12]. Lack of detection of chlorpyrifos oxon in the human serum following a severe intoxication with chlorpyrifos could be a consequence of the fast degradation of the greater part of chlorpyrifos before its transformation to the oxon as well as of the rapid rate of chlorpyrifos oxon hydrolysis relative to the rate of oxon formation which would prevent its passage into the circulation. A conclusion about the primary route of chlorpyrifos biotransformation could hardly be drawn on the basis of the DETP to DEP concentration ratio in the serum and urine of poisoned persons. The total diethylphosphorus metabolites in the serum of person A during the first six days after poisoning consisted of an average of 67% of DETP and 33% of DEP. After this period DEP was not detected. On the contrary, the total metabolites in the serum of person B at 'zero time' consisted of nearly equal parts of DEP and DETP and at a later time the DEP became predominant (57-85%). In person C, DEP was the predominant metabolite (59 - 86%) in all analysed serum samples. DEP was also the predominant metabolite (54 - 82%) in all but one analysed urine sample. The only exception was the 'zero time' urine sample of person B (Table I). The presence of DETP in all analysed serum and urine samples confirmed that in all persons part of chlorpyrifos was hydrolysed before its oxidation. DETP predominance in total diethylphosphorus metabolites would suggest a faster hydrolysis than oxidation of chlorpyrifos. DETP was indeed the predominant metabolite in serum samples of the person having the highest and in the 'zero time' urine sample of the person having the second highest concentrations of total metabolites and intact pesticide. However DEP may be produced not only by hydrolysis of chlorpyrifos oxon but also by oxidation of DETP. Thus, its appearance in most serum and urine

322

samples in concentrations higher than those of DETP could not be used as an indicator of the faster chlorpyrifos oxon formation related to the rate of chlorpyrifos degradation. REFERENCES 1 J. Tafuri and J. Roberts, Organophosphate poisoning, Ann. Emerg. Med., 16 (1987) 193-202. 2 R.A. Fenske and K.P. Elkner, Multi-route exposure assessment and biological monitoring of urban pesticide applicators during structural control treatments with chlorpyrifos, Toxicol. Ind. Health, 6 (1990) 349-371. 3 R.J. Nolan, D.L. Rick, N.L. Freshour and J.H. Saunders, Chlorpyrifos: pharmacokinetics in human volunteers, Toxicol. Appl. Pharmacol., 73 (1984) 8-15. 4 Z. Vasili~:, V. Drevenkar, V. Rumenjak, B. Stengl and Z. Fr6be, Urinary excretion of diethylphosphorus metabolites in persons poisoned by quinalphos or chlorpyrifos, Arch. Environ. Contam. Toxicol., 22 (1992) 351-357. 5 G.L. Ellman, K.D. Courtney, V. Andres and R.M. Featherstone, A new and rapid colorimetric determination of acethylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 6 V. Rumenjak and V. Simeon, Measurement of acetylcholinesterase activity in human red blood cells using an adapted assay kit, Jugoslav. Med. Biokem., 8 (1989) 9-12 (in Croatian). 7 V. Drevenkar, R. Kova~i6 and B. Stengl, Microanalysis of dialkyl phosphorothio- and -dithioates in human blood, European Conference on Analytical Chemistry EUROANALYIS Vll, Book of Abstracts, in: A. Rizzi and G. Sontag (Eds.), Vol. 1, p. A 2.2 P-Fr-6, ASAC in the Austrian Chemical Society, Vienna, 1990. 8 B. Straus, Medicinska biokemija, JUMENA Zagreb (in Croatian), 1988, p. 490. 9 M. Lotti, A. Moretto, R. Zoppellary, R. Dainese, N. Rizzuto and G. Barusco, Inhibition of lymphocytic Neuropathy Target Esterase predicts the development of organophosphate-induced delayed polyneuropathy, Arch. Toxicol., 59 (1986) 176-179. 10 H.J. Stan, B. Abraham, J. Jung, M. Kellert and K. Steinland, Nachweis von Organophosphorinsecticiden durch Gas Chromatographie-Massenspektrometrie, Fresenius Z. Anal. Chem., 287 (1977) 271-285. 11 L.G. Sultatos, M. Shao and S.D. Murphy, The role of hepatic biotransformation in mediating the acute toxicity of the phosphorothionate insecticide chlorpyrifos, Toxicol. Appl. Pharmacol., 73 (1984) 60-68. 12 L.G. Sultatos, Metabolic activation of organophosphorus insecticides chlorpyrifos and fenithrothion by perfused rat liver, Toxicology 68 (1991) 1-9.