Increased Urinary Excretion of Xanthurenic Acid as a Biomarker of

Chapter 7

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Increased Urinary Excretion of Xanthurenic Acid as a Biomarker of Exposure to Organophosphorus Insecticides Josef Seifert Department of Environmental Biochemistry, University of Hawaii, Honolulu, HI 96822

This paper considers increased urinaryexcretion of xanthurenic acid caused by organophosphorous insecticides (OPI's) as a possible biomarker of exposure to OPI's. Urinary xanthurenic acid is assayed by highperformance l i q u i d chromatography. Pyrimidinyl phosphorothioates and crotonamide phosphates are the most potent OPI's, increasing xanthurenic acid urinary excretion up to sixfold. Elevation of xanthurenic acid occurs within 6 hr of OPI administration and persists for 48 hr following a single OPI dose. Age, dietary regimen and several drugs related to OPI action or L-tryptophan metabolism do not affect OPI-increased xanthurenic acid urinary excretion, while i t s onset and extent is gender-dependent. The magnitude of a single OPI dose for increasing xanthurenic acid excretion (5 mg of diazinon/kg) indicates importance of this phenomenon for occupational and accidental poisonings.

Organophosphorous i n s e c t i c i d e s (OPI's) a r e the major type of i n s e c t i c i d e s used i n the U.S.A. and worldwide. The mechanism of t h e i r a c t i o n i s based on i n h i b i t i o n o f acetylcholinesterase with toxic consequences due t o accumulation o f a c e t y l c h o l i n e (1) . The i n h i b i t i o n o f a c e t y l c h o l i n e s t e r a s e i s r o u t i n e l y used as a b i o c h e m i c a l marker o f exposure t o OPI's ( 2 ) . Some t o x i c e f f e c t s of OPI's a r e caused by mechanisms other than i n h i b i t i o n o f a c e t y l c h o l i n e s t e r a s e (3-5) and have the p o t e n t i a l f o r use as complementary biomarkers o f exposure t o OPI's. Recently, we discovered that p y r i m i d i n y l phosphorothioate and crotonamide phosphate

0097"6156/96/0643-0094$15.00/0 © 1996 American Chemical Society In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

7. SEIFERT

Xanthurenic Acid and OP Insecticides

95

i n s e c t i c i d e s i n c r e a s e d u r i n a r y e x c r e t i o n o f xanthurenic a c i d i n mice (6,7). I n h i b i t i o n o f kynurenine formamidase ( a r y l formylamine hydrolase, EC 3.5.1.9), an enzyme i n the L-kynurenine pathway o f L-tryptophan metabolism (Figure 1), was proposed as the t r i g g e r f o r changes i n Ltryptophan metabolism. T h i s paper summarizes our f i n d i n g s on t h i s novel phenomenon and p r o v i d e s the i n f o r m a t i o n needed f o r i t s use as a biomarker o f exposure t o OPI's.


Experimental Animals and T h e i r Treatment. Mice (Swiss Webster; 8-12 week-old males except f o r the study o f the e f f e c t s o f age and gender) were used throughout t h i s p r o j e c t . OPI's and other t e s t e d drugs were administered i n t r a p e r i t o n e a l l y i n t o mice i n m e t h o x y t r i g l y c o l f o r the nonpolar compounds and i n i s o t o n i c s a l i n e f o r the p o l a r ones. C h o l i n e r g i c t o x i c i t y o f the potent a c e t y l c h o l i n e s t e r a s e i n h i b i t o r s (£.g./ parathion) was a l l e v i a t e d by a t r o p i n e . D i a z i n o n [0,0-diethy 1 0- (2 -isopropyl-4-methyl-6 - p y r i m i d i n y l ) phosphorothioate] was used as a model OPI s i n c e i t i s both a potent kynurenine formamidase i n h i b i t o r and an inducer o f xanthurenic a c i d u r i n a r y e x c r e t i o n (6-8). Assay of Liver Kynurenine Formamidase Activity. Kynurenine formamidase activity was assayed s p e c t r o p h o t o m e t r i c a l l y with N-formyl-L-kynurenine as a s u b s t r a t e (9) . The enzyme was prepared from l i v e r s o f c o n t r o l and/or OPI-treated mice by homogenization and subsequent centrifugations a t 10,000 and 150,000xg. Increase i n absorbancy due t o e n z y m a t i c a l l y r e l e a s e d Lkynurenine was monitored at 365 nm. Assay o f U r i n a r y Xanthurenic A c i d . U r i n e c o l l e c t e d from 2-4 mice was preserved with the a d d i t i o n o f h y d r o c h l o r i c a c i d . A c i d i f i e d u r i n e can be s t o r e d a t -5°C without s u b s t a n t i a l changes i n xanthurenic a c i d c o n c e n t r a t i o n f o r several days. Urine samples were cleared by c e n t r i f u g a t i o n and evaporated t o dryness on a vacuum r o t a r y evaporator a t 40°C. The r e s i d u e was d i s s o l v e d i n 10 mM sodium hydroxide i n 80% methanol and the p r e c i p i t a t e formed a f t e r 2 h r - s t a n d i n g a t 0°C was removed by c e n t r i f u g a t i o n . The r e s u l t i n g supernatant was f i l t e r e d through a m i c r o f i l t e r p r i o r t o high-performance liquid chromatography (HPLC) . The sample was separated on a reversed-phase C i g column with 1 mM monosodium phosphate b u f f e r , pH 2.3 as a mobile phase. Xanthurenic a c i d was d e t e c t e d a t 254 nm (Figure 2). Results OPI E f f e c t s on L i v e r Kynurenine Formamidase. Liver kynurenine formamidase was almost completely i n h i b i t e d by a s i n g l e dose o f d i a z i n o n w i t h i n 30 min a f t e r i t s administration (Figure 3) . In vivo spontaneous r e a c t i v a t i o n o f the i n h i b i t e d enzyme was slow. Full

In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

96

BIOMARKERS FOR AGROCHEMICALS AND TOXIC SUBSTANCES

CH CHCOOH NH 2

2

H L-TRYPTOPHAN

^^^&-CH CHCOOH Downloaded by STANFORD UNIV GREEN LIBR on September 23, 2012 | http://pubs.acs.org Publication Date: September 27, 1996 | doi: 10.1021/bk-1996-0643.ch007

2

I X *"»

N-FORMYL-L-KYNURENINE

(

Kynurenine formamidase O

H

( X ** X^NH

2

L-KYNURENINE

I

6-CH CHCOOH NH 2

2

NH

COOH

2

3-HYDROXY-L-KYNURENINE

XANTHURENIC ACID

1

NAD

a

C0

2

K

COOH

PICOLINIC ACID F i g u r e 1. The L-Kynurenine Pathway o f L-Tryptophan Metabolism. Decreased formation of l i v e r L-kynurenine due to i n h i b i t i o n of kynurenine formamidase by OPI's i s compensated by i n c r e a s e i n L-kynurenine plasma p o o l (6, 7) .



7. SEIFERT


control

DIA

F i g u r e 2. Assay o f U r i n a r y Xanthurenic A c i d by H P L C . Mice were administered 20 mg of d i a z i n o n / k g . Xanthurenic a c i d i n the u r i n e c o l l e c t e d f o r 20 hr was separated on a reversed-phase CIQ column (4.6x30 mm, 3 urn CR Pecosphere, Perkin-Elmer) and d e t e c t e d a t 254 nm. DIA - d i a z i n o n - t r e a t e d . Adapted from r e f . 5.


97


98


TIME (days) F i g u r e 3. L i v e r Kynurenine Formamidase i n Mice T r e a t e d w i t h Diazinon. Mice were administered a s i n g l e (1 o r 10 mg/kg) o r m u l t i p l e doses of d i a z i n o n (1 mg/kg on days 0, 3, 6 and 9 ) . L i v e r kynurenine formamidase was assayed s p e c t r o p h o t o m e t r i c a l l y w i t h N-formyl-L-kynurenine. Values a r e the means o f two assays with the range i n d i c a t e d by b a r s . A c t i v i t y a t time 0 - 424±75 (N=14) nmol. (mg p r o t e i n ) .min" . Adapted from r e f . 6. - 1


1

7.

SEIFERT

99



recovery of the enzyme a c t i v i t y i n mice t r e a t e d w i t h 10 mg of diazinon/kg has not occured even a f t e r 10 days. A single dose of pirimiphos-ethyl, diazinon, pirimiphos-methyl, etrimfos, dicrotophos and monocrotophos, cited i n descending order of their potency, i n h i b i t e d l i v e r kynurenine formamidase by more than 80% (Table I ) . M a j o r i t y of OPI's were i n t e r m e d i a t e kynurenine formamidase i n h i b i t o r s that reduced the enzyme a c t i v i t y by 28-78%. Ronnel, t r i c h l o r f o n and leptophos d i d not i n h i b i t l i v e r kynurenine formamidase.

Table I . Potency o f Organophosphorous I n s e c t i c i d e s t o I n h i b i t Mice L i v e r Kynurenine Formamidase and Increase Xanthurenic A c i d U r i n a r y E x c r e t i o n OPI

Kynurenine formamidase inhibition, %

a

Xanthurenic a c i d , % of c o n t r o l (N)

Potent i n h i b i t o r s / i n c r e a s e d xanthurenic a c i d Pirimiphos-ethyl Diazinon Pirimiphos-methyl Etrimfos Dicrotophos Monocrotophos

576±195 (2) 445±213 (14) 387 184 294±22 (2) 183±22 (3)

99 97*3 95 89 89 86

Intermediate infrjbjLtQr-s/nQ ingrgfrgq j Diazoxon Parathion-methy1 Profenophos Parathion-ethyl Malathion Chlorpyriphos-methyl

78 76 73 69 58 28

Q

xanthurenic a c i d 50±11 (2) 104 69 72 95±2 (2) 56

N o n i n h i b i t o r s / n o i n c r e a s e i n xanthurenic a c i d Leptophos Trichlorfon Ronnel

11 8 1

100±12 (2) 72 113

L i v e r kynurenine formamidase was assayed 12-18 hr a f t e r a d m i n i s t e r i n g 20 mg/kg of a t e s t compound (10 mg of diazoxon/kg) to mice. Xanthurenic a c i d was determined i n u r i n e c o l l e c t e d f o r 15-24 hr. Values are means±S.D. f o r U independent experiments (a range f o r two experiments). Basal values f o r l i v e r kynurenine formamidase a c t i v i t y and u r i n a r y xanthurenic a c i d were 424±75 (N=14) nmol Lkynurenine.min" . (mg protein)" and 737 ± 256 (N=27) nmol.(24 h r ) , r e s p e c t i v e l y . A s i m i l a r values were obtained when xanthurenic a c i d was expressed i n nmol.(ml of u r i n e ) " . Adapted from r e f . 7. 1

1

_ 1

a

1



100


1

2 3 4 TIME (days)

5

F i g u r e 4. U r i n a r y E x c r e t i o n o f Xanthurenic A c i d i n Control and Diazinon-Treated Mice. Mice were administered 40 mg of diazinon/kg. Urinary xanthurenic acid was assayed by HPLC. Standard d e v i a t i o n s i n d i c a t e d by bars were c a l c u l a t e d from 3-4 independent experiments. The i n s e t shows xanthurenic a c i d e x c r e t i o n f o r the i n i t i a l p e r i o d . Key: (o) c o n t r o l ; (e) d i a z i n o n - t r e a t e d . Reproduced from r e f . 6 w i t h k i n d permission from E l s e v i e r Science L t d , The Boulevard, Langford Lane, K i d l i n g t o n 0X5 1GB, UK.



7.

SEIFERT

101


OPI E f f e c t s on Xanthurenic A c i d U r i n a r y Excretion. Xanthurenic a c i d increased two to s i x - f o l d i n the u r i n e of mice t r e a t e d with a s i n g l e dose (20 mg OPI/kg) of the s i x most potent l i v e r kynurenine formamidase i n h i b i t o r s (Table I) . The highest increase was obtained with p i r i m i p h o s - e t h y l and d i a z i n o n . E l e v a t e d xanthurenic a c i d e x c r e t i o n was detected as e a r l y as s i x hours a f t e r a d i a z i n o n treatment, reaching a maximum i n 24- and 48u r i n e c o l l e c t i o n s (Figure 4) . The dose-dependence study e s t a b l i s h e d that 5 mg of diazinon/kg was a t h r e s h o l d f o r i n c r e a s i n g xanthurenic a c i d u r i n a r y e x c r e t i o n (Table I I ) . Diazinon at doses higher than 10 mg/kg d i d not f u r t h e r i n c r e a s e xanthurenic a c i d .

Table I I . E f f e c t o f V a r y i n g D i a z i n o n Doses on Xanthurenic A c i d U r i n a r y E x c r e t i o n Diazinon, mg.kg1

Xanthurenic a c i d , nmol.(24 h r ) (N)

0 1 5 10 20 40 80 Urine was c o l l e c t e d f o r 24 a d m i n i s t r a t i o n . Values are independent experiments (a experiments). Adapted from

1

737±256 855±199 1754 3913*479 2417 3670±752 3169

(27) (3) (2) (13)

hr a f t e r d i a z i n o n means!S.D. f o r & range f o r two r e f . 6.

E f f e c t s of Endogenous and Exogenous F a c t o r s on D i a z i n o n Induced Increase i n Xanthurenic A c i d U r i n a r y E x c r e t i o n . Increases i n xanthurenic a c i d u r i n a r y e x c r e t i o n , caused by d i a z i n o n , were not a l t e r e d by the mice age (Table I I I ) . The i n c r e a s e was s i m i l a r i n p r e p u b e r t a l , a d u l t and "aging" mice. The d i e t a r y regimen was not c r i t i c a l f o r elevation of u r i n a r y xanthurenic acid (Table III) . Diazinon-induced increases were p r o p o r t i o n a l to the changes i n b a s a l l e v e l s of xanthurenic a c i d that were caused by d i e t a r y d e p r i v a t i o n . Gender was the o n l y f a c t o r that a f f e c t e d xanthurenic a c i d u r i n a r y e x c r e t i o n a f t e r diazinon treatment (Table III). The increase in xanthurenic a c i d occured e a r l i e r and at higher r a t e s i n female mice, a maximal 1 1 - f o l d i n c r e a s e compared to 4 - f o l d i n c r e a s e i n males, r e l a t i v e to c o n t r o l l e v e l s . The increase i n xanthurenic a c i d u r i n a r y e x c r e t i o n caused by d i a z i n o n was not a f f e c t e d by any of the t e s t e d drugs (Table I I I ) . Replacement of s u l f u r by oxygen (i.£., P=S i n d i a z i n o n to P=0 i n diazoxon) reduced i n v i v o l i v e r



102


f 800 3 J

J600 o

diazinon administration

S e

^ 400

**' basal excretion 0

-L

10 20 TIME (days)

F i g u r e 5. E f f e c t o f a M u l t i p l e A d m i n i s t r a t i o n o f D i a z i n o n on U r i n a r y E x c r e t i o n o f Xanthurenic A c i d . Mice were administered 20 mg o f diazinon/kg a t i n d i c a t e d times. Urine was c o l l e c t e d f o r 24 h r and xanthurenic a c i d assayed by HPLC.


7.

SEIFERT


103

kynurenine formamidase i n h i b i t i o n and a b o l i s h e d the OPIenhanced xanthurenic a c i d u r i n a r y e x c r e t i o n (Table I ) . Table I I I . E f f e c t s of Age, Gender, D i e t and Drugs on Diazinon-Increased U r i n a r y E x c r e t i o n of Xanthurenic A c i d


Factor

Xanthurenic a c i d % of c o n t r o l

Aae (weeks) 3-4 8-12 55-60

513±85 445±213 411±135

Gender - u r i n e c o l l e c t e d a f t e r d i a z i n o n administration for: $-13 [hr] 2n£ 0=1 Male 385 255 137 200 604±154 1104±73 Female Diet Normal Restricted

445±213 402±39

a

Drugs Atropine Cycloheximide Pyridine-2-aldoxime Phenylmethylsulfonyl fluride

519 464132 4 9 614 8 408

Urine was c o l l e c t e d f o r 24 hr except f o r the gender examination. Values are meansirange f o r two independent experiments (meaniSD, N=27, f o r 8-12 week-old mice maintained on a normal d i e t ) . Adapted from r e f . 7. a

L i v e r Kynurenine Formamidase and U r i n a r y Xanthurenic A c i d i n M u l t i p l e and Prolonged Exposures o f Mice t o D i a z i n o n . Multiple administration of diazinon maintained the i n h i b i t i o n of l i v e r kynurenine formamidase f o r the e n t i r e p e r i o d of treatment (Figure 3) . U r i n a r y xanthurenic a c i d e x c r e t i o n increased r a p i d l y f o l l o w i n g mice treatment and returned to b a s a l values i n 24 to 48 hours a f t e r each d i a z i n o n a d m i n i s t r a t i o n (Figure 5). Discussion P y r i m i d i n y l phosphorothioates and crotonamide phosphates are the two groups of OPI that increased urinary xanthurenic a c i d e x c r e t i o n with a s i n g l e dose of 20 mg/kg. U r i n a r y xanthurenic a c i d i s e l e v a t e d only when i n h i b i t i o n of l i v e r kynurenine formamidase exceeds 80%.



104


Since the majority of OPI's inhibit kynurenine formamidase to some extent [Table I, r e f e r e n c e (8)], it i s l i k e l y that even l e s s potent OPI i n h i b i t o r s , a t higher doses, have the potential to inhibit kynurenine formamidase to the c r i t i c a l l e v e l with the consequences of i n c r e a s e d u r i n a r y e x c r e t i o n of xanthurenic a c i d . Changes i n u r i n a r y xanthurenic a c i d caused by a s i n g l e dose of OPI can be monitored from s e v e r a l hours up to two days a f t e r exposure to OPI. Response to a m u l t i p l e d i a z i n o n a d m i n i s t r a t i o n follows the p a t t e r n of a s i n g l e dose and allows one to monitor exposure to the OPI f o r the e n t i r e p e r i o d of treatment. The magnitude of the d i a z i n o n dose that i s needed f o r e l e v a t i o n of xanthurenic a c i d e x c r e t i o n implies the importance of t h i s phenomenon i n a c c i d e n t a l and occupational poisonings. The L-tryptophan metabolism and its biochemical components can be a l t e r e d by numerous endogenous and exogenous f a c t o r s (10). Gender was the only observed factor that affected OPI-induced enhancement of xanthurenic a c i d u r i n a r y e x c r e t i o n and t h e r e f o r e needs to be considered when u s i n g u r i n a r y xanthurenic a c i d f o r the assessment of OPI exposure. Abolishment of the d i a z i n o n e f f e c t on xanthurenic a c i d e x c r e t i o n a f t e r a replacement of s u l f u r by oxygen i s probably due to r e d u c t i o n of diazoxon metabolic stability and consequently lower i n h i b i t i o n of kynurenine formamidase [Table I, r e f e r e n c e (7)] . Increase i n xanthurenic a c i d u r i n a r y e x c r e t i o n i s a unique consequence of OPI i n t e r f e r e n c e with the Ltryptophan metabolism. However, there are other conditions and environmental factors under which xanthurenic a c i d u r i n a r y e x c r e t i o n i s elevated. For instance, u r i n a r y e x c r e t i o n of xanthurenic a c i d i s r a i s e d i n s e v e r a l mental d i s o r d e r s (11) and during pregnancy (12) i n humans, and i n poisoning by carbon d i s u l f i d e i n r a t s (13). Thus, when the medical h i s t o r y of s u b j e c t s t e s t e d i s unknown, use of u r i n a r y xanthurenic a c i d concentrations will also require measurement of c h o l i n e s t e r a s e a c t i v i t i e s f o r a comprehensive assessment of OPI exposures. Two lines of biochemical evidence support e x t r a p o l a t i o n of OPI-increased xanthurenic a c i d u r i n a r y e x c r e t i o n from mice to humans. F i r s t , the mouse Lkynurenine pathway of the L-tryptophan metabolism i s s i m i l a r to that i n man (14) . Second, the s u s c e p t i b i l i t y of l i v e r kynurenine formamidases from v a r i o u s mammals and avians to OPI's i s an e s t a b l i s h e d phenomenon (15). I t i s reasonable to speculate that the b a s i c assumption f o r OPI-increased xanthurenic a c i d e x c r e t i o n can be met, ±..£. that the human l i v e r enzyme i s i n h i b i t e d by OPI to the c r i t i c a l l e v e l . Use of u r i n a r y xanthurenic a c i d as a biomarker of exposure to OPI's needs to be v a l i d a t e d by examining workers exposed to higher amounts of OPI's, such as p e s t i c i d e formulators and applicators, farm workers and survivors of accidental or suicidal poisonings.


7. SEIFERT


105

Acknowledgments T h i s work was supported i n p a r t by the USDA S p e c i a l Grants Program f o r T r o p i c a l and S u b t r o p i c a l A g r i c u l t u r e Research. I t i s c o n t r i b u t e d as J o u r n a l S e r i e s no. 4104 by the Hawaiian I n s t i t u t e of T r o p i c a l A g r i c u l t u r e and Human Resources. I thank Drs. B.M. Brennan, R. Dashwood and J.W. H y l i n f o r t h e i r reading and c r i t i c i s m o f t h i s manuscript.


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Increased Urinary Excretion of Xanthurenic Acid as a Biomarker of

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