Disposition, biotransformation, and detoxication of molinate (Ordram) in whole blood of the common carp (Cyprinus carpio)

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

31, 24-35 (1988)

Disposition, Biotransformation, and Detoxication of Molinate (Ordram) in Whole Blood of the Common Carp (Cyprinus carpio) RONALD S.TJEERDEMA' AND DONALD G.CROSBY Department of Environmental

Toxicology, University of California, Davis. California 95616

Received August 12, 1987; accepted March 3, 1988 Disposition and biotransformation of molinate and its sulfoxide in whole blood of the common carp (Cyprinus carpio) were separately compared in vitro; analysis was by high-pressure liquid chromatography. Accumulation of both compounds by erythrocytes was nearly complete within 4 hr although molinate was absorbed to a greater extent. Molinate was oxidized by erythrocytes to the sulfoxide, and possibly the sulfone, then conjugated with glutathione (GSH) or L-cysteine and cleaved to form the mercapturic acid in both erythrocytes and plasma. Molinate sulfoxide was conjugated similarly; however, its larger initial level may have saturated the GSH conjugation pathway. Cellular debris residues (indicating possible hemoglobin carbamylation) following sulfoxide exposure were substantially greater than after molinate exposure. Molinate does not react with either GSH or amines in aqueous solution. Therefore, conjugation and possible hemoglobin carbamylation occur only after molinate sulfoxidation. Different from cyanate, molinate did not cause significant changes in whole blood oxygen dissociation, pH, or the Bohr shift. Therefore. at observed toxic levels, molinate is effectively detoxified within erythrocytes; it must exert its toxic effect elsewhere. c 1988 Academic Pw. IIW

INTRODUCTION

served (6). Similar effects have been observed in common carp (7). The anemia is The thiocarbamate herbicide molinate ameliorated by N-ethyl-N-benzyldichloro(Ordram) is applied to California rice fields acetamide and N,N-diallyldichloroacetfor control of barnyard grass (Echinochloa amide, which increase hepatic glutathione spp). (1); water discharge into agricultural (GSH)’ levels and mercapturic acid producdrains has resulted in die-offs of primarily tion (8). In mammals, GSH conjugation folcommon carp (Cyprinus carpio) (2-5). At lowing hepatic sulfoxidation represents a 0.32 mg liter-‘, molinate causes hemorthiocarbamate detoxication pathway (9). rhagic anemia in Japanese carp (C. carpio Cyanate, a strong carbamylating agent, var. Yamato Koi) (6). It is characterized by binds to the terminal amino groups of the gill microaneurysm and punctate hemora-chains of mammalian hemoglobin, reducrhage and a 40% decrease in mature erything CO, and proton binding and thus the rocytes and hemoglobin; no erythrocytic cytopathological changes have been ob- Bohr effect by 25% (10); hemoglobin becomes less responsive to plasma proton and CO, levels, and oxygen delivery to active ’ Present address and to whom reprint requests tissues is reduced. As hemoglobin represhould be addressed: Aquatic Toxicology Program, Insents up to 85% of blood buffering capacity stitute of Marine Sciences, University of California, (1 l), that function is also reduced. The reSanta Cruz, CA 95064. ’ Abbreviations used: GSH, glutathione, 4-HM, 4- sult may be erythrocyte lysis and anemia. Molinate sulfoxide and/or sulfone may hydroxymolinate; 4-KM, 4-ketomolinate; HHA, hexamethyleneimine; MSO, molinate sulfoxide; MS02, also carbamylate hemoglobin to cause simmolinate sulfone; CBM, carboxymolinate: MMA, mo- ilar effects; however, both are more polar linate mercapturic acid; MCYS, molinate-r-cysteine; and reactive than molinate. Therefore, moMGSH, molinate glutathione; MBA, molinate benzyllinate may cross erythrocyte membranes amine; CAD, collisionally activated decomposition: prior to being sulfoxidized; products MIKE, mass-analyzed ion kinetic energy; HCT, hematocrit: BCF, bioconcentration factor. formed intracellularly then react with either 24 0048-3575/88 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

DETOXICATION

OF

MOLINATE

hemoglobin, other proteins, or GSH, which is present to maintain hemoglobin in its active state and possibly protect erythrocytes from oxidative damage (12, 13). The objectives of this study were to create an in vitro system for exposing whole blood to volatile chemicals while providing aeration; to compare the uptake and biotransformation of molinate and its sulfoxide by erythrocytes of common carp, and, if GSH conjugation occurs, to demonstrate its saturability; to compare the reactivity of molinate, its sulfoxide, and its sulfone with GSH and benzylamine, a model neuleophile to simulate the terminal amino groups of hemoglobin; and to determine whether molinate and/or its metabolites, like cyanate, can alter oxygen dissociation and reduce the Bohr effect by carp hemoglobin, explaining its toxicity. MATERIALS

AND

METHODS

Chemicals Structures of the metabolically important standards are presented in Fig. 1. [ring-14C]Molinate, S-ethyl hexahydro-[2-14C]azepine-l-carbothioate (10.5 mCi mmol - ‘), reference standard molinate, 4-hydroxymolinate (4-HM), and 4-ketomolinate (4-KM) were provided by Stauffer Chemical Co. (Richmond, CA); [‘4C]molinate was purified to 98% by preparative thin-layer chromatography (TLC; Table l), Hexahydroazepine (hexamethyleneimine, HHA), benzyl isocyanate, ben-

IN

COMMON

CARP

2.5

zylamine, and potassium cyanate (KCNO) were purchased from Aldrich Chemical Co. (Milwaukee, WI), and L-cysteine (L-Cys, free base), reduced GSH, Drabkin’s reagent, and standard hemoglobin were from Sigma Chemical Co. (St. Louis, MO). Molinate sulfoxide (MSO, both 14C, 98%, and unlabeled) was prepared by oxidation of molinate with sodium hypochlorite (5.3%), while molinate sulfone (MSO,) required MS0 and hydrogen peroxide (90%) (14). Two other metabolite standards, carboxymolinate (CBM) and molinate mercapturic acid (MMA) (Table l), were synthesized as previously described (15, 16). Reference standard identities were confirmed by TLC (Table l), high-pressure liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), or solid probe MS (15, 16). Molinate-L-cysteine (S-(hexahydro-lHazepine-1-carbonyl)-L-cysteine, MCYS) was prepared by reacting MSO, with L-Cys in ethanol, triethylamine, and water, while molinate glutathione (S-(hexahydrolti-azepinel-carbonyl)-y-glutamylcysteinylglycine, MGSH) substituted GSH for L-Cys (17, 18). The benzylamine conjugate, molinate benzylamine (S-(hexahydro-lH-azepine-1-carbonyl)benzylamine, MBA), was prepared by reacting HHA with equimolar benzyl isocyanate in benzene (19, 20). The identities of MCYS, MGSH, and MBA were confirmed by fast atom bombardment mass spectrometry (FAB MS) on a ZAB-HS-2F reverse-geometry double-focusing mass spectrometer (VG Analytical, Wythenshawe, UK) using a xenon atom beam. Samples were dissolved in dithiothreitol and dithioerythritol (3: l), and identification was by collisionally activated decomposition (CAD) mass-analyzed ion kinetic energy (MIKE) spectra, using helium as the collision gas, of the following characteristic (M + H)+ions: MCYS, m/z 247 (M+H)+, 230 (C,H,* NCOSC,H,CO,H), 201 (C,H,,NCOSC,H, NH,), 158 (C6H,,NCOS), 126 (C6H,*NC O), 121 (SC,H,(NH,)CO,H), 98 (C6H,,N).

26

TJEERDEMA

AND

TABLE Reference

CROSBY

1

Slandard

TLC R+ Values

TLC Rf values” Component

A

B

C

Molinate 4-Hydroxymolinate (4-HM) 4-Ketomolinate (4-KM) Hexahydroazepine (HHA) Molinate sulfoxide (MSO) Molinate sulfone (MSO,) Carboxymolinate (CBM) Molinate mercapturic acid (MMA) Molinate-L-cysteine (MCYS) Molinate glutathione (MGSH) Molinate benzyiamine (MBA)

0.62 0.33 0.43 0.12 0.17 0.53 0.49 0.41 0.64 0.61 0.35

0.73 0.42 0.53 0.15 0.14 0.69 0.65 0.59 0.67 0.66 0.44

0.73 0.13 0.28 0.09 0.05 0.64 0.53 0.33 0.71 0.68 0.22

D .-__ 0.69 0.61 0.56 0.09 0.31 0.65 0.63 0.57

0.62

E

F

0.67 0.60 0.54 0.12 0.39 0.56 0.63 0.45 0.66

0.71 0.61 0.63 0.19 0.35 0.67 0.65 0.25

-

-

a (A) Acetone-hexane, 1:l; (B) ethyl acetate-formic acid, 3O:l; (C) toluene-ether, 2:3; (D) butanol-acetic acid-water, 6: 1:1; (E) butanolethanol-water, 4: 1:1; (F) ethyl acetate-methanol-pyridine-water, 13:3:3: 1.

MGSH, m/z 433 (M + H)+, 358 (C6H,, NCOSC,H3(CO)NHCOC,,H,(C0,H) NH,), 304 (C,H,,NCOSC,H,(NH)CON HCH,CO,H), 274 (H,NCH(CO,H)C,H, CONHCH(CH,)CONHCH,CO,H), 228 (C6H12NCOSC2H,(NH)CO), 201 (H*NCH (C0,H)C2H,CONHCH(CH2)CO), 172 (CONHCH(CH,)CONHCH,CO,H), 126 (C,H,WOL 98 (C,H,,N). MBA, m/z 233 (M + H)+, 141 (&,H,,N CONH), 126 (C6H,,NCO), 106 (C,H&H, NH), 100 (C,H,,NH,), 98 (C,H,,N), 91 GH,CH,). Animals Thirty-four common carp (C. carpio), average age 2 years (mean fork length, 38.1 t 4.2 cm; mean weight, 1.2 * 0.1 kg), were purchased from Frank W. Maurer, Jr. (Davis, CA) and Harland’s Sunset Acres (Wilton, CA). They were housed in a continuous-flow Fiberglas tank, fed BioDiet Grower (BioProducts Inc., Worrenton, OR), and acclimated for 30 days at 18°C prior to the study. Blood Collection Fish were anesthetized with Finquel (tricaine methanesulfonate, MS-222; Argent Chemical Laboratories, Redmond, WA) until equilibrium loss occurred. Blood samples (7-8 ml) were collected via cardiac

puncture using Monoject lo-ml heparinized vacuum blood collection tubes (Sherwood Medical, St. Louis, MO) and 20-ga needles. Hematocrit (HCT) values and hemoglobin concentrations were measured prior to in vitro chemical exposure using conventional means (21), and blood was kept at 4°C for use within 8 hr; clotting was negligible. Exposure System To prevent chemical volatilization while still providing aeration, whole blood (5 ml) was exposed in IO-ml Warburg flasks sealed with rubber scepta (13 x 18 mm). The sidearm vents were connected by silicone rubber tubing to compressed gas cylinders, and evolved CO, was absorbed by 20% KOH on paper wicks in the center wells (22). Flasks were agitated at 18°C. and samples were drawn using a l-ml Hamilton gas-tight syringe with a 22-ga, 2-in needle. Disposition

and Metabolism

Based on a molinate bioconcentration factor (BCF) in common carp, 30.5 ? 6.6 (16), and the LC& (28 days, 0.21 mg liter-‘) (7), pooled blood was dosed in triplicate with 50 ~1 of either radiolabeled molinate (0.64 2 0.05 mg ml- ‘> or MS0 (0.69 -+ 0.05 mg ml- ‘>, both in methanol, to produce a blood concentration of 34.17 + 2.50 nmol

DETOXICATION

OF

MOLINATE

ml-’ (molinate, 6.40 2 0.47 pg ml-‘; MSO, 6.94 ? 0.51 p,g ml-‘); plasma and saline (0.9%) controls were also run. The flasks were pressurized with air, and OS-ml samples were drawn at intervals of 0, 0.25, 0.5, 1, 2, 4, 8, 16, and 24 hr and placed in Eppendorf centrifuge tubes, quantitated by liquid scintillation counting (LSC) to check i4C recovery, and frozen. To precipitate proteins, 1 ml of methanol was added to each plasma sample (23). Following centrifugation for 20 min at 10,000 rpm and 4°C (24) in a Savant Model HSC1Ok high-speed centrifuge, plasma controls were analyzed by HPLC; saline samples were analyzed directly. Whole blood samples were centrifuged at 1000 rpm and 4°C for 1.5min to separate cells and plasma (24), and the plasma was aspirated, 14C quantitated by LSC, and analyzed by HPLC cochromatography, LSC, and TLC. Erythrocytes were resuspended in saline, gently agitated, and again centrifuged (24); this was repeated three times. To lyse cells and precipitate proteins, 1 ml of methanol was added to each cell pack, and they were rapidly frozen and thawed (four cycles) between acetone/CO, and a 50°C water bath (25). The lysate was centrifuged at 10,000 rpm and 4°C for 20 min to remove cellular debris (24) and analyzed by HPLC, LSC, and TLC. The debris was air-dried for 48 hr weighed, and oxidized with a Packard TriCarb Model B-0306 sample oxidizer. Metabolites were identified from 24-hr samples by HPLC cochromatography with unlabeled reference standards on an Altex programmed HPLC system equipped with an ISCO Model V4 variable wavelength detector (220 nm) (26) and an Eldex Model 1044 fraction collector. An Alltech reversephase Cis column (250 x 4.6 mm i.d., 10 TV) and a 40-min gradient elution (Altex Curve 2) with a mobile phase of water-methanol (initial, 95:5; final, 40:60), 1.0 ml min-‘, were used (Fig. 2). Fractions were collected for later 14C quantitation in Liquistint scintillation cocktail (National Diagnostics, Somerville, NJ) with a Packard

IN

M

COMMON

MMA MGSH

I 0

4-KM

I

I

21

CARP

\4-H

I 20

I 40

I 60

I 80

Time(h) FIG. 2. Chromatogram of a reference standard mixture (see Table 1 for key to abbreviations). Mobile phase: gradient water-methanol (95.3 to 40:60), 40 min, Curve 2, 1.0 ml min-‘. Column, reverse-phase C,,. Detector, uv at 220 nm.

Tri-Carb 2450 scintillation spectrometer: samples were corrected for background, quenching, and efficiency. Amounts as small as 0.3 nmol could be detected accurately and reproducibly. Identities were confirmed by TLC on silica gel 60 F-254 plates (E. Merck, Darmstadt, FRG) in at least three solvent systems (Table 1) (9, 17, 18,27,28). Developed plates were scanned with a Bioscan System 200 imaging scanner, and Rf values were compared to those of standards (Table 1). Silica gel was removed, extracted with methanol, volume reduced to 100 ul, and the metabolites reconfirmed by HPLC and LSC. Disposition differences were statistically analyzed by a one-tail t test (29). Binding

Reactions

Molinate, MSO, and MSO, were separately combined with equimolar amounts (50 Fmol) of GSH or benzylamine in either organic (0.6 ml ethanol and 40 l~,l triethylamine) or aqueous (0.3 ml water, 0.3 ml ethanol, and 40 ~1 triethylamine) solution at 20°C for 18 hr. Parallel controls, containing only molinate, MSO, or MSO,, were run to

28

TJEERDEMA

AND

determine hydrolytic stability, and all reaction mixtures were stored at -5°C until analysis. MGSH was identified by gradient HPLC (as above), while MBA required isocratic conditions (water-methanol, 40:60; 1.O ml min-‘) and comparison with reference standard retention times (Fig. 3); quantities were determined from standard curves (Y, 0.981-0.996). Identities were confirmed by TLC, in at least three solvent systems (Table l), and FAB MS, and data were compared by a one-tail t test (29). Oxygen Dissociation Pooled blood, in four flasks, was dosed in triplicate with 50 pJ of either molinate (as above) or KCNO (0.28 + 0.02 mg ml-‘), both in diethyl ether, to create a blood concentration of 34.17 + 2.50 nmol ml-’ (molinate, as above; KCNO, 2.77 * 0.20 pg ml- ‘); parallel diethyl ether controls were also run. Following I-hr incubation, each flask was pressurized with either a deoxygenated gas (100% N, or 99% N, + 1% CO,) or an oxygenated gas (100% air or 99% air + 1% COZ) (30, 31). After 1-hr equilibration, blood samples from two flasks were mixed in a single syringe, using MS0 , 2 MBA

MS0

I 0

I

I

10

Molinate

I

Time FIG. 3. Chromurogram

I

30

20

I

40

(h)

of u reference standard mixfure (see Table I for key to ubbreviutions). Mobile phase: isocratic water-methanol (40:60), 1 ml min-‘. Column, reverse-phase C,,. Detector, uv at 220 nm.

CROSBY

a metal mixing bead, to simulate “arterial” (0% CO,) or “venous” (1% CO,) blood (30, 3 1). Blood volumes were adjusted for bead, syringe, and needle deadspaces to give 0, 20, 50, 80, 95, and 100% oxygenated mixtures after 30-set mixing (30, 31). Oxygen tension (PO,) and pH measurements were made using an IL Model 113 pH/blood gas analyzer (Instrumentation Laboratory, Boston, MA) and an IL Model 127 thermostatted (1S’C) P,* and pH/ reference electrode system. The PO, and pH electrodes were calibrated with air/N, and an aneroid barometer to -to. 1 Torr and Harleco precision buffers (Gibbstown, NJ) to ?O.OOl pH units, respectively; data were compared using a one-tail t test (29). RESULTS

Disposition

and Metabolism

The measured whole blood 14C exposure concentrations were (in parent molar equivalents, N = 3): molinate, 33.54 +- 2.50 nmol ml-‘, MSO, 32.78 + 3.55 nmol ml-‘. They did not significantly differ (P > 0.05), and the average recoveries after 24 hr were: molinate, 98.16 ? 2.32%; MSO, 95.93 +2.20%. Mean HCTs, measured prior to chemical exposure, were 36.7 + 1.5% (for molinate exposure) and 35.0 ? 2.4% (for MS0 exposure), and mean hemoglobin concentrations, also measured prior to exposure, were 10.58 I 1.24 g% (for molinate exposure) and 9.82 + 1.58 g% (for MS0 exposure); differences were not significant (both, P > 0.05). While whole blood, plasma, and debris 14C levels were measured directly, erythrocyte levels were calculated as follows: C, = (C, - C,)( 1 - HCT)HCT-i; C,, concentration in erythrocytes; C,, whole blood concentration; and C,, plasma concentration (32). Accumulation of both molinate and MS0 by erythrocytes was rapid, nearing completion within

4 hr (Figs.

4 and 5); after I6 hr

apparent steady-state conditions prevailed. The erythrocyte 14C concentration following 24-hr exposure to molinate (52.48 +

DETOXICATION

04

: 0

: 2

4

: 6

8

, 10

12

14

OF

;

7

16

18

MOLINATE

r 20

IN

COMMON

0

2

29

CARP

, 22

24

Time (h)

FIG. 4. “C disposition between plasma, etyrhrocytes, and protein (cellular debris) by common turp whole blood daring molinate exposure. Bars represent standard deviation (N = 3).

1.51 nmol ml- ‘) was significantly higher than after exposure to MS0 (48.53 + 1.62 nmol ml- ‘; P < O.OS), while the plasma concentration (molinate, 22.51 + 1.26 nmol ml-‘, MSO, 24.30 rt 0.53 nmol ml-‘) was significantly lower (P < 0.05). For better comparison, the erythrocyte/plasma (E/P) 14C concentration ratios were plotted (Fig. 6) (32). Noticeable divergence occurred after 1 hr; after 24 hr molinate uptake was significantly greater than that of MS0 (molinate, 2.33 t 0.12; MSO, 2.00 + 0.13; P < 0.05). Cellular debris 14C levels (in nmol ml-’ of erythrocytes) also reached steady state rapidly (Figs. 4 and 5), although the

FIG. 5. 14C disposition between plasma. erythrocyte.s. and prorein (cellular debris) by common carp whole blood daring molinate sulfoxide exposure. Bars represent standard deviation (N = 3).

4

6

8

10

12

Ii

16

I.8

2.0

22

24

Time (h)

FIG. 6. Differences in the eryrhrocytelplasma concentration ratio during exposure to molinate .&oxide. Bars represent standard deviation (N

(E/P) and its = 3).

level after 24-hr exposure to MS0 (1.45 + 0.15 nmol ml-‘) was significantly greater than after exposure to molinate (0.32 + 0.04 nmol ml-‘, P < 0.01). Covalent binding was not determined. No breakdown products were detected in the saline or molinate-exposed plasma controls; however, in the MSO-exposed plasma controls (total 14C, 37.51 2 3.41 nmol ml- ‘) the following metabolites were found: MMA, 1.85 t 0.08 nmol ml- ‘, 4.93% of total 14C; MGSH, 0.44 + 0.21 nmol ml-‘, 1.17%; and MCYS, 0.23 t 0.13 nmol ml-‘, 0.62%. Tables 2 and 3 present metabolite profiles from molinate and MS0 exposure, respectively. After 24 hr in order of decreasing total amount, the following were found: from molinate exposure, MGSH, MCYS, MSO, and MMA; from MS0 exposure, MGSH, MMA, and MCYS. In erythrocytes, the main product was MGSH, while in plasma it was MGSH (from molinate) or MMA (from MSO). Total production of MMA, MCYS, and MGSH after MS0 exposure (5.18 + 1.79 nmol ml-‘, 15.8%) was significantly greater than following molinate exposure (2.92 + 0.27 nmol ml-‘, 8.71%, P < 0.05). Also, assuming only sulfoxidized forms were conjugated, the percentage of free MS0 following molinate exposure (10.70 ? 1.74%) was significantly lower than after MS0 exposure (84.20 ? 8.90%; P < 0.01).

30

TJEERDEMA

Molinate Metabolite

AND CROSBY

TABLE 2 Profiles following 24-hr Disposition by Common Carp Whole Blood “‘C disposition Erythrocyte

Component

nmol ml-‘”

Plasma %

Molinate Molinate sulfoxide Molinate mercapturic acid Molinate-L-cysteine Molinate glutathione

45.48 0.82 0.42 0.82 4.94

(8.44) (0.19) (0.24) (0.21) (0.16)

86.67 1.56 0.80 1.56 9.41

Totals

52.48 (3.51)

100.00

Total blood

nmol ml-‘” 21.42 0.08 0.11 0.61 0.29

%

(2.35) (0.04) (0.02) (0.21) (0.06)

95.15 0.36 0.49 2.71 1.29

22.51 (2.46)

100.00

nmol ml-‘” 30.27 0.35 0.22 0.69 2.00

%

(4.59) (0.10) (0.10) (0.21) (0.10)

90.25 1.04 0.66 2.06 5.99

33.54 (2.50)

100.00

a Mean (SD), N = 3.

Binding Reactivities

In organic solvent, recoveries of molinate (49.12 + 0.58 kmol, 98.24%), MS0 (48.83 k 0.21 prnol, 97.66%), and MSO,? (48.62 +- 0.34 pmol, 97.24%) were all uniformly high. In aqueous solvent, and when compared to the organic recoveries, while molinate was very stable (48.22 k 0.49 Fmol, 96.44%; P > O.OS),MS0 (4.31 k 0.02 pmol, 8.62%) and MS02 (0.00 * 0.00 p,mol, 0.00%) were extensively hydrolyzed (both, P < 0.01); differences between aqueous recoveries were significant (all, P < 0.01). Therefore, reactions of MS0 or MSO, in aqueous solvent compete with efficient hydrolysis. Molinate did not react measurably with either GSH or benzylamine (Tables 4 and 5). While both MS0 and MSO, readily formed MGSH and MBA, reactions were

Molinate

Sulfoxide Metabolite

significantly more efficient in organic solvent (all, P < 0.01; Tables 4 and 5). Also, MSO, always reacted more completely than MS0 (all, P < 0.01; Tables 4 and 5), and both MS0 and MSO, reacted most efficiently with GSH in either solvent (all, P < 0.01; Tables 4 and 5). Oxygen Dissociation

The mean HCTs and hemoglobin concentrations were: for the control, 37.0 + 1.0% and 10.76 + 0.14 g%; with KCNO, 38.3 + 0.8% and 10.62 * 0.10 g%; and with molinate, 36.3 + 1.5%and 10.51 + O.l6g%. The differences were not significant (all, P > 0.05). Oxygen dissociation curves are shown in Figs. 7 and 8. None of the arterial P,,‘s (oxygen tensions at 50% saturation) differed significantly (all, P > 0.05; Table 6); however, the venous P,, during KCNO

TABLE 3 ProjZes following 24-hr Disposition by Common Carp Whole Blood “‘C disposition Erythrocyte

Component

nmol ml-‘”

Plasma %

Molinate sulfoxide Molinate mercapturic acid Molinate-L-cysteine Molinate glutathione

35.28 0.42 0.21 12.62

(2.77) (0.21) (0.11) (4.49)

72.70 0.87 0.43 26.00

Totals

48.53 (9.62)

100.00

n Mean (SD), N = 3.

nmol ml ‘U 23.47 0.57 0.11 0.15

Total blood %

(0.27) (0.29) (0.02) (0.02)

96.58 0.49 0.45 0.62

24.30 (0.53)

100.00

nmol ml-‘” 27.60 0.52 0.15 4.51

%-

(1.15) (0.26) (0.05) (1.59)

84.20 1.58 0.46 13.76

32.78 (3.55)

100.00

DETOXICATION TABLE Reaction

Glutathione

OF

sulfoxide sulfone

%

Km01

%

0.00 (0.00) 30.14 (1.53)

0.00 60.28

0.00 (0.00) 24.56 (0.20)

0.00 49.12

34.83

69.66

26.21

52.42

(0.13)

(40 p1).

exposure was lower than that for either the control or molinate (both, P < 0.05; Table 6), which were not significantly different (P > 0.05; Table 6). Both arterial and venous pH (at 50% saturation) during KCNO exposure were reduced when compared to those of the control or molinate (all, P < 0.01; Table 6), which also did not significantly differ (both, P > 0.05; Table 6). To compare dissociation characteristics, Bohr shifts were calculated as follows: B = (Alog P,,)(ApH)’ (Table 6) (31, 33). The control shift (-0.98 + 0.17) was within the range of those previously reported (-0.91- 1.0) (34-40). The KCNO-induced shift ( -0.72 2 0.11) was substantially lower than those from the control (26.5%, P < 0.05) and from molinate exposure (-0.95 t 0.10, 24.2%, P < 0.05); the reduction was similar to that for horse hemoglobin (25%) (10). The control and molinate shifts were not significantly different (P > 0.05).

Reaction

blood oxygen during expoand molinate. ranged as and molinate,

fol-

DISCUSSION

Investigation of xenobiotic disposition and biotransformation in whole blood usually incorporates in vivo exposure (32, 41, 42), which is influenced by hepatic metabolism and excretion, or in vitro exposure, where chemical loss by volatilization may occur (24, 25, 43). As an aerated environment is essential, whole blood is usually aerated by agitation and CO, removed by a stream of humidified, compressed air (30, 31, 33, 44); for volatile chemicals such as molinate (vapor pressure, 5.6 x 10W3 Tot-r) (I), this is unsatisfactory. Therefore, whole

effkiency”,b

Organic solution‘

sulfoxide sulfone

FIG. 7. “Arterial” (0% CO,) whole dissociation curves from common carp sure to diethyl ether (contro& KCNO, Standard deviations (in Torr, N = 3) low: control, 0.0-3.9; KCNO, 0.1-5.2; 0.14.7.

TABLE 5 Reaction Efficiencies

Benzylamine

Molinate Molinate Molinate

31

Aqueous solutiond

u 100% efficiency = 50 )*mol. b Mean (SD). N = 3. c Ethanol (0.6 ml) and triethylamine (40 ~1). d Water (0.3 ml). ethanol (0.3 ml), and triethylamine

Component

CARP

effkiency”,b

+mol

(0.69)

COMMON

Ffjciencies

Organic solutionC

Molinate Molinate Molinate

IN

4

Reaction

Component

MOLINATE

Aqueous solutiond

pm01

%

pmol

5%

0.00 (0.00) 23.78 (0.23)

0.00 47.56

0.00 (0.00) 3.88 (0.04)

0.00

27.28

54.56

(0.34)

10.58

(0.13)

u 100% efficiency = 50 kmol. b Mean (SD). N = 3. c Ethanol (0.6 ml) and triethylamine (40 ~1). ’ Water (0.3 ml), ethanol (0.3 ml), and triethylamine

7.76 21.16

(40 ~1).

FIG. 8. “Venous” (1% CO?) whole dissociation curves from common carp sure to diethyl ether (control), KCNO, Standard deviations (in Torr, N = 3) lows: control, 0.1-4.7; KCNO, 0.2-4.4; 0.2-3.7.

blood oxygen during expoand molinate. ranged as folund molinute,

32

TJEERDEMA

AND CROSBY

TABLE P,, und pH

Values

of Common

Carp

6

Whole

Blood

following

Chemical

Exposure

Chemical exposure” Control

Cyanate

Molinate

Bloodb

P,, (Tom)

pH’

P,, (TOW

PH’

Arterial Venous

10.9 (0.4) 27.5 (5.7)

7.81 (0.01) 7.40 (0.01)

12.1 (3.6) 23.1 (4.9)

7.74 (0.03) 7.35 (0.01)

P,,

(Torr)

11.2 (2.4) 26.8 (2.2)

PH’ 7.82 (0.01) 7.42 (0.02)

a Mean (SD), N = 3. b Arterial, 0% CO,; venous, 1% CO?. ’ Blood pH at half-saturation (P,,).

blood in sealed Warburg flasks was held under compressed air while CO, was removed with KOH (22); the 14C recoveries and control Bohr shift indicate both control of volatilization and adequate blood aeration. Also, both gradient and isocratic HPLC optimized peak resolution and minimized analysis time. Since several metabolic products were thermally unstable, HPLC provided a nondestructive alternative to gas chromatography with greater sensitivity than TLC. While erythrocytes accumulated both molinate and MSO, molinate was absorbed to a significantly greater extent (Tables 2 and 3). Molinate was oxidized by erythrocytes to MSO, and possibly MSO?, then efficiently conjugated with GSH. While MMA probably resulted from enzymatic cleavage and N-acetylation of MGSH, MCYS was formed either by cleavage of MGSH or by direct conjugation. MS0 was conjugated similarly by both erythrocytes and control plasma (without erythrocytes); although metabolites of both molinate and MS0 originated mainly from erythrocytes (Tables 2 and 3), substantial levels of both GSH and L-cys (both oxidized and reduced) have been found in rat plasma, where they may be transported between tissues (4547). They may also contribute to xenobiotic conjugation in carp plasma. Xenobiotic oxidation can be catalyzed by oxyhemoglobin (48. 49); while not considered an enzyme because required concentrations are high and iron chelates can re-

place it (50), it has been implicated in styrene oxidation (25), lipid peroxidation (51), dealkylation of N,N-dimethylamineN-oxides (52, 53), dopa decarboxylation (54, 55), aniline hydroxylation (50, 56. 57), and retinoic acid 5,6-epoxidation (58). GSH occurs in human erythrocytes at about 2.08 pmol ml-‘; approximately 90% is in the reduced form (59-61). In its oxidized form, GSSG, it is transported out of erythrocytes (62); GSH may protect erythrocytes from oxidative damage, and transport may reduce cellular toxic effects (62). Also, glutathione S-transferase, which catalyzes GSH conjugation with electrophilic compounds, is in human erythrocytes (63), and y-glutamyl transpeptidase, which cleaves glutamate from GSH in the first degradative step toward L-Cys, and finally mercapturic acid, conjugate formation, is in human plasma (64). Erythrocytic glutathione S-transferase differs from hepatic forms in amino acid composition (63); both may be present in carp blood. Molinate did not react with GSH or benzylamine in either aqueous or organic solution; only the sulfoxidized forms were conjugated nonenzymatically. Therefore, conjugates formed in blood were probably derived from sulfoxidized molinate. Most of the molinate sulfoxidized by erythrocytes were conjugated: however, following MS0 exposure most of it remained unchanged (active). The difference may indicate saturability of the enzyme conjugation system; although the sulfoxidized products

DETOXICATION

OF

MOLINATE

are efficient carbamylators, the GSH pool was probably too large to be exhausted. Also, the debris residue, largest following MS0 exposure, may represent amine carbamylation. Finally, while KCNO reduced blood pH, oxygen dissociation, and the Bohr shift, molinate did not. Therefore, molinate at levels normally toxic to carp appears to exert its toxic effect by a different mechanism. Stable thioethers formed by GSH conjugation are transported out of human erythrocytes 10 times faster than GSSG (65). In addition to their gas transport function, erythrocytes may act as circulating detoxication packets which remove harmful substances from plasma, detoxify them with GSH, and return the conjugates to be further metabolized elsewhere (65). Molinate is detoxified by erythrocytes in the same manner. High levels of MS0 or MSO, saturate the system; hemoglobin carbamylation, producing toxic effects similar to those caused by KCNO, may result. This may explain why MS0 and MSO, are more toxic than molinate at very high levels (4 mg liter - ‘) (9). Throughout this investigation sample sizes were limited to three replicates; while the minimum sizes may have introduced a significant degree of uncertainty to the results, for practicality and efficiency they were deemed appropriate. Variations between individuals were not very large, as can be determined from the standard deviations. In conclusion, molinate does not produce significant reductions in carp whole blood oxygen dissociation, pH, or the Bohr shift at normally toxic levels. In erythrocytes it is sulfoxidized, then efficiently detoxified with GSH; significant hemoglobin carbamylation cannot occur. MSO, at high levels, saturates GSH conjugation, leaving a substantial amount for possible hemoglobin carbamylation. Therefore, toxic effects may result. At observed toxic levels, however, another mechanism of toxicity is active and should be investigated.

IN

COMMON

CARP

33

ACKNOWLEDGMENTS

We thank Joseph J. Cech, Jr. (UCD) for equipment and advice; Jeffrey L. Miller (Stauffer Chemical Co.) for samples of molinate, 4-hydroxymolinate, and 4ketomolinate; and Jeffrey M. Macdonald (UCD) for assistance. Support was provided by NIEHS Training Grant ES 07059. REFERENCES

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