The effect of phenobarbital induction on glutathione conjugation of diazinon in susceptible and resistant house flies

PESTICIDE

BIOCHEMISTRY

The Effect

AND

PHYSIOLOGY

19, 344-349 (1983)

of Phenobarbital Induction on Glutathione Conjugation Diazinon in Susceptible and Resistant House Flies T. HAYAOKAAND W.C.

of

DAUTERMAN

Toxicology Program/Department of Entomology, North Carolina State University, Raleigh, North Carolina 27650 Received November 9, 1982; accepted January 12, 1983 The relationship between glutathione S-transferase activity toward 3,Qdichloronitrobenzene and 0-alkyl or 0-aryl conjugation of diazinon was investigated in eight strains of house flies. No significant difference was found in the amount of 0-aryl conjugation. In contrast, house flies which had higher glutathione S-transferase activity toward 3,4-dichloronitrobenzene also had higher 0-alkyl conjugating activity toward diazinon. The glutathione S-transferase(s) in phenobarbital-pretreated flies degraded diazinon faster than those in the nontreated ones. The present results showed that the formation of the 0-alkyl conjugate was enhanced by phenobarbital pretreatment, while the formation of the 0-aryl conjugate was not affected by induction. Based on these findings, it would appear that one of the multiple forms of glutathione S-transferase is specitically induced and responsible for the increase in 0-alkyl conjugation. INTRODUCTION

Glutathione S-transferases (EC 2.5.1.18) play an important role in the degradation of organophosphorus insecticides (1). These enzymes catalyze the 0-alkyl and 0-aryl conjugation of diazinon with reduced glutathione (GSH) in mammals (2, 3) and insects (3, 4). Lewis (5) reported that diazinon was metabolized by the soluble fraction from house flies and suggested the degradation was one of the important mechanisms responsible for resistance. A subsequent study by Motoyama and Dauterman (4) confirmed the findings that more rapid degradation of diazinon by the soluble fraction from the resistant strains than from the susceptible strains occurred in the presence of GSH. They also found the formation of desethyl diazinon paralleled glutathione S-transferase activity toward 3 ,Cdichloronitrobenzene (DCNB). Furthermore, Motoyama et al. (6) reported that high levels of DCNB conjugation and 0-alkyl conjugation of diazinon were controlled by gene(s) on chromosome II, while the amount of 0-aryl conjugation of diazinon was at the same level in all substrains tested. These inter344 0048-3575183 $3.00 Copyright AU rights

@ 1983 by Academic Press, Inc. of reproduction in any form reserved.

strain comparisons and genetic studies revealed a certain relatinship between glutathione S-transferase activity and 0-alkyl conjugation of diazinon. Recently, like many drug metabolizing enzymes, the inducibility of house fly glutathione S-transferase has been demonstrated (7). Hayaoka and Dauterman (8) reported that pretreatment of house flies with phenobarbital (PB) resulted in an increase in the LD,, values of the insecticides evaluated. They assumed that the induced glutathione S-transferases played some role in the reduction of toxicity of these insecticides. It is important to evaluate the in vitro effect of phenobarbital pretreatment on the metabolism of insecticides in order to confirm this assumption. The present study was initiated to clarify the relationship between glutathione S-transferase activity toward DCNB and 0-alkyl conjugation of diazinon using phenobarbital induction as a tool. MATERIALS

AND

METHODS

House flies. Properties and background of the various house fly strains used in the present study have been described pre-

PHENOBARBITAL

INDUCTION

EFFECT

viously (g-10). The Takatsuki and CSMA are susceptible strains which have low levels of glutathione S-transferase activity toward DCNB. The Orlando-DDT, Rugers, Fc, and Dimethoate 49r2 strains are resistant to organophosphorus insecticides and have higher levels of glutathione S-transferase activity than the susceptible strains. The Hirokawa and Cornell-R are resistant to organophosphorus insecticides and have much higher levels of glutathione S-transferase activity than the other resistant strains. The adults were maintained on a diet of milk and sugar. Chemicals. DCNB was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin, and GSH was obtained from Sigma Chemical Company, St. Louis, Missouri. Ring-labeled 14C diazinon (O,Odiethyl - 0 - [2 - [14C]isopropyl - 4 - methyl - 6pyrimidinyl] phosphorothioate) was kindly supplied by Ciba-Geigy Corporation, Greensboro, North Carolina, and had a sp act of 36.3 &i/mg. All other reagents and chemicals were of analytical grade. Treatment with phenobarbital. Adult house flies were fed 0.25% sodium phenobarbital (PB) in milk (w/v) for 24 hr on the first and third day after emergence (8). Twenty-four hours after the final treatment, house flies were used for assays. To avoid variations due to difference in size and vigor, the same generation was used for the treatment as for the control. Enzyme preparation. Female house flies were homogenized in 0.1 M Tris-HCl buffer, pH 8.0 (100 flies/l0 ml), using a motor-driven glass homogenizer. The homogenate was centrifuged at 10,OOOg for 10 min, and the supematant was recentrifuged at 100,000g for 1 hr. The supematant was filtered through glass wool and used as the enzyme source. In vitro metabolism of diazinon. The incubation mixture consisted of 1 ml of enzyme (equivalent to 10 female house flies), 0.5 pmol of the substrate in 10 ,xI of acetone, and 1 ml of 8 mM GSH. The reaction mixture was incubated for 1 hr at 37°C. The

ON

GLUTATHIONE

ACTIVITY

345

reaction was stopped by the addition of 2 ml of chloroform, and the two layers were separated by centrifugation. All reactions were corrected for nonenzymatic reaction using the enzyme denatured by heating. To identify the water-soluble metabolites, the components of the water phase were separated by thin-layer chromatography (TLC) of Polygram silica gel plates. The plates were developed to a height of 15 cm with acetonitrile and water (85 : 15 v/v) (4). Three components of the water phase were identified by cochromatography with known standards as GS-pyrimidinyl, desethyl diazinon, and diazinon. The amount of individual metabolites was quantitated in a liquid scintillation counter by scraping the silica gel from tic plates. Glutathione S-transferase assay. Glutathione S-transferase activity was determined using DCNB as substrate according to the spectrophotometric method of Booth et al. (11) as modified by Motoyama and Dauterman (12). The reaction mixture consisted of 20 ~1 of 60 mM DCNB, 100 ~1 of enzyme solution, 1.5 ml of 8 mM GSH, and 0.1 M Tris-HCl buffer, pH 9.0, in a total volume 3 ml. The reaction was run at 25°C. The change in absorbance at 344 nm was measured using a Beckman Acta V spectrophotometer and converted to nanomoles of DCNB conjugated using the extinction coefficient (E = 10 mM-’ cm-‘) for S-(2chloro-4-nitrophenyl) glutathione (13). All reactions were corrected for nonenzymatic conjugation. RESULTS

AND

DISCUSSION

The degradation of 14C ring-labeled diazinon via 0-aryl conjugation results in the formation of radiolabeled S-(Zisopropyl-C methyl-6-pyrimidinyl) glutathione (GS-pyrimidinyl) and nonradioactive diethyl phosphorothioic acid, while 0-alkyl conjugation results in the formation of radiolabeled desethyl diazinon and nonradioactive ethyl glutathione. The amount and percentage of the two radiolabeled metabolites formed in the vari-

346

HAYAOKA

Formation

of Water-Soluble from

AND

DAUTERMAN

TABLE 1 Metabolites of Diazinon by 100,OOOg Various House Fly Strains”

Water-soluble

metabolites

GS-pyrimidinyl Strain Takatsuki CSMA Orlando-DDT Rutgers Fc Dimethoate 49rz Hirokawa Cornell-R

nmoVlOO/hr 10.8 11.4 11.9 10.9 9.8

Supernatant

Desethyl diazinon

& SD

%

f 0.5 -+ 0.1 + 0.1 + 0.5 -+ 0.2

nmoUlO?/hr

43.9 45.7 36.2 32.0 26.4 23.2 15.9 11.9

11.1 + 0.7 10.1 2 0.4

10.5 * 0.3

13.8 15.9 21.0 23.3 27.5 36.9 53.5 77.4

-+ SD

f 0.4 + 0.2 f 0.3 it 0.4 k 0.1 c 0.6 +- 0.7 k 0.9

% 56.1 54.3 63.8 68.0 73.6 76.8 84.1 88.1

a Three replicates were used to determine means 2 SD.

ous house fly strains are presented in Table 1. No significant difference was found in the amount of GS-pyrimidinyl formed in the eight house fly strains studied. In contrast, about a twofold increase in desethyl diazinon formation was observed in the Orlando-DDT, Rutgers, Fc, and Dimethoate 49r, and up to a 5.6-fold increase in desethyl formation was observed in the Hirokawa and Cornell-R strains when compared to the susceptible Takatsuki strain. The amount of metabolite formed in each house fly strain was plotted against their individual glutathione S-transferase activity toward DCNB (Fig. 1). All strains evaluated produced GS-pyrimidinyl at the same rate, while the formation of desethyl diazinon increased in accordance with an increase in glutathione S-transferase activity toward DCNB. Recently, Clark and Dauterman (14) observed some qualitative differences in partially purified glutathione S-transferase between moderately resistant and highly resistant house fly strains using isoelectrofocusing. They postulated that the various strains might possess a multiplicity of glutathione S-transferase. Their finding might explain the difference in the ratio of 0-alkyl to O-aryl conjugation of diazinon in the various resistant strains if different forms of this enzyme catalyze either 0-alkyl or Oaryl conjugation.

Although glutathione S-transferase activity toward DCNB is inducible, the effect of induction on the conjugation of diazinon has not been investigated. The rate of formation of water-soluble metabolites of diazinon by the 100,OOOg supernatant from phenobarbital-treated and nontreated flies of the CSMA strain is shown in Fig. 2. The

I 0

I 40

I I I 60 120 I60 GSH S-TRANSFERASE (nmollindividuollmin)

I 200 ACTIVITY

I 240

I 260

FIG. 1. Relationship between DCNB conjugating activity in various house jZy strains and the formation of diazinon metabolites. a, Takatsuki; b, CSMA; c, Orlando-DDT; d, Rutgers, e, Fe; f, Dimethoate 49r,; g. Hirokawa; h, Cornell-R.

PHENOBARBITAL

0 20

40 INCUBATION

60

INDUCTION

80 TIME

EFFECT

(min)

FIG. 2. Rute offormation of water-soluble metabolites of diazinon by phenobarbital-pretreuted (0) and control (nontreated) (0) CSMA strain of houseflies.

results show that the 100,OOOg supernatant from pretreated flies degraded diazinon faster than those from nontreated flies. As these data only show the total amount of water-soluble metabolites of diazinon formed, the question as to which type of conjugation is inducible still remains. Therefore, the amount of individual metabolites was evaluated. The effect of phenobarbital pretreatment on the formation of the two glutathione TABLE Effect

of Phenobarbital

Pretreatment

ACTIVITY

on the Formation

2 of Water-Soluble

Water-soluble

Metabolites

CSMA Control Phenobarbital Rutgers Control Phenobarbital Fc Control Phenobarbital

nmol/lO?/hr

-c SD

of Diazinon”

metabolites

GS-pyrimidinyl Strain treatment

347

conjugates of diazinon in three house fly strains is summarized in Table 2. No significant difference was found in the amount of GS-pyrimidinyl formed by the induced enzyme and noninduced enzyme found in three house fly strains. In contrast, the formation of desethyl diazinon was enhanced by phenobarbital preteatment in all the strains evaluated. The formation of desethyl diazinon increased 2.5fold in the phenobarbital pretreated CSMA strain when compared to the control. In the Rutgers and Fc strains, the effect of phenobarbital pretreatment on the formation of desethyl diazinon was less than that in the CSMA strain. This result agrees with the previous report (8) which showed glutathione S-transferase activity toward DCNB in the CSMA strain is more inducible than in the Fc and Rutgers strains. The above results suggest that 0-alkyl conjugation of diazinon and DCNB conjugation are catalyzed by the same glutathione S-transferase. Although it is unknown whether 0-alkyl and 0-aryl conjugation of diazinon are mediated by distinct glutathione S-transferases in the house fly, the present results indicate that more than one enzyme is involved in 0-aryl and 0-alkyl conjugations. Is there a multiplicity of glutathione Stransferase in the house fly? Motoyama and Dauterman (15) reported that both 0-aryl

I20

100

ON GLUTATHIONE

Desethyl diazinon 9%

nmol/lOP/hr

2 SD

9.7 2 0.2 8.9 f 0.4

48.1 25.5

10.5 k 1.1 26.0 2 2.2

74.5

9.1 i

10.0 + 0.5

27.8 23.4

25.2 32.8

e 0.6 2 0.8

72.2 76.b

8.4 f 1.3 7.7 k 0.5

23.1 17.5

27.9 k 0.4 36.3 A 1.3

76.9 82.5

0.5

” Three replicates were used to determine means k SD.

51.9

348

HAYAOKA

AND

and U-alkyl conjugation were catalyzed by a form of glutathione S-transferase which was partially purified using DCNB as the substrate. They showed the house fly glutathione S-transferase has a broad substrate spectrum .and the ratio of 0-aryl and Oalkyl conjugation varied depending upon the structure of the compound. Broad or overlapped substrate specificity for organophosphorus insecticides have been reported with partially purified glutathione Stransferases in mammals and insects (16, 17). Oppenoorth et al. (18) reported that at least four different glutathione S-transferases active for I-chloro-2,4-dinitrobenzene (CDNB) could be separted on carboxymethyl cellulose columns. More recently, Clark and Dauterman (14) proposed that various house fly strains might possess a multiplicity of glutathione S-transferases, which has been substantiated by Motoyama et al. (19). If multiple forms of this enzyme exist in the house fly, phenobarbital might induce a specific form of glutathione Stransferase. Such a phenomenon has been reported by Stockstill and Dauterman (20), who demonstrated that one of the three GSH S-transferase peaks for DCNB conjugation obtained by isoelectrofocusing was more inducible than the others in the mouse. Therefore, it is possible that one of the multiple forms is specifically induced by phenobarbital and is responsible for an increase in 0-alkyl conjugation. To confirm this possibility, it will be necessary to separate the multiple forms and study the effect of phenobarbital pretreatment on them. ACKNOWLEDGMENTS

This is paper No. 8562 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650. The work was supported in part by Public Health Service, Research Grant ES00044, from the National Institute of Environmental Health Sciences. REFERENCES

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(1980).

DAUTERMAN

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Physiffl.

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EFFECT

thione S-transferase: Separation of transferases from fat bodies of American cockroaches active on organophosphorus triesters, Pestic. Biothem. Physiol. 7, 249 (1977). 17. K. Usui, T. Shishido, and T. Fukami, Glutathione S-transferases of rat liver active on organophosphorus triesters, Agric. Biol. Chem. 41, 2491 (1977). 18.

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strain of housefly and their influence on resistance. Pestic. Biochem. Physiol. 11, 176 (1979). 19. N. Motoyama, A. Hayashi, and W. C. Dauterman, The presence of two forms of ghrtathione S-transferases with distinct substrate specificity in OP-resistant and -susceptible housefly strains, in “Proceedings, 5th International Congress Pesticide Chemistry,” in press. 20. M. E. Stockstill and W. C. Dautermam, Studies on the induction of ghrtathione S-transferases in mouse liver, Drug Chem. Toxicol. 5,427 (1982).