Toxicology, 76 (1992) 69-77 Elsevier Scientific Publishers Ireland Ltd.

69

Hepatotoxicity of trichlorfon and dichlorvos in isolated rat hepatocytes Tetsuo Yamano and Shigeru Morita Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543 (Japan) (Received May 14th, 1992; accepted July 17th, 1992)

Summary Hepatotoxicity of organophosphorus insecticides, trichlorfon and dichlorvos, a dechlorinated form of the former, was examined in isolated hepatocytes from untreated control and phenobarbital-pretreated (80 mg/kg, i.p., for 3 days) rats. These compounds produced toxic effects on hepatocytes as evidenced by malondialdehyde production and lactate dehydrogenase leakage in a dose-dependent manner up to the concentration of 2 mM, dichlorvos being more toxic than trichlorfon. Hepatocytes from phenobarbitalpretreated rats were more sensitive to these organophosphates than those from control rats. Dichloroacetaldehyde and dichloroacetic acid, metabolites of dichlorvos, did not injure hepatocytes. The toxic effects of dichlorvos on hepatocytes were enhanced by increasing oxygen concentration during the incubation, or by addition of glycolytic substrates (pyruvate, lactate or fructose) to the incubation mixtures. On the other hand, addition of antioxidants (diethyldithiocarbamate or N,N'-diphenyl-pphenylenediamine), or cytochrome P-450 inhibitors (SKF-525A or metyrapone) to the incubation mixtures attenuated malondialdehyde production caused by dichlorvos and protected cells from death. Addition of dichlorvos to the incubation mixtures of hepatic microsomes stimulated lipid peroxidation in the presence of NADPH, which was inhibited by further addition of superoxide dismutase but not catalase. These results suggest that hepatotoxicity of trichlorfon and dichlorvos are related to their peroxidative property in microsomes which is accelerated by oxygen.

Key words: Lipid peroxidation; Organophosphates; Microsome; Liver

Introduction Organophosphorus insecticides are replacing organochlorine insecticides because the former are biodegraded more rapidly than the latter [1]. Numerous works have been made with respect to the toxicity of organophosphates, especially to their abilities to inhibit cholinesterase and their alkylating properties linked to a genotoxic risk [2]. Although liver has an important role in degradation or in some cases bioactivation of organophosPhates, the effects of these substances on liver have not been so well understood. The main reason is that doses of these substances to animals, to examine their effects on liver, are restricted because of their lethal action due to neurotoxicity. Gibel et al. [3,4] have reported liver damage including necrosis, Correspondence to: Tetsuo Yamano, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543, Japan. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

70 steatosis and cirrhosis after oral treatment of rats with trichlorfon (2,2,2-trichloro-1hydroxyethyl dimethyl phosphonate) and these findings have brought about a new view on risk evaluation of organophosphates. When incorporated into organism [5,6], trichlorfon is rapidly dechlorinated to dichlorvos (2,2-dichlorovinyl dimethyl phosphate), a much more potent cholinesterase inhibitor than trichlorfon [7]. There are several reports about increased activities in serum phosphatases and/or transaminases after acute or subchronic dichlorvos treatment in experimental animals [8,9]. Damage to liver organelles has been observed under electron microscopy after a single oral administration of dichlorvos to mice [10]. Ellinger [11] has concluded in his review that the risk of dichlorvos to health through its adverse effects on liver should not be ignored on the basis of increases in plasma levels of transaminases and other enzymes or alterations of lipid contents in liver after administration of the compound. In this study, the direct effects of two organophosphorus insecticides, trichlorfon and dichlorvos, on isolated rat hepatocytes were examined. The results showed a close relationship between hepatotoxicity and lipid peroxidation caused by these compounds. The role of lipid peroxidation in dichlorvos hepatotoxicity was studied in detail. Materials and methods

Materials Trichlorfon (2,2,2-trichloro-l-hydroxyethyl dimethyl phosphonate) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate), (99.5% and 99.0% purity by gas chromatography, respectively) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dichloroacetaldehyde and dichloroacetic acid were purchased from Tokyo Kasei Co., Ltd. (Tokyo, Japan). All other reagents used were of superior grade. Animals Male Sprague-Dawley rats (Slc:S.D., Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing between 180 and 250 g were used. They were maintained in a room of controlled temperature (23 ± 2°C) and relative humidity (60 4- 10%) with a 12-h light/dark cycle. They were allowed free access to food (MF, Oriental Yeast Co., Ltd., Osaka, Japan), if not stated otherwise. Tap water was freely available throughout the experiments. Sodium pentobarbital (50 mg/kg, i.p.) was used to anesthetize the rats. Experiments with hepatocytes Hepatocytes were isolated from either untreated-control or phenobarbital(PB)pretreated (80 mg/kg, i.p., for 3 days) rats after 24-h starvation using the method of Seglen [12]. The isolated cells were washed and suspended in Krebs-Henseleit buffer (pH 7.4). Cell death was indirectly determined by lactate dehydrogenase (LDH) leakage from cells into cell-free medium. Cell suspensions with more than 90% cell viability were used. Each 3 ml of the cell suspension (6-8 mg protein/ml) containing appropriate concentrations of various agents (see legends to figures and table) was incubated in 50-ml conical flasks with rubber stoppers at 37°C. The incubation atmosphere was set at either 95% 02:5% CO2, air, or 95% N2:5% CO2 in the flasks.

71

Experiments with microsomes Hepatic microsomes were isolated by the method of Cinti et al. [13] from PBpretreated (80 mg/kg, i.p., for 3 days) rats. Each 2 ml of the microsomal suspension (2 mg protein/ml 0.1 M Tris-HCl buffer, pH 7.5) containing appropriate concentrations of various agents (see legends to Fig. 4) was incubated for 20 min at 37°C for the determination of malondialdehyde production. Assays Non-protein sulfhydryl (NPSH) contents in hepatocytes were measured as an indication of glutathione using the method of Ellman [14] and expressed as nmol NPSH/mg protein, with reduced glutathione used as a standard. Activity of LDH was measured with Lactate Dehydrogenase CII-Test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and the activity in hepatocytes is reported as the percentage of the activity in cell suspensions. Lipid peroxidation in hepatocytes and microsomes was measured by the thiobarbituric acid method [15] and expressed in terms of malondialdehyde (MDA, nmol/mg protein), with tetramethoxypropane used as a standard. Cellular and microsomal protein was determined by a modified biuret method [161, with bovine serum albumin used as a standard. Statistics Comparisons between groups were made using Student's t-test or by one-way analysis of variance followed by Dunnett's or Scheff6's test. Results

Effects of trichlorfon, dichlorvos and their metabolites on isolated hepatocytes Incubation of isolated hepatocytes for 90 min with more than 1 mM trichlorfon or dichlorvos resulted in a significant loss of cell viability on the basis of LDH leakage from the cells (Table I). Dichlorvos was more toxic than trichlorfon at the same concentrations. Hepatocytes from PB-pretreated rats were more sensitive to both compounds than those from control rats, therefore, the subsequent experiments were carried out by using the former. Effects of these compounds and their metabolites on isolated hepatocytes from PB-pretreated rats are shown in Fig. 1. Contents of NPSH in hepatocytes decreased to less than 5 nmol/mg protein within 30 min of incubation with 2 mM trichlorfon, dichlorvos or dichloroacetaldehyde. On the other hand, an increase in MDA production followed by LDH leakage occurred by addition of 2 mM trichlorfon or dichlorvos, the latter causing more toxic effects on hepatocytes. Neither 2 mM dichloroacetaldehyde nor dichloroacetic acid affected cell viability. Effects of oxygen status, glycolytic substrates, antioxidants and cytochrome P-450 inhibitors on the toxicity of dichlorvos in isolated hepatocytes Contents of NPSH were rapidly depleted in isolated hepatocytes to less than 8% of the initial value within 30 min of incubation with 2 mM dichlorvos in all atmosphere conditions tested (data not shown). As shown in Fig. 2(A), incubation of hepatocytes with 2 mM dichlorvos in the atmosphere of 95% 02 caused a marked

72 TABLE I EFFECTS O F T R I C H L O R F O N A N D D I C H L O R V O S O N T H E VIABILITY OF H E P A T O C Y T E S I S O L A T E D F R O M C O N T R O L OR P H E N O B A R B I T A L - P R E T R E A T E D RATS Addition

Viability of hepatocytes %

None Trichlorfon

0.5 1.0 2.0 0.5 1.0 2.0

Dichlorvos

mM mM mM mM mM mM

Control

Phenobarbital

92 88 86 80 84 43 41

89 85 76 71 81 29 17

4444444-

1.2 1.3 3.9 a 1.7 a 3.8 5.5 a 2.8 a

4- 2.6 -4- 3.7 4- 5.3 a 4- 2.3 a,b 4- 1.7 4- 5.9 b 4- 2.1 a'b

Hepatocytes isolated from either untreated control or phenobarbital-pretreated rats were incubated for 90 min with 0.5-2.0 m M of trichlorfon or dichlorvos. Cell viability was determined indirectly on the basis of L D H leakage from cells into cell-free medium. Data represent mean 4- S.D. of four hepatocyte preparations, aSignificantly different from the hepatocytes from the corresponding control rats incubated without organophosphates, P < 0.05. bSignificantly different from the hepatocytes from untreated control rats incubated with the same concentration of organophosphate, P < 0.05. 100 r LDH

6 o r \ 0 12.5 •=

30

i 60

MD^

/

1o.o

90 •

~. 7.5 5.o

j 2.s 0.0 0 30 25 [ ~ ©

.E ~~. ~

60

90

20~ 10 13,~U~ C~ 15

o

5[

:

olo

~A

^

-..------_,,

30

A

60

90

Time ( m i n )

© control • dichlorvos • trichlorfon [] dichloroacotic acid • dichloroacataldehydo

Fig. 1. Effects of trichlorfon, dichlorvos, dichloroacetaldehyde and dichloroacetic acid on L D H leakage, M D A production and NPSH contents in isolated hepatocytes. Hepatocytes from phenobarbitalpretreated rats were incubated without any addition (control) or with 2 m M of each substrate. One experiment typical of three.

73 (A)

(B)

100 • LDH

100 " LDH

80

X'° o

6O 4O

40

2O

20

20 •~

60

I

I

!

30

60

90



0

0 20

MDA

15

"-=

I

I

!

30

60

90

A

©

MDA

15

B. lO

5

5

0

30 60 Time (min)

90

0

30 60 Time (min)

• dichlorvos (95~z:5~COz)

• dichlorvos

0 dichlorvos (air)

0

+ pyruvate

[] dichlorves (95~z:5%COz)

[]

+ lactate

A

+ fructose

90

Fig. 2. Effects of oxygen status (A) and glycolytic substrates (B) on dichlorvos hepatotoxicity in isolated hepatocytes. (A): Hepatocytes from phenobarbital-pretreated rats were incubated with 2 mM dichlorvos in the atmosphere of either 95% 02:5% CO2, air, or 95% N2:5%CO2. (B): Hepatocytes from phenobarbital-pretreated rats were incubated with 2 mM dichlorvos and with either 10 mM fructose, lactate or pyruvate. One experiment typical of three.

increase in LDH leakage from hepatocytes and MDA production. Dichlorvos in air (21% 02) had weaker effects on these parameters than in 95% 02. Dichlorvos in 95% N2 (no oxygen) slightly increased LDH leakage, but did not affect MDA production. Addition of several glycolytic substrates to the incubation mixtures of isolated hepatocytes has been reported to increase the cellular NADPH/NADP + ratio [17]. As shown in Fig. 2(B), addition of 10 mM fructose, lactate or pyruvate to the incubation mixtures potentiated both MDA production and LDH leakage caused by dichlorvos in order of decreasing potency. On the other hand, addition of antioxidants (20 /zM diethyldithiocarbamate or 2 #M N,N'-diphenyl-pphenylenediamine), or cytochrome P-450 inhibitors (50/~M SKF-525A or 500 ttM metyrapone) inhibited MDA production completely and protected cells from death (Fig. 3(A,B)).

74 (a)

(A)

lOO . LDH

100" LDH ,.© L1

80

60

6o

\.

40 2O o 12.5

20

I

I

I

30

60

90

o 12.5

MDA

.E IO.C

~-7.5

,/

5.0

"\

40

./



30 60 Time (min)



!

9O

MDA

o

~. 7.5

•/

5.0 2.5 /

0

I

6O

._ 1 0 . 0

2.5 0.0

!

30

90

dichlorvos

O

+ diethyldithiocarbamta

5

+ N,N'-diphanyl-p-phenylenediamine

./

~

o.o

~

0

30 60 Time (min)

@

90

• dichlorvos 0

+ SKF 525A + metyrapona

Fig. 3. Effects of antioxidants (A) and cytochrome P-450 inhibitors (B) on dichlorvos hepatotoxicity in isolated hepatocytes. (A): Hepatocytes from phenobarbital-pretreated rats were incubated with 2 mM dichlorvos and with either 20/~M diethyldithiocarbamate or 2 #M N,N'-diphenyl-p-phenylenediamine. (B) Hepatocytes from phenobarbital-pretreated rats were incubated with 2 mM dichlorvos and with either 50 #M SKF 525A or 500/zM metyrapone. One experiment typical of three.

Microsomal lipid peroxidation by dichlorvos Addition o f 200 # M dichlorvos to the suspension o f liver microsomes isolated from PB-pretreated rats did not increase inherent lipid peroxidation. However, coadministration o f dichlorvos with N A D P H significantly increased lipid peroxidation, which was markedly inhibited in the presence o f superoxide dismutase (02scavenger) but not catalase (H202 scavenger) (Fig. 4). Discussion

Trichlorfon and dichlorvos are both widely used organ©phosphorus c o m p o u n d s as insecticides and anthelmintics. Trichlorfon is rapidly converted in 'a nonenzymatic manner to dichlorvos [5,6], a more potent cholinesterase inhibitor than the parent c o m p o u n d [7]. Dichlorvos is metabolized in the liver to desmethyl

75

3-0

u

Ta

2.5--

"~

~. CD.

2.0

--

1.5

m

-rL T

U;

-

1

.~

o,0 s,.

•~

0.5

-IIII T

T

--

+

0.0 Dichlorvos NADPH

SOD Catalase

-

+

+

+

+

+

+

+

-

-

+

-

-

-

-

+

Fig. 4. Effects of NADPH, superoxide dismutase and catalase on hepatic microsomal lipid peroxidation by dichlorvos. Microsomes isolated from phenobarbital-pretreated rats were incubated for 20 min at 37°C with appropriate agents shown in the figure. The concentration of each agent was as follows: dichlorvos, 200 #M; NADPH, 1 mM; superoxide dismutase (SOD), 1500 units/ml; catalase, 1000 units/ml. Data represent mean 4- S.D. of three microsome preparations, asiguificantly different from microsomes incubated with NADPH, P < 0.05. bSignificantly different from microsomes incubated with dichlorvos and NADPH, P < 0.05.

dichlorvos in a glutathione-dependent or cytochrome P-450-dependent manner. Further, desmethyl dichlorvos is changed to dichloroacetaldehyde by liver soluble enzymes and then to dichloroacetic acid or dichloroethanol by alcohol dehydrogenase in the presence of NADH [18]. These metabolites are known to have less or no inhibitory effect on cholinesterase and higher LDs0 values than dichlorvos [19]. In this study, both trichlorfon and dichlorvos were toxic to isolated hepatocytes, the latter being more toxic. On the other hand, their metabolites produced no apparent hepatotoxicity. These findings suggest that hepatotoxicity of trichlorfon seems to be mediated by dichlorvos, which exerts its toxic effects directly or through metabolism to dichloroacetaldehyde. The present study revealed that incubation of isolated hepatocytes with dichlorvos caused loss of cell viability on the basis of LDH leakage into the medium in association with a marked decrease in glutathione and an increase in MDA production. LDH leakage and MDA production by dichlorvos were enhanced by increasing oxygen concentration during the incubation, but were completely inhibited by addition of antioxidants, diethyldithiocarbamate and N,N'-diphenyl-p-phenylenediamine. Many kinds of hepatotoxins have been shown to cause lipid peroxidation in liver, though lipid peroxidation is not always related to their toxicities [20]. Intracellular sources of reactive oxygen formation include xanthine oxidase, damaged mitochondria or microsomes [21-23]. In this study, PB-pretreatment increased the sensitivity

76

of hepatocytes to dichlorvos. Addition of glycolytic substrates to hepatocytes for increasing the NADPH/NADP ÷ ratio enhanced dichlorvos toxicity, whereas cytochrome P-450 inhibitors, SKF-525A and metyrapone inhibited its toxicity. Furthermore, microsomal lipid peroxidation was stimulated by a combined use of dichlorvos and NADPH. Ellinger [11] has reported an increase in cytochrome P-450 contents in liver after oral treatment of male rats with dichlorvos. Dichlorvos and other chlorinated organophosphates have been shown to change a paramagnetic form of hepatic cytochrome P-450 in rats [24]. Thus, it is probable that microsomal metabolism of dichlorvos generates active oxygen (maybe 02-) which causes lipid peroxidation related to cellular death. This conclusion is supported by the observation of damage of hepatic endoplasmic reticulum after a single oral treatment of mice with dichlorvos reported by Lee et al. [10]. However, the relationship between dichlorvos hepatotoxicity and lipid peroxidation deserves further examination, since MDA production, determined in this study, does not sufficiently reflect cellular lipid peroxidation [25] and in vivo studies there have not been reported any evidences of an increase in hepatic lipid peroxidation by trichlorfon or dichlorvos toxicities. In general, organophosphates are rapidly degraded in organisms and excreted. It is not probable that trichlorfon and dichlorvos cause liver disorder in usual environmental concentrations. However, as shown in this study, these compounds seem to have properties to enhance the toxicity of other hepatotoxins which are metabolized in a cytochrome P-450-dependent or glutathione-dependent manner. References 1

2 3

4 5 6 7 8 9 10 11 12 13

M.A. Gallo and N.J. Lawryk, Classification of organic phosphorus compounds, in W.J. Hayes Jr. and E.R. Laws Jr. (Eds.), Handbook of Pesticide Toxicology, Academic Press, New York, 1991, pp. 917-920. WHO, Organophosphorus insecticides: A general introduction. WHO, Geneva, 1986. W. Gibel, K. Lohs, G.P. Wildner and D. Ziebarth, Experimental studies on the hepatotoxic and carcinogenic activity of organophosphorus compounds. I. Trichlorfon. Arch. Geschwulstforsch., 37 (1971) 303. W. Gibel, K. Lohs, G.P. Wildner, D. Ziebarth and R. Stieglitz, Experimental studies on carcinogenic and hepatotoxic activity of organophosphate pesticides. Arch. Geschwulstforsch., 41 (1973) 311. T. Villrn, Determination of metrifonate and dichlorvos in whole blood using gas chromatography and gas chromatography-mass spectrometry. J. Chromatog., 529 (1990) 309. Y.A. Adbi and T. Villrn, Pharmacokinetics ofmetrifonate and its rearrangement product dichlorvos in whole blood. Pharmacol. Toxicol., 68 (1991) 137. J. Pachecka, A. Sulinski and K. Traczykiewicz, The effect of acute intoxication by dichlorvos and trichlorfon on the activities of some rat brain esterases. Neuropathol. Pol., 15 (1977) 85. A.K. Srivastava, R. Rainka, R.K. Chaudhary and J.K. Malik, Acute toxicity and biochemical alterations in rats after single oral exposure to dichlorvos. Pesticide, 23 (1989) 35. I.G. Kim, K.H. Koo and J.M. Lee, An experimental study on the influence of organophosphorus pesticides upon the liver. Hanyang Uidae Haksulchi, 5 (1985) 65. S.H. Lee, K.J. Lee, H.S. Chung and K.S. Lee, An electron microscopic study on the organelles of hepatocytes in dichlorvos treated mice. Hanyang Uidae Haksulchi, 7 (1987) 15. C.H. Ellinger, Is dichlorvos hepatotoxic? Z. Gesamte Hyg., 30 (1984) 156. P.O. Seglen, Preparation of isolated rat liver cell, in L. Wilson (Ed.), Methods in Cell Biology, Vol. 13, Academic Press, New York, 1976, pp. 29-83. D.L. Cinti, P. Moldeus and J.B. Schenkman, Kinetic parameters of drug-metabolizing enzymes in Ca2÷-sedimented microsomes from rat liver. Biochem. Pharmacol., 21 (1972) 3249.

77 14 15 16 17 18 19 20

21

22 23

24

25

G.L. Ellman, Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82 (1959) 70. J.A. Buege and S.D. Aust, Microsomal lipid peroxidation, in S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 52, Academic Press, New York, 1978, pp. 302-310. L. Szarkowska and M. Klingenberg, The role of ubiquinone in mitochondria. Biochem. Z., 338 (1963) 674. P. Moldeus, R. Grundin, H. Vadi and S. Orrenius, A study of drug metabolism linked to cytochrome P-450 in isolated rat-liver cells. Eur. J. Biochem., 46 (1974) 351. E. Hodgson and J.E. Casida, Mammalian enzymes involved in the degradation of 2,2-dichlorovinyl dimethyl phosphate. J. Agric. Food Chem., 10 (1962) 208. J.E. Casida, L. McBride and R.P. Niedermeier, Metabolism of 2,2-dichlorovinyl dimethyl phosphate in relation to residues in milk and mammalian tissues. J. Agric. Food Chem., 10 (1962) 370. P. Dogterm, J.F. Nagelkerke, J. Steveninck and G.J. Mulder, Inhibition of lipid peroxidation by disulfiram and diethyldithiocarbamate does not prevent hepatotoxin-induced cell death in isolated rat hepatocytes. A study with allyl alcohol, tert-butyl hydroperoxide, diethyl maleate, bromoisovalerylurea and carbon tetrachloride. Chem-Biol. Interact., 66 (1988) 251. H. Jaeschke and J.R. Mitchell, Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury. Biochem. Biophys. Res. Commun., 160 (1989) 140. B.J. Keller, H. Yamanaka, D. Liang and R.G. Thurman, Hepatotoxicity due to clofibrate is oxygendependent in the perfused rat liver. Toxicol. Appl. Pharmacol., 104 (1990) 259. S. Takekoshi, K. Sugioka, M. Nakano and K. Watanabe, Lipid peroxidation of microsomal membranes as the mechanism of 4-aminopyrazolopyrimidine-induced cell injury in the rat liver. J. Clin. Biochem. Nutr., 10 (1991) 4t. N.V. Kokshareva, L.M. Ovsyannikova and I.I. Samusenko, Effect of phenobarbital on the activity of a paramagnetic form of cytochrome P-450 and the survival of animals poisoned with some organophosphorus and chlorinated organic pesticides. Farmakol. Toksikol., 15 (1980) 120. L. Gabriel and H.P. Witschi, Chemicals, drugs and lipid peroxidation. Annu. Rev. Pharmacol. Toxicol., 16 (1976) 125.

Hepatotoxicity of trichlorfon and dichlorvos in isolated rat hepatocytes.

Hepatotoxicity of organophosphorus insecticides, trichlorofon and dichlorvos, a dechlorinated form of the former, was examined in isolated hepatocytes...
462KB Sizes 0 Downloads 0 Views