Studies on the interaction of methomyl and ethanol in rats

OOIS-h?M 79 0801.033310?.00 0

STUDIES ON THE INTERACTION OF METHOMYL AND ETHANOL IN RATS M.ANTAL,M.BED&G.

CONSTANTINOVITS, K.NAGY

and J.SZkPVi)LGYI Institute of Nutrition,

Cyrili

ut 3/a, 1097 Budapest,

(Received I? December

Hungary

197X)

Abstract-The effects of feeding methomyl (200 mg/kg diet) and ethanol (10% aqueous solution) both separately and together were studied in R/Amsterdam rats. Response to the combined administration of the two agents produced evidence of some interactions. resulting in a decrease in growth in both male and female rats and an increase in the relative weights of the adrenals. Sex differences were apparent, however, in some of the responses to the combined treatment: only in the male rats were hepatic levels of triglyceride and free fatty acids increased and the cholinesterase activity of the brain decreased to a level significantly below those associated with administration of either agent separately, whereas in the females the relative weights of the kidneys and the fasting level of blood-glucose were increased.

INTRODUCTION Methomyl (I-methylthioacetaldehyde-O-(methylcarbamoyl)oxime; Lannate@) is a broad-spectrum insecticide. In Hungary, the granting of approval for the widespread application of methomyl is now under consideration. According to the Information Bulletin issued by the Du Pont Company, the oral LDso is 17-24mg/kg for starved male or female rats and 37 mg/kg for fed rats. Studies on labelled methomyl have indicated rapid metabolism and no accumulation of the insecticide, which is eliminated from the organism in 24 hr as carbon dioxide, acetonitrile and an unidentified urinary metabolite. A potential acute hazard is the inhibition of cholinesterase. In an earlier study in this laboratory, the LDSo determined for methomyl in ‘rats of our own stock was the same as that reported by the Du Pont Company. In a subacute feeding study in rats, the no-effect level for methomyl has been calculated as 100 mg/kg diet, a value also in agreement with corresponding data from Du Pont. On feeding a dietary level of 200 mg methomyl/kg, serum levels of total lipid and cholesterol were raised in the rats. The purpose of the work now described was to study the combined effects of methomyl and a second toxic agent, ethanol, on growth and some metabolic parameters of rats. EXPERIMENTAL Chemicals. Methomyl 0-(methylcarbamoyl)oxime; bamoyl)oxy]thioacetimidate), was a generous gift from and Co. Inc., Industrial ment, Wilmington, DE, white crystalline powder

(l-methylthioacetaldehydeS-methyl-N-[(methylcarwith a purity of 90x, E. I. Du Pont de Nemours and Biochemicals DepartUSA. The substance was a with a mild odour of sul-

@‘Registered trade-name of the Du Pont Company. Wilmington, DE, USA. 333

phur, melting at 78-79% and readily soluble in water and organic solvents. Ethanol (96%) was glass-distilled before use. Animals and diet. Lean R/Amsterdam albino rats, which are not prone to gain weight, were housed in wire cages. Room temperature was 20 f 1°C with a relative humidity of 50-60%. The animals were fed a semi-synthetic diet containing (per kg): casein 180 g, sucrose 368 g, wheat starch 368 g, sunflower oil 40 g, salt mixture 34 g and vitamin mixture 10 g. The 34 g of salt mixture consisted of CaHP04.2H20 25.76 g, KHCOJ 3.417g. KH2P0, 1.625g, NaCl 1.27g. MgC03 1.387 g, CuS0,.5Hz0 19.6 mg, KI 0.196 mg, C,HSFe0,.3Hz0 133.8 mg, MnCI, 114.5 mg and ZnCl, 25 mg. The vitamin mixture comprised choline chloride 2000 mg, m-inositol 300 mg, DL-calcium pantothenate 20 mg, p-aminobenzoic acid 5 mg, nicotinic acid 20mg, biotin O.lOmg, folic acid 1.5 mg, thiamine. HCI 4 mg, pyridoxine. HCI 3 mg, riboflavin 6 mg, a-tocopherol 120 mg, menadione 30 mg. retinol 12 mg and cholecalciferol 1000 IU, made up to 10 g by the addition of glucose. Experimental design and conduct. R/Amsterdam albino rats were divided into four groups of ten males (weighing 175-220 g) and ten females (110-150 g). The control group received the semi-synthetic diet and tap-water, a second group was fed basal diet to which 200mg methomyl/kg had been added, the third was given untreated diet and drinking fluid containing 10% ethanol, while the fourth received the 200mg methomyl/kg diet and 10% ethanol as drinking-fluid. The semi-synthetic diet was made semi-solid by addition of fluid (0.5-O6ml/g), tap-water for the first two groups.and 10% aqueous ethanol for the other two. Food and fluid intakes were recorded daily and the rats were weighed fortnightly. At week 12. haematocrit and fasting blood-glucose levels were determined, using the Galenopharm Gluco GOD/POD/ PAP enzymatic test method, and at the end of week 12 the animals were killed by decapitation and the liver, kidneys, adrenals, heart and spleen were weighed and studied by light microscopy. After rapid removal

334

M. ANTAL.

M.

BEDS, G. CONSTANT~NOV~~

and weighing of the’liver, samp s were dissected out, weighed and processed for bi d”chemical studies. Brain tissue was also removed for study. Studies of liver biochemistry. Liver glywgen was estimated by the enzymatic (Galenopharm) determination of glucose formed following hydrolysis with sulphuric acid (Good, Kramer & Somogyi, 1933). Liver samples were extracted by the method of Folch, Lees & Sloane-Stanley (1957) for the estimation of triglycerides (Vartinyi Nagy. 1974) and free fatty acids (Mosinger, 1965). For the determination of gluwse&phosphate dehydrogenase (G6PDase) and glucase-dphosphatase (G6Pase),the liver was homogenized in Q25 ~-sucrose in a glass-teflon tube. G6PDase activity was assayed in the 105,OOOgsupematant by the method described by Glock & McLean (1953), as modified by Liihr & Waller (1974), and G6Pase activity was determined in the microsomal fraction (Baginski, Foil & Zak, 1974). For mixed-function oxygenase assays,liver was homogenized in 1.15% KC3 and aminopyrine demethylase and aniline hydroxylase activities were determined (Kato & Gillette, 1965). Protein contents of the different subcellular fractions of liver were estimated by the method of Lowry, Rosebrough, Farr & Randall (1951). Brain cholinesterase.The activity of brain cholinesterase was determined by the method of Ellmann, Courtney, Andres % Featherstone (1961) as modified by Simon & Winter (1976). Statistical analyses. Data were analysed for statistical significance by the Student’s t test.

K. NAGY

and J. Szti~viSu;ur RESULTS

The rate of body-weight gain for both the male and female groups of rats receiving both methomyl and ethanol was considerably lower than that for the other groups, the response of the female rats being the more marked. The mean weight of the male rats became significantly different from that of the controls from week 4 (P < 001 at week 4 and
35a IMales 300

-

1‘ / ,

I4 ,

2

0

4 Duration

Fig. 1. Growth diet

(0)

or diet

curves of containing

6

6 of

feeding,

10

12

wk

groups of rats given, for 12wk, tap-water nnd either a semi-syntheticcontrol 200 mg

methomyl/kg (V).

control diet ( x ) or the methomyl diet

(o),

10%

ethanol

as drinking-fluid

with

either

the

Interaction

of methomyl

and

ethanol

in rats

335

6

aa:: .-E

6-

z 2 E

5-

.g

3-

4

-

2

-

I

0

2

I

4

6

Duration Fig.

2. Intake

of drinking-fluid

by groups

of

of rats

feeding, given (0).

diet (0) or diet containing 200mg methomyl/kg the control diet (x) or the methomyl diet (V).

females of the methomyl/ethanol-treated group were significantly higher than those for the other groups (Table 3). In the males the relative weights of the spleen were augmented by ingestion of ethanol or methomyl separately, but no further significant increase occurred in response to combined administration. The relative weight of the liver was significantly increased in the female rats given ethanol or methomy1 plus ethanol, and the relative weight of the kidney was increased in females by the separate administration of the two agents and was increased further by the combined administration (Table 3). No pathoTable 1.

Mean

dtrily Total

juid fluid

and food intake*

Food

body

Group

Control Methomyl Ethanol

and

either

qfrats

weight/day)

treated

with

methomyl

and/or

Methomyl intake (me/1OCJg body

weight/day)

Males 5.15

6.19 6.60

a semi-synthetic

or 10% ethanol as drinking-fluid

consumption*

body

w k

tap-water

(!ivl~g

weight/day)

12

control

with either

logical changes were seen in either organ by light microscopy. The fasting blood-glucose levels were similar in all the male groups, whereas in the females ethanol treatment resulted in a lower value and the methomyl plus ethanol treatment was followed by a significantly higher fasting blood-glucose level than those in controls (Table 2). No appreciable differences \?rere seen in hepatic glycogen among the various groups (Table 4). The triglyceride content of the liver was significantly higher than the control level in the methomyl-treated males and was further increased follow-

consumption

(ml/100 g

10

8

ethunol

Ethanol intake body

-

two !z weight/day)

5.41

I .08

-

544 5.70

I.14

0.46 0.42

Methomyl/ethanol

5.73 5.25

Control Methomyl

7.70 760

Females 6.15 6.45

I .29

-

Ethanol

7.20 5.52

6.00 6.42

I ,28

057 044

Methomyl/ethanol *Total

fluid the means

(drinking fluid and dietary for groups of ten animals.

diluent)

and

food

consumption

values

are

those

recorded

at week

I2 and

are

336

M. ANTAL, M. B~ti, G. CONSTANTINOVITS,K. NAGY and J. s~epvi.jL~v~

Table

2. Effect of ingestion of methomyl undlor erhunol for I2 wk on the haemutocrit and blood-glucose leuels of ruts

Group

Haematocrit (%I

Glucose bgP ml)

Control Methomyl Ethanol Methomyl/ethanol

Males 49.7 + 1.47 48.5 If: 1.72 48.8 f 1.48 49.0 f 1.73

107 f 116 f 111 f 109 *

8.1 10.7 14.3 8.3

Control Methomyl Ethanol Methomyl/ethanol

Females 49.0 + 1.59 51.1 * 1.91 51.6 f 1.57 51.5 & 2.00

96 f 83 f 68 * 127 f

9.1 7.9 14.3** 24.9**

Values are expressed as the means + SD for groups of ten rats, and those marked with asterisks differ significantly (Student’s I test) from the control value: **p < 0.01.

Table 3.

Relative

weights

organ

of rats

ing the combined administration. The free fatty acid content was decreased by methomyl but increased significantly by combined administration of methomyl and’ethanol. No changes in lipid metabolism were apparent in the treated female rats (Table 4). G6PDase activity was reduced significantly in the livers of methomyl-treated male and female rats (Table 5). The effect of methomyl appeared to account for a similar depression of G6PDase activity in the methomyl/ethanol-treated groups. Microsomal G6Pase activity was increased only in the females treated with ethanol or with methomyl plus ethanol. Of the drug-metabolizing enzymes tested, only aniline hydroxylase was increased both in males and females given ethanol or methomyl plus ethanol (Table 5). The increases in both of these enzymes following the combined treatment appeared to be attributable to the ethanol (Table 5). Brain cholinesterase activity was decreased in the male rats by administration of methomyl or ethanol and a further reduction occurred on combined admini-

treared

with

methomyl

and/or

ethanol

for

I2 wk

Relative organ weight (g/100 g body weight) Liver

Group

f f f +

Kidneys

Control Methomyl Ethanol Methomyl/ethanol

3.58 3.73 3.81 3.71

041 0.81 0.32 0.22

Control Methomyl Ethanol Methomyl/ethanol

Z90 & 0.26 3.02 +_ 0.18 3.18 f 0.12* 3.28 + 0.32*

Adrenalsj

Heart

Spleen3

0.78 0.80 0.74 0.87

f f f +

Males 0.12 0.12 0.03 0.04

15.21 k 15.26 + 15.50 + 19.48 f

D94 1.82 1.18 2.15**

0.32 0.32 0.33 0.35

+ + + +

0.03 0.05 0.03 0.03

97.00 118.90 116.90 124.00

f k + k

11.46 15.76** 7.90*’ 14.90**

0.67 0.74 0.74 0.90

k + f +

Females 0.03 0.03** 0.04** 0.07t

24.60 25.63 24.83 28.80

2.90 3.98 3.37 2.64

0.34 0.36 0.36 0.37

f k f f

0.03 0.03 0.03 0.01

159.40 164.00 16920 17480

+ f + +

23.99 35.88 23.88 38.86

f + + f

SWeights of these organs are expressed in mg/lOO g body weight. Values are expressed as the means I!Z SD for groups of ten rats, and those marked with asterisks differ significantly (Student’s t test) from the control value: *P < O-05; **P < 0.01. The value marked with an arrow differs significantly from the’ values from the methomyl- and ethanol-treated groups: tP < 0.01.

Table

4. Hepatic

triglyceride,

free

farry

acid and glycogen levels ethanol for I2 wk

in rats

treated

with

methomyl

and/or

Triglyceride (mgig liver)

Free fatty acid (pmol/g liver)

Glycogen (mg/g liver)

Control Methomyl Ethanol Methomyl/ethanol

Males 23.50 +_ 4.87 33.00 f 5.42* 26.25 f 4.78 39-90 + 10.40**

33.90 27.39 38.08 43.30

+_ 5.50 + 600* ?; Il.20 f 2.89”

14.00 + 14.00 + 12.20 + 14.90 +

0.06 0.45 0.60 0.97

Control Methomyl Ethanol Methomyl/ethanol

32.13 31.20 30.28 34.93

27.82 27.28 27.10 25.70

f f + +

15.02 k 14.70 & 14.10 * 14.00 +

2.00 I.80 2.10 I.90

Group

+ f f +

Females 5.50 7.70 7.10 8.32

3.94 4.50 5.30 5.20

Values are expressed as the means f SD for groups of ten rats, and those marked with asterisks differ significantly (Student’s t test) from the control value: ‘P < 0.05; l *P -C 0.01.

Interaction of methomyl and ethanol in rats Table 5.

Chonyes

ill rhe

aclioilies

of certain

hepotic

enzymes in ruts 1-7 Wk

ufier

treatment

337 with

merhomyl

und/or

e~honol

for

Enzyme activities? Group

G6PDase

G6Pase

Control Methomyl Ethanol Methomyl/ethanol

0.088 0,062 0.076 0.063

k f + +

0.006 0.009** 0,015 0408**

Males 0.23 0.26 0.25 0.23

Control Methomyl Ethanol Methomyl/ethanol

0144 0,105 0.142 0,094

+ + f *

0.016 0,022*** 0.018 0.013***

Females 0.11 f 0.12 + 0.18 + 0.19 *

f. f f f

AH0

APDM

0.02 0.03 0.03 0.02

oJJO9 * 0.001 0~010 + 0.002 0.013 f 0.002** 0.013 + 0.003**

0072 0064 0.073 0.070

f 0.014 k 0.014 &- 0.005 + 0008

0.02 0.03 0,03** 0.0321

0.006 k 0401 0006 & 0001 0008 k OQOl* 0,009 * 0.002**

0036 f O-034 i: 0.03 1 + 0.041 f

0.012 04Kl9 0.009 0.013

tG6PDase = Glucose-6-phosphate dehydrogenase (pmol NADPH/mg protein/min); G6Pase = glucose-&phosphatase (pmol Pi/mg protein/min); AH0 = aniline hydroxylase (pmol p-aminophenol/mg protein/hr); APDM = aminopyrine demethylase (pmol formaldehyde/mg protein/hr). Values are expressed as the means + SD for groups of ten rats and those marked with asterisks differ significantly (Student’s t test) from the control value: *P < 0.05; **f < 0.01; ***P < 0401. stration (Table 6). No such additive effect was seen with the females, in which a similar reduction in cholinesterase activity occurred in ail the treated groups. DISCUSSION

Our previous studies showed that methomyl interfered slightly with lipid metabolism. In the present investigations the dose of methomyl was chosen accordingly. Earlier results from this laboratory also showed that ethanol-the so-called second toxic agent-failed to influence metabolic functions in the liver of this strain of rat when offered as the drinkingfluid even in a concentration of 15%. The responses to treatment with either agent applied separately were essentially the same in the present studies as were observed previously. At the same time, results of the combined administration of mkthomyl and ethanol suggest that the actions of these two agents are additive in some respects. Although the animals were pair fed, it is notable that the rates of body-weight gain for both males and females given the combined treatTable 6. Chunyes in bruin trcerylcholinesterrrse uctiuiry iu rats following ingestion oJmerhomy/ tmdlor e~hrrnoljir I? wk

Brain acetylcholinesterase activity1 Group

Males

Females

Control

2.09 f @06

I.93 f 0.12

Methomyl Ethanol

1.80 f 0.04*** I.88 + 0.14**

I.49 I.52

Methomyl/ethanol

I.36 + O,IOt

I.46 f 0.1 I+**

f 0,04*** + 0,27**

$Expressed as bmol acetylthiocholine iodide/mg brain/min. Values are expressed as the means + SD for groups of ten rats, and those marked with asterisks differ significantly (Student’s t test) from the control values: **P < 0.01: ***P < 0.001. The value marked with an

arrow diRers significantly from those for the methomyland ethanol-treated

groups:

tP

< OCOI.

ment were significantly lower than for the other groups. The relative weights of adrenals were increased in both males and females given the two compounds. Mallov & Bloch (1956). using a higher ethanol dose than we did, were able to induce adrenal hypertrophy in a long-term study and suggested the involvement of the adrenal cortical hormones in the development of fatty liver. Under the experimental conditions now described, ethanol per se failed to influence the hepatic triglyceride content in the male rats while methomyl increased it, and a combination of the two agents resulted in a marked increase. It is possible that the adrenals are involved in the mechanism underlying this effect. The increase in the relative liver weight in both groups of female rats given ethanol was not considered to be of toxicological significance and. like the increase in relative kidney weights seen in all the treated females, it was not accompanied by any pathological changes detectable by light microscopy. In well-fed humans and animals, ethanol is known to induce a slight and transient hyperglycaemia which has been attributed in part to the release of epinephrine. Ethanol ingestion also causes hypoglycaemia during starvation, by blocking gluconeogenesis (Lundquist, 1971). a point that may account for the lower fasting level of blood glucose in ethanol-treated female rats. The cause of the higher blood-glucose level in the females treated with methomyl plus ethanol remains to be elucidated, but the role of catecholamines is a factor to be taken into consideration. Smyth, Martin, Moss & Beck (1967) have shown that the long-term administration of ethanol decreases brain-cholinesterase activity, and the same effect was observed with ethanol in the present study. The cholinesterase-inhibiting action of methomyl is well known. A striking finding in the work now reported, however, was the difference in the response of male and female rats to the combined administration of the two agents (Table 6). While the cholinesterase activity in the brain of male rats was significantly lower after the combined administration than

338

M. ANTAL, M. BED& G. CONSTANTINOVITS,K. NAGY and J. SZBPV~~LGYI

after the ingestion of either agent lone, no such additive action was seen in the feIwf les. The results of this study indicate that ingestion of these two agents for 3 months may result in interactions, even when the ethanol is administered in a relatively low concentration. REFERENCES Baginski, E. S., FOB P. P. & Zak. B. (1974). Methods for determination of enzyme activity. In Methods of Enzymatic Analysis. Edited by H. U. Bergmeyer. 2nd Ed. p. 876. Verlag Chemie, Weinheim Academic Press, Inc., New York. London. Ellmann, G. L., Courtney, K. D, An&es, V. J. & Featherstone, R. M. (1961). A new and rapid calorimetric determidation of acetylcholinesterase activity. Biochem Pharmat. 1, 88. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem 226, 497. Glock, G. E. & McLean, P. (1953). Further studies on the properties and assay of gkJcose-Cphosphate dehydrogenase and .6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55, 400. Good, C. A.. Kramer, H. & Somogyi. M. (1933). The determination of glycogen. .J. biol. Chem. 100, 485.

Kato, R. & Gillette, J. R. (1965). Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmac. exp. Thu. 150, 279. Lihr, G. W. & Wailer, H. D. (1974). Methods for determination of enzyme activity. In Methods of Enzymatic Analysis. Edited by H. U. Bergmeyer. 2nd Ed. p. 636. Verlae Chemie. Weinheim Academic Press. Inc.. New YorkrLondon. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 11951). Protein measurement with the Folin Dhenol reagent. J.’ biol. Chem. 193, 265. Lundquist, F. (1971). Influence of ethanol on carbohydrate metabolism. Q. J. Stud. Akohol 32, 1. Mallov, S. & Bloch, J. L. (1956). Role of hypophysis and adrenals in fatty infiltration of liver resulting from acute ethanol intoxication. Am. J. Physiol. 184, 29. Mosineer. F. (1965). Photometric adautation of Dole’s micrid&erminatidn of FFA. J. Lipid kes. 6, 157. Simon, G. & Winter, M. (1976). The effect of sympatholytic and sympathomimetic agents on acetylcholinesterase and cholinesterase activity. in vitro. Biochem. Pharmac. 25, 881. Smyth, R. D., Martin, G. J., Moss, J. N. & Beck, H. (1967). The modification of various enzyme parameters in brain acetylcholine metabolism by chronic ingestion of ethanol. Expl Med. Surg. 25, 1. Varsinyi Nagy, M. (1974). Serum triglicerid m&&e. Oruosi Hetilap

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